WO2019030706A1

WO2019030706A1 - Anti-thrombin antibody molecules and methods for use in orthopedic surgery

Info

Publication number: WO2019030706A1
Application number: PCT/IB2018/056011
Authority: WO
Inventors: Gary Peters; Juliane BERNHOLZ; Thomas Connolly; Anasuya HAZRA; Michael Lee
Original assignee: Janssen Pharmaceutica Nv
Priority date: 2017-08-10
Filing date: 2018-08-09
Publication date: 2019-02-14

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 antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other conditions mediated by thrombin. In particular, the present invention relates to the clinically proven safe and clinically proven effective use of the anti-thrombin antibody molecules in patients undergoing orthopedic surgery, including total knee replacement (TKR) surgery and total hip replacement (THR) surgery.

Description

ANTI- THROMBIN ANTIBODY MOLECULES AND METHODS FOR USE IN ORTHOPEDIC SURGERY

TECHNICAL FIELD

[ 0001 ] 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 antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other conditions mediated by thrombin. In particular, the present invention relates to the clinically proven safe and clinically proven effective use of the anti-thrombin antibody molecules in patients undergoing orthopedic surgery, including total knee replacement (TKR) surgery and total hip replacement (THR) surgery.

BACKGROUND OF THE INVENTION

[ 0002 ] 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.

[ 0003 ] 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.

[ 0004 ] 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 clinically proven safe and clinically proven effective treatment and prevention of thrombosis, embolism and other thrombin-mediated conditions.

SUMMARY OF THE INVENTION

[ 0005 ] 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.

[ 0006 ] In one embodiment, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 2.5 mg/kg.

[ 0007 ] In one embodiment, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe dose in the range of 0.03 mg/kg to 2.5 mg/kg.

[ 0008 ] In one embodiment, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 2.0 mg/kg.

[ 0009 ] In one embodiment, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg.

[ 0010 ] In one embodiment, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.3 mg/kg to 1.8 mg/kg.

[ 0011 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery.

[ 0012 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0013 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein the anti-thrombin antibody is administered at a clinically proven safe dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0014 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

[ 0015 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery.

[ 0016 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the single IV infusion of the anti-thrombin antibody is administered the day after the surgery.

[ 0017 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin-mediated condition is thrombosis or embolism.

[ 0018 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).

[ 0019 ] In another embodiment, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 2.5 mg/kg.

[ 0020 ] In another embodiment, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe dose in the range of 0.03 mg/kg to 2.5 mg/kg.

[ 0021 ] In another embodiment, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 2.0 mg/kg.

[ 0022 ] In another embodiment, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg.

[ 0023 ] In another embodiment, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.3 mg/kg to 1.8 mg/kg.

[ 0024 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery.

[ 0025 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single IV infusion at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0026 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single IV infusion at a clinically proven safe dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0027 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single IV infusion at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

[ 0028 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the single IV infusion of the anti-thrombin antibody is administered the day after surgery.

[ 0029 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin-mediated condition is thrombosis or embolism. [ 0030 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE). 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 HCDRl 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 HCDRl, HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising a LCDRl, 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 HCDRl, HCDR2 and/or HCDR3 to be replaced or lack a HCDRl, 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 HCDRl, 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 LCDRl having the amino acid sequence of SEQ ID NO: 7 with one or more amino acid substitutions, deletions or insertions and wherein said LCDRl 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; (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 HCDRl, HCDR2 and HCDR3,

wherein the parent VH domain HCDRl, 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) 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 HCDRl, HCDR2 and/or HCDR3 to be replaced or lack a HCDRl, 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 HCDRl, 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 CDRl, 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.

[ 0031 ] 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.

[ 0032 ] 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 HCDRl 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.

[ 0033 ] 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.

[ 0034 ] 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.

[ 0035 ] 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 HCDRl 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 HCDRl, 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.

[ 0036 ] An aspect of the invention provides an isolated antibody molecule that specifically binds to exosite 1 of thrombin.

[ 0037 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti -thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti -thrombin antibody administered at a dose in a range of 0.1 mg/kg to 2.5 mg/kg.

[ 0038 ] In certain embodiments, the present invention provides a clinically proven safe dose of an anti -thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.03 mg/kg to 2.5 mg/kg.

[ 0039 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 2.0 mg/kg. [ 0040 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg.

[ 0041 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.3 mg/kg to 1.8 mg/kg.

[ 0042 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg, and wherein the single intravenous (IV) infusion of the anti-thrombin antibody is administered the day after TKR surgery.

[ 0043 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin- mediated condition is thrombosis or embolism.

[ 0044 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin- mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).

[ 0045 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0046 ] In certain embodiments, the present invention provides a clinically proven safe dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg. [ 0047 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

[ 0048 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the single intravenous (IV) infusion of the anti- thrombin antibody is administered the day after TKR surgery.

[ 0049 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the thrombin-mediated condition is thrombosis or embolism.

[ 0050 ] In certain embodiments, the present invention provides a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15 for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, and wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).

[ 0051 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous

(IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 2.5 mg/kg.

[ 0052 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.03 mg/kg to 2.5 mg/kg.

[ 0053 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 2.0 mg/kg.

[ 0054 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg.

[ 0055 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.3 mg/kg to 1.8 mg/kg.

[ 0056 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg, and wherein the single intravenous (IV) infusion of the anti-thrombin antibody is administered the day after TKR surgery.

[ 0057 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous

(IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin-mediated condition is thrombosis or embolism.

[ 0058 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose in a range of 0.1 mg/kg to 1.8 mg/kg, and wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).

[ 0059 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0060 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.75 mg/kg, 1.8 mg/kg, 2.125 mg/kg, and 2.5 mg/kg.

[ 0061 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

[ 0062 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the single intravenous (IV) infusion of the anti-thrombin antibody is administered the day after TKR surgery.

[ 0063 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said safe and effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the thrombin -mediated condition is thrombosis or embolism.

[ 0064 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery, comprising administering a clinically proven safe and clinically proven effective dose of an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said clinically proven safe and clinically proven effective dose is a single intravenous (IV) infusion of the anti-thrombin antibody administered at a dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg, and wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).

[ 0065 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg.

[ 0066 ] In certain embodiments, the present invention provides a composition comprising an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one

pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin- mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

[ 0067 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg.

[ 0068 ] In certain embodiments, the present invention provides a method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose selected from the group consisting of 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

[ 0069 ] In certain embodiments, the invention as defined herein, comprises a clinically proven safe and clinically proven effective dose in a range of 0.1 mg/kg to 2.5 mg/kg, and preferably comprises a dose of 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.025, 1.05, 1.075, 1.1, 1.125, 1.15, 1.175, 1.2, 1.225, 1.25, 1.275, 1.3, 1.325, 1.35, 1.375, 1.4, 1.425, 1.45, 1.475, 1.5, 1.525, 1.55, 1.575, 1.6, 1.625, 1.65, 1.675, 1.7, 1.725, 1.75, 1.775, 1.8, 1.825, 1.85, 1.875, 1.9, 1.925, 1.95, 1.975, 2, 2.025, 2.05, 2.075, 2.1, 2.125, 2.15, 2.175, 2.2, 2.225, 2.25, 2.275, 2.3, 2.325, 2.35, 2.375, 2.4, 2.425, 2.45, 2.475 or 2.5 mg/kg.

[ 0070 ] In certain embodiments, the invention as defined herein, comprises a clinically proven safe and clinically proven effective dose in a range of 0.1 mg/kg to 2.0 mg/kg, and preferably comprises a dose of 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.025, 1.05, 1.075, 1.1, 1.125, 1.15, 1.175, 1.2, 1.225, 1.25, 1.275, 1.3, 1.325, 1.35, 1.375, 1.4, 1.425, 1.45, 1.475, 1.5, 1.525, 1.55, 1.575, 1.6, 1.625, 1.65, 1.675, 1.7, 1.725, 1.75, 1.775, 1.8, 1.825, 1.85, 1.875, 1.9, 1.925, 1.95, 1.975, or 2.0 mg/kg.

[ 0071 ] In certain embodiments, the invention as defined herein, comprises a clinically proven safe and clinically proven effective dose in a range of 0.1 mg/kg to 1.8 mg/kg, and preferably comprises a dose of 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.025, 1.05, 1.075, 1.1, 1.125, 1.15, 1.175, 1.2, 1.225, 1.25, 1.275, 1.3, 1.325, 1.35, 1.375, 1.4, 1.425, 1.45, 1.475, 1.5, 1.525, 1.55, 1.575, 1.6, 1.625, 1.65, 1.675, 1.7, 1.725, 1.75, 1.775, or 1.8 mg/kg.

[ 0072 ] In certain embodiments, the invention as defined herein, comprises a clinically proven safe and clinically proven effective dose in a range of 0.3 mg/kg to 1.8 mg/kg, and preferably comprises a dose of 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.025, 1.05, 1.075, 1.1, 1.125, 1.15, 1.175, 1.2, 1.225, 1.25, 1.275, 1.3, 1.325, 1.35, 1.375, 1.4, 1.425, 1.45, 1.475, 1.5, 1.525, 1.55, 1.575, 1.6, 1.625, 1.65, 1.675, 1.7, 1.725, 1.75, 1.775, or 1.8 mg/kg.

[ 0073 ] In certain embodiments, the invention as defined herein, comprises a clinically proven safe and clinically proven effective dose in a range of 0.3 mg/kg to 1.8 mg/kg, and preferably comprises a dose of 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8 mg/kg.

[ 0074 ] In certain embodiments, the invention as defined herein, comprises a clinically proven safe dose in a range of 0.03 mg/kg to 2.5 mg/kg, and preferably comprises a dose of 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.1, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.2, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.3, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.4, 2.41, 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49, or 2.5 mg/kg.

[ 0075 ] According to the invention as defined herein, the term "clinically proven safe", as it relates to a dose or treatment with the anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, refers to a favorable risk:benefit ratio with a relatively low or reduced frequency and/or low or reduced severity of adverse events, including adverse bleeding events, infusion or hypersensitivity reactions, or wound or joint complications compared to the standard of care or to a comparator, e.g., apixaban. Adverse bleeding events are the primary safety endpoint and include, for example, major bleeding, minor bleeding, and the individual components of the composite endpoint of any bleeding event.

[ 0076 ] According to the invention as defined herein, the terms "clinically proven effective" or "clinically proven efficacy", as they relate to terms such as dose, dosage regimen, or treatment with the anti-thrombin antibody or with a composition comprising the anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, refer to the inhibition of a thrombin- mediated condition, for example venous thromboembolism (VTE), deep vein thrombosis (DVT), and/or pulmonary embolism (PE), including wherein the primary efficacy endpoint is inhibition of total VTE, defined as the composite of proximal and/or distal DVT (asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic), nonfatal PE, or any death assessed through the Day 10-14 visit. Such inhibition can be observed, for example, as a reduction in the frequency of occurrence or severity of the thrombin-mediated condition in patients treated with the anti-thrombin antibody. [ 0077 ] As used herein, unless otherwise noted, the term "clinically proven" (used independently or to modify the terms "safe" and/or "effective") shall mean that it has been proven by a clinical trial wherein the clinical trial has met the approval standards of U.S. Food and Drug Administration, EMEA or a corresponding national regulatory agency. For example, the clinical study may be an adequately sized, randomized, double- blinded study used to clinically prove the effects of the drug.

[ 0078 ] 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.

[ 0079 ] Exosite 1 (also known as 'anion binding exosite Γ 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).

[ 0080 ] 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, PARI and fibrin (the co-factor for factor XIII activation).

[ 0081 ] 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).

[ 0082 ] 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.

[ 0083 ] 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.

[ 0084 ] 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, K149e, 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.

[ 0085 ] 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.

[ 0086 ] 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.

[ 0087 ] 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.

[ 0088 ] 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. [ 0089 ] 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.

[ 0090 ] 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 ΙΟηΜ, or less than InM. 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.

[ 0091 ] 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.

[ 0092 ] 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.

[ 0093 ] 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 Holliger & Hudson, Nature Biotechnology 23(9) : 1126-1136 (2005).

[ 0094 ] 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.

[ 0095 ] 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.

[ 0096 ] Anti-exosite 1 antibody molecules may be chimeric, humanised or human antibodies. [ 0097 ] 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.

[ 0098 ] 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.

[ 0099 ] 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 al, 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.

[ 00100 ] 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.

[ 00101 ] Other anti-exosite 1 antibody molecules may be identified by screening patient serum for antibodies which bind to exosite 1.

[ 00102 ] 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.

[ 00103 ] 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.

[ 00104 ] 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.

[ 00105 ] Suitable antibody molecules may be tested for activity using a fibrinogen clotting or thrombin time assay. Suitable assays are well-known in the art.

[ 00106 ] 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.

[ 00107 ] 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).

[ 00108 ] Each of the VH and VL domains typically comprise three complementarity determining regions (CDRs) responsible for antigen binding, interspersed by framework regions.

[ 00109 ] In some embodiments, binding to exosite 1 may occur wholly or substantially through the VHCDR3 of the anti-exosite 1 antibody molecule.

[ 00110 ] 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.

[ 00111 ] 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.

[ 00112 ] The VH domain of an anti -exosite 1 antibody molecule may further comprise an HCDRl 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 HCDRl 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.

[ 00113 ] In some embodiments, an antibody molecule may comprise a VH domain comprising a HCDRl, 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.

[ 00114 ] 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.

[ 00115 ] In some embodiments, the VL domain may comprise Tyr49. [ 00116 ] 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.

[ 00117 ] 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.

[ 00118 ] 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.

[ 00119 ] 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.

[ 00120 ] 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.

[ 00121 ] 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.

[ 00122 ] 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 LCDRl, LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8 and 9 respectively.

[ 00123 ] 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 heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 14 and a light chain (LC) comprising the 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 (LCDRl), SEQ ID NO:8 (LCDR2), and SEQ ID NO:9 (LCDR3).

[ 00124 ] 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 HCDRl, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and LCDRl, 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. [ 00125 ] 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, PARI and/or hirugen and hirudin analogues to thrombin.

[ 00126 ] 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-1 and coagulation factor FVIII. For example, binding of the antibody molecule to thrombin may result in an at least 5-fold, at least 10-fold, or at least 15-fold decrease in the hydrolysis of fibrinogen, PAR-1, 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.

[ 00127 ] 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.

[ 00128 ] 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.

[ 00129 ] 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)

[ 00130 ] Anti-exosite 1 antibody molecules may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

[ 00131 ] 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.

[ 00132 ] 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.

[ 00133 ] 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.

[ 00134 ] 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.

[ 00135 ] 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).

[ 00136 ] 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.

[ 00137 ] 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.

[ 00138 ] Administration is normally in a "therapeutically 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 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.

[ 00139 ] A typical antibody dose will be in the range 100 μg to 1 g for systemic applications, and 1 μg 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.

[ 00140 ] 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.

[ 00141 ] 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.

[ 00142 ] 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.

[ 00143 ] Anti-exosite 1 antibody molecules described herein inhibit thrombin and may be useful in the treatment of thrombin- mediated conditions. [ 00144 ] 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.

[ 00145 ] Anti-exosite 1 antibody molecules may have no effect or substantially no effect on haemostasis i.e. they do not promote bleeding or haemorrhage.

[ 00146 ] 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.

[ 00147 ] 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.

[ 00148 ] Thrombin plays a key role in haemostasis, coagulation and thrombosis.

Thrombin-mediated conditions include thrombotic conditions, such as thrombosis and embolism.

[ 00149 ] 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).

[ 00150 ] 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.

[ 00151 ] 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.

[ 00152 ] 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.

[ 00153 ] Thrombosis and embolism may occur in any vein, artery or other blood vessel within the circulatory system and may include microvascular thrombosis.

[ 00154 ] 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.

[ 00155 ] 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.

[ 00156 ] Thrombotic conditions may include thrombophilia, thrombotic stroke and coronary artery occlusion.

[ 00157 ] 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.

[ 00158 ] 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.

[ 00159 ] 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.

[ 00160 ] 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; 19(1) : 11-22; Tsopanoglou et al Eur Cytokine Netw. 2009 Dec 1;20(4) : 171-9).

[ 00161 ] 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.

[ 00162 ] 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.

[ 00163 ] 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.

[ 00164 ] 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.

[ 00165 ] The chromogenic signal may be measured directly without defibrination of the sample.

[ 00166 ] 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 lnc OH USA). [ 00167 ] 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.

[ 00168 ] 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.

[ 00169 ]

[ 00170 ] 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.

[ 00171 ] 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. [ 00172 ] 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.

[ 00173 ] The VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDRl 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.

[ 00174 ] A method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin may comprise:

recovering said antibody molecule or nucleic acid encoding it.

[ 00175 ] Suitable techniques for the maturation and optimisation of antibody molecules are well-known in the art.

[ 00176 ] 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. [ 00177 ] 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.

[ 00178 ] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[ 00179 ] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[ 00180 ] Unless stated otherwise, antibody residues are numbered herein in accordance with the Kabat numbering scheme.

[ 00181 ] "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.

[ 00182 ] 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.

[ 00183 ] Thus, the features set out above are disclosed in all combinations and permutations.

BRIEF DESCRIPTION OF THE DRAWINGS

[ 00184 ] Certain aspects and embodiments of the present invention will now be illustrated by way of example only, with reference to the figures described below.

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 IB shows a native blue gel showing total IgA load, flow-through from the human thrombin column and eluate following elution at low pH.

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.

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.

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

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 12nM.

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.

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

Figure 8 shows detail of crystal structure showing interaction between specific residues of thrombin exosite 1 and HCDR3 of the Fab fragment.

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)

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 (ΙΟΟμΙ in PBS).

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 (ΙΟΟμΙ in PBS), and a region outside of injury site for comparison. 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 (ΙΟΟμΙ in PBS), and a region outside of injury site for comparison.

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 (ΙΟΟμΙ in PBS).

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 4μΜ (final concentration in mouse blood, equivalent to a dose of approximately 60 mg/Kg) anti-exosite 1 IgA (ΙΟΟμΙ in PBS).

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

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.

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.

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).

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.

Figure 20 shows the binding of synthetic IgG to immobilized thrombin (on ForteBio Octet Red instrument). Figure 21 shows a typical Octet trace for the binding of 24nM S 195 A thrombin to immobilized IgG showing the on phase, followed by an off phase. The black line is the fit.

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.

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

Figure 24 shows the potency of IgG and IgG S3 OA in an ex vivo activated partial thromboplastin time (APTT) coagulation assay.

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

Figure 26 shows total bleeding time data for IgG S3 OA and IgG in the rat tail clip bleeding model.

Figure 27 shows total hemoglobin lost data for IgG S30A and IgG in the rat tail clip bleeding model.

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.

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

Figure 30 shows a schematic overview of the study design for the clinical trial.

Figure 31 shows the probability to flag a higher event rate in the JNJ-64179375 group than in the apixaban group at a 1 -sided, 10% a-Level. Event rates in apixaban are shown for 0.05 (bottom line), 0.075 (darker middle line), and 0.1 (upper line). In the legend, JNJ- 9375=JNJ-64179375.

Figure 32 shows candidate models for total VTE (Efficacy). VTE=venous

thromboembolism.

Figure 33 shows candidate models for any bleeding (Safety). DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[ 00185 ] 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.

Examples

[ 00186 ] 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

[ 00187 ] 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.

[ 00188 ] 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.

[ 00189 ] Thrombin time was not corrected by heparinase, indicating that heparin treatment or contamination was not responsible.

[ 00190 ] 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.

[ 00191 ] 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).

[ 00192 ] The binding site of the IgA on the thrombin molecule was then investigated.

[ 00193 ] 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).

[ 00194 ] 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).

[ 00195 ] 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.

[ 00196 ] 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).

[ 00197 ] The dissociation constant (Kd) of the IgA for thrombin was initially estimated based on rate of S2238 hydrolysis to be approximately 12nM (Figure 5). The Kd for the binding of the IgA to SI 95 A thrombin (inactivated by mutation of the catalytic serine) was determined to be 2nM using the ForteBio Octet Red instrument (Table 4).

[ 00198 ] 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 1.9A; 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).

[ 00199 ] 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). [ 00200 ] 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 (Kabat numbering): 30, 51, 52a, 53-55, 96, 98, 99, 100, 100a, 100b, 100c, 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 A% Glu 100a 80.6 A², Phe lOOc 141.7 A^J.

[ 00201 ] 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 A', Arg73 44.5 A2, Thr74 60.1 A', TYI-76 78.4 A², Arg77a 86.9 A².

[ 00202 ] 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

[ 00203 ] 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.

[ 00204 ] 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.

[ 00205 ] 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).

[ 00206 ] The bleeding times of the mice were also measured. Bleeding times were assessed as time to cessation of blood flow after a tail 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). [ 00207 ] 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

[ 00208 ] 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

[ 00209 ] 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

[ 00210 ] The IgA molecule identified in the patient described above was re-formatted as an IgG using standard techniques.

[ 00211 ] 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.

[ 00212 ] This was confirmed by measuring the binding of synthetic IgG to immobilized SI 95 A thrombin using a ForteBio™ Octet Red instrument. Thrombin was attached to the probe and the binding of the antibodies (at various concentrations) was monitored. [ 00213 ] On-rates and off-rates were determined. Both antibodies gave similar on-rates of approximately 3x10⁵ M^"1 s^"1 and off-rates of approximately 5x10^"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.

[ 00214 ] 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

[ 00215 ] 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 fucosylated 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.

[ 00216 ] 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.

[ 00217 ] 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. [ 00218 ] 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

[ 00219 ] 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).

[ 00220 ] 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 ΔΚ 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 CHOKISV GS KO cells for initial expression at a volume of 200 ml and then subsequently at a scaled-up volume of 2.5 litres.

[ 00221 ] The methods used will be described below. Where manufacturer' s instructions were followed, this will be indicated.

6.2.1 Vector Construction

[ 00222 ] 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 ΔΚ 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 μΐ of lyophilised shuttle vectors, as produced by GeneArt AG, were resuspended in 50 μΐ endotoxin free, sterile water. 1 μg of DNA was digested with the relevant restriction enzymes in a total volume of 50 μΐ and samples were incubated for 2 hours at 37°C. 8.3 μΐ 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 μΐ Lonza Simply Load Tandem DNA ladder was used as reference ladder. [ 00223 ] 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 μΐ T4 quick ligase, 10 μΐ of 2x T4 quick ligation buffer, reaction volume adjusted to 21 μΐ with endotoxin-free, sterile water when necessary and samples incubated at room temperature for 10 minutes. 10 μΐ 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 μg/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-HF and Hindlll-HF) and nucleotide sequencing of the gene of interest through a 3^rd party provider.

6.2.2 DNA Amplification

[ 00224 ] A single bacterial colony was picked into 15 ml Luria Bertani (LB) medium (LB Broth, Sigma-Aldrich, L7275) containing 50 μΐ/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 μΐ/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

[ 00225 ] 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

[ 00226 ] 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.

[ 00227 ] 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 μg DNA (1 : 1 ratio of heavy and light chain SGVs) and 700 μΐ cell suspension were aliquoted into each cuvette/well. Cells were electroporated at 300 V, 900 μΡ for the Gene Pulse XCell system and 300 V, 1300 μΡ 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

[ 00228 ] 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 μπι 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 μπι filter. 6.2.6 SE-HPLC Analysis

[ 00229 ] Duplicate samples were analysed to SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 μιη 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 μπι filter prior to injection. 80 μΐ 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

[ 00230 ] 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 μΐ 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 μΐ of each sample at 1 mg/ml were loaded onto the gel. Once electrophoresed, gels were stained with InstantBlue (TripleRed, ISBOIL) for 30 min at room temperature. Images of the stained gels were analysed on a BioSpectrum Imagine System (UVP).

6.2.8 Endotoxin Analysis

[ 00231 ] 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

[ 00232 ] 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.

[ 00233 ] 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 S3 OA with a small trace of high molecular weight impurity (1.8%) and an endotoxin level below the detectable scale of <0.02 EU/mg.

[ 00234 ] 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.

[ 00235 ] 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.

[ 00236 ] Using a standard ex vivo activated partial thromboplastin time (APTT) coagulation assay, IgG S30A was found to be equivalent or more potent than IgG.

[ 00237 ] 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).

[ 00238 ] Table 6 shows that both IgG S3 OA 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.

[ 00239 ] Figure 24 shows the potency of IgG and IgG S30A in an ex vivo activated partial thromboplastin time (APTT) coagulation assay. IgG S3 OA is equivalent or more potent than IgG.

[ 00240 ] 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.

[ 00241 ] 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. [ 00242 ] 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.

[ 00243 ] 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 S3 OA and IgG causing total prevention of thrombus formation.

[ 00244 ] 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 S3 OA and IgG causing similar reduction of thrombus formation.

[ 00245 ] 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.

[ 00246 ] Specific anti-exosite 1 antibody molecules disclosed herein include the following:

i. a wild-type anti-exosite 1 IgA antibody;

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

iii. 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.

[ 00247 ] 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.

[ 00248 ] 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

[ 00249 ] 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 S3 OA following stable cell transfection using standard techniques is also possible.

7.2 Materials and Methods

[ 00250 ] 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 ΔΚ 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.

[ 00251 ] The methods used will be described below. Where manufacturer' s instructions were followed, this will be indicated.

7.2.1 Vector Construction

[ 00252 ] Single gene vectors (SGVs) established in Lonza' s GS Xceed vectors from the previous transient production of ichorcumab S3 OA (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 μΐ and incubated at 37°C for 2 hours. 4.0 μΐ 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 μΐ Lonza Simply Load Tandem DNA ladder was used as a reference ladder. The agarose gel was imaged using BioSpectrum Imaging System (IVP).

[ 00253 ] 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 μΐ T4 quick ligase, 10 μΐ of 2x T4 quick ligation buffer, 2 μΐ of lOx DNA dilution buffer, reaction volume adjusted to 21 μΐ with endotoxin-free, sterile water when necessary and samples incubated at room temperature for 10 minutes. 10 μΐ 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.

[ 00254 ] Cells were spread onto ampicillin-containing (50 μg/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

[ 00255 ] 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 μg/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

[ 00256 ] 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

[ 00257 ] 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 μg/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 1.43x 10⁷ cells/ml. 100 μΐ linearized DNA at a concentration of 400 μg/ml was aliquoted into a 0.4 cm gap electroporation cuvette and 700 μΐ cell suspension added. Three cuvettes of cells and DNA were electroporated at 300 V, 900 μΡ 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.

[ 00258 ] 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 μΜ MSX (L- Methionine Sulfoximine, Sigma-Aldrich, M5379) and 10 ml/L SP4. Cell growth and viability were periodically checked post-transfection.

[ 00259 ] 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 μΜ MSX/ lOml/L SP4, in a 500ml vented Erlenmeyer flask (Fisher Scientific (Corning), 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

[ 00260 ] 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 μΐ aliquots of supernatant samples, 0.22 μπι 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

[ 00261 ] 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

[ 00262 ] 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

[ 00263 ] 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 μπι 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

[ 00264 ] 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 μπι filter.

7.2.10 SE-HPLC Analysis

[ 00265 ] Duplicate samples were analysed by SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 μπι 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 μπι filter prior to injection. 80 μΐ 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

[ 00266 ] 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 μΐ 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 μΐ of each sample at 1 mg/ml were loaded onto the gel. Once electrophoresed, gels were stained with InstantBlue (TripleRed, ISBOIL) for 30 min at room temperature. Images of the stained gels were analysed on a BioSpectrum Imaging System (UVP).

7.2.12 Endotoxin Analysis

[ 00267 ] 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

[ 00268 ] 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 I 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.

[ 00269 ] 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.

[ 00271 ] Amino acid sequence of anti-exosite 1 IgA and IgG VH domain with Kabat

Numbering (CDRs underlined): (SEQ ID NO:2).

[ 00272 ] Amino acid sequence of anti-exosite 1 IgA and IgG HCDRl: (SEQ ID NO:3).

GYTLTEAAIH

[ 00273 ] Amino acid sequence of anti-exosite 1 IgA and IgG HCDR2: (SEQ ID NO:4).

GLDPQDGETVYAQQFKG

[ 00274 ] Amino acid sequence of anti-exosite 1 IgA and IgG HCDR3: (SEQ ID NO:5).

GDFSEFEPFSMDYFHF

[ 00275 ] Amino acid sequence of anti-exosite 1 IgA and IgG VL domain with Kabat Numbering (CDRs underlined): (SEQ ID NO:6).

[ 00276 ] Amino acid sequence of anti-exosite 1 IgA and IgG LCDR1: (SEQ ID NO: 7).

[ 00277 ] Amino acid sequence of anti-exosite 1 IgA and IgG LCDR2: (SEQ ID NO: 8).

[ 00278 ] Amino acid sequence of anti-exosite 1 IgA and IgG LCDR3: (SEQ ID NO: 9).

QQRRSWPPLT

[ 00279 ] 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.

[ 00280 ] 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.

[ 00281 ] 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 LSLSPGEPAT LSCRASQNVA SFLAWYQHKP GQAPRLLIYD ASSRATDIPI 61 RFSGSGSGTD FTLTI SGLEP EDFAVYYCQQ RRSWPPLTFG GGTKVEIKR [ 00282 ] Amino acid sequence of anti-exosite 1 IgG4 (JNJ-64179375) LCDRl: (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.

.

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").

CLINICAL TRIAL PROTOCOL

[ 00283 ] A Randomized, Double-blind. Double-dummy. Multicenter, Adaptive Design, Dose Escalation (Part 1) and Dose-Response (Part 2) Study to Evaluate the Safety and Efficacy of Intravenous JNJ-64179375 Versus Oral Apixaban in Subjects Undergoing Elective Total Knee Replacement Surgery.

[ 00284 ] Protocol 64179375THR2001 ; Phase 2

[ 00285 ] EudraCT NUMBER: 2016-004550-15

1. INTRODUCTION

[ 00286 ] 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-1 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.¹ JNJ- 64179375 has a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 14 and a light chain (LC) comprising the 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). The JNJ-64179375 sequences include an S30A substitution in the LC to remove a glycosylation site and a serine 228 to proline substitution (S228P, as numbered according to the EU numbering system) in the HC to stabilize the hinge region.¹⁷-¹⁸

[ 00287 ] JNJ-64179375 acts as an anticoagulant by blocking only exosite-1 -mediated fibrinogen binding to thrombin (and also blocks proteinase -activated receptor- 1 binding), but does not inhibit the catalytic activity of the protease. 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) and from other mechanisms that inhibit thrombin generation (eg, Factor Xa [FXa] inhibitors). The primary goal of the clinical program will be to demonstrate noninferior efficacy with a reduced bleeding risk versus active comparators. However, the possibility of demonstrating superior efficacy may be considered based on better compliance with a once-monthly dosing regimen and/or the ability to achieve more effective drug levels due to a reduced risk of bleeding (ie, doses not limited by bleeding risk).

[ 00288 ] An estimated 920,000 patients in Japan and Europe experience venous thromboembolism (VTE) annually, which includes deep vein thrombosis (DVT) and pulmonary embolism (PE).^{6 19} Orthopedic joint replacement surgery of the lower extremities, including total knee replacement (TKR) and total hip replacement (THR), carry a high risk of VTE due to prothrombotic processes such as soft tissue and bone injury during surgery, which causes coagulation activation from thromboplastin release, venous stasis from peri- and postoperative immobilization, and inflammation from the healing process.³² This has led to recommendations in the guidelines that all patients undergoing total knee replacement (TKR) should receive pharmacologic and/or mechanical VTE prophylaxis.^10,19 It has also been demonstrated that there is not a statistically significant difference in the relative treatment effects for anticoagulants used for prevention of VTE based on type of surgery for TKR and THR.³⁵ Furthermore, analyses suggested that the outcome of a single study in one type of surgery with one comparator dose regimen, used in conjunction with the model-based meta-analysis, would be sufficient to determine the optimal dose for both types of surgery and compared with both regimens of comparator.³⁵ Low molecular-weight heparin (LMWH) and several direct-acting oral anticoagulants (DOACs), including apixaban, have been used in the prevention of VTE in patients undergoing TKR surgery. However, limitations of these drugs include once- or twice-daily dosing regimens and the possibility of an increased bleeding risk at their recommended dosages.

[ 00289 ] JNJ-64179375 is being developed for multiple thrombosis-mediated conditions, including the prevention of VTE after TKR and THR surgery, and may offer the potential for equivalent (or superior) efficacy to currently available anticoagulant drugs with a reduced risk of bleeding and a simpler dosing regimen. A novel

antithrombotic agent requires the demonstration of both efficacy and safety in relevant populations. Phase 2 studies are frequently performed in subjects undergoing TKR surgery because of the well -documented high incidence of DVT in the absence of adequate thromboprophylaxis, as well as the increased risk of bleeding after recent surgery.⁷ An assessment of the effect on efficacy and safety of JNJ-64179375 compared with apixaban in this study will be used to identify an appropriate dose for evaluation in the Phase 3 clinical development program that could include both TKR and THR surgery.

Subjects who do not receive the full infusion of JNJ-64179375 or prematurely discontinue dosing (ie, EW) with apixaban or matching apixaban placebo before the end of the double-blind dosing phase will be instructed to return to the study site at the originally scheduled Day 10-14 visit to conduct assessments, including the venography assessment of the operated leg (unless a pulmonary embolism [PE] or symptomatic proximal deep vein thrombosis [DVT] has been diagnosed), and to complete the remaining visits through the Week 18 assessments.

At the discretion of the investigator, subjects may return to the study site between scheduled visits. Subjects should return to the study site for the assessment of any potential bleeding or efficacy endpoint events. Unscheduled PK and PD samples (except D- dimer) should be collected as soon as practically possible for any subject who experiences symptomatic thrombotic or bleeding events.

At the time of informed consent, 2 alternative means of contact for each subject will be collected (eg, contact information of the subject's children, spouse, significant other, caretaker, legal representative, or health care professional).

The investigator will need to determine if the subject is medically appropriate for postoperative anticoagulant prophylaxis on the basis of physical examination, medical history, vital signs measurements, and clinical laboratory tests performed as part of screening for elective TKR surgery and any examination performed as part of standard postoperative care following surgery.

Procedures should be conducted prior to the first dose of the study drug.

Details regarding the total knee replacement (TKR) surgery and the post-surgery management (eg, type of anesthesia, procedure duration, cement use, tourniquet use and duration, drain use and volume, use of all mechanical venous thromboembolism [VTE] prophylaxis methods) will be collected in the electronic case report form (eCRF).

The day after TKR surgery (Day 1, 0 hours), while the subject is still hospitalized and within a minimum of 12 hours and a maximum of 24 hours after the end of the TKR surgery, defined as the time of wound closure, all subjects will receive a single IV infusion of JNJ-64179375 or JNJ-64179375 placebo and oral apixaban or matching apixaban placebo. The first dose of apixaban or matching apixaban placebo will be administered while the subject is hospitalized, with up to a maximum of 60 minutes between the start of the IV infusion and the first dose of oral study drug. Subjects will receive apixaban or matching apixaban placebo twice a day while hospitalized and will be given a supply of apixaban or matching apixaban placebo at the time of discharge or transfer to an alternate facility, with instructions to take the study drug orally, twice a day at approximately the same times each day to complete a total of 10 to 14 days of dosing.

Height and weight should be obtained at the screening visit, with weight only on Day -1 or prior to dosing on Day 1 and the final visit. An assessment of the wound will be made at all visits as part of the adverse event assessment and the final physical examination will assess the range of motion of the operated joint.

Blood pressure and pulse/heart rate (HR) measurements will be assessed with subjects in the supine position with a completely automated device and should be preceded by at least 5 minutes of rest. The subject's temperature should also be obtained.

Subjects who complete dosing with apixaban or matching apixaban placebo will return to the study site for final EOD assessments (at the Day 10-14 visit, EOD), at which time a unilateral venography assessment of the operated leg will be performed within 24 hours of the last dose of apixaban or matching apixaban placebo. If dosing with apixaban or matching apixaban placebo is prematurely discontinued, the venography assessment should be completed on the originally scheduled Day 10-14 (EOD) visit, not earlier. If a subject has suspected symptomatic DVT prior to the Day 10-14 visit, an ultrasound will be performed. If the ultrasound confirms symptomatic proximal DVT, a subsequent venography assessment is not required. If the ultrasound is negative or confirms a distal DVT, the venography assessment should be conducted on the Day 10-14 visit. In addition, if the subject is diagnosed with a PE, a venography assessment of the operated leg is not required.

Suspected symptomatic efficacy (thrombotic) events (DVT, PE, death, myocardial infarction, ischemic stroke, peripheral arterial embolism) will be reported by the investigator and reviewed by the Clinical Events Committee to ascertain if a thrombotic event has occurred.

All sites in Part 1 will collect PK blood samples for subjects at all visits (hereafter referred to as dense PK sample collection) until approximately up to the first 200 subjects have been randomized. The Day 1 samples will be drawn at 1 hour and 4 hours from the start of the IV infusion. The Day 2 blood sample will be drawn 24 hours after the start of the IV infusion and may be done with the subject as an inpatient or outpatient. The Day 3 (48 hours) and Day 7 (144 hours) blood samples are only required for those subjects who are still hospitalized.

After approximately up to the first 200 randomized subjects in Part 1 have initiated dense PK sampling, the remaining subjects in Part 1 and all subjects in Part 2 will have PK blood samples collected at a limited number of visits (hereafter referred to as sparse PK sample collection). The Day 2 sample will be drawn 24 hours after the start of the IV infusion and may be done with the subject as an inpatient or outpatient.

The PK sample will only be collected at this visit if it was not collected at the Week 10 visit.

Pharmacodynamic evaluations will include the coagulation assays (ie, thrombin time [TT], ecarin clotting time [ECT], prothrombin time [PT], and activated partial thromboplastin time [aPTT]). Samples on Day 1 will be obtained before the study drug is administered and at 1 hour after the start of the study drug infusion.

All adverse events, whether serious or nonserious, will be reported from the time a signed and dated informed consent form (ICF) is obtained until the completion of the subject's last study-related procedure. Adverse events of special interest are bleeding events, infusion reactions, hypersensitivity reactions, and wound or joint complications). All suspected symptomatic efficacy (thrombotic) events will also be captured as adverse events of special interest.

1.1. Background

1.1.1. Nonclinical Studies

1.1.1.1. Nonclinical Pharmacology

[ 00290 ] JNJ-64179375 binds to human, mouse, rat, and monkey thrombin with high affinity (i.e., equilibrium dissociation constants <2 nM) and does not bind to prothrombin or other serine proteases. Spiking of JNJ-64179375 in human plasma in vitro resulted in concentration-dependent prolongation of clotting in various coagulation assays (thrombin time [TT] [dilute and normal], ecarin clotting time [ECT], prothrombin time [PT], and activated partial thromboplastin time [aPTT]), with TT being the most sensitive for this compound.

[ 00291 ] The antithrombotic efficacy of JNJ-64179375 was evaluated in several different thrombosis models in rats and cynomolgus monkeys, and in a human translational ex-vivo flow-chamber model (Badimon chamber). JNJ-64179375 inhibited thrombosis in rat ferric chloride (FeCb) and arteriovenous (AV)-shunt models of venous thrombosis in a dose-dependent manner (0.1 to 1 mg/kg, intravenous [IV]). At 0.3 and 1 mg/kg, JNJ-64179375 produced antithrombotic efficacy accompanied by approximately 1.5- and 2-fold increases in TT ex vivo, respectively (serum level approximately 7 and 23 μg/mL, respectively). JNJ-64179375 (0.3 and 1 mg/kg, IV) also inhibited thrombosis in a dose-dependent manner in a FeCb model of venous thrombosis in cynomolgus monkeys. At the 1-mg/kg dose, serum levels were approximately 40 μg/mL.

[ 00292 ] The Badimon chamber is a clinical ex-vivo model of thrombosis that mimics the arterial flow conditions within the coronary circulation. Under these conditions, higher concentrations of JNJ-64179375 added extracorporeally to the blood

(approximately 250 μg/mL) were required to demonstrate efficacy. Overall, the pharmacology data suggests that the therapeutic range of JNJ-64179375 for the prevention of venous thrombosis may be between approximately 7 μg/mL and 40 μg/mL (ie, the concentrations in rats at the 0.3-mg/kg dose and in monkeys at the 1-mg/kg dose, respectively).

[ 00293 ] The ability of 4 marketed nonspecific reversal agents (Beriplex^®, FEIBA, Novo Seven^®, and tranexamic acid [TXA]) to reverse the bleeding induced by JNJ- 64179375 was evaluated in the rat-tail transection model. JNJ-64179375 at a dose of 10 mg/kg IV, (ie, a 10-fold higher dose than the effective dose in the FeCb and AV-shunt models of venous thrombosis), resulted in increases in bleeding time and blood loss in this model; however, these increases in bleeding parameters were significantly attenuated by pretreatment with either Beriplex (a 4-factor prothrombin complex concentrate [PCC]) or FEIBA (an activated PCC). In contrast, TXA and NovoSeven did not shorten the bleeding times after dosing with JNJ-64179375 at the doses studied.

1.1.1.2. Pharmacokinetic and Product Metabolism in Animals

[ 00294 ] Single-dose pharmacokinetic (PK) studies were conducted in rats and cynomolgus monkeys and multiple-dose TKs were assessed in the 1 -month non-GLP and GLP rat and cynomolgus monkey toxicology studies. In the single-dose PK studies in rats, the drug exposure increased with dose in an approximately dose-proportional manner following a single IV dose in the dose range from 0.1 to 10 mg/kg. In cynomolgus monkeys, the Cmax increased with dose in an approximately dose-proportional manner, but the dose-normalized area under the concentration-time curve from time zero to infinity (AUCM) increased with dose in a more than dose-proportional manner following a single IV dose ranging from 0.3 to 10 mg/kg. The maximum concentration occurred at 3 to 5 days postdose and the estimated absolute bioavailability was 61.40% to 70.40% following administration of a single SQ dose of JNJ-64179375 of 1 mg/kg and 3 mg/kg, respectively, in rats. Following administration of a single SQ dose of JNJ-64179375 (3 mg/kg) in monkeys, the maximum concentration occurred at 3 days postdose, and the absolute bioavailability was nearly complete.

[ 00295 ] In the single-dose PK studies in rats, the mean total clearance of drug after IV administration (CL) of JNJ-64179375 from the 0.1- to 10-mg/kg IV-dose groups ranged from 3.95 to 6.44 mL/day/kg, and the mean terminal half-life (ti/2) ranged from 9.64 to 13.44 days across all dose groups. The mean CL of JNJ-64179375 from the IV-dose groups ranged from 1.81 to 4.82 mL/day/kg, and the mean values of ti/2 ranged from 7.2 to 17.3 days across all dose groups in monkeys. The mean apparent volume of distribution in the terminal phase (Vz) of JNJ-64179375 ranged from 53.07 to 94.46 mL/kg across the 0.1- to 10-mg/kg single IV dose groups in rats. The mean Vz of JNJ- 64179375 following a single IV dose ranged from 26.32 to 62.26 mL/kg across all dose groups in monkeys. The Vz of JNJ-64179375 in rats and cynomolgus monkeys was similar to the blood volume in each species, suggesting that JNJ-64179375 was mainly distributed in the intravascular space in rats and monkeys.

1.1.2. Clinical Studies

[ 00296 ] The safety, tolerability, PK, immunogenicity, and pharmacodynamics (PD) of single-ascending IV and SQ doses of JNJ-64179375 was evaluated in a first-in-human study (Protocol 64179375EDI1001) and is also currently being evaluated in a second study (Protocol 64179375EDI1002). The 64179375EDI1001 study (SAD) was a 3-part study in healthy male non- Japanese subjects (mainly Caucasian subjects) that was conducted at a single study site in Belgium. The 64179375EDI1002 study (JSAD) is a 2- part study in healthy Japanese subjects that was designed as a bridging study in healthy Japanese subjects. The JSAD study is being conducted at two study sites (HMR in London, UK and Hakata Clinic, in Fukuoka, Japan).

[ 00297 ] Race/ethnicity can affect PK and PD of drugs due to intrinsic factors (eg, genetics, metabolism, elimination), which may affect their safety, efficacy dosage and dose regimen of the drug. Thus, evaluating the potential ethnic differences in PK and clinical dose-response relationships between Japanese and non-Japanese has been an indispensable step for the Pharmaceuticals and Medical Devices Agency (PMDA).

1.1.2.1. Protocol 64179375EDI1001 (SAD)

1.1.2.1.1. Overall Study Design

[ 00298 ] The study has 3 parts, and was conducted in healthy male subjects: Part 1 assessed the safety, tolerability, PK, immunogenicity, and PD of single-ascending IV doses of JNJ-64179375; Part 2 assessed the reversibility of the PD effects of JNJ- 64179375 following a single IV dose of a 4-factor PCC, as well as safety, tolerability, PK, and immunogenicity; and Part 3 assessed the safety, tolerability, PK,

immunogenicity, and PD of a single SQ dose of JNJ-64179375.

[ 00299 ] In Part 1, up to 6 cohorts of 8 subjects each were to be enrolled to evaluate successively increasing doses of JNJ-64179375 over a dose range of 0.03 to 5.0 mg/kg (planned doses of 0.03, 0.1, 0.3, 1.0, 2.5, and 5.0 mg/kg). Up to 2 additional optional cohorts could be enrolled to repeat a dose or examine other doses. In Parts 2 and 3, 1 cohort of 8 subjects was to be enrolled in each. [ 00300 ] In Parts 1 and 3 of the study, 6 of the 8 subjects to be enrolled in each cohort were to be randomly assigned to treatment with JNJ-64179375 and 2 subjects were to be randomly assigned to treatment with placebo. In Part 2, all 8 subjects were to receive JNJ- 64179375 followed by either 4-factor PCC (n=6) or placebo (n=2). For each subject in each part, the study consists of a screening period (up to 28 days), an in-house period (15 days/14 nights), and an outpatient period (approximately 99 days), with the total study duration up to approximately 22 weeks for each subject.

1.1.2.1.2 Part 1

1.1.2.1.2.1 Demographic Characteristics

[ 00301 ] In Part 1, the mean age of the subjects was 32.7 years (range 19 to 45 years) and the mean weight was 81.24 kg (range 60.2 to 100.3 kg). Over 90% of the subjects were Caucasian. There were no meaningful differences between the cohorts for any demographic parameter.

1.1.2.1.2.2. Summary of Part 1 Results

[ 00302 ] Results, in brief, are described below for the single-ascending dose (SAD), randomized, double-blind, placebo-controlled study in healthy male subjects (Part 1). The actual dose sequence in Part 1 (8 cohorts) was 0.03, 0.1, 0.3, 1.0, 2.5, repeat 2.5, 1.75, and 2.125 mg/kg (total n=63 subjects: JNJ-64179375=48 subjects, placebo=15 subjects). The planned 5 mg/kg dose of JNJ-64179375 was not studied.

[ 00303 ] JNJ-64179375 was generally well tolerated over the dose range of 0.03 to 2.5 mg/kg. There were no reported Treatment-Emergent Adverse Events (TEAEs) leading to treatment discontinuation and there were no deaths. All non-bleeding TEAEs were assessed as mild in intensity, except for three serious adverse events in two subjects, one subject in the placebo group (transient HaNDL syndrome) and one subject in the 1.75 mg/kg IV JNJ-64179375 group (Panic attack and metabolic acidosis). These serious adverse events in the two subjects were considered by the investigator to be doubtfully related to the study drug. In addition, there was no evidence of a dose relationship for the non-bleeding adverse events. There were also no bleeding TEAEs that were major bleeding events or clinically relevant non-major bleeding events according to

International Society on Thrombosis and Haemostasis (ISTH). All observed bleeding TEAEs were BARC Type 1 according to Bleeding Academic Research Consortium (BARC) classification, e.g., bleeding that is not actionable and does not cause the patient to seek unscheduled performance of studies, hospitalization, or treatment by a healthcare professional {Circulation. 2011;123:2736-2747). These non-major bleeding TEAEs were predominantly associated with mild trauma (i.e., venipuncture, brushing teeth/gums, blowing nose), all were mild or moderate in intensity, and all resolved. The criteria for moderate intensity were defined as an episode of epistaxis lasting more than 5 minutes or a skin bruise event with a maximum dimension of 10 cm or larger. The non-major bleeding TEAEs appeared to be dose-dependent with increased incidence and severity in the 2 highest doses studied (2.125 and 2.5 mg/kg). The non-major bleeding events considered to be of moderate intensity at the 2.125 mg/kg and 2.5 mg/kg doses included 1 event of ecchymosis at 2.125 mg/kg and 4 skin-bruising or hematoma events at 2.5 mg/kg. The dose-dependent increase in non-major bleeding TEAEs at the highest doses of 2.125 mg/kg and 2.5 mg/kg suggested a potential bleeding risk signal and is referred to herein as a bleeding signal.

[ 00304 ] JNJ-64179375 administered as an IV infusion also exhibited a favorable PK profile in healthy subjects, and the observed PK parameters were close to those predicted based on the animal PK data. Mean Cmax and AUCM values for JNJ-64179375 increased in an approximately dose-proportional manner in the studied dose range and variations of the PK concentration and parameters were low, indicating PK predictability. Also, consistent with its mechanism of action and pharmacodynamic and efficacy assessments in nonclinical findings, JNJ-64179375 produced a dose-dependent prolongation of the coagulation parameters (PT, aPTT, and TT) over the studied dose range and effect-time curves for all coagulation parameters seemed parallel to the plasma drug concentration- time curves of JNJ-64179375 with no apparent lag time.

[ 00305 ] Dosing for the Phase 2 study and for Part 2 and Part 3 of the Phase 1 study was guided in part based on the data from the results of Phase 1, Part 1.

1.1.2.1.3. Parts 2 and 3

[ 00306 ] Parts 2 and 3 of the study (1 single cohort in each part) have also been completed. A blinded data review is briefly summarized below. Eight subjects were dosed intravenously with 1.0 mg/kg of JNJ-64179375 in Part 2 followed by either placebo (n=2) or 50 IU/kg of the 4-factor PCC Confidex^® (n=6). Confidex did not appear to reverse the effects of JNJ-64179375 on either aPTT or TT. In Part 3, 8 subjects were dosed subcutaneously with either 1.0 mg/kg of J J-64179375 (n=6) or placebo (n=2). The PK data showed the expected delay to peak drug levels. For both cohorts, the bleeding adverse events were mostly mild skin-site events as observed in Part 1. One moderate intensity epistaxis bleeding event was reported in Part 2.

[ 00307 ] In the nonclinical studies, 4-factor PCC and activated PCC were able to normalize rat-tail bleeding times after the administration of JNJ-64179375 but did not reverse TT or aPTT prolongation. Therefore, the lack of reversal of TT and aPTT with 4- factor PCC in Part 2 of the Phase 1 study was not unexpected and likely reflects the insensitivity of these coagulation assays for measuring the potential hemostatic effects of PCC administration.

1.1.2.2. Protocol 64179375EDI1002 (JSAD)

1.1.2.2.1. Overall Study Design and Subjects

[ 00308 ] Eligible criteria for healthy Japanese subjects included male or female of non- childbearing potential aged 20-45 years, body mass index between 18 and 27 kg/m² and body weight greater than 50 kg but less than 100 kg. All subjects were generally in good health based on physical examination, medical history, vital signs, laboratory tests, and ECGs performed at screening and/or prior to administration of the study drug and signed an informed consent form.

[ 00309 ] Part 1 is a double-bind, randomized, placebo-controlled, single ascending dose study to assess the safety, tolerability, PK, immunogenicity and PD of single ascending IV doses of JNJ-9375. Subjects were randomly assigned, in a 3: 1 ratio, to receive JNJ-9375 or matching placebo, administered as a single IV infusion over a period of 30 minutes. Three dose escalation cohorts and 2 optional cohorts were planned for Part 1. Each dose cohort had 8 subjects (6 active, 2 placebo).

[ 00310 ] Part 2 is a double-blind, randomized, placebo-controlled, single cohort study that evaluated a single SC dose of JNJ-9375 (n=6) or matching placebo (n=2) to assess the safety, tolerability, PK, immunogenicity and PD of a single SC dose of JNJ-9375.

[ 00311 ] For each eligible subject in each part, the study consisted of a Screening Period (up to 28 days), an Inpatient Period (10 days), and an Outpatient Period

(approximately 103 days). [ 00312 ] Qualified subjects were admitted to the clinical research unit (CRU) in a fasting state on the morning of Day -1 for baseline safety assessments. Subjects received the study drug on Day 1 in the morning at least 30 minutes after a standardized light breakfast. Subjects were domiciled continuously at the CRU for safety, tolerability, PK, immunogenicity, and PD assessments until Day 10, when upon completion of all study evaluations, subjects were discharged at the discretion of the investigator or appropriate designee. If the prothrombin time (PT) or activated partial thromboplastin time (aPTT) for a subject was elevated >2X the upper limit of normal (ULN) on Day 10, then the subject would remain as an inpatient until the value fell below this level. Subjects were to return to the CRU on Days 14, 22, 29, 43, 57, 85, and 113 for safety, tolerability, PK, immunogenicity and PD assessments. Completion of the Day 113 visit constitutes a subject's completion.

1.1.2.2.2. Interim Analysis Results Summary (Part 1)

[ 00313 ] Results of this interim analysis were based on data analysis from the data cut- off of January 31, 2018. A total of 3 cohorts of subjects were studied in Part 1. Cohort 1 was conducted at the study site in UK and Cohorts 2 and 3 were studied at the study site in Japan. The actual dose levels evaluated are 0.3, 1 and 2.5 mg/kg IV of JNJ-9375 as planned. Based on safety/tolerability, PK and PD results from the 3 cohorts, the DRC recommended not to perform the 2 optional cohorts. Twenty-four (24) subjects (Cohorts 1-3) were randomized and completed up to 14 days of Part 1. There were no major protocol deviations identified.

[ 00314 ] Baseline demographic characteristics were similar among the treatment groups. Subjects are all male Asian (Japanese) ethnicity. The mean (SD) of age, body weight and BMI were 29.2 (3.97) years, 63.95 (7.18) kg, and 21.564 (2.27) kg/m2, respectively. The total mean body weight of the Japanese subjects in Part 1 of this study is approximately 21% lower than that of the non-Japanese subjects (81.24 kg) in Part 1 of the 64179375EDI1001 FIH SAD study.

[ 00315 ] The summary, in brief, similar safety and tolerability were observed in Part 1 of the JSAD study in healthy Japanese subjects and the FIH SAD study in healthy non- Japanese male subjects. JNJ-9375 was well tolerated in this JSAD study in Japanese subjects. There were no dose related non-bleeding TEAEs observed. The bleeding TEAEs, predominantly skin related, were all assessed by the investigators to be mild in severity except for one example of infusion site bruising in the placebo group that was assessed by the investigator to be moderate in severity. The difference in the incidence of the mild bleeding TEAEs between the combined JNJ-9375 groups vs the placebo group was attributed to the higher incidence in the highest 2.5mg/kg dose group compared to the other treatment groups, suggesting a potential bleeding risk signal at the top dose of 2.5 mg/kg IV (83.3% in the 2.5 mg/kg IV dose group vs 16.7% in each of the 0.3 mg/kg and the 1 mg/kg IV dose groups).

[ 00316 ] The similar safety/tolerability profiles in both studies were likely attributed to the similar PK (after the difference in body weight has been taken into account) and similar PD profiles between the Japanese and the non-Japanese populations. The PK of JNJ-9375 was predictable, approximately dose-proportional and variation of PK was low (CV < 30%). The effects of JNJ-9375 on coagulation parameters (TT, PT and aPPT) were consistent with its mechanism of action of direct effect on thrombin mediated fibrin formation, and consistent with PD and efficacy assessments in animal models. The similarity of safety/tolerability, PK and PD profiles of JNJ-9375 in Japanese and non- Japanese subjects support that same dosing regimens may provide similar clinical benefit in these two ethnic groups in the planned Phase 2 total knee replacement study.

1.2. Comparator Drug - Eliquis^® (apixaban)

[ 00317 ] The following information, taken from the European Union Summary of Product Characteristics, is intended to provide a brief, representative overview of apixaban.⁸

[ 00318 ] Eliquis^® (apixaban) is a potent, oral, reversible, direct and highly selective active-site inhibitor of FXa. Apixaban does not require antithrombin III for

antithrombotic activity. It inhibits free and clot-bound FXa and prothrombinase activity. Apixaban has no direct effects on platelet aggregation but indirectly inhibits platelet aggregation induced by thrombin. By inhibiting FXa, apixaban prevents thrombin generation and thrombus development. Apixaban does not have any effects on TT but does prolong PT and aPTT although the changes in these assays are small and variable at therapeutic doses.

[ 00319 ] Apixaban is indicated in the European Union for the prevention of VTE in adult patients who have undergone elective hip or knee replacement surgery. For the prevention of VTE after TKR surgery, the recommended dose of apixaban is 2.5 mg taken orally twice daily, with the initial dose taken 12 to 24 hours after surgery. The recommended duration of treatment is 10 to 14 days.

[ 00320 ] The ADVANCE- 1 and ADVANCE-2 studies included subjects who had undergone a TKR for one or both knees.

[ 00321 ] In the ADVANCE-1 study, which included 3, 195 randomized subjects and 3,184 subjects in the safety population, apixaban 2.5 mg twice daily following TKR was compared with enoxaparin 30 mg twice daily. Both medications were started 12 to 24 hours after surgery and continued for 10 to 14 days. The primary efficacy endpoint was total VTE defined as the composite of adjudicated asymptomatic and symptomatic DVT, nonfatal PE, or all-cause death. The primary safety outcome was major bleeding during the treatment period or within 2 days after the last dose of the study drug. The total VTE rates were similar for apixaban and enoxaparin although statistically apixaban did not meet 1 of the 2 prespecified criteria for noninferior efficacy. Bleeding events were lower in the apixaban group.²³ (Table 8 and Table 9)

[ 00322 ] In the ADVANCE-2 study, which included 3,057 randomized subjects and 3,009 subjects in the safety population undergoing elective TKR surgery, subjects received either apixaban 2.5 mg given orally twice daily or enoxaparin 40 mg SQ once daily. Apixaban was started 12 to 24 hours after surgery, while enoxaparin was started 9 to 15 hours before surgery, with both drugs given for 10 to 14 days. The primary efficacy endpoint was total VTE and the primary safety outcome was major bleeding reported during the treatment period. Apixaban demonstrated a statistically superior reduction in total VTE and in the major VTE endpoint, a composite of proximal DVT, nonfatal PE, and VTE-related death, compared with enoxaparin. The safety endpoints of major bleeding, the composite of major and clinically relevant nonmajor bleeding, and all bleeding were not statistically different but were numerically lower for subjects treated with apixaban 2.5 mg twice daily compared with enoxaparin 40 mg SQ once daily.²⁴ (Table 8 and Table 9)

1.3. Overall Rationale for the Study

[ 00323 ] Low molecular-weight heparins have a long and well-established role in the prevention of VTE in subjects undergoing TKR surgery, and while very effective with an acceptable bleeding risk, SQ dosing once or twice a day is required. More recently, several DOACs (eg, apixaban, rivaroxaban, dabigatran, edoxaban) have been approved for use in TKR, based on comparisons with the LMWH, enoxaparin, and their use has become more widespread in this patient population. Apixaban starting 12 to 24 hours after TKR surgery demonstrated superior efficacy compared with enoxaparin 40 mg once daily and similar efficacy to enoxaparin 30 mg twice daily, with numerically less bleeding than both enoxaparin regimens.^23,24 Apixaban was chosen as the active comparator for this study because it is orally administered and compares favorably with enoxaparin for both efficacy and bleeding endpoints.

[ 00324 ] JNJ-64179375 is a first-in-class, recombinant, fully human IgG4 monoclonal antibody that binds reversibly with high affinity and specificity to the exosite-1 region on thrombin. By only blocking exosite-1, the catalytic activity of the protease is maintained. Therefore, this unique mechanism of action of JNJ-64179375 may offer the potential for noninferior (or superior) efficacy compared with currently available anticoagulant drugs (eg, vitamin K antagonists, enoxaparin, and DOACs) with a reduced risk of bleeding.

[ 00325 ] Given that TKR surgery carries a high risk of VTE combined with the hemostatic challenges of surgery, it provides a good setting to evaluate the relative efficacy and safety (bleeding) characteristics of novel anticoagulants.⁷ As a monoclonal antibody, JNJ-64179375 has an expected duration of action of approximately 4 weeks, thereby allowing for the postoperative administration of a single IV dose to be used for VTE prophylaxis after TKR surgery. Based on the preclinical and Phase 1 studies conducted to date, JNJ-64179375 is anticipated to have a favorable safety profile with respect to bleeding risk. Therefore, JNJ-64179375 offers the potential for an efficacious treatment that has limited bleeding, with a simpler dosing regimen compared with currently available oral or parenteral treatments. 2. OBJECTIVES, ENDPOINTS, AND HYPOTHESIS

2.1. Objectives and Endpoints

2.1.1. Objectives

Part 1

In men and women undergoing primary unilateral TKR surgery, after single-ascending IV doses of JNJ-64179375 or 10 to 14 days of oral apixaban:

Primary Objective

The primary objective is to assess the safety and tolerability of JNJ-64179375 for each dose level for dose escalation within Part 1 and any bleeding events (the composite of major, clinically relevant nonmajor, and minimal bleeding events) for the selection of doses for Part 2.

Secondary Objectives

· To assess the dose response of JNJ-64179375 for the occurrence of the composite endpoint of any bleeding events, the composite endpoint of major or clinically relevant nonmajor bleeding events, and the individual components of the composite endpoint of any bleeding event

• To assess the dose response of JNJ-64179375 for the prevention of total VTE (proximal and/or distal DVT [asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic], nonfatal PE, or any death) and the individual components of total VTE

Part 2

In men and women undergoing primary unilateral TKR surgery, after a single IV dose of JNJ-64179375 or 10 to 14 days of oral apixaban:

Primary Objective

The primary objective is to assess the efficacy dose response of JNJ-64179375 for the prevention of total VTE (proximal and/or distal DVT [asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic], nonfatal PE, or any death).

Key Secondary Objectives

• To assess the dose response of JNJ-64179375 for the occurrence of the composite endpoint of any bleeding events, the composite endpoint of major or clinically relevant nonmajor bleeding events, and the individual components of the composite endpoint of any bleeding event

• To assess the dose response of JNJ-64179375 for the prevention of major VTE (asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic proximal DVT, nonfatal PE, or any death) and the individual components of the total VTE endpoint

Common Objectives in Parts 1 and 2

Other Secondary Objectives

· To assess the effect of individual doses of JNJ-64179375 compared with apixaban for both efficacy and safety endpoints, with the goal to identify a studied or model- predicted dose with the most promising benefit-risk profile for a more extensive evaluation in Phase 3

• To assess the effect of JNJ-64179375 compared with apixaban on wound or joint complications in the operated leg

Exploratory Objectives

• To assess the effect of JNJ-64179375 compared with apixaban on other thrombotic events (ie, myocardial infarction [MI], ischemic stroke, peripheral arterial embolism)

• To evaluate the cost effectiveness of JNJ-64179375 compared with apixaban based on health resource utilization

• To assess the PK, PD, and PK/PD relationships of JNJ-64179375 in men and women undergoing primary unilateral TKR surgery and the relation of these measures to efficacy and safety endpoints (eg, exposure-response analyses)

• To evaluate the PD assays (TT, ECT, PT, aPTT, and D-dimer) to determine the most appropriate tests to measure the effect of JNJ-64179375

2.1.2. Endpoints

The endpoints of the study will be the same for Parts 1 and 2 although the focus of Part 1 will be primarily dose escalation based on safety while the focus of Part 2 will primarily be the assessment of dose response in both safety and efficacy.

Common Endpoints in Parts 1 and 2

Primary Safety Endpoint

The primary safety endpoint is any bleeding event defined as the composite of major, clinically relevant nonmajor, and minimal bleeding events assessed through the Day 10-14 visit.

Primary Efficacy Endpoint

The primary efficacy endpoint is total VTE, defined as the composite of proximal and/or distal DVT (asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic), nonfatal PE, or any death assessed through the Day 10-14 visit.

Key Secondary Endpoints

The key secondary endpoints are the assessment of the primary endpoints through the Week 18 visit, and: • All individual components of the primary safety endpoint (major bleeding, clinically relevant nonmajor bleeding, and minimal bleeding)

• Composite of major and clinically relevant nonmajor bleeding

• Major VTE, a composite of proximal DVT (asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic), nonfatal PE, or any death

• All individual components of the primary efficacy endpoint (ie, proximal and/or distal DVT [asymptomatic confirmed by venography assessment of the operated leg or objectively confirmed symptomatic], nonfatal PE, any death)

Other Secondary Endpoints

• Any wound or joint complication in the operated leg

Exploratory Endpoints

• Other thrombotic events (ie, MI, ischemic stroke, peripheral arterial embolism)

• The total length of the initial hospitalization, including the level of care and discharge destination

• The incidence of rehospitalization for any reason

• The number of scheduled and unscheduled visits, including the associated cost to healthcare providers for study outcomes, any other medical reasons, and the diagnostic procedures used in relation to study endpoints

· Calculation of PK parameters, eg, CL, volume of distribution (Vd), tin, AUCinf, and

• Changes in PD by dose: TT, ECT, PT, aPTT, and D-dimer

• Changes in PD assays as listed above by concentration metrics (AUCinf, Cmax, or time -matched concentrations)

Refer to Section 9, Study Evaluations for evaluations related to endpoints.

2.2. Hypothesis

[ 00326 ] In Part 1, a range of doses of JNJ-64179375 will be assessed to determine if it is sufficiently safe to proceed with Part 2 of the study. No formal hypothesis testing will be conducted for Part 1. In Part 2, the clinical hypothesis is to demonstrate proof-of- efficacy based on the total VTE endpoint. This can be achieved by either showing that JNJ-64179375 has a statistically significant dose-response trend with respect to the total VTE endpoint, or the combined dose groups of JNJ-64179375 have a total VTE event rate of less than 30%. 3. STUDY DESIGN AND RATIONALE

3.1. Overview of Study Design

[ 00327 ] This is a randomized, double-blind, double-dummy, active-controlled, multicenter, dose-escalation and dose-response study of JNJ-64179375. The study has 2 parts, dose-escalation and dose-response evaluation, and will be conducted in subjects undergoing primary unilateral elective TKR surgery. Part 1 will assess the safety of single-ascending IV doses of JNJ-64179375 while Part 2 will confirm the safety of the doses selected from Part 1 and will assess the efficacy dose-response relationship with respect to the selected doses in the parallel groups. Subjects will be men or women of non-childbearing potential≥50 years of age, considered medically appropriate for postoperative anticoagulant prophylaxis based on the study- and local standard-of-care- required assessments for pre- and postoperative evaluation. Subjects will participate in either Part 1 or Part 2 of the study only. A total of approximately 1,500 subjects combined for Parts 1 and 2 are planned to be enrolled. Part 1 will include approximately

300 subjects and Part 2 will include approximately 1,200 subjects as described in Section 3.1.1, Part 1 : Single-Ascending Dose and Section 3.1.2, Part 2: Dose-Response

Confirmation, respectively.

[ 00328 ] For each subject in each part, the study will be conducted in 3 phases: an up to 30-day screening phase before surgery, a 14-day double-blind dosing phase, and a 16- week follow-up phase. Unscheduled visits may be performed at the discretion of the investigator for the assessment of any potential bleeding or efficacy endpoint events. The total duration of the subject's participation in Part 1 or Part 2 after randomization will be approximately 18 weeks.

[ 00329 ] Screening for eligible subjects in Parts 1 and 2 may be done up to 30 days before randomization, pre- or postoperatively. Following primary unilateral elective TKR surgery, eligible subjects will be randomly assigned to treatment with either JNJ- 64179375 or apixaban. All subjects will receive a single IV infusion of JNJ-64179375 or JNJ-64179375 placebo and oral apixaban or matching apixaban placebo the day after TKR surgery (Day 1, 0 hours), while the subject is still hospitalized and within a minimum of 12 hours and a maximum of 24 hours after the end of the TKR surgery, defined as the time of wound closure. Details regarding the timing of oral dosing in relation to the start of the IV infusion are provided in Section 0, Dosage and

Administration.

[ 00330 ] Following discharge or transfer to an alternate facility, subjects in both parts will be reminded to continue to take apixaban or matching apixaban placebo twice daily to complete a total of 10 to 14 days of dosing as described in Section 6, Dosage and Administration. Unilateral venography assessment of the operated leg will be performed after the last dose of apixaban or matching apixaban placebo is taken as described in Section 9.2.1, Assessments for DVT.

[ 00331 ] Subjects in both Parts 1 and 2 will return to the site for study -related procedures 5, 10, and 18 weeks after TKR surgery as described in the Time and Events Schedule. Safety evaluations will include the monitoring of all nonserious and serious adverse events, (including adverse events of special interest: bleeding events, infusion reactions, hypersensitivity reactions, and wound or joint complications), clinical laboratory tests (ie, hematology, clinical chemistry, urinalysis), vital signs measurements (blood pressure, pulse/HR, temperature), and physical examinations. Pharmacokinetics (dense and sparse), PD, and immunogenicity samples will be collected and health resource utilization will also be assessed at the time points indicated in the Time and Event Schedule.

[ 00332 ] An unblinded data review is planned for Part 1 after all subjects are expected to have completed the Day 10-14 visit to determine the dose range and doses of JNJ-

64179375 for Part 2. Two planned, unblinded interim analysis will be conducted in Part 2 by the IDMC as part of the adaptive approach that will be used to guide decisions to drop and/or add doses of JNJ-64179375 and adjust the randomization ratio based on the available efficacy, safety, PK, and PD data. Additional details are provided in Section 0, Interim Analyses. An unblinded administrative interim analysis is planned after all subjects are expected to have completed the Day 10-14 visit in Part 2 to facilitate additional study planning as described in Section 0, Interim Analyses.

[ 00333 ] A diagram of the study design is provided in Figure 30.

3.1.1. Part 1: Single-Ascending Dose

[ 00334 ] Eligible subjects in Part 1 will be randomly assigned to a single-ascending IV dose of JNJ-64179375 or apixaban 2.5 mg given orally twice daily for 10 to 14 days. Six cohorts of up to approximately 50 unique subjects per cohort (total of up to approximately 300 unique subjects) are planned but the number of cohorts and the size of each cohort may be adjusted based on the ongoing unblinded data review by the OC. Within each cohort, subjects will be randomized in a 4: 1 ratio to JNJ-64179375 or apixaban, respectively (ie, approximately 40 subjects to JNJ-64179375: approximately 10 subjects to apixaban). JNJ-64179375 will be administered in a dose-escalation manner, with planned doses of 0.3, 0.6, 1.2 mg/kg, and 1.8 mg/kg in Cohorts 1, 2, and 3, respectively. In Part 1, the OC will be responsible for reviewing ongoing safety and efficacy data by unblinded subject treatment assignments approximately every 1 to 3 weeks. Subjects enrolled in the 3 optional cohorts will receive doses of JNJ-64179375 in the range of 0.1 to 1.8 mg/kg, which will be dependent on the available preliminary safety, tolerability, efficacy, PK, and PD data obtained from the preceding cohorts (refer to Section 6, Dosage and Administration for additional details). After all of the subjects in Part 1 are expected to have completed the Day 10-14 visit, an unblinded data review will be conducted by the OC, SC, IDMC, and sponsor to determine the dose range and doses for Part 2.

3.1.2. Part 2: Dose-Response Evaluation

[ 00335 ] Part 2 of this study has an adaptive design, with the intent to optimize data collection for the dose-response evaluation using multiple comparison procedure and modeling (MCP-Mod). Eligible subjects prior to the first interim analysis in Part 2 will be randomly assigned equally to 1 of up to 5 parallel treatment groups, including up to 4 dose levels of JNJ-64179375, given as a single, active IV infusion, or oral

apixaban 2.5 mg twice daily for 10 to 14 days. The number of doses and randomization ratio after the 2 interim analyses will depend on the interim analysis results. However, the number of ongoing doses of JNJ-64179375 in the study is not expected to exceed 4 doses. The Part 2 sample size is estimated to be 1,200 subjects. In Part 2, the IDMC will be responsible for monitoring ongoing safety and efficacy and for conducting the 2 planned interim analyses to apply the adaptive design rules. Subjects will be randomized to 1 of the treatment groups with a balanced randomization ratio until the first interim analysis. After the review of each planned interim analysis, the IDMC will make a

recommendation to declare futility, adjust the study drug doses, or modify the randomization ratio (see Section 11.10, Interim Analyses for details). The SC and sponsor will make a decision whether to implement the IDMC's recommendation. The interim analyses will be performed after approximately 400 and 800 subjects are enrolled and are expected to have completed the Day 10-14 visit. The final number of subjects in each dose group will depend on the results of the interim analyses.

3.2. Study Design Rationale Blinding, Control, Study Phase/Periods, Treatment Groups

[ 00336 ] Randomization will be used to minimize bias in the assignment of subjects to treatment groups, to increase the likelihood that known and unknown subject attributes (eg, demographic and baseline characteristics) are evenly balanced across treatment groups, and to enhance the validity of statistical comparisons across treatment groups. Blinded treatment will be used to reduce potential bias during data collection and evaluation of clinical endpoints.

[ 00337 ] The treatment phase duration of up to 14 days is the approved duration of therapy with apixaban after TKR surgery. JNJ-64179375 has an estimated human plasma t½ of approximately 25 days based on preliminary data in healthy subjects. The 16-week follow-up duration (18 weeks from dosing) in this study is approximately 5 times the calculated ti/2 of JNJ-64179375, and is therefore adequate for PK analysis and is consistent with the safety reporting period.

[ 00338 ] The dose-escalation design in Part 1 will allow for a careful stepwise approach to assess bleeding risk in subjects undergoing TKR surgery to determine the dose range for the parallel design in Part 2, which will confirm the safety and efficacy dose response of JNJ-64179375.

Study Population

[ 00339 ] The risk of VTE following major orthopedic surgery, specifically joint replacement surgery, has been well documented and the use of postoperative prophylactic anticoagulant therapy is widely accepted as standard of care.^10,19 Over the past 20 years, Phase 2 studies of injectable anticoagulants (LMWH, fondaparinux) and all of the newer oral anticoagulants (apixaban, rivaroxaban, dabigatran, and edoxaban) were conducted in this population using venography to detect asymptomatic DVT. Subsequently, all of these oral medications were evaluated in large, similarly designed Phase 3 studies and have been approved by regulatory agencies for the prevention of VTE in TKR surgery because they were found to be at least as effective as LMWH, have a similar or improved bleeding profile, and have the convenience of easy administration.⁹'¹³'¹⁵'²³'²⁴-³⁰ Therefore, the TKR study population is appropriate to evaluate the efficacy and safety of JNJ-64179375.

[ 00340 ] Total knee replacement surgery is most commonly performed due to arthritic disease of the joint, which occurs primarily in older men and women. The risks of VTE and bleeding also increase with age. Therefore, the intended population, which includes both men and women who are at least 50 years of age, is also appropriate. Women of childbearing potential are excluded from this study because reproductive toxicology testing of JNJ-64179375 has not been conducted. Dose Selection

[ 00341 ] The Part 1 planned doses of JNJ-64179375 (0.3, 0.6, or 1.2 mg/kg) were chosen based on preclinical data and data from the Phase 1 study in healthy subjects (Protocol 64179375EDI1001). In preclinical venous thrombosis models in both rats and monkeys, JNJ-64179375 at a dose of 0.3 mg/kg demonstrated good efficacy, which increased even further at 1 mg/kg. In healthy subjects, the 0.3-mg/kg dose was associated with a maximum mean blood level of approximately 7 μg/mL and TT prolongation of approximately 3 -fold from baseline while the 1 -mg/kg dose had maximum mean blood levels of about 30 μg/mL, with TT prolongations of greater than approximately 5 -fold from baseline. Both doses demonstrated an acceptable safety profile. Therefore, the starting dose of 0.3 mg/kg was selected as it should be effective for VTE prevention after TKR surgery and provides a wide safety margin from the Phase 1 doses that showed a bleeding signal at 2.125 and 2.5 mg/kg (as described supra, Section 1.1.2.1.2.2., Summary of Part 1 Results). Dose escalation will then be guided by the OC based on accumulating data for both safety and efficacy. The highest planned dose in Part 1 of 1.2 mg/kg has been selected because it is similar to the 1 -mg/kg dose studied in the Phase 1 study and is approximately 50% of the lowest dose with a bleeding signal

(ie, 2.125 mg/kg) in healthy subjects. If the 0.3-mg/kg dose appears to be fully effective in Part 1, there is the option to study a lower dose (as low as 0.1 mg/kg); likewise, if the 1.2-mg/kg dose appears well tolerated and incremental benefit is expected from a higher dose, there is the option to study a higher dose (up to 1.8 mg/kg). The dose selection in Part 2 will be based on the safety and efficacy results/comprehensive analyses from Part 1, with the goal being to study the widest possible dose range supported by the data. Choice of Comparator

[ 00342 ] An active control (apixaban) will be used because pharmacologic VTE prophylaxis is considered standard-of-care in subjects undergoing TKR surgery. Given that VTE and bleeding event rates vary across studies, the active control will also provide an internal reference for comparison with JNJ-64179375 in this study.

[ 00343 ] Apixaban was chosen as the comparator in this study because it is an approved oral drug for this indication, has a start time after surgery that is preferred by many surgeons, and has demonstrated favorable efficacy and safety (ie, bleeding) results compared with enoxaparin. Refer to Section 1.2, Comparator Drug for additional details regarding apixaban.

[ 00344 ] The 2.5-mg dose of apixaban, taken twice daily for 10 to 14 days beginning 12 to 24 hours after the end of TKR surgery will be used in this study as it is the approved dosage regimen (in approved countries) for the indication being studied.⁸

Choice of Efficacy Measures

[ 00345 ] Total VTE is a standard efficacy measure for Phase 2 TKR VTE prophylaxis studies. The use of venography to detect asymptomatic DVT and standardized definitions to assess bleeding events are specifically recommended as the best approach for the Phase 2 orthopedic surgery setting.⁷ The assessment time between Days 10-14 is appropriate given that it is the approved duration of therapy for apixaban.

[ 00346 ] Ultrasound is a noninvasive, widely available technique, with a high sensitivity and specificity for symptomatic DVT that has replaced venography in clinical practice for the diagnosis of DVT events (all DVT sensitivity 88%, specificity 96%).²⁰ However, ultrasound has repeatedly been shown to have very low sensitivity compared with venography for detecting asymptomatic DVT in the postoperative setting.^20,27 In a meta-analysis of 15 studies, the sensitivity of ultrasound compared with venography for detecting asymptomatic DVT was 47%.²⁰ More recent data are from the Phase 2 studies of rivaroxaban in DVT prophylaxis following hip and knee replacement surgeries, where a substudy (VENUS study) was conducted comparing venography to ultrasound. Despite rigorous methodology, including separate adjudication sites for each technique and a large number of matching pairs of evaluable venography assessments and ultrasounds, the authors concluded that ultrasound cannot replace venography for DVT diagnosis in this setting. The observed frequency of any DVT was 18.9% with venography and 11.5% with ultrasound. The sensitivity of ultrasound compared with venography was

31.1% (95% confidence interval [CI]: 23.4, 38.9) for any DVT, 21.0% (95% CI: 2.7, 39.4) for proximal DVT, and 30.8% (95% CI: 23.1, 38.6) for distal DVT. The results for specificity were 93.0% (95% CI: 91.0, 95.1), 98.7% (95% CI: 98.0, 99.5), and 93.3% (95% CI: 91.5, 95.3), respectively.²⁷

[ 00347 ] Therefore, venography is still considered the gold standard and the only reliable method for diagnosing asymptomatic DVT after TKR surgery. The most likely explanations for the poor performance of ultrasound compared with venography in this setting are the nature of the clots that form early after surgery (small and compressible) compared with symptomatic clots (larger and noncompressible) and the distortion of the veins produced by the postoperative swelling.

[ 00348 ] Although research into new anticoagulants has slowed in recent years, all proof-of-concept studies using the postoperative orthopedic model have continued to use venography to assess the primary endpoint.⁵'²²-³¹ Venography produces plausible and reliable results in clinical studies⁷'^14,29 and no anticoagulant has been approved by a health authority for postoperative orthopedic DVT prophylaxis without venography data.

Despite the challenges with performing venography, it remains the standard for detecting DVT after TKR surgery and is the most appropriate method for use in this study.

[ 00349 ] Bilateral venography has been used in most but not all previous Phase 3 studies.^11,13 However, as unilateral venography of the operated leg detects over 90% of DVTs after TKR surgery¹² and exposes subjects to less risk (radiation, contrast dye, venipuncture) and less discomfort, only venography of the operated leg will be performed in this study.

[ 00350 ] Suspected symptomatic efficacy (thrombotic) events (ie, DVT, PE, death, MI, stroke, peripheral arterial embolism) will also be specifically assessed in this study as these are clinically important events from the perspective of both the subject and the surgeon.

Choice of Safety Measures/Assessments

[ 00351 ] Bleeding events are the standard primary safety endpoint in studies of anticoagulant VTE prophylaxis after TKR surgery. Because the occurrence of major bleeding events is infrequent and previous dose-ranging studies have demonstrated that all categories of bleeding events increase with dose in a similar manner, the any bleeding event composite will be the primary safety endpoint in this study.²¹ Published guidelines that describe how to define major bleeding events after major orthopedic surgery will be followed.²⁸ For nonmajor bleeding events, standardized definitions, as utilized in the Phase 3 studies of apixaban, will be followed.²³-²⁴ All wound or joint complications will also be specifically assessed in this study as these are important from the perspective of both the subject and the surgeon.

Choice of PK and Immunogenicity Measures

[ 00352 ] Pharmacokinetic blood samples will be collected from all subjects and analyzed for subjects who are randomly assigned to treatment with JNJ-64179375 to further understand the PK characteristics and variability of JNJ-64179375 in the TKR patient population. The combination of dense and sparse PK samples in this patient population will be sufficient for the development of a population PK model and predicted concentrations from such a model will allow for the development of exposure-response models for various endpoints related to safety and efficacy. Immunogenicity blood samples will be collected from all subjects and analyzed for subjects randomly assigned to JNJ-64179375 to assess for the development of any ADAs.

Choice of Pharmacodynamic/Biomarker and PK/PD

[ 00353 ] The PD assays (TT, ECT, PT, aPTT, and D-dimer) will be used to evaluate the mechanism of action and pharmacologic activity of JNJ-64179375. A battery of coagulation assays with different inhibition profiles for JNJ-64179375 has been chosen to evaluate those assays that might be most appropriate for assessing both pharmacologic activity and any relationships with PK and/or efficacy and/or safety endpoints. The goal of the PD analyses is to evaluate the PD effects of JNJ-64179375, the PK/PD relationship, and PD outcome relationships. Additional details regarding the collection of the PD assessment blood samples and the test procedures will be included in the laboratory manuals. It is expected that apixaban will have no effect or minimal effects on all of the coagulation assays and therefore, analysis of the apixaban samples is not planned.

[ 00354 ] Biomarker samples may be used to help address emerging issues and enable the development of safer, more effective, and ultimately individualized therapies. An exploratory objective will be to evaluate and compare the performance of the various PD assays. A central specialty laboratory will be used to provide more robust quality control for these assays by performing them in batches and to maintain the study blind. No local PD sample collection is planned. Detailed descriptions of each of the assays are provided in Section 9.4, Pharmacodynamic/Biomarker Evaluations.

Choice of Health Resource Utilization Data Collection

[ 00355 ] With the growing demand on limited healthcare resources and concern about healthcare expenditures, the collection of health resource utilization data will be integrated into the study. Health resource utilization endpoints collected from this study will be key inputs for conducting exploratory cost-effectiveness analyses.

4. SUBJECT POPULATION

[ 00356 ] It is estimated that approximately 1,500 subjects will be randomly assigned to treatment in this study as described in Section 3.1.1, Part 1 : Single-Ascending Dose and Section 3.1.2, Part 2: Dose-Response Evaluation. Subjects who are withdrawn early from the study will not be replaced. For a discussion of the statistical considerations of subject selection, refer to Section 11.2, Sample Size Determination.

[ 00357 ] The inclusion and exclusion criteria for enrolling subjects in this study are described in the following 2 subsections. If there is a question about the inclusion or exclusion criteria below, the investigator must consult with the appropriate sponsor representative and resolve any issues before enrolling a subject in the study. Waivers are not allowed.

[ 00358 ] Screening for eligible subjects in Parts 1 and 2 may be done up to 30 days before randomization, pre- or postoperatively. Subjects will be eligible for rescreening only in those cases when TKR surgery is rescheduled outside of the 30-day window. Subjects may only be rescreened on 1 occasion.

4.1. Inclusion Criteria

[ 00359 ] Each potential subject must satisfy all of the following criteria to be enrolled in the study:

1. Male or female of non-childbearing potential

2. At least 50 years of age or older

3. Weight≥40 kg to < 150 kg Medically appropriate for postoperative anticoagulant prophylaxis as determined by the investigator on the basis of the physical examination, medical history, and vital signs measurements performed as part of screening for elective TKR surgery and any examinations performed as part of standard postoperative care following surgery Medically appropriate for postoperative anticoagulant prophylaxis on the basis of clinical laboratory tests performed as part of screening for elective TKR surgery and any examinations performed as part of standard postoperative care following surgery. If the results of the laboratory tests are outside the normal reference ranges, the subject may be included only if the investigator judges the abnormalities or deviations from normal to be not clinically significant or to be appropriate and reasonable for the population under study. This determination must be recorded in the subject's source documents.

Has undergone an elective primary unilateral TKR

Must sign an informed consent form (ICF) indicating that he or she understands the purpose of, and procedures required for, the study and is willing to participate in the study. Before randomization, a woman must not be of childbearing potential defined as:

o Postmenopausal

A postmenopausal state is defined as no menses for 12 months without an alternative medical cause. A high follicle stimulating hormone (FSH) level (>40 IU/L or mlU/mL) in the postmenopausal range may be used to confirm a postmenopausal state in women not using hormonal contraception or hormonal replacement therapy, however in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient.

o Permanently sterile

Permanent sterilization methods include hysterectomy, bilateral salpingectomy, bilateral tubal occlusion/ligation procedures, and bilateral oophorectomy.

If reproductive status is questionable, additional evaluation should be considered in consultation with the sponsor.

A woman must agree not to donate eggs (ova, oocytes) for the purposes of assisted reproduction during the study

Contraceptive use by men should be consistent with local regulations regarding the use of contraceptive methods for subject participating in clinical studies.

During the study from Day 1 through the Week 18 visit, a man

• Who is sexually active with a woman of childbearing potential must agree to use a barrier method of contraception (eg, condom with spermicidal foam/gel/film/cream/suppository)

• Who is sexually active with a woman who is pregnant must use a condom

• Must agree not to donate sperm • Must agree not to father a child

11. Willing and able to adhere to the prohibitions and restrictions specified in this protocol.

4.2. Exclusion Criteria

[ 00360 ] Any potential subject who meets any of the following criteria will be excluded from participating in the study:

1. Any condition for which the use of apixaban is not recommended in the opinion

of the investigator (eg, spinal anesthesia procedure with bleeding or significant trauma, postoperative epidural analgesia with an epidural catheter within 5 hours of oral or IV study drug administration, previous allergic reaction, creatinine clearance <15 mL/minute or on dialysis)

2. Bilateral, revision or unicompartmental procedure

3. Known or suspected hypersensitivity or intolerance to any biologic medication

or known allergies or clinically significant reactions to murine, chimeric, or human proteins, monoclonal antibodies or antibody fragments, or any of the excipients of JNJ-64179375

4. Unable to undergo venography (eg, due to contrast agent allergy, poor venous

access, or impaired renal function that would increase the risk of contrast- induced nephropathy)

5. Known previous DVT in either lower extremity

6. Chronic anticoagulation therapy for any condition (eg, atrial fibrillation,

mechanical heart valve) is required

7. Any preplanned invasive procedure (eg, surgery, colonoscopy) up to Week 18

for which anticoagulant or antiplatelet therapy would be interrupted

8. Planned use of intermittent pneumatic compression after randomization

9. Received an investigational drug (including investigational vaccines,

experimental antibody or biologic therapy) within the previous 6 months or 5 half-lives, whichever is longer, or received any other experimental therapy, or used an invasive investigational medical device within 60 days before the planned first dose of study drug or is currently enrolled in an investigational study

Previous randomized subject in this study or participated in previous studies with JNJ-64179375 11. Any condition for which, in the opinion of the investigator, participation would not be in the best interest of the subject (eg, compromise the well-being) or that could prevent, limit, or confound the protocol-specified assessments

12. Employee of the investigator or study site, with direct involvement in the proposed study or other studies under the direction of that investigator or study site, as well as family members of the employees or the investigator

13. At the time of informed consent, the subject does not agree to following up with scheduled study visits or allowing a telephone contact to the subject's alternative means of contact (eg, subject's children, spouse, significant other, caretaker, legal representative, or healthcare professional), as necessary, until the end of the study, should he or she discontinue prematurely

NOTE: Investigators should ensure that all study enrollment criteria have been met at screening. If a subject's clinical status changes (including any available laboratory results or receipt of additional medical records) after screening but before the first dose of study drug is given such that he or she no longer meets all eligibility criteria, then the subject should be excluded from participation in the study. Section 9.1.2, Screening Phase, describes options for retesting.

5. TREATMENT ALLOCATION AND BLINDING Treatment Allocation

[ 00361 ] Appropriate measures were taken for randomization of subjects in this study based on a computer-generated randomization schedule. Blinding

[ 00362 ] Appropriate measures were taken for blinding and unblinding information related to this study.

6. DOSAGE AND ADMINISTRATION

[ 00363 ] In both Parts 1 and 2, subjects will be randomly assigned to receive an active single IV dose of JNJ-64179375 or apixaban 2.5 mg given orally twice daily for 10 to 14 days. The administration of the study drugs will begin the day after the TKR surgery (Day 1, 0 hours), while the subject is still hospitalized and within a minimum of 12 hours and a maximum of 24 hours after the end of the TKR surgery, defined as the time of wound closure. Within that timeframe, both the single IV infusion and the oral study drug should be administered as close as possible to each other, with up to a maximum of 60 minutes between the start of the IV infusion and the first dose of the oral study drug. Subjects will be randomly assigned to the treatment groups as follows:

Part 1

• Cohort 1 : JNJ-64179375 0.3 mg/kg IV/JNJ-64179375 placebo (saline) IV infusion and matching apixaban placebo/apixaban, orally twice a day for 10 to 14 days

• Cohort 2: JNJ-64179375 0.6 mg/kg IV/JNJ-64179375 placebo (saline) IV infusion and matching apixaban placebo/apixaban, orally twice a day for 10 to 14 days

• Cohort 3: JNJ-64179375 1.2 mg/kg IV/JNJ-64179375 placebo (saline) IV infusion and matching apixaban placebo/apixaban, orally twice a day for 10 to 14 days

Optional Cohorts (JNJ-64179375 in the Range of 0.1 to 1.8 mg/kg)

[ 00364 ] The doses of JNJ-64179375 to be used in the optional cohorts will be dependent on the available preliminary safety, tolerability, efficacy, PK, and PD data obtained from the preceding cohorts as described in Section 3.1.1, Part 1, Single- Ascending Dose. Doses within the optional cohorts will either be new doses within the range of 0.1 to 1.8 mg/kg not previously administered in the preceding cohorts, or doses from the preceding cohorts, which may be repeated, as needed.

• Cohort 4: JNJ-64179375 Dose to be determined 1.8 mg/kg IV/JNJ-64179375 placebo (saline) IV infusion and matching apixaban placebo/apixaban, orally twice a day for 10 to 14 days

· Cohort 5: JNJ-64179375 Dose 1.8 mg/kg IV/JNJ-64179375 placebo (saline) IV infusion and matching apixaban placebo/apixaban, orally twice a day for 10 to 14 days

• Cohort 6: JNJ-64179375 Dose 1.8 mg/kg IV/JNJ-64179375 placebo (saline) IV infusion and matching apixaban placebo/apixaban, orally twice a day for 10 to 14 days

Part 2

· Group A: JNJ-64179375 Dose A mg/kg IV and apixaban placebo orally twice a day for 10 to 14 days

• Group B: JNJ-64179375 Dose B mg/kg IV and apixaban placebo orally twice a day for 10 to 14 days

• Group C: JNJ-64179375 Dose C mg/kg IV and apixaban placebo orally twice a day for 10 to 14 days

• Group D: JNJ-64179375 Dose D mg/kg IV and apixaban placebo orally twice a day for 10 to 14 days

• Group E: JNJ-64179375 placebo (saline) IV and apixaban 2.5 mg orally twice a day for 10 to 14 days

[ 00365 ] Doses of JNJ-64179375 may change after the interim analyses in Part 2 as described in Section 11.10, Interim Analyses. [ 00366 ] For both parts, JNJ-64179375 will be prepared according to the subject's weight and treatment assignment by the pharmacist or other appropriately licensed and authorized health professional who is not blinded to the treatment assignment. All study drug administrations of JNJ-64179375 must be calculated based on the subject's weight on Day -1 or prior to dosing on Day 1. The single infusion of JNJ-64179375 or JNJ- 64179375 placebo (saline) will be administered under the supervision of the investigator or his/her designee over a period of approximately 30 minutes by infusion pump or by gravity flow using a flow regulator. A physician must be immediately available at the study site at all times during the administration of the study drug infusion. Detailed instructions for dose preparation, dosing procedures, and storage conditions of the study drug will be provided to the study site in the IPPI. These documents may be revised as needed and should be maintained in the study files.

[ 00367 ] The first dose of apixaban or matching apixaban placebo will be administered as previously described. Subjects will receive apixaban or matching apixaban placebo twice a day while hospitalized and will be given a supply of apixaban or matching apixaban placebo at the time of discharge or transfer to an alternate facility, with instructions to take the study drug orally, twice a day at approximately the same times each day to complete a total of 10 to 14 days of dosing. Apixaban or matching apixaban placebo should be swallowed with water, with or without food.

[ 00368 ] If a dose of apixaban or matching apixaban placebo is missed, the missed dose should be taken as soon as possible and then the subject should continue with the twice- daily intake as previously instructed. The dose should not be doubled to make up for a missed dose and more than 2 doses should not be taken on the same day.

[ 00369 ] Refer to Section 13, Study Drug Information for details regarding a physical description of the study drugs, packaging, labeling, and preparation, handling, and storage.

7. TREATMENT COMPLIANCE

[ 00370 ] Drug supplies will be inventoried and accounted for throughout the study. The IWPvS will track the study drug dispensed to (JNJ-64179375 or apixaban/matching apixaban placebo) and returned by (apixaban or matching apixaban placebo only) subjects. [ 00371 ] JNJ-64179375 or JNJ-64179375 placebo will be administered as a single IV infusion by qualified study site personnel and the details of the administration will be recorded. For apixaban or matching apixaban placebo, subjects will be required to return empty study drug containers and unused study drug at their Day 10-14 visit, at which time study drug accountability will be performed.

8. PRESTUDY AND CONCOMITANT THERAPY

[ 00372 ] Modification of an effective preexisting therapy should not be made for the explicit purpose of entering a subject into the study.

[ 00373 ] The use of the following medications/therapies is not permitted from randomization through Week 18:

• Additional anticoagulant(s) (eg, vitamin K antagonists, Factor Ila or FXa inhibitors) .

The blinded study drug (apixaban) will be discontinued in subjects who develop any condition that requires long-term anticoagulation (eg, DVT, atrial fibrillation). Refer to Section 9.2, Efficacy Evaluations for details regarding the management approach for subjects who develop a symptomatic VTE event or who have asymptomatic DVT detected by venography.

• Antiplatelet therapies (eg, platelet adenosine diphosphate P2Yn receptor antagonist [eg, clopidogrel, ticagrelor]) during the study except for aspirin <100 mg/day

• Nonsteroidal anti-inflammatory drugs (NSAIDs) should be avoided, if possible, during the study because their use can increase the risk of bleeding and may interfere with collagen formation. If NSAID use is necessary, it is recommended that the minimum dose is used for the shortest possible duration.

• Intermittent pneumatic compression after randomization (all other mechanical VTE prevention methods such as foot pumps, graduated compression stockings and continuous passive motion devices are permitted)

[ 00374 ] In addition, the use of the following medications is not permitted

concomitantly with apixaban or matching apixaban placebo:

• Concomitant systemic treatment with strong inhibitors of both cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp), such as azole-antimycotics (eg, ketoconazole, itraconazole, voriconazole, and posaconazole) and human immunodeficiency virus protease inhibitors (eg, ritonavir). These medicinal products may increase apixaban exposure by 2-fold or greater in the presence of additional factors that increase apixaban exposure (eg, severe renal impairment).⁸

• Strong CYP3A4 and P-gp inducers (eg, rifampicin, phenytoin, carbamazepine, phenobarbital, or St. John's Wort). The concomitant use with apixaban may lead to an approximately 50% reduction in apixaban exposure.⁸ [ 00375 ] Only selected medications administered up to 7 days before randomization will be recorded in the electronic case report form (eCRF) as prestudy therapies (eg, TXA, antiplatelet therapies, anticoagulant therapies, and NSAIDs).

[ 00376 ] All concomitant pharmacologic therapies, including prescription or over-the- counter medications, products to manage bleeding, vaccines, vitamins, and herbal supplements different from the study drug must be recorded throughout the study from the time of randomization through the last follow-up visit on Week 18.

[ 00377 ] All non-pharmacologic therapies such as intermittent pneumatic compression devices, foot pump devices, continuous passive motion devices, compression stockings, electrical stimulation, acupuncture, special diets, exercise regimens, including physical and occupational therapy must also be recorded in the eCRF.

[ 00378 ] Recorded information will include a description of the type of therapy, duration of use, dosing regimen, route of administration, and its indication.

[ 00379 ] The sponsor must be notified in advance (or as soon as possible thereafter) of any instances in which prohibited therapies are administered.

9. STUDY EVALUATIONS

9.1. Study Procedures

9.1.1. Overview

[ 00380 ] The Time and Events Schedule summarizes the frequency and timing of efficacy, PK, immunogenicity, PD/biomarker, health resource utilization, and safety measurements applicable to this study. The Time and Events Schedule, and the description of the study phases that follows, are applicable to subjects participating in either Part 1 or Part 2 of the study, unless otherwise noted.

[ 00381 ] If multiple assessments are scheduled for the same time point, it is recommended that procedures be performed in the following order: ECG (as applicable), vital signs, and blood draw.

[ 00382 ] Blood collections for PK and PD assessments should be kept as close to the specified time as possible. Other measurements may be done earlier than specified time points, if needed. Actual dates and times of assessments will be recorded in the source documentation. Blood samples for PK and PD assessments on Day 1 postdose should be collected in the opposite arm as the infusion of JNJ-64179375 or placebo. Blood samples will be collected in the following order: safety (serum chemistry, hematology), PK, immunogenicity, and PD.

[ 00383 ] Venous blood will be collected for all blood-based analysis. Blood will be drawn using a cannula or by venipuncture. Only saline (not heparin) can be used for flushing the cannula. If blood samples are collected via an indwelling cannula, an appropriate amount (ie, 1 mL) of fluid slightly greater than the dead space volume of the lock will be removed from the cannula and discarded before blood samples are taken.

[ 00384 ] The total blood volume to be collected from each subject will be

approximately 95.4 mL for subjects with dense PK sampling and approximately 80.4 mL for subjects with sparse PK sampling as described in Table 10. It is important to record the exact date and time for PK sample collection even if the time deviates slightly from the scheduled time of collection.

[ 00385 ] Additional blood samples may be collected, if necessary, for additional safety, PK, or PD assessments based on emerging data, but the total blood volume collected from an individual subject during this study will not exceed 200 mL without prior Independent Ethics Committee (IEC) or Institutional Review Board (IRB) and health authority approvals. Repeat or unscheduled samples may be taken for safety reasons or for technical issues with the samples and do not require prior IEC/IRB and health authority approvals.

[ 00386 ] Health resource utilization data will be collected. Refer to Section 0, Health Resource Utilization for details.

9.1.2. Screening Phase

[ 00387 ] The screening phase will occur up to 30 days before randomization, pre- or postoperatively. Potential subjects may be evaluated for eligibility before or after their unilateral elective TKR surgery. Final eligibility must be confirmed after surgery, prior to randomization.

[ 00388 ] Prior to conducting any study-related procedure, the investigator (or designated study personnel) will review and explain the written ICF to each subject. No study procedures can be performed until the subject signs the ICF. At the time of informed consent, 2 alternative means of contact for the each subject will be collected (eg, contact information of the subject's children, spouse, significant other, caretaker, legal representative, or health care professional). Details regarding the TKR surgery and the post-surgery management (eg, type of anesthesia, procedure duration, cement use, tourniquet use and duration, drain use and volume, and use of all mechanical

VTE prophylaxis methods) will be collected in the eCRF. In addition, the subject will respond to questions regarding his or her family and personal bleeding history, including any bleeding from previous surgical procedures.

[ 00389 ] Subjects will be eligible for rescreening only in those cases when TKR surgery is rescheduled outside of the 30-day window. Subjects may only be rescreened on 1 occasion.

9.1.3. Double-Blind Dosing Phase Day 1/Day of Randomization

[ 00390 ] On Day 1, subjects who meet all of the eligibility criteria will be randomly assigned to study drug, either JNJ-64179375 or apixaban as described in Section 6, Dosage and Administration. JNJ-64179375 or JNJ-64179375 placebo will be administered under the supervision of the investigator or his/her designee. Subjects will also begin to take oral apixaban or matching apixaban placebo twice daily. All procedures specified for the 0-hour time point should be conducted prior to the first dose of the study drug. The postdose samples will be drawn at 1 hour (±10 minutes) and 4 hours from the start of the IV infusion. Dosing Visits

[ 00391 ] Subjects will be discharged from the hospital or transferred to an alternate facility at an appropriate time as determined by the managing physician. The Day 2 blood sample will be drawn 24 hours after the start of the IV infusion and may be done with the subject as an inpatient or outpatient. Study procedures will be conducted on Days 3 (48 hours) and 7 (144 hours) only for those subjects who are still hospitalized. At the time of hospital discharge or transfer to an alternate facility, the subject will be reminded to continue to take the oral apixaban or matching apixaban placebo twice daily to complete a total of 10 to 14 days of dosing, the duration of which will be determined by the managing physician. Subjects who complete dosing with apixaban or matching apixaban placebo will return to the study site for final assessments in the double-blind dosing phase (Day 10-14 visit, end of dosing [EOD]), at which time a unilateral venography assessment of the operated leg will be performed within 24 hours of the last dose of apixaban or matching apixaban placebo. Further details regarding the venography are provided in Section 9.2.1 , Assessments for DVT. Unscheduled Visits

[ 00392 ] At the discretion of the investigator, subjects may return to the study site between scheduled visits. Subjects should return to the study site for the assessment of any potential bleeding or efficacy endpoint events. Unscheduled PK and PD samples (except D-dimer) should be collected as soon as practically possible for any subject that experiences symptomatic thrombotic or bleeding events.

Early Withdrawal

[ 00393 ] Subjects who do not receive the full infusion of JNJ-64179375 or prematurely discontinue dosing with apixaban or matching apixaban placebo before the end of the double-blind dosing phase will be instructed to return to the study site at the originally scheduled Day 10-14 visit (not earlier) to conduct assessments, including the venography assessment of the operated leg. If a subject has a suspected symptomatic DVT prior to the Day 10-14 visit, an ultrasound will be performed. If the ultrasound confirms symptomatic proximal DVT, a subsequent venography assessment is not required. In addition, if the subject is diagnosed with a PE as defined in Section 9.2.2, Assessments for PE, a venography assessment is not required. Refer to Section 9.2, Efficacy Evaluations for further details regarding anticoagulation treatment. If the ultrasound is negative or confirms a distal DVT, the venography assessment should be conducted on the Day 10-14 visit. Subjects should complete the remaining visits through the Week 18 assessments as indicated in the Time and Events Schedule.

[ 00394 ] If a subject withdraws from the study before the end of the follow-up phase and is unwilling or unable to return for follow-up visits in person or have follow-up contacts, the study site should collect as much follow-up visit information as possible, including contacting the subject or the subject's representative or health care professional by telephone or by mail to determine vital status and to collect medical information related to endpoint events, as agreed to by the subject during the initial informed consent process. For subjects who withdraw consent from study participation, the reasons for the withdrawal of consent should be documented in the source documents and entered in the eCRF. If applicable, vital status may be obtained by reviewing the subject's medical or public records unless this contact is not permitted per local regulations. Refer to Section 10.2, Discontinuation of Study DrugAVithdrawal from the Study for additional details.

9.1.4. Follow-Up Phase

[ 00395 ] The first follow-up visit will occur approximately 3 weeks following the Day 10-14 visit, with subsequent follow-up visits occurring until Week 18 as described in the Time and Events Schedule. Reasonable attempts, as defined in Section 10.2,

Discontinuation of Study DrugAVithdrawal from the Study should be made to conduct the follow-up visit(s) at the scheduled time points. Subjects will continue to be monitored for safety, including bleeding events and efficacy events, according to the time points indicated in the Time and Events Schedule.

9.2. Efficacy Evaluations

[ 00396 ] Efficacy evaluations, including the unilateral venography assessment of the operated leg and assessments of symptomatic DVT, PE, death, or other thrombotic events (ie, MI, ischemic stroke, and peripheral arterial embolism) will be performed according to the time points in the Time and Events Schedule to assess the primary, secondary, and exploratory efficacy outcomes. All venography assessments, ultrasound evaluations for suspected symptomatic DVT, as applicable, and evaluations for suspected PE or other thrombotic events will be performed and read locally and sent for central adjudication by the independent CEC. The central adjudication evaluations will not be provided to the local sites. The CEC-adjudicated and investigator-reported results on efficacy and safety outcomes will be provided for the unblinded data reviews and the interim analyses. The CEC-adjudicated events will be used in the final analysis.

[ 00397 ] If an event or condition requiring anticoagulant treatment occurs during the double-blind dosing phase then the blinded apixaban or matching apixaban placebo should be discontinued. Antithrombotic therapy management for conditions requiring anticoagulation (eg, new onset atrial fibrillation, symptomatic DVT, or symptomatic PE) will be at the discretion of the local investigator. It is recommended that the event be treated with either a DOAC only or LMWH with a transition to a DOAC. If knowledge of the likely treatment group would change the planned event management strategy then local laboratory coagulation testing may also be considered, with a normal TT likely indicating the absence of JNJ-64179375. For asymptomatic distal DVT detected by venography, a possible management approach would be to use serial ultrasound to assess for any proximal progression and withhold anticoagulant therapy unless proximal progression is observed. If it is decided that treatment is to be given, the same approach as suggested for proximal DVT or PE is recommended.

[ 00398 ] In this study, all suspected symptomatic efficacy (thrombotic) events will also be captured as adverse events of special interest, which are defined in Section 9.7.2, Adverse Events of Special Interest.

9.2.1. Assessments for DVT Unilateral Venography

[ 00399 ] Venography assessments of the operated leg will be performed by injecting contrast agent into a foot vein and obtaining x-ray images of the proximal and distal leg veins consistent with the technique described by Rabinov and Paulin.²⁶ Evaluable venography assessments require the visualization of all of the deep veins except for the muscular, anterior tibial, and deep femoral veins. A diagnosis of DVT will be made if a constant intraluminal filling defect is observed in at least 2 images. An ultrasound will be performed in those subjects with suspected symptomatic DVT prior to the Day 10-14 visit. In these cases, if the ultrasound confirms symptomatic proximal DVT, a subsequent venography assessment is not required. If the ultrasound is negative or confirms a distal DVT, the venography assessment should be conducted on the Day 10-14 visit. Details regarding the timing of the venography assessment are provided in Section 9.1.3, Double- Blind Dosing Phase.

[ 00400 ] Study-specific venography assessment training will be provided to each study site. Each study site will be responsible for identifying at least 1 primary person to perform the venography assessments for subjects. Evaluability of the venography assessments based on centrally adjudicated data will be monitored for each site on an ongoing basis. If venography assessment performance is considered not acceptable, then further randomization by the investigator may be suspended until additional training or retraining is provided. Additional details regarding the venography procedure and study- specific training requirements will be provided in a venography manual, which will be provided separately to the study sites.

9.2.2. Assessments for PE

[ 00401 ] For all subjects with symptoms of PE, spiral computed tomography (CT), pulmonary angiography, or perfusion/ventilation lung scintigraphy combined with chest radiography will be performed. A diagnosis of PE will be made only if the subject has symptoms of PE (eg, sudden onset of dyspnea, chest pain, or fainting), and 1 of the following criteria is met:

• Positive spiral CT scan of the chest

• Positive direct pulmonary arteriogram

• High probability ventilation/perfusion lung scan (defined as 1 or more segmental or large [>75% of a segment] subsegmental perfusion defects associated with ventilation mismatch)

• Intermediate probability ventilation/perfusion lung scan and ultrasound or venographic evidence of DVT

• Autopsy confirmation

[ 00402 ] If a subject is diagnosed with a PE meeting the above definitions, a subsequent venography assessment of the operated leg is not required. 9.2.3. Assessments for Other Thrombotic Events

[ 00403 ] Other thrombotic events will include MI, ischemic stroke, and peripheral arterial embolism. In addition, events that appear suggestive of study endpoints (eg, transient ischemic attack [TIA], unstable angina) will be reported by the investigator and reviewed by the CEC to ascertain if a thrombotic event has occurred. The investigator will use his/her medical judgment based upon the definitions below to determine if a thrombotic event has occurred.

Myocardial Infarction

[ 00404 ] The diagnosis of MI requires the combination of:

• Evidence of myocardial necrosis (either changes in cardiac biomarkers or postmortem pathologic findings); and

• Supporting information derived from the clinical presentation, ECG changes, or myocardial or coronary artery imaging.

Stroke

[ 00405 ] Stroke is defined as a new, sudden, focal neurologic deficit resulting from a presumed cerebrovascular cause and not due to a readily identifiable cause such as trauma, a tumor, or seizure.

[ 00406 ] If an event matching this definition lasts less than 24 hours it will be considered a TIA unless imaging confirmation of infarction is obtained. Investigators will further classify the strokes based upon imaging studies.

· Primary ischemic infarction - stroke without focal collections of intracranial blood confirmed by imaging. The occurrence of hemorrhagic conversion of a primary ischemic infarction will be recorded.

• Primary hemorrhagic - stroke with focal collections of intraparenchymal blood. The diagnosis of primary hemorrhagic stroke can only be made with imaging studies. It may include intraventricular hemorrhage.

• Subarachnoid hemorrhage - the diagnosis requires documentation by an imaging study

• Uncertain - no imaging or autopsy available

[ 00407 ] Other events of intracranial bleeding such as subdural hematoma and epidural hematoma will not be considered to be a stroke as they are usually traumatic in nature. Peripheral Arterial Embolism

[ 00408 ] A peripheral arterial embolism is defined as an abrupt vascular insufficiency associated with clinical or radiologic evidence of arterial occlusion in the absence of other likely mechanisms (eg, trauma, atherosclerosis, instrumentation). In the presence of atherosclerotic peripheral vascular disease, diagnosis of embolism to the lower extremities should be made with caution and requires angiographic demonstration of abrupt arterial occlusion.

9.3. Pharmacokinetics and Immunogenicity

[ 00409 ] Plasma samples will be used to evaluate the PK, as well as the development of ADAs (immunogenicity).

9.3.1. Evaluations

[ 00410 ] Samples for analysis of JNJ-64179375 plasma concentration will be collected for all subjects over time as specified in the Time and Events Schedule but will only be analyzed for subjects randomly assigned to JNJ-64179375. Dense PK sampling will be conducted at all sites for subjects at all visits in Part 1 until approximately up to the first 200 subjects have been randomized. Thereafter, the remaining subjects in Part 1 and all subjects in Part 2 will have PK blood samples collected at a limited number of visits (ie, sparse PK sample collection). The exact date and time of each PK blood sample collection will be recorded even if the time deviates slightly from the scheduled time of collection. Subjects who experience a bleeding event or symptomatic thrombotic event should have PK samples collected as soon as practically possible after the event occurs.

[ 00411 ] The timing of PK samples may be changed to ensure thorough PK

monitoring; however, the total number of samples and/or the volume of blood drawn will not exceed the amount stated in Section 9.1.1, Overview without prior IEC/IRB and health authority approvals. Of note, additional blood samples may be collected for immediate safety monitoring of a subject and do not require prior IEC/IRB and health authority approvals.

[ 00412 ] Samples collected for analyses of JNJ-64179375 plasma concentration and antibodies may additionally be used to evaluate safety or efficacy aspects that address concerns arising during or after the study period, or for further characterization of immunogenicity. Genetic analyses will not be performed on these plasma samples.

Subject confidentiality will be maintained. Additional information about the collection, handling, and shipment of biological samples will be provided in the laboratory manual. 9.3.2. Analytical Procedures Pharmacokinetics

[ 00413 ] Blood samples will be collected from all subjects according to the Time and Events Schedule and analyzed to determine plasma concentrations of JNJ-64179375 in appropriate samples using a validated, specific, and sensitive immunoassay method by or under the supervision of the sponsor.

Immunogenicity

[ 00414 ] The detection and characterization of antibodies will be performed using a validated assay method by or under the supervision of the sponsor. All samples collected for detection of antibodies will also be evaluated for plasma concentration to enable interpretation of the antibody data. The Day 1, 0 hours (predose) ADA sample may be used for baseline characterization for both the PK and immunogenicity assays.

9.3.3. Pharmacokinetic Parameters

[ 00415 ] The PK parameters to be calculated following the single IV dose of JNJ- 64179375 will include, but are not limited to, the following:

Cmax maximum concentration during a dosing interval

AUCinf area under the plasma concentration versus time curve from time 0 to infinity

ti/2 terminal half-life

CL total clearance of drug after IV administration

Vz apparent volume of distribution in the terminal phase

[ 00416 ] Additional PK parameters may be determined, as appropriate.

[ 00417 ] Based on the individual plasma concentration-time data using the actual dose taken and the actual sampling times, PK parameters and exposure information of JNJ- 64179375 will be derived using population PK modeling. Baseline covariates (eg, body weight, age, sex, creatinine clearance, race) may be included in the model, if relevant. A separate population PK modeling plan will be developed before the first subject is consented and the population PK modeling results will be reported separately, in a document other than the clinical study report (CSR). 9.3.4. Immunogenicity Assessments

[ 00418 ] Blood samples for antibodies will be collected from all subjects according to the Time and Events Schedule but will only be analyzed in plasma samples for subjects randomized to JNJ-64179375. The exact date and time of each blood sample collection will be recorded. Additionally, samples should also be collected at the final visit from subjects who are discontinued from treatment or withdrawn from the study. These samples will be tested by the sponsor or sponsor's designee.

[ 00419 ] Plasma samples will be screened for antibodies binding and the titer of confirmed positive samples will be reported. Other analyses (eg, neutralization capacity) may be performed to further characterize the immunogenicity.

9.4. Pharmacodynamic/Biomarker Evaluations

[ 00420 ] All subjects will have plasma samples collected to assess PD markers as specified in the Time and Events Schedule. The assay plan is designed to assess target engagement and the mechanism of action with a battery of PD assessments.

Pharmacodynamic evaluations will include the coagulation assays (ie, TT, ECT, PT, aPTT) and a D-dimer assessment. Subjects who experience a bleeding event or symptomatic thrombotic event should have PD samples (except D-dimer) collected as soon as practically possible after the event occurs. Samples on Day 1 will be obtained before the study drug is administered and at 1 hour after the start of the IV infusion and all assays will be performed by a central specialty laboratory. Additional information about the collection, processing, storage, and shipment of PD samples will be provided in a separate laboratory manual. It is expected that apixaban would have no effect or minimal effects on all of the coagulation assays and therefore, analysis of the apixaban samples is not planned.

[ 00421 ] To maintain the blind of the study, the investigator should not measure coagulation assays locally unless considered necessary for subject clinical care. In addition, the investigator will not receive the results of the PD parameters from the central specialty laboratory during the conduct of the study.

Thrombin Time

[ 00422 ] The thrombin clotting time (designated TT) is a simple test that measures the time for clot formation in citrated plasma after the addition of thrombin. It reflects the effect of thrombin to cleave fibrinogen to form fibrin. Heparin and direct thrombin inhibitors such as hirudin, argatroban, and dabigatran are inhibitory, LMWH is partially inhibitory, and apixaban is inactive.

Ecarin Clotting Time

[ 00423 ] The ECT test is based on the cleavage of prothrombin by ecarin, a highly purified metalloprotease isolated from the venom of the saw-scaled viper Echis carinatus. Ecarin generates meizothrombin from prothrombin, which is proteolytically active and can cleave fibrinogen to form a fibrin clot. Meizothrombin, like thrombin, is inactivated by hirudin and other direct thrombin inhibitors, but is unaffected by heparin (as the heparin-antithrombin complex cannot inhibit meizothrombin due to steric hindrance). JNJ-64179375 is able to block the binding of meizothrombin to fibrinogen and therefore inhibit clot formation. This assay may offer potential advantages in sensitivity and useful range over the TT test.

Prothrombin Time

[ 00424 ] Prothrombin time is a global clotting test that is used for the assessment of the extrinsic pathway of the blood coagulation cascade. It is a 1 -stage test based upon on the time required for a fibrin clot to form after the addition of Tissue Factor (historically known as tissue thromboplastin), phospholipid, and calcium to decalcified, platelet-poor plasma. The test is sensitive for deficiencies of Factors II, V, VII, and X, with sensitivity being best for Factors V, VII, and X and less pronounced for Factor II. Prothrombin time and the normalized version, international normalized ratio, have been used to monitor warfarin therapy. It should be noted that currently, 3 types of PT reagents are used: recombinant thromboplastins, tissue thromboplastins (which are usually from rabbit brain or human placenta), or combined thromboplastins (tissue thromboplastin diluted into fibrinogen). These reagents differ in factor sensitivity, heparin responsiveness, lot-to-lot consistency, and absolute value of the clotting times.

Activated Partial Thromboplastin Time

[ 00425 ] Activated partial thromboplastin time is a measure of the intrinsic and final common pathways of the coagulation cascade. It represents the time, in seconds, for plasma to clot after addition of phospholipid, an intrinsic pathway activator, and calcium. The name 'Activated Partial Thromboplastin Time' comes from the original form of the test in which only the phospholipid concentration of the test was controlled (as opposed to the phospholipid and the surface activator concentrations) and the name 'partial thromboplastin' was applied at the time to phospholipid preparations that accelerated clotting but did not correct the prolonged clotting times of hemophilic plasma. The term 'partial' means phospholipid is present but no tissue factor. The normal and reference ranges vary depending on reagent and instrument combinations, particularly with the phospholipid composition. It is used to evaluate the coagulation Factors XII, XI, IX, VIII, X, V, II (prothrombin), and I (fibrinogen) as well as prekallikrein and high molecular weight kininogen. Heparin and direct thrombin inhibitors, including hirudin, argatroban, and dabigatran have an effect on the assay.

D-dimer

[ 00426 ] The various states of coagulation activation that occur in vivo lead to the production of thrombin and then, fibrin. What follows is a reactive fibrinolysis, during which plasmin breaks down fibrin. The D-dimer is the ultimate degradation product of fibrin. The presence of D-dimer in plasma is an indirect marker of a coagulation activation followed by a reactive thrombolysis. Increased levels can be found in patients with DVT, PE, disseminated intravascular coagulation, hemorrhages, surgery, cancers, and severe infections. The D-dimer assay is an enzyme immunoassay procedure for the quantitative determination of D-dimer.

9.5. Pharmacokinetic/Pharmacodynamic Evaluations

00427 I The PD variables that will be evaluated for possible PK/PD relationships are

TT, PT, and aPTT (refer to Section 9.4, Pharmacodynamic/Biomarker Evaluations for a description of the PD variables). The PK of JNJ-64179375 to be used as predictor variables are Cmax and AUCinf (but not limited to) obtained by population PK analysis as described in Section 9.3.3, Pharmacokinetic Parameters. The relationship between TT, PT, and aPTT and plasma exposure metrics (Cmax and AUCinf) will be evaluated using models that will further be detailed in a separate population PK modeling plan.

9.6. Health Resource Utilization

[ 00428 ] Health resource utilization data, associated with medical encounters, will be collected in the eCRF by the investigator and study-site personnel for all subjects throughout the study. Protocol-mandated procedures, tests, and encounters are excluded. Key parameters of healthcare resource utilization will be collected for all subjects and compared between the treatment groups. The data collected will include the following:

• Total length of initial hospitalization and length of stay for each specialized unit by level of care (eg, ward type, intensive care unit and cardiac care unit stays)

· Rehospitalization for any reason through 18 weeks of follow up

• Any scheduled and unscheduled visits to healthcare providers for study outcomes, any other medical reasons, and diagnostic procedures used in relation to study outcomes through 18 weeks of follow up

• Discharge destination from the initial or subsequent hospitalization(s) (eg, home, rehabilitation)

• Details related to the management of any bleeding complications, (eg, procedures, extension of hospital stay, blood and/or plasma products)

• Details regarding the management of subjects who develop symptomatic VTE 9.7. Safety Evaluations

[ 00429 ] The study will include the following evaluations of safety and tolerability according to the time points provided in the Time and Events Schedule: adverse events, including serious adverse events, adverse events of special interest (ie, bleeding events, infusion reactions, hypersensitivity reactions, and wound or joint complications), clinical laboratory tests (ie, hematology, clinical chemistry, urinalysis), vital signs measurements (blood pressure, pulse/HR, temperature), and physical examinations. Safety evaluations may also be performed at unscheduled time points, if deemed by the investigator or appropriate designee as necessary to ensure the safety of the subject.

[ 00430 ] Any clinically relevant changes occurring during the study must be recorded on the adverse event section of the eCRF.

[ 00431 ] Any clinically significant abnormalities persisting at the end of the study /early withdrawal will be followed by the investigator until resolution or until a clinically stable endpoint is reached.

9.7.1. Adverse Events

[ 00432 ] Adverse events, including serious adverse events, will be reported by the subject (or, when appropriate, by a caregiver, surrogate, or the subject's legally acceptable representative) for the duration of the study. Adverse events will be followed by the investigator as specified in Section 12, Adverse Event Reporting. [ 00433 ] All adverse events, whether serious or nonserious, will be reported from the time a signed and dated ICF is obtained until the completion of the subject's last study- related procedure. Refer to Section 12.3.1, All Adverse Events for additional details.

[ 00434 ] Any wound or joint complications occurring after the TKR surgery will be specifically evaluated as described in Section 9.7.2.3, Wound or Joint Complications. In addition, knee joint range of motion will be evaluated at Week 18 as described in Section 9.7.6, Physical Examination.

9.7.2. Adverse Events of Special Interest

[ 00435 ] In this study, adverse events of special interest are:

• Bleeding events (Section 0, Bleeding Events)

· Infusion or hypersensitivity reactions (Section 0, Infusion or Hypersensitivity Reactions)

• Wound or joint complications (Section 0, Wound or Joint Complications)

[ 00436 ] As previously noted in Section 0, Efficacy Evaluations, all suspected symptomatic efficacy (thrombotic) events will also be captured as adverse events of special interest.

9.7.2.1. Bleeding Events

9.7.2.1.1. Classification of Bleeding Events

[ 00437 ] All subjects will be observed for signs and symptoms of bleeding events throughout the study and at each study visit as indicated in the Time and Events Schedule.

[ 00438 ] The investigator's classification of bleeding events according to the protocol classification will be collected in the eCRF. Additional information, including but not limited to the list below, will be collected for subjects with bleeding events and entered into the eCRF:

• Location of the bleeding

· Provocation of the bleeding

• Association with any procedure

• Action taken regarding the study drug

• Concomitant or additional treatment given for this bleeding event

• Any associated hemoglobin (and/or hematocrit) levels

· Any associated blood transfusions and products • Hospitalization (or prolonged hospitalization) due to bleeding events

• Outcome

[ 00439 ] All available information related to the classification of bleeding events will be collected and adjudicated by the independent CEC (refer to Section Clinical Events Committee for additional details).

9.7.2.1.2. Approach to Subjects With a Bleeding Event

[ 00440 ] There is no antidote for either study drug. If a subject has a bleeding event requiring intervention during the study, the following measures should be considered for both study drugs:

• Discontinue the study drug (refer to Section 10.2, Discontinuation of Study Drug/ Withdrawal from the Study)

• Usual treatment measures for bleeding events, including local pressure, fluid replacement and hemodynamic support, blood transfusion, and fresh frozen plasma, if physical examination and laboratory testing suggest that benefit could be obtained

• Other causes besides antithrombotic medication can be contributory to the seriousness of the bleeding event (ie, rule out disseminated intravascular coagulation, thrombocytopenia, and other coagulopathies, kidney and liver dysfunction, concomitant medications) and should be treated accordingly

· Depending on local availability, consultation with a coagulation expert should be considered

[ 00441 ] If bleeding cannot be controlled by these measures, consider administration of 1 of the following procoagulants according to the dosages and dosing schedules that are recommended in their respective package inserts (Note: consultation with a coagulation expert is recommended before use):

• PCC

• Activated PCC

• Factor Vila

[ 00442 ] Of note, protamine sulfate and vitamin K are not expected to affect the anticoagulant activity of either study drug. In a rat-tail transection bleeding model,

Beriplex (4-factor PCC) and FEIBA (activated 4-factor PCC) returned bleeding times to control levels while TXA and activated Factor Vila (NovoSeven) did not shorten the bleeding times after dosing with JNJ-64179375 at the doses studied. See Section 1.1.2.3, Parts 2 and 3 for reversal of PD effects for JNJ-64179375. Reversal of the PD effects of apixaban, as demonstrated by changes in the thrombin generation assay, was evident at the end of the infusion and reached baseline values within 4 hours after the start of a 30- minute infusion of 4-factor PCC in healthy subjects. However, there is little clinical experience with the use of 4-factor PCC products to reverse bleeding in individuals who have received apixaban. Currently, there is no experience with the use of recombinant Factor Vila in individuals receiving apixaban.

[ 00443 ] Due to its high molecular weight, JNJ-64179375 is not expected to be dialyzable. Hemodialysis decreased the AUC of apixaban by 14% in subjects with end- stage renal disease when a single dose of apixaban 5 mg was administered orally.

Therefore, hemodialysis is unlikely to be an effective means of managing an overdose with apixaban or to manage bleeding with JNJ-64179375.⁸

[ 00444 ] In those cases where approaches to reduce the blood levels of the study drug are being considered:

• In healthy subjects, administration of activated charcoal 2 and 6 hours after ingestion of a 20-mg dose of apixaban reduced mean AUC of apixaban by 50% and 27%, respectively, and had no impact on Cmax. The mean half-life of apixaban decreased from 13.4 hours when apixaban was administered alone to 5.3 hours and 4.9 hours, respectively, when activated charcoal was administered 2 and 6 hours after apixaban. Therefore, administration of activated charcoal may be useful in the management of an overdose with apixaban or accidental ingestion.⁸

· JNJ-64179375 has a plasma half-life of approximately 25 days as estimated from preliminary PK data in healthy subjects. Administration of IV immunoglobulins would be expected to increase the clearance of JNJ-64179375 by saturation of neonatal Fc receptors. A therapeutic plasma exchange to remove JNJ-64179375 could also be considered (estimated removal would be about 60% to 70% with 1 plasma volume exchange).³³

9.7.2.2. Infusion or Hypersensitivity Reactions

[ 00445 ] Infusion reactions or hypersensitivity reactions (eg, anaphylaxis, urticaria, dyspnea, hypotension) have been observed with the administration of monoclonal antibodies and may occur at any time during the administration of the IV study drug or within the first few hours after administration. Serum sickness-like reactions (also known as delayed hypersensitivity reactions) have been observed between 1 to 14 days after treatment. Symptoms associated with these reactions include fever, rash, headache, sore throat, myalgia, polyarthralgia, hand and facial edema, and/or dysphagia.

[ 00446 ] The IV study drug must not be administered to individuals with known or suspected intolerance or hypersensitivity to any biologic medication or known allergies or clinically significant reactions to murine, chimeric, or human proteins, monoclonal antibodies or antibody fragments, or to any of the excipients of the JNJ-64179375 formulation used in this study (refer to Section 4.2, Exclusion Criteria).

[ 00447 ] A physician must be immediately available at the site at all times during the administration of the study drug infusion as noted in Section 0, Dosage and

Administration. All subjects will be carefully observed for symptoms of an infusion reaction during the infusion and for at least 90 minutes after the start of the IV infusion and for the development of any allergic reaction during the study. The investigator should use clinical judgment in assessing the intensity of any infusion reactions. If an infusion reaction is observed, treatment such as oral paracetamol/acetaminophen and/or oral antihistamines may be administered. Details regarding the infusion reaction, including any medications administered, should be captured in the eCRF.

[ 00448 ] The following precautions should be applied during a study infusion:

• Before an infusion is started, the appropriate personnel, medications (eg, epinephrine, inhaled β-agonists, antihistamines, and corticosteroids), and other requirements to treat allergic reactions, including anaphylaxis, must be readily available.

• If a subject has a moderate or severe infusion reaction, the infusion should be terminated immediately and the subject should be treated appropriately according to institutional guidelines.

· Subjects discontinuing the study drug due to an infusion reaction will be asked to return for required assessments at all scheduled visits through the Week 18 visit. Refer to Section 10.2, Discontinuation of Study Drug/Withdrawal from the Study for additional details regarding subject discontinuation. 9.7.2.3. Wound or Joint Complications

[ 00449 ] All subjects will be observed for signs and symptoms of wound or joint complications throughout the study and at each study visit as indicated in the Time and Events Schedule. The wound will be evaluated by the investigator for any abnormal bleeding, swelling, redness, drainage, or infection. Any joint complications such as bleeding/hematoma, infection, or prosthesis malfunction (eg, limited range of motion) will also be recorded as adverse events of special interest. Any medical or surgical treatments for wound or joint complications will be recorded as well. 9.7.3. Clinical Laboratory Tests

[ 00450 ] Blood samples for serum chemistry and hematology and a urine sample for urinalysis will be collected. The investigator or appropriate designee must review the laboratory results, document this review, and record any clinically relevant changes occurring during the study in the adverse event section of the eCRF. The laboratory reports must be filed with the source documents.

[ 00451 ] At screening, the investigator will need to determine that the subject is medically appropriate for postoperative anticoagulant prophylaxis on the basis of clinical laboratory tests performed as part of local standard-of-care as part of screening for elective TKR surgery and as part of standard postoperative care following surgery. Hematology and chemistry laboratory tests and a urine sample for urinalysis will be obtained before dosing (0 hour) and performed by the central laboratory. The following tests will be performed by the central laboratory:

•

[ 00452 ] Note: A WBC evaluation may include any abnormal cells, which will then be reported by the laboratory. A RBC evaluation may include abnormalities in the RBC count, RBC parameters, or RBC morphology, which will then be reported by the laboratory. In addition, any other abnormal cells in a blood smear will also be reported.

•

[ 00453 ] Note: If dipstick result is abnormal, flow cytometry will be used to measure sediment. In case of discordance between the dipstick results and the flow cytometric results, the sediment will be examined microscopically.

9.7.4. Electrocardiogram

[ 00454 ] A 12-lead ECG will be performed during screening. During the collection of ECGs, subjects should be in a quiet setting without distractions (eg, television, cell phones). Subjects should rest in a supine position for at least 5 minutes before

ECG collection and should refrain from talking or moving arms or legs. If blood sampling or vital signs measurements are scheduled for the same time point as ECG recording, it is recommended that the procedures be performed in the following order: ECG, vital signs, and blood draw.

9.7.5. Vital Signs (temperature, heart rate, blood pressure)

[ 00455 ] Vital signs measurements will be obtained at the time points indicated in the Time and Events Schedule.

[ 00456 ] Blood pressure and pulse/HR measurements will be assessed with subjects in the supine position with a completely automated device. Manual techniques will be used only if an automated device is not available. Single measurements at each time point will be made. However, if any clinically significant abnormal values are detected (systolic blood pressure > 160 or <90 mmHg, diastolic blood pressure >100 mmHg, HR <40 or >100 beats per minute) then repeat measurements will be performed at the discretion of the investigator.

[ 00457 ] Blood pressure and HR measurements should be preceded by at least 5 minutes of rest in a quiet setting without distractions (eg, television, cell phones).

[ 00458 ] The subject's temperature should also be obtained.

9.7.6 Physical Examination

[ 00459 ] The physical examination consists of a routine medical examination that includes general appearance and a review of the following systems: neurologic, eyes/ears/nose/throat, thyroid, cardiovascular, respiratory, abdominal/gastrointestinal, hepatic, musculoskeletal, and dermatologic. Any bleeding observed during the examination (eg, skin, gingiva, nares) will be recorded as a potential bleeding event as described in Section 9.7.2.1.1, Classification of Bleeding Events. Additional body systems or further detailed physical examinations (eg, rectal examinations) should be performed if considered clinically appropriate by the investigator. Physical examinations will be performed by the investigator or a designated health care professional who is licensed and/or certified in accordance with applicable local laws to perform physical examinations.

[ 00460 ] Height and weight should be obtained at the screening visit, with weight only on Day - 1 or prior to dosing on Day 1 and the final visit. Scales used should be calibrated according to the standard operating procedures at the study site to assure weight is accurate.

[ 00461 ] The physical examination will be conducted at the time points indicated in the Time and Events Schedule. An assessment of the wound will be made at all visits as part of the adverse event assessment and the final physical examination will assess the range of motion of the operated joint. Any new, clinically significant findings (in the opinion of the investigator) that were not noted at the time of the screening visit must be captured as adverse events and will be followed to resolution.

9.8. Sample Collection and Handling

[ 00462 ] The actual dates and times of sample collection must be recorded on the laboratory requisition form. If blood samples are collected via an indwelling cannula, an appropriate amount (1 mL) of serosanguineous fluid slightly greater than the dead space volume of the lock will be removed from the cannula and discarded before each blood sample is taken. After blood sample collection, the cannula will be flushed with 0.9% sodium chloride, United States Pharmacopeia (USP) (or equivalent) and charged with a volume equal to the dead space volume of the lock. Blood samples for PK and PD assessments on Day 1 post dosing should be collected in the opposite arm as the infusion of JNJ-64179375 or placebo.

[ 00463 ] Refer to the Time and Events Schedule for the timing and frequency of all sample collections.

[ 00464 ] Instructions for the collection, handling, storage, and shipment of all blood and urine samples are found in the separate laboratory manual that will be provided. Collection, handling, storage, and shipment of samples must be under the specified, and where applicable, controlled temperature conditions as indicated in the laboratory manual.

10. SUBJECT COMPLETION/Discontinuation of Study DRUG/ WITHDRAWAL From the STUDY

10.1. Completion

[ 00465 ] A subject will be considered to have completed the study if he or she has completed the Week 18 visit of the follow-up phase.

[ 00466 ] Subjects who prematurely discontinue the study drug for any reason before completion of the double-blind dosing phase will be considered to have completed the study if they have completed the Week 18 visit. A subject who dies will be considered to have completed the study.

10.2. Discontinuation of Study Drug/Withdrawal from the Study

[ 00467 ] It is imperative for the integrity of the study and results to have complete data. If a subject withdraws from the study before the end of the follow-up phase and is unwilling or unable to return for follow-up visits in person, the study site should collect as much follow-up visit information as possible, by contacting the subject by telephone or by mail. If applicable, vital status may be obtained by reviewing the subject's medical or public records unless this contact is not permitted per local regulations. [ 00468 ] Study drug assigned to a subject that discontinues study drug or withdraws may not be assigned to another subject. Subjects who withdraw will not be replaced.

Discontinuation of Study Drug

[ 00469 ] A subject will not be automatically withdrawn from the study if he or she has to discontinue the study drug before the end of the double-blind dosing phase.

[ 00470 ] Subjects who discontinue the study drug before the end of the double-blind dosing phase will be instructed to return to the study site, if possible, to conduct assessments, including venography assessments of the operated leg as described in Section 0, Double-Blind Dosing Phase. If a subject withdraws from the study before the end of the double-blind dosing phase, EOD and follow-up assessments should be obtained.

[ 00471 ] A subject's study drug must be discontinued for any of the following reasons:

• The investigator believes that for safety reasons or tolerability reasons (eg, adverse event) it is in the best interest of the subject to discontinue study drug

· The subject requests to discontinue the study drug permanently

• Development of any condition that requires open-label treatment with an anticoagulant or other prohibited medication

• Bleeding into a critical site, (eg, intracranial, intraspinal, intraocular, pericardial, intra-articular, intramuscular with compartment syndrome, retroperitoneal)

· Active uncontrolled hemorrhage

• At the (exceptional) request of the sponsor

Withdrawal From the Study

[ 00472 ] A subject will be withdrawn from the study for any of the following reasons:

• Lost to follow up

· Withdrawal of consent

[ 00473 ] Withdrawal of consent should be a very unusual occurrence in a clinical study. Subjects who elect to stop the study drug are not automatically considered to have withdrawn consent. The investigator should make every effort to maintain a good relationship with subjects to avoid this occurrence. The recording of withdrawal of consent in the eCRF for this study will occur after a discussion between the investigator and the appropriate sponsor representative has taken place. [ 00474 ] At the time of signing the ICF, a subject will agree to be contacted to obtain poststudy drug follow-up information should he or she decide to withdraw from the study. If a subject withdraws from the study before the end of the follow-up phase and is unwilling or unable to return for follow-up visits in person, he or she should be contacted and the study site should collect as much follow-up information as possible. This includes contacting the subject or the subject's alternative means of contact (eg, subject's children, spouse, significant other, caretaker, legal representative or health care professional), by telephone or mail to determine vital status and if an endpoint event has occurred, as agreed to by the subject during the initial informed consent process. If applicable, vital status may be obtained by reviewing the subject's medical or public records, unless this process is not allowed by local regulations.

[ 00475 ] For subjects who withdraw consent and are not agreeable to any follow-up contact, it is recommended that the subject withdraw consent in writing, and the subject will be asked to supplement the withdrawal of consent with a signed written statement documenting refusal from all subsequent contact. If the subject refuses or is physically unavailable, the study site should document and sign the reason for the subject's failure to withdraw consent in writing and maintain it with the subject's source records.

[ 00476 ] When a subject withdraws consent before completing the study, it is not required for he/she to give a reason. If the reason for the withdrawal is known, it should be documented in the eCRF and in the source document.

[ 00477 ] If a subject is lost to follow up, every reasonable effort must be made by the study-site personnel to contact the subject and determine their status and the reason for discontinuation/withdrawal, including the possible use of locator agencies to determine vital status, as local law permits. The measures taken to follow up must be documented. A subject will be considered lost to follow up only after all means of all subsequent contact have been exhausted.

11. STATISTICAL METHODS

[ 00478 ] Statistical analysis will be done by the sponsor or under the authority of the sponsor. A general description of the statistical methods to be used to analyze the efficacy and safety data is outlined below. Specific details will be provided in a statistical analysis plan (SAP), which will be prepared for Part 1 before the first subject is consented for Part I . For Part 2, a draft SAP will be prepared before the first subject is consented into Part 1 and an updated Part 2 SAP based on the Part 1 data will be prepared before the first subject is consented in Part 2.

[ 00479 ] In Part 1, the intent is to escalate across the planned doses (0.3 to 0.6 to 1.2 mg/kg) of JNJ-64179375 based on the OC review of the totality of the data but with a focus on bleeding events. Specific analyses to be reviewed (eg, continual reassessment or escalation with overdose control methods) and dose-escalation decision guidelines will be included in the OC charter. After all subjects are expected to have completed the Day 10- 14 visit for Part 1, an unblinded data review is planned and the selection of the dose range and doses for Part 2 will be based on the evaluation of the totality of the data but with a focus on the any bleeding and total VTE endpoints. Analyses to aid in dose selection will be prespecified in the OC charter and the Part 1 SAP. No formal hypothesis testing is planned for Part 1 due to the limited sample size. Historical apixaban bleeding and VTE event rates may also be considered in making decisions.

[ 00480 ] In Part 2, this study will use an adaptive design intended to optimize data collection for dose-response modeling. In Part 2, the primary goal is to assess the efficacy (total VTE endpoint) dose-response relationship of JNJ-64179375. A hybrid methodology that combines aspects of multiple testing with modeling techniques (MCP-Mod) will be used for evaluating dose-response trends and estimating the dose-response relationships for the efficacy and bleeding endpoints for JNJ-64179375.²'³-²⁵ The sample size for Part 2 may be adjusted based on the observed bleeding and total VTE event rates in Part 1. Additional details are provided in Section 11.3, Efficacy and Bleeding Analyses.

[ 00481 ] If a sufficient number of JNJ-64179375 doses (ie, 4 or more) are studied in Part 2 then dose-response modeling using the MCP-Mod approach will be the primary analysis strategy. If the number of JNJ-64179375 doses is not sufficient to support the MCP-Mod approach then linear dose-response modeling (if 3 doses are studied), or pairwise comparisons (if 2 doses are studied), will be the primary analysis strategy.

[ 00482 ] An unblinded data review is planned for Part 1 after all subjects are expected to have completed the Day 10-14 visit to determine the dose range and doses of JNJ- 64179375 for Part 2. Two planned, unblinded interim analyses and 1 unblinded administrative interim analysis are intended for Part 2 of this study. Refer to Section

I I .10, Interim Analyses for further details. 11.1. Analysis Datasets

[ 00483 ] The 2 elements that define an analysis data set are:

• The analysis population, which specifies those subjects who will be included in an analysis

• The observation period, which specifies the time window within which data will be included in an analysis

Efficacy Analyses

[ 00484 ] Two populations will be used in the efficacy analyses:

• Modified Intent to Treat (ITT): all randomized subjects with an evaluable venography assessment or a confirmed symptomatic VTE event, or any death.

· Efficacy Per Protocol: all randomized subjects with no major protocol deviations (to be defined in the SAP) and an evaluable venography assessment within 24 hours after the last dose of the oral study drug or with confirmed symptomatic VTE within 2 days of the last dose of oral study drug, or any death within 2 days of the last dose of oral study drug. Subjects without symptomatic VTE or death must also have received the single complete infusion of J J-64179375 or at least 18 doses of oral apixaban.

[ 00485 ] The 2 observation periods to be used in the efficacy analyses include the following:

• Up to Day 10-14 visit (venography assessment)

· Up to the Week 18 visit

[ 00486 ] The primary efficacy endpoint analysis (total VTE) will use the modified ITT population up to the Day 14 observation period. Additional details will be provided in the SAP. Bleeding Event Analyses

[ 00487 ] Two populations will be used in the bleeding event analyses:

• Safety: all randomized subjects who received at least 1 dose (partial or complete) of active study drug

• Safety Per Protocol: all randomized subjects with no major protocol deviations who received the single complete infusion of JNJ-64179375 or at least 18 doses of oral apixaban or with a bleeding event centrally adjudicated by the CEC within 2 days of the last dose of oral study drug

[ 00488 ] The 3 observation periods to be used in the bleeding event analyses include the following: • Up to Day 10-14 visit (venography assessment)

• Up to the Week 5 visit

• Up to the Week 18 visit [ 00489 ] The primary bleeding event analysis (any bleeding) will use the safety population up to the Day 10-14 visit observation period.

11.2. Sample Size Determination

[ 00490 ] A total of 1,500 subjects are planned for this study, of which up to 300 subjects will be enrolled in Part 1 and the remainder for the entire study will be enrolled in Part 2. In Part 1, the sample size is not based on hypothesis testing but rather on making a preliminary assessment of the bleeding risk for the doses of JNJ-64179375. The event rate of any bleeding up to 14 days for apixaban 2.5 mg twice daily is expected to be between 5% and 10%. Figure 31 shows the probability to flag a higher event rate compared to apixaban for any dose of JNJ-64179375 in Part 1, assuming that the true underlying bleeding event rate in the apixaban group is 5%, 7.5%, or 10%. The figure presents the numbers of subjects in the JNJ-64179375 and apixaban treatment groups in rows and columns, respectively. The assumed event rate in the JNJ-64179375 group is presented as the relative risk to apixaban in the horizontal axis; the statistical comparison is based on the difference between event rates.

[ 00491 ] When the true underlying event rate in the apixaban group is 7.5%, there is a 67% probability for a dose of JNJ-64179375 to have the lower bound of the 1 -sided 90% CI for between-treatment difference exclude 0 if the true relative risk (or risk ratio) is 3 (ie, the true underlying event rate in the JNJ-64179375 group is 22.5%), with 40 subjects in the JNJ-64179375 group (ie, any 1 planned cohort of JNJ-64179375 subjects) and 30 subjects in the apixaban group (ie, pooled apixaban subjects from 3 planned cohorts). The probability will increase to 81% when the dose of JNJ-64179375 is repeated in another planned or optional cohort, where there will be 80 subjects in the JNJ-64179375 treatment group and 40 subjects in the pooled apixaban group.

[ 00492 ] In Part 2, it is anticipated that 10% to 20% of subjects will either not have the venography assessment of the operated leg performed as scheduled or will not have an evaluable venography assessment for any DVT or proximal DVT. Assuming that 80% of venography assessments are evaluable and the true underlying total VTE event rates are 15%, 10%, 8%, and 6% at potential doses of JNJ-64179375 of 0.3, 0.6, 0.9, 1.2 mg/kg, and 1.8 mg/kg, respectively, with a total sample size of 1,200 subjects, the study is expected to have over 90% power to declare proof-of-efficacy, which is defined as either a statistically significant dose-response trend or a total VTE event rate lower than 30% in the pooled doses of JNJ-64179375 at a 1-sided, 2.5% ot-level. The exact power will vary because of the nature of the adaptive study design.

[ 00493 ] For comparison with apixaban in Part 2, if the true underlying total VTE and any bleeding event rates are as specified in Table 11, a simulation study demonstrates that there will be approximately 90% probability to identify a dose within the dose range of 0.3 to 1.2 mg/kg, of which both the model-predicted total VTE and any bleeding event rates met the prespecified noninferiority (NI) criterion (the upper bound of the 1 -sided 90% CI for the odds ratio [JNJ-64179375 to apixaban] is equal to or lower than 1.5 for total VTE and any bleeding events).

Table 11: Assumed Efficacy and Safety Event Rates by Treatment Group in Sample Size Determination for Part 2

11.3. Efficacy and Bleeding Analyses

[ 00494 ] A dose-response trend will be assessed with respect to the primary efficacy (total VTE) and safety (any bleeding) endpoints in Part 2. Depending on the number of doses studied, a set of up to 7 candidate models (doses are in logarithm scale) will be used to cover a suitable range of possible efficacy dose-response shapes (linear, Emax, exponential, sigEmaxl, sigEmax2, sigEmax3, and sigEmax4) (see Figure 32).

[ 00495 ] Similarly, a set of up to 6 candidate models will be used to cover a suitable range of safety dose-response shapes (linear, Emax, exponential, sigEmaxl, sigEmax2, and sigEmax3) (see Figure 33). The set of safety candidate models used will be similarly chosen as for the efficacy candidate models, which is dependent on the number of doses studied in Part 2. [ 00496 ] Each of the dose-response shapes in the candidate set will be tested using the corresponding contrast /-test statistic, employing a critical value derived for the maximum of the /-test statistics (based on the associated multivariate /-distribution) to ensure appropriate multiplicity correction that preserves the Type I error rate. A dose-response trend is established for an endpoint when the maximum of the /-test statistics for the corresponding endpoint exceeds the critical value.

[ 00497 ] Predicted event rates at a specific dose will be derived for each candidate model. The weighted average of these predicted event rates (weights are determined by the model goodness-of-fit), will then be used to estimate the target dose.^4,16 The minimum dose that meets the NI efficacy criterion (defined as the upper bound of the 1 -sided 90% CI for model-predicted JNJ-64179375/apixaban odds ratio less than 1.5) is considered as the minimum effective dose. The highest dose to be considered to proceed is the maximum dose that meets the NI safety criterion (defined as the upper bound of the 1- sided 90% CI for model-predicted JNJ-64179375/apixaban odds ratio less than 1.5). Any dose between the minimum effective dose and the highest dose to proceed is considered an eligible dose to be further studied in a subsequent study(ies). The final dose selection will be based on the consideration of the total benefit-risk balance.

[ 00498 ] The determination of the NI criterion above is based on 2 considerations: 1) distribution of the point estimate for the odds ratio for the given sample size when the NI criterion is met; and 2) the balance between the false-positive rate (incorrectly conclude an inferior dose to be comparable to apixaban) and the false-negative rate (incorrectly conclude an efficacious dose to be inferior to apixaban). With the given sample size and event rates as specified in Table 11, the median corresponding odds ratio in 10,000 simulated studies is approximately 1.0 for total VTE and 0.94 for any bleeding. With the same assumptions, these NI criteria will yield approximately 90% probability to correctly identify an eligible dose for subsequent study(ies).

[ 00499 ] The details of the adaptive decision guidelines for adding and/or dropping treatment groups or changing the randomization ratio in Part 2 will be specified in the IDMC charter and the Part 2 SAP.

[ 00500 ] Prespecified clinical variables of interest for the efficacy subgroup analyses include region, age, sex, weight/body mass index, renal function, surgery duration, TXA use, tourniquet use, and mechanical VTE prophylaxis device use. The same variables will be used for safety subgroup analyses with the addition of aspirin/NSAID use.

11.4. Pharmacokinetic Analyses

[ 00501 ] Plasma concentrations of JNJ-64179375 will be listed for all subjects by time of collection and dose level. For each dose, descriptive statistics, including mean, median, standard deviation (SD), and coefficient of variation will be calculated for the plasma concentrations at each nominal sampling time. Plasma concentrations of JNJ-64179375 versus time profiles will be plotted for each subject based on actual sampling times, and mean concentration versus time profiles will be plotted for each dose level based on planned sampling times.

[ 00502 ] Data will be listed for all subjects with available plasma concentrations per treatment group. Subjects will be excluded from the PK analysis if their data do not allow for accurate assessment of the PK (eg, incomplete administration of the study drug;

missing information of dosing and sampling times; concentration data not sufficient for PK parameter calculation).

[ 00503 ] All plasma concentrations below the lowest quantifiable concentration or missing data will be labeled as such in the concentration database. Concentrations below the lower quantifiable concentration will be treated as zero in the summary statistics. All subjects and samples excluded from the analysis will be clearly documented in the study report.

[ 00504 ] In addition, a population PK model will be developed and the dependence of PK of JNJ-64179375 on population covariates (eg, demographics, laboratory variables) will be evaluated. This will be performed using nonlinear mixed-effects modeling with the software package NONMEM^®. The mean (and variance) values for specific PK parameters (eg, CL and Vz) will be estimated and the statistical significance of the relationships between the covariates and PK parameters will be evaluated. The results of this population PK (PK parameters signifying relationship between PK and various covariates) analysis will be reported separately, in a document other than the CSR. 11.5. Immunogenicity Analyses

[ 00505 ] The incidence of antibodies will be summarized for all subjects who receive a dose and have at least 1 appropriate sample obtained after study drug administration for the detection of antibodies.

[ 00506 ] A listing of subjects who are positive for antibodies will be provided. The maximum titers of antibodies will be summarized for subjects who are positive for antibodies.

[ 00507 ] Analyses of the impact of immunogenicity on PK, PD, PK/PD, efficacy, and safety endpoints will be performed to further characterize the immune responses that are generated.

11.6. Pharmacodynamic/Biomarker Analyses

[ 00508 ] The PD parameters are TT, ECT, PT, aPTT, and D-dimer. Descriptive statistics including mean, median, SD, minimum, and maximum will be provided for the PD parameters. The parameters of TT, PT, and aPTT may be statistically analyzed using mixed models. The mixed model for the change from baseline PD parameters will include dose and visit as fixed effects and subjects as random effects. The least-squares means for change from baseline and associated 95% CIs will be provided. Other PD parameters may be analyzed similarly.

11.7. Pharmacokinetic/Pharmacodynamic Analyses

[ 00509 ] The PK/PD relationships will be investigated graphically and, if appropriate, may be further analyzed using suitable statistical methods.

[ 00510 ] Plasma PK exposures (Cmax, AUCinf, and/or time-matched concentration) from the population PK model versus PD variables (eg, TT and aPTT) and safety/efficacy endpoints will be graphically examined. This can be accomplished by plotting relevant PD measures versus PK exposure parameters (quantiles/deciles) for preliminary assessment and possible model identification. If the graphical representation of the relationship indicates possible correlation, then these data may be analyzed statistically using a suitable model. For example, a mixed-effects linear model may be used to identify the possible relationship between PK and PD. Other models using time-matched concentrations with PD may also be used for exploring the relationship further, as appropriate. Other PD variables (eg, ECT) may be analyzed in a similar fashion. The details of the analyses plan will be presented in a population PK modeling plan that is separate from the SAP.

[ 00511 ] Preliminary assessments of these relationships will be made available to the IDMC at both interim analyses to support their evaluations.

11.8. Health Resource Utilization Analyses

[ 00512 ] Health resource utilization will be descriptively summarized by treatment group.

11.9. Safety Analyses

[ 00513 ] All safety data will be fully listed. The reporting of the safety data of all subjects receiving at least 1 active dose of JNJ-64179375 or apixaban will include the incidence and type of adverse events, plus absolute values and changes in blood pressure (systolic and diastolic), HR, clinical laboratory data, and physical examinations from predose to the final postdose time point.

Adverse Events

[ 00514 ] The verbatim terms used in the eCRF by investigators to identify adverse events will be coded using the MedDRA. 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. All reported adverse events will be included in the analysis. For each adverse event, the percentage of subjects who experience at least 1 occurrence of the given event will be summarized by treatment group. In addition, comparisons between treatment groups will be provided if appropriate.

[ 00515 ] Summaries, listings, datasets, or subject narratives may be provided, as appropriate, for those subjects who die, who discontinue treatment due to an adverse event, or who experience a severe or a serious adverse event.

[ 00516 ] Based on the mechanism of action of JNJ-64179375 and given that it is a monoclonal antibody, adverse events of special interest will include bleeding events, infusion reactions, hypersensitivity reactions, and wound or joint complications as described in Section 9.7.2, Adverse Events of Special Interest. Subjects with adverse events of special interest may be counted or listed using MedDRA SMQs (eg, hemorrhage excluding laboratory terms SMQ). All suspected symptomatic efficacy (thrombotic) events will also be captured as adverse events of special interest.

Clinical Laboratory Tests

[ 00517 ] Laboratory data will be summarized by type of laboratory test. Reference ranges and markedly abnormal results (specified in the Part 1 and Part 2 SAP) will be used in the summary of laboratory data. Descriptive statistics will be calculated for each laboratory analyte at baseline and for observed values and changes from baseline at each scheduled time point. Frequency tabulations of the changes from baseline will be presented in pre- versus posttreatment cross-tabulations (with classes for below, within, and above normal ranges). Frequency tabulations of the abnormalities will be made. A listing of subjects with any laboratory results outside the reference ranges will be provided. A listing of subjects with any markedly abnormal laboratory results will also be provided.

Vital Signs

[ 00518 ] Descriptive statistics of temperature, HR, and blood pressure (systolic and diastolic) (supine) values and changes from baseline will be summarized at each scheduled time point. The percentage of subjects with values beyond clinically important limits will be summarized.

Physical Examination

[ 00519 ] Descriptive statistics of changes from baseline will be summarized at each scheduled time point.

[ 00520 ] Physical examination findings will be summarized at each scheduled time point. Descriptive statistics will be calculated at baseline and for observed values and changes from baseline at each scheduled time point. Frequency tabulations of the abnormalities will be made.

11.10. Interim Analyses

[ 00521 ] Two planned, unblinded interim analyses will be conducted in Part 2 by the IDMC as part of the adaptive approach that will be used to guide decisions to drop and/or add doses of JNJ-64179375 and adjust the randomization ratio based on available efficacy, safety, PK, and PD data. The objectives of the interim analyses are to: • Test futility

• Test dose-response trends to determine if the dose range being studied is appropriate for both efficacy and safety

• Determine if a dose(s) should be added and/or dropped to optimize data collection for dose-response modeling

• Determine how to allocate the remaining subjects optimally (based on observed efficacy, safety, or balanced efficacy/safety results)

[ 00522 ] These 2 interim analyses will be performed after approximately 400 and 800 subjects are enrolled and are expected to have completed the Day 10-14 visit. Depending on interim analysis results, possible modification options to study conduct are stopping the study prematurely for futility, adding/dropping a dose of JNJ-64179375, and adjusting randomization ratio based on optimization of the dose-response model estimation.

[ 00523 ] Subject randomization will be balanced until the first interim analysis.

Adaptation guidelines, including futility, adding/dropping a dose, modification of the randomization ratio, will be specified in the IDMC charter and the Part 2 SAP.

Administrative Interim Analysis

[ 00524 ] An unblinded administrative interim analysis is planned to be conducted by the sponsor after all subjects are expected to have completed the Day 10-14 visit in Part 2 to facilitate additional study planning. The conduct and integrity of the study will not be altered by the results of this interim analysis.

12. ADVERSE EVENT REPORTING

[ 00525 ] Timely, accurate, and complete reporting and analysis of safety information from clinical studies are crucial for the protection of subjects, investigators, and the sponsor, and are mandated by regulatory agencies worldwide. The sponsor has established standard operating procedures in conformity with regulatory requirements worldwide to ensure appropriate reporting of safety information; all clinical studies conducted by the sponsor or its affiliates will be conducted in accordance with those procedures.

[ 00526 ] Adverse events of special interest have been identified for this study and are described in Section 9.7.2, Adverse Events of Special Interest. Method of Detecting Adverse Events and Serious Adverse Events

[ 00527 ] Care will be taken not to introduce bias when detecting adverse events or serious adverse events. Open-ended and nonleading verbal questioning of the subject is the preferred method to inquire about adverse event occurrence. For some studies, subjects are not always able to provide valid verbal responses to open-ended questions. In these circumstances, events will be solicited. In this study, subjects will be queried directly to solicit potential bleeding events using general questions about any bleeding and specific questions about common minor bleeding events that could be overlooked by the subject (eg, bruising, gingival bleeding, epistaxis). Solicited Adverse Events

[ 00528 ] Solicited adverse events are predefined local and systemic events for which the subject is specifically questioned. In this study, bleeding events will be solicited.

Unsolicited Adverse Events

[ 00529 ] Unsolicited adverse events are all adverse events for which the subject is specifically not questioned.

12.1. Definitions

12.1.1. Adverse Event Definitions and Classifications Adverse Event

[ 00530 ] An adverse event is any untoward medical occurrence in a clinical study subject administered a medicinal (investigational or non-investigational) product. 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 (investigational or non-investigational) product, whether or not related to that medicinal (investigational or non-investigational) product. (Definition per International Conference on Harmonisation [ICH])

[ 00531 ] 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. [ 00532 ] A laboratory test abnormality that is considered by the investigator to be clinically relevant (eg, causing the subject to discontinue the study drug, requiring treatment, or causing apparent clinical manifestations) should be reported as an adverse event.

[ 00533 ] Note: The sponsor collects adverse events starting with the signing of the ICF (refer to Section 12.3.1, All Adverse Events, for time of last adverse event recording).

Serious Adverse Event

[ 00534 ] A serious adverse event based on ICH and European Union Guidelines on Pharmacovigilance for Medicinal Products for Human Use is any untoward medical occurrence that at any dose:

• Results in death

• Is life threatening

(The subject was at risk of death at the time of the event. It does not refer to an event that hypothetically might have caused death if it were more severe.)

· Requires inpatient hospitalization or prolongation of existing hospitalization

• Results in persistent or significant disability/incapacity

• Is a congenital anomaly/birth defect

• Is a suspected transmission of any infectious agent via a medicinal product

• Is Medically Important*

[ 00535 ] *Medical and scientific judgment should be exercised in deciding whether expedited reporting is also appropriate in other situations, such as important medical events that may not be immediately life threatening or result in death or hospitalization but may jeopardize the subject or may require intervention to prevent one of the other outcomes listed in the definition above. These should usually be considered serious.

[ 00536 ] If a serious and unexpected adverse event occurs for which there is evidence suggesting a causal relationship between the study drug and the event (eg, death from anaphylaxis), the event must be reported as a serious and unexpected suspected adverse reaction even if it is a component of the study endpoint (eg, all-cause mortality). Unlisted (Unexpected) Adverse Event/Reference Safety Information

[ 00537 ] An adverse event is considered unlisted if the nature or severity is not consistent with the applicable product reference safety information. Adverse Event Associated With the Use of the Drug

[ 00538 ] An adverse event is considered associated with the use of the drug if the attribution is possible, probable, or very likely by the definitions listed in Section 12.1.2, Attribution Definitions.

12.1.2. Attribution Definitions Not Related

[ 00539 ] An adverse event that is not related to the use of the drug.

Doubtful

[ 00540 ] An adverse event for which an alternative explanation is more likely, eg, concomitant drug(s), concomitant disease(s), or the relationship in time suggests that a causal relationship is unlikely.

Possible

[ 00541 ] An adverse event that might be due to the use of the drug. An alternative explanation, eg, concomitant drug(s), concomitant disease(s), is inconclusive. The relationship in time is reasonable; therefore, the causal relationship cannot be excluded. Probable

[ 00542 ] An adverse event that might be due to the use of the drug. The relationship in time is suggestive (eg, confirmed by dechallenge). An alternative explanation is less likely, eg, concomitant drug(s), concomitant disease(s).

Very Likely

[ 00543 ] An adverse event that is listed as a possible adverse reaction and cannot be reasonably explained by an alternative explanation, eg, concomitant drug(s), concomitant disease(s). The relationship in time is very suggestive (eg, it is confirmed by dechallenge and rechallenge).

12.1.3. Severity Criteria

[ 00544 ] The investigator should use clinical judgment in assessing the severity of events not directly experienced by the subject (eg, laboratory abnormalities).

[ 00545 ] An assessment of severity grade will be made using the following general categorical descriptors: Mild:

[ 00546 ] Awareness of symptoms that are easily tolerated, causing minimal discomfort and not interfering with everyday activities.

Moderate:

[ 00547 ] Sufficient discomfort is present to cause interference with normal activity. Severe:

[ 00548 ] Extreme distress, causing significant impairment of functioning or incapacitation. Prevents normal everyday activities.

13. STUDY DRUG INFORMATION 13.1. Physical Description of Study Drugs JNJ-64179375 or JNJ-64179375 Placebo

[ 00549 ] The JNJ-64179375 supplied for this study is a sterile, preservative-free liquid with a concentration of 50 mg/mL of monoclonal antibody in a solution composed of 10 mM sodium phosphate, 8.5% (w/v) sucrose, 0.04% (w/v) polysorbate 20, and 10 μg/mL EDTA at a pH of 7.1. JNJ-64179375 will be provided frozen in a glass vial with an approximately 3-mL fill volume. JNJ-64179375 has a molecular mass of 149,139 Dalton for the G0F/G0F glycoform and isoelectric points, ranging from 6.1 to 6.4. The absorptivity constant for JNJ-64179375 at 280 nm was determined to be 1.37 (mg/mL)^" ^m^"1. It will be manufactured and provided under the responsibility of the sponsor.

[ 00550 ] Placebo infusions will be 0.9% normal saline. Apixaban or Matching Apixaban Placebo

[ 00551 ] Apixaban will be supplied for this study as over-encapsulated 2.5-mg tablets in a red-colored, hard, gelatin capsule. The over-encapsulated tablet will be backfilled with microcrystalline cellulose to prevent the tablet from rattling in the capsule shell.

[ 00552 ] Matching apixaban placebo capsules will consist of microcrystalline cellulose within a red-colored, hard, gelatin capsule. 13.2. Packaging

[ 00553 ] JNJ-64179375 will be packaged in individual kits. Each kit will consist of 1 vial of JNJ-64179375 frozen liquid in a carton. JNJ-64179375 will not be dispensed in child-resistant packaging. It will be stored in a locked pharmacy and only dispensed after preparation for an IV infusion.

[ 00554 ] Apixaban or matching apixaban placebo will be packaged in bottles containing 30 capsules. The bottles will be child resistant.

13.3. Labeling

[ 00555 ] Study drug labels will contain information to meet the applicable regulatory requirements.

13.4. Preparation, Handling, and Storage

[ 00556 ] JNJ-64179375 must be stored at -40°C ± 10°C (-30°C to -50°C) or -70°C ± 20°C (-50°C to -90°C), and protected from light. Before use, the drug product should be diluted to the required dose strength using 0.9% nonnal saline. Diluted drug product for IV administration must be administered with in-line filtration.

[ 00557 ] Apixaban and matching apixaban placebo must be stored at controlled temperatures, as indicated on the product-specific labeling.

[ 00558 ] Refer to the phannacy manual/IPPI for additional guidance on study drug preparation, handling, and storage.

REFERENCES

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an anti -thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, and at least one pharmaceutically acceptable carrier or diluent, for use in treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, wherein said composition is administered via a single intravenous (IV) infusion such that the anti- thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 2.5 mg/kg.

2. The composition of claim 1, wherein the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg.

3. The composition of claim 1, wherein the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose in the range of 0.3 mg/kg to 1.8 mg/kg.

4. The composition of claim 1, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery.

5. The composition of claim 3, wherein the anti-thrombin antibody is administered at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

6. The composition of claim 3, wherein the single IV infusion of the anti-thrombin antibody is administered the day after the surgery.

7. The composition of any one of claims 1-6, wherein the thrombin-mediated condition is thrombosis or embolism.

8. The composition of claim 6, wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).

9. A method for treating or inhibiting a thrombin-mediated condition in a patient undergoing orthopedic surgery, comprising administering an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 2.5 mg/kg.

10. The method of claim 9, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.1 mg/kg to 1.8 mg/kg.

11. The method of claim 9, wherein said anti-thrombin antibody is administered via a single intravenous (IV) infusion at a clinically proven safe and clinically proven effective dose in the range of 0.3 mg/kg to 1.8 mg/kg.

12. The method of claim 9, wherein the orthopedic surgery is total knee replacement (TKR) surgery or total hip replacement (THR) surgery.

13. The method of claim 11, wherein said anti-thrombin antibody is administered via a single IV infusion at a clinically proven safe and clinically proven effective dose selected from the group consisting of: 0.3 mg/kg, 0.6 mg/kg, 1.2 mg/kg, and 1.8 mg/kg.

14. The method of claim 11, wherein the single IV infusion of the anti-thrombin antibody is administered the day after surgery.

15. The method of any one of claims 9-14, wherein the thrombin-mediated condition is thrombosis or embolism.

16. The method of claim 15, wherein the thrombin-mediated condition is selected from the group consisting of: venous thromboembolism (VTE), deep vein thrombosis (DVT), and pulmonary embolism (PE).