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WO2019035055A1 - Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires - Google Patents

Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires Download PDF

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Publication number
WO2019035055A1
WO2019035055A1 PCT/IB2018/056198 IB2018056198W WO2019035055A1 WO 2019035055 A1 WO2019035055 A1 WO 2019035055A1 IB 2018056198 W IB2018056198 W IB 2018056198W WO 2019035055 A1 WO2019035055 A1 WO 2019035055A1
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Prior art keywords
thrombin
seq
antibody
exosite
antiplatelet agents
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Inventor
Madhu Chintala
Juliane BERNHOLZ
Thomas Connolly
Gary Peters
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to isolated anti-thrombin antibody molecules that recognize the exosite 1 epitope of thrombin and selectively inhibit thrombin without promoting bleeding. These anti-thrombin antibody molecules may be useful in the treatment and prevention of thrombotic and/or embolic disorders and other conditions mediated by thrombin.
  • the present invention relates to use of the anti- thrombin antibody molecules in combination with one or more antiplatelet agents.
  • Blood coagulation is a key process in the prevention of bleeding from damaged blood vessels (haemostasis).
  • 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.
  • 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.
  • the present invention relates to the unexpected finding that anti-thrombin antibody molecules which recognise the exosite 1 epitope of thrombin selectively inhibit thrombin without promoting bleeding. These antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other thrombin-mediated conditions.
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) an anti-thrombin antibody having a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising SEQ ID NO: 15.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 therapeutically effective amount is a sub-therapeutic dosage of the one or more antiplatelet agents or the anti- thrombin antibody.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 therapeutically effective amount is a sub-therapeutic dosage of the one or more antiplatelet agents and the anti- thrombin antibody.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the one or more antiplatelet agents and the anti-thrombin antibody are administered simultaneously.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the one or more antiplatelet agents and the anti-thrombin antibody are administered sequentially.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the one or more antiplatelet agents are selected from the group consisting of: aspirin and clopidogrel.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the one or more antiplatelet agents is aspirin.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the one or more antiplatelet agents is clopidogrel.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein said therapeutically effective amount is a sub-therapeutic dosage of the one or more antiplatelet agents or the anti-thrombin antibody.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein said therapeutically effective amount is a sub-therapeutic dosage of the one or more antiplatelet agents and the anti-thrombin antibody.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents and the anti-thrombin antibody are administered simultaneously.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents and the anti-thrombin antibody are administered sequentially.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents are selected from the group consisting of: aspirin and clopidogrel.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents is aspirin.
  • HC heavy chain
  • LC light chain
  • the present invention provides a method for treating or inhibiting a thrombotic and/or embolic disorder in a patient, comprising administering a combination of therapeutically effective amounts of: (a) one or more antiplatelet agents and (b) 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 the combination comprising the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents is clopidogrel.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder.
  • a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein at least the one or more antiplatelet agents or the anti-thrombin antibody are present in a sub-therapeutic dosage.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the one or more antiplatelet agents and the anti -thrombin antibody are present in sub-therapeutic dosages.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the one or more antiplatelet agents are selected from the group consisting of: aspirin and clopidogrel.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the one or more antiplatelet agents is aspirin.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the one or more antiplatelet agents is clopidogrel.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the composition comprising the combination of the therapeutically effective amounts is associated with reduced adverse bleeding events.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the composition comprising the combination of the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein at least the one or more antiplatelet agents or the anti-thrombin antibody are present in a sub-therapeutic dosage.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the composition comprising the combination of the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents and the anti-thrombin antibody are present in sub-therapeutic dosages.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the composition comprising the combination of the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents are selected from the group consisting of: aspirin and clopidogrel.
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the composition comprising the combination of the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents is aspirin.
  • HC heavy chain
  • LC light chain
  • the present invention provides a composition comprising a combination of therapeutically effective amounts of (a) one or more antiplatelet agents and (b) 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 thrombotic and/or embolic disorder, wherein the composition comprising the combination of the therapeutically effective amounts is associated with reduced adverse bleeding events, and wherein the one or more antiplatelet agents is clopidogrel.
  • HC heavy chain
  • LC light chain
  • the invention also encompasses the following items:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • An antibody molecule which competes with an antibody molecule according to any one of items 5 to 12 for binding to exosite 1.
  • a pharmaceutical composition comprising an antibody molecule according to any one of items 1 to 18 and a pharmaceutically acceptable excipient.
  • a method of treatment of a thrombin-mediated condition comprising
  • a method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin comprising;
  • 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.
  • a method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin comprises:
  • 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;
  • 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;
  • 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.
  • the antibody molecule according to item 29 that inhibits thrombin activity.
  • the antibody molecule according to item 30 which causes minimal inhibition of haemostasis and/or bleeding.
  • 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.
  • 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.
  • 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.
  • the antibody molecule according to item 29 which is a whole antibody.
  • the antibody molecule according to item 41 which is an IgA or IgG.
  • a pharmaceutical composition comprising an antibody molecule according to item 29 and a pharmaceutically acceptable excipient.
  • a method of treatment of a thrombin-mediated condition comprising
  • a method of treatment of a thrombin-mediated condition comprising
  • thrombotic-mediated condition is thrombosis or embolism.
  • thrombotic-mediated condition is inflammation, infection, tumour growth, tumour metastasis or dementia.
  • a method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin comprising;
  • 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,
  • a method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin comprises:
  • 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;
  • 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.
  • expression vectors and their uses are well known to those of skill in the art.
  • 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.
  • the expression vector may provide for protein expression in CHO cells.
  • a recombinant expression vector encoding for an isolated antibody molecule that specifically binds to the exosite 1 region of thrombin.
  • 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.
  • 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.
  • 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 S3 OA, and optionally one or more additional amino acid substitutions, deletions or insertions.
  • the recombinant expression vector according to item 56 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 7 and 8, respectively.
  • 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.
  • 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.
  • recombinant cells may comprise recombinant expression vectors that provide for the expression of the antibody molecules of the present invention in such cells.
  • Recombinant cells may be prokaryotic cells such as bacteria, as well as eukaryotic cells such as mammalian cells.
  • the recombinant cells may be CHO cells such as those described in the working examples of the specification.
  • a recombinant cell expressing an antibody molecule that specifically binds to the exosite 1 region of thrombin.
  • 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.
  • 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.
  • 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.
  • 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.
  • the recombinant cell according to item 66 comprising a VH domain comprising an HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising an LCDR2 and LCDR3 having the sequences of SEQ ID NOs 8 and 9, respectively.
  • 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.
  • a recombinant cell comprising the expression vector according to items 55-64.
  • An aspect of the invention provides an isolated antibody molecule that specifically binds to exosite 1 of thrombin.
  • 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).
  • 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.
  • Exosite 1 (also known as 'anion binding exosite ⁇ 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).
  • Exosite 1 is involved in recognising thrombin substrates, such as fibrinogen, but is remote from the catalytic active site.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an anti-exosite 1 antibody binds to an epitope which consists entirely of exosite 1 residues.
  • 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.
  • the epitope may comprise the thrombin residues Q38, R73, T74, Y76 and R77a and optionally one or more additional residues.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ , or less than InM.
  • 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.
  • 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.
  • 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.
  • 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). [ 0056 ] In some preferred embodiments, the anti-exosite 1 antibody molecule may be a whole antibody.
  • 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.
  • 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.
  • Anti-exosite 1 antibody molecules may be chimeric, humanised or human antibodies.
  • 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.
  • 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.
  • a mammal e.g. mouse, rat, rabbit, horse, goat, sheep or monkey
  • 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.
  • 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.
  • anti-exosite 1 antibody molecules may be identified by screening patient serum for antibodies which bind to exosite 1.
  • 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.
  • 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.
  • an anti-exosite 1 antibody molecule 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.
  • Suitable antibody molecules may be tested for activity using a fibrinogen clotting or thrombin time assay. Suitable assays are well-known in the art.
  • the effect of an antibody molecule on coagulation and bleeding may be determined using standard techniques.
  • 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.
  • 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).
  • VH immunoglobulin heavy chain variable domain
  • VL immunoglobulin light chain variable domain
  • Each of the VH and VL domains typically comprise three complementarity determining regions (CDRs) responsible for antigen binding, interspersed by framework regions.
  • CDRs complementarity determining regions
  • binding to exosite 1 may occur wholly or substantially through the VHCDR3 of the anti-exosite 1 antibody molecule.
  • 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.
  • 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.
  • SEFEPF amino acid residues at positions 4 to 9 of SEQ ID NO: 5
  • D and SEFEPFS amino acid residues at positions 2, and 4 to 10 of 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the VL domain may comprise Tyr49.
  • 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.
  • 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.
  • 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.
  • an antibody may comprise one or more substitutions, deletions or insertions which remove a glycosylation site.
  • a glycosylation site in VL domain of SEQ ID NO 6 may be mutated out by introducing a substitution at either N28 or S30.
  • the anti-exosite 1 antibody molecule may be in any format, as described above.
  • the anti-exosite 1 antibody molecule may be a whole antibody, for example an IgG, such as IgGl or IgG4, IgA, IgE or IgM.
  • 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
  • (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, LCDRl, 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.
  • a further aspect of the present invention provides an antibody molecule comprising an antibody antigen-binding site that competes with an antibody molecule, for example an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of the parent antibody described above for binding to exosite 1 of thrombin.
  • a suitable antibody molecule may comprise an antibody antigen- binding site which competes with an antibody antigen-binding site for binding to exosite 1 wherein the antibody antigen- binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and 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.
  • An anti-exosite 1 antibody molecule as described herein may inhibit the binding of thrombin-binding factors, including factors which bind to exosite 1.
  • 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.
  • An anti-exosite 1 antibody molecule as described herein may inhibit one or more activities of thrombin.
  • 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.
  • 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.
  • binding of thrombin by the anti-exosite 1 antibody molecule may result in no detectable cleavage of the thrombin substrate by thrombin.
  • 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.
  • the effect of an anti-exosite 1 antibody molecule on coagulation and bleeding may be determined using standard techniques.
  • 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)
  • Anti-exosite 1 antibody molecules may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.
  • 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.
  • anti-exosite 1 antibody molecules may be provided in a lyophilised form for reconstitution prior to administration.
  • lyophilised antibody molecules may be re-constituted in sterile water and mixed with saline prior to administration to an individual.
  • 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.
  • 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.
  • 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.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer' s Injection.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a typical antibody dose will be in the range 100 ⁇ g to 1 g for systemic applications, and 1 ⁇ g to 1 mg for topical applications.
  • An initial higher loading dose, followed by one or more lower doses, may be administered.
  • 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.
  • 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.
  • 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.
  • the therapeutic effect of the anti-exosite 1 antibody molecule may persist for several half- lives, depending on the dose.
  • 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.
  • Anti-exosite 1 antibody molecules described herein inhibit thrombin and may be useful in the treatment of thrombin- mediated conditions.
  • 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.
  • Anti-exosite 1 antibody molecules may have no effect or substantially no effect on haemostasis i.e. they do not promote bleeding or haemorrhage.
  • 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.
  • 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.
  • Thrombin plays a key role in haemostasis, coagulation and thrombosis.
  • Thrombin-mediated conditions include thrombotic conditions, such as thrombosis, embolism, and stroke.
  • 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).
  • 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.
  • 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.
  • 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.
  • Thrombosis and embolism may occur in any vein, artery or other blood vessel within the circulatory system and may include microvascular thrombosis.
  • 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.
  • 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.
  • Thrombotic conditions may include thrombophilia, thrombotic stroke and coronary artery occlusion.
  • 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.
  • 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.
  • 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.
  • the risk of haemorrhage in patients treated with anti-exosite 1 antibody molecules may not be increased or substantially increased relative to untreated individuals.
  • 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)
  • 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.
  • 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.
  • 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.
  • the chromogenic signal may be measured directly without defibrination of the sample.
  • 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).
  • 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.
  • 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.
  • the treated blood may be subsequently administered to an individual.
  • inventions 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.
  • a method for producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin may comprise;
  • 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;
  • VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations
  • 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.
  • 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.
  • a method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin may comprise:
  • 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;
  • 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;
  • 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.
  • 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.
  • 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.
  • 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 ( ⁇ 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 ( ⁇ 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 ( ⁇ 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 ( ⁇ 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 4 ⁇ (final concentration in mouse blood, equivalent to a dose of approximately 60 mg/Kg) anti-exosite 1 IgA ( ⁇ 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 18 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
  • 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.
  • APTT partial thromboplastin time
  • 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 representations thrombin and different thrombin binding sites, including the catalytic site, exosite 1 and exosite 2 and also shows the different binding modes for Hirudin, Bivalrudin, Dabigatran, and J J-64179375.
  • Figure 31 shows correlations between plasma concentrations of JNJ-64179375
  • Figure 32 shows the effect of JNJ-64179375 (Compound) on ex vivo platelet activation.
  • Extra-corporeal administration of JNJ-64179375 inhibited thrombin-mediated [A] p- selectin expression and [B] platelet-monocyte aggregates in a dose-dependent manner, but had no effect on ADP activity.
  • Data shown are statistical means ⁇ 95% confidence intervals. Comparisons are versus placebo; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • ADP adenosine diphosphate
  • PMA platelet-monocyte aggregates
  • GMFI geometric mean fluorescent intensity
  • Figure 33 shows the effect of JNJ-64179375 (Compound) on ex vivo total thrombus formation as compared to placebo.
  • Extra-corporeal administration of JNJ-64179375 inhibited total thrombus formation in a dose-dependent manner at both [A] low shear stress (212 s-1) and [B] high shear stress (1690 s-1) shear stress.
  • Data shown are the mean change (%) in total thrombus area as compared to placebo ⁇ 95% confidence intervals; * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • Figure 34 shows the effect of JNJ-64179375 (Compound) on fibrin-rich and platelet-rich thrombus formation as compared to placebo.
  • Extra-corporeal administration of JNJ- 64179375 inhibited fibrin-rich thrombus deposition in a dose-dependent manner at both [A] low shear stress (212 s-1) and [C] high shear stress (1690 s-1) shear stress, as compared to placebo.
  • JNJ-64179375 had no effect on platelet-rich thrombus deposition under either shear stress.
  • Bivalirudin reduced fibrin-rich thrombus deposition at low and high shear stress, and platelet-rich thrombus deposition at high heart stress. Data shown are the absolute change in area ( ⁇ m 2 /mm) ⁇ 95% confidence intervals; * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001. Abbreviation used: Bival., bivalirudin.
  • Figure 36 shows bar graphs of the blood coagulation test results from the rat AV shunt model with different doses of JNJ-64179375 and reference agents including Apixaban, Dabigatran, Bivalirudin, and Heparin.
  • the tests included Thrombin Time (TT), activated Partial Thrombin Time (aPTT), Prothrombin Time (PT), and Ecarin Clotting Time (ECT). Doses are mg/kg except for heparin which is U/kg.
  • Figure 37 shows graphs of the plasma concentrations in the rat AV shunt model with different doses of Apixaban, Dabigatran, and Bivalirudin. Plasma concentrations are on the y-axis in mg/ml and dose levels are on the x-axis in mg/kg by intravenous administration (IV).
  • IV intravenous administration
  • Figure 38 shows graphs of mean arterial blood flow over time in the rat Arterial FeCb model with different doses of JNJ-64179375 and reference agents including Apixaban, Dabigatran, Bivalirudin, and Heparin.
  • FIG 39 shows graphs of Time to Occlusion (TTO) in the rat Arterial FeCb model with different doses of JNJ-64179375 and reference agents including Apixaban, Dabigatran, Bivalirudin, and Heparin.
  • TTO Time to Occlusion
  • Figure 40 shows graphs of Area Under Curve (AUC) for mean blood flow in the rat Arterial FeCb model with different doses of JNJ-64179375 and reference agents including Apixaban, Dabigatran, Bivalirudin, and Heparin.
  • AUC Area Under Curve
  • Figure 41 shows graphs of coagulation parameters in blood samples from the rat Arterial FeCb model with different doses of JNJ-64179375, the coagulation factors including Thrombin Time (TT), activated Partial Thrombin Time (aPTT), Prothrombin Time (PT), and Ecarin Clotting Time (ECT).
  • TT Thrombin Time
  • aPTT activated Partial Thrombin Time
  • PT Prothrombin Time
  • ECT Ecarin Clotting Time
  • Figure 42 shows graphs of the results for the rat tail transection model for JNJ-64179375 and Apixaban (Figure 42).
  • Figures A and B show treatments with Vehicle (negative control) and different doses (mg/kg) of JNJ-64179375.
  • Figure C shows Vehicle (negative control) with different doses (mg/kg) of JNJ-64179375 (IchorS30A) and Apixaban (Apix). Treatments were administered as an intravenous bolus.
  • Figure 43 shows graphs for the results for platelet aggregation studies performed in platelet rich plasma with different doses of JNJ-64179375 and various platelet agonists, including: AA (Arachidonic Acid at 1.5mM), human thrombin (hthrombin at 80 nM), rat thrombin (rthrombin at 80 nM), ADP (Adenine di-Phosphate at 20 ⁇ ), and collagen at 10 ⁇ g/ml.
  • the results for the different doses of JNJ-64179375 are in order from lower concentration to higher concentration: 0 mg/kg (vehicle), 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg.
  • Figure 44 shows results from the rat Arterial FeCb model with Vehicle (control), and different doses of clopidogrel (lmg/kg, 2mg/kg, 3mg/kg, and lOmg/kg).
  • Figure (A) shows graphs of mean arterial blood flow over time
  • (B) shows Time to Occlusion (TTO) and (C) Area Under Curve (AUC).
  • Figure 45 shows results from the rat Arterial FeCb model with Vehicle (control), and different doses of aspirin (3mg/kg, lOmg/kg, and 30mg/kg).
  • Figure (A) shows graphs of mean arterial blood flow over time
  • (B) shows Time to Occlusion (TTO) and (C) Area Under Curve (AUC).
  • Figure 46 shows graphs of mean arterial blood flow over time in the rat Arterial FeCb model with Vehicle (control), clopidogrel (2mg/kg), JNJ-64179375 (3mg/kg), and a combination of clopidogrel (2mg/kg) plus JNJ-64179375 (3mg/kg).
  • B shows graphs of mean arterial blood flow over time in the rat Arterial FeCb model with Vehicle (control), clopidogrel (lmg/kg), JNJ-64179375 (3mg/kg), and a combination of clopidogrel (lmg/kg) plus JNJ-64179375 (3mg/kg).
  • Figure (A) shows graphs of mean arterial blood flow over time
  • (B) shows Time to Occlusion (TTO) and
  • the proportion of total number that occluded in each group were: Vehicle (10/10), clopidogrel lmg/kg (5/5), aspirin 30mg/kg (4/6), JNJ-9375 0.3mg/kg (6/6), and triple combination (3/6).
  • Figure (A) shows graphs of mean arterial blood flow over time
  • (B) shows Time to Occlusion (TTO) and (C) Area Under Curve (AUC).
  • the proportion of total number that occluded in each group were: Vehicle (10/10), clopidogrel lmg/kg (5/5), aspirin 30mg/kg (4/6), JNJ-9375 lmg/kg (12/12), and triple combination (2/6).
  • Figure (A) shows graphs of mean arterial blood flow over time
  • (B) shows Time to Occlusion (TTO) and
  • the term "safe”, as it relates to a dose, dosage regimen or treatment with combinations comprising one or more antiplatelet agents and an 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 relatively low or reduced frequency and/or low or reduced severity of adverse events, including reduced adverse bleeding events, reduced infusion or hypersensitivity reactions, or reduced wound or joint complications compared to the standard of care or to another comparator.
  • the relatively low or reduced frequency and/or low or reduced severity of adverse events of the present invention are compared to adverse events caused by combinations comprising one or more antiplatelet agents and other anticoagulants, including for example direct acting oral anticoagulants (DOACs), e.g., factor Xa (FXa) inhibitors (e.g., apixaban), thrombin inhibitors (e.g., dabigatran), or factor XIa (FXIa) inhibitors.
  • DOACs direct acting oral anticoagulants
  • FXa factor Xa
  • apixaban e.g., apixaban
  • thrombin inhibitors e.g., dabigatran
  • FXIa factor XIa
  • the invention as defined herein comprises a safe dose of JNJ-64179375 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
  • reduced adverse bleeding events includes low or reduced frequency and/or low or reduced severity of adverse bleeding events.
  • Adverse bleeding events may include, for example, major bleeding events, nonmajor clinically relevant bleeding events, and/or an any bleeding event composite. Bleeding events are a standard primary safety endpoint in studies of anticoagulants, but there is substantial heterogeneity in bleeding definitions.
  • TIMI Thrombolysis in Myocardial Infarction
  • Apixaban defines major bleeding as clinically overt bleeding that was accompanied by one or more of the following: a decrease in hemoglobin of 2 g/dL or more; a transfusion of 2 or more units of packed red blood cells; bleeding that occurred in at least one of the following critical sites: intracranial, intraspinal, intraocular, pericardial, intra-articular, intramuscular with compartment syndrome, retroperitoneal; or bleeding that was fatal.
  • Intracranial hemorrhage included intracerebral (hemorrhagic stroke), subarachnoid, and subdural bleeds.
  • the label for Pradaxa defines major bleeding as bleeding accompanied by one or more of the following: a decrease in hemoglobin of >2 g/dL, a transfusion of >2 units of packed red blood cells, bleeding at a critical site or with a fatal outcome.
  • Intracranial hemorrhage included intracerebral (hemorrhagic stroke), subarachnoid, and subdural bleeds.
  • bleeding events are based on the ISTH bleeding scale, e.g., "major bleeding" in non-surgical patients is defined as, 1. fatal bleeding and/or; 2.
  • symptomatic bleeding in a critical area or organ such as intracranial, intraspinal, intraocular, retroperitoneal, intraarticular or pericardial, or intramuscular with compartment syndrome; and/or 3. bleeding causing a fall in hemoglobin level of 2 g/dL (1.24 mmol/L) or more, or leading to transfusion of two or more units of whole blood or red cells.
  • treatment with combinations comprising one or more antiplatelet agents and JNJ-64179375 compared to treatment with combinations comprising one or more antiplatelet agents and other DOACs is associated with significantly reduced adverse bleeding events of >5%, >10%, >15%, >20%, >25%, >30%, >35%, >40%, >45%, or > 50%.
  • treatment with combinations comprising one or more antiplatelet agents and JNJ-64179375 is associated with significantly reduced adverse bleeding events of 35-50% reduction compared to treatment with combinations comprising one or more antiplatelet agents and the DOAC.
  • the significantly reduced adverse bleeding events is a >35% reduction compared to combinations comprising one or more antiplatelet agents and a DOAC. In other embodiments, the significantly reduced adverse bleeding events is a >40% reduction compared to combinations comprising one or more antiplatelet agents and the DOAC. In other embodiments, the significantly reduced adverse bleeding events is a >45% reduction compared to combinations comprising one or more antiplatelet agents and the DOAC. In other embodiments, the significantly reduced adverse bleeding events is a >50 % reduction compared to combinations comprising one or more antiplatelet agents and the DOAC.
  • treatment with combinations comprising one or more antiplatelet agents and JNJ-64179375 is associated with significantly reduced adverse bleeding events of 35-50% reduction compared to combinations comprising one or more antiplatelet agents and the FXa inhibitor apixaban. In certain embodiments, treatment with combinations comprising one or more antiplatelet agents and JNJ- 64179375 is associated with significantly reduced adverse bleeding events of 35-50% reduction compared to combinations comprising one or more antiplatelet agents and the thrombin inhibitor dabigatran.
  • An "adverse event” is any untoward medical occurrence in a clinical study subject administered a medicinal product.
  • Treatment-emergent adverse events are adverse events with onset during the treatment phase or that are a consequence of a preexisting condition that has worsened since baseline, but an adverse event does not necessarily have a causal relationship with the treatment.
  • An adverse event can therefore be any unfavorable and unintended sign (including an abnormal finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not related to that medicinal product.
  • ICH Interpretition per International Conference on Harmonisation
  • a laboratory test abnormality that is considered by the investigator to be clinically relevant (e.g., causing the subject to discontinue the study drug, requiring treatment, or causing apparent clinical manifestations) should be reported as an adverse event.
  • TI therapeutic index
  • therapeutic ratio a comparison of the amount of a therapeutic agent that causes the therapeutic effect (e.g., inhibition of a thrombin- mediated condition) to the amount that causes adverse bleeding events (e.g., major bleeding, minor clinically relevant bleeding, and/or individual components of the composite endpoint of any bleeding event).
  • efficacy refers to terms such as amounts, dose, dosage regimen, or treatment with a combination comprising one or more antiplatelet agents and an 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 treatment or inhibition of a thrombotic and/or embolic disorder. Such inhibition can be observed, for example, as a reduction in the frequency of occurrence and/or severity of the thrombin- mediated condition in patients treated with the anti-thrombin antibody.
  • HC heavy chain
  • LC light chain
  • effective is intended to refer to agents that are effective when administered in combination to treat a desired disease or condition, e.g., JNJ-64179375 in combination with one or more antiplatelet agents used to treat a thrombotic and/or embolic disorder.
  • a desired disease or condition e.g., JNJ-64179375
  • antiplatelet agents used to treat a thrombotic and/or embolic disorder.
  • the preferred combinations can have an additive effect or a synergistic effect, wherein such
  • combinations provide improved or comparable efficacy with reduced adverse bleeding events, wherein the reduced adverse bleeding events include low or reduced frequency and/or low or reduced severity of adverse bleeding events compared to the standard of care or treatment with a comparator, including, for example, treatment with one or more antiplatelet agents, treatment with an anticoagulant other than JN J-64179375 , or treatment with a combination of one or more antiplatelet agents and an anticoagulant other than JNJ-64179375.
  • Non-limiting examples of anticoagulants other than JNJ-64179375 include, for example, heparin, warfarin (Coumadin), rivaroxaban (Xarelto), dabigatran (Pradaxa), apixaban (Eliquis), Bivalirudin (Angiomax or Angiox) edoxaban (Savaysa), enoxaparin (Lovenox), and fondaparinux (Arixtra).
  • the combinations of agents could allow for lower dosages of each individual agent used in the combination or the combinations described herein could have enhanced efficacy for the treatment of thrombotic and/or embolic disorders with low or reduced frequency and/or low or reduced severity of adverse bleeding events.
  • a non-limiting example where an additive effect could be preferred is in patients that have "arterial" indications such as acute coronary syndromes (ACS).
  • ACS acute coronary syndromes
  • Antiplatelet agents are the standard of care for patients with ACS and it would be of benefit to be able to add a second agent that inhibits thrombosis or embolism without an increase in the risk of bleeding.
  • an additive effect could also be preferred in patients with coronary artery disease (CAD).
  • CAD coronary artery disease
  • Synergy occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent [50]. In general, a synergistic effect is most clearly demonstrated at sub-optimal concentrations of the compounds (i.e., a "sub-therapeutic dose"). Synergy can be in terms of lower cytotoxicity, increased antithrombotic effect, improved therapeutic index, reduced frequency and or severity of adverse bleeding events, or some other beneficial effect of the combination compared with the individual components.
  • a thrombotic and/or embolic disorder is a circulatory disease or condition caused by thrombosis or embolism which can involve the effects of platelet activation and/or platelet aggregation.
  • the term "thrombotic and/or embolic disorder” as used herein includes arterial cardiovascular thrombotic and/or embolic disorders, venous cardiovascular thrombotic and/or embolic disorders, arterial cerebrovascular thrombotic and/or embolic disorders, and venous cerebrovascular thrombotic and/or embolic disorders.
  • Non-limiting examples of "thrombotic and/or embolic disorders” include, for example, unstable angina, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures in which blood is exposed to an artificial surface that promotes thrombosis.
  • thrombosis includes occlusion (e.g., after a bypass) and reocclusion (e.g., during or after percutaneous transluminal coronary angioplasty).
  • the term thrombotic and/or embolic disorders also includes conditions such as acute coronary syndrome, coronary artery disease, peripheral artery disease, unstable angina, refractory angina, occlusive coronary thrombus occurring post-thrombolytic therapy or post-coronary angioplasty, a thrombotically mediated cerebrovascular syndrome, embolic stroke, thrombotic stroke, transient ischemic attacks, venous thrombosis, deep venous thrombosis, pulmonary embolus, coagulopathy, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, thromboangiitis obliterans, thrombotic disease associated with heparin-induced thrombocytopenia, thrombotic complications associated with extracorporeal
  • J J-64179375 in combination with one or more antiplatelet agents preferably affords an efficacy advantage over the agents alone (i.e., an additive combination or a synergistic combination), and may permit use of lower doses of each of JNJ-64179375 and/or the one or more antiplatelet agents (i.e., sub- therapeutic dosages).
  • a lower dosage of the JNJ-64179375 and/or the one or more antiplatelet agents could minimize the potential of side effects, such as adverse bleeding events, thereby providing an increased margin of safety.
  • JNJ-64179375 and/or the one or more antiplatelet agents are administered in a sub-therapeutic dose.
  • subtherapeutic is intended to mean an amount of a therapeutic agent that by itself does not give the desired therapeutic effect for the disease being treated.
  • Synergistic combination is intended to mean that the observed effect of the combination is greater than the sum of the individual agents administered alone.
  • JNJ-64179375 in combination with one or more antiplatelet agents may be administered at the same time or sequentially in any order at different points in time.
  • the combination of JNJ-64179375 and the one or more antiplatelet agents may be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • the combination of JNJ-64179375 and the one or more antiplatelet agents may also be formulated into a single pharmaceutical composition.
  • antiplatelet agents or “platelet inhibitory agents”, as used herein, denotes agents that inhibit platelet function, for example by inhibiting the aggregation, adhesion, or granular secretion of platelets.
  • Agents include, for example, but are not limited to, the various known non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, piroxicam, and pharmaceutically acceptable salts or prodrugs thereof.
  • NSAIDS non-steroidal anti-inflammatory drugs
  • aspirin acetylsalicyclic acid or ASA
  • ASA acetylsalicyclic acid
  • Suitable platelet inhibitory agents include Ilb/IIIa antagonists (e.g., tirofiban, eptifibatide, and abciximab), thromboxane -A2 -receptor antagonists (e.g., ifetroban), thromboxane-A2-synthetase inhibitors, PDE-III inhibitors (e.g., dipyridamole), thrombin receptor antagonists that are also referred to as PAR-1 antagonists, e.g., Vorapaxar (trade name Zontivity), P2Yn inhibitors, e.g., Ticagrelor (trade name Brilinta and others), clopidogrel (brand name Plavix among others), and Cangrelor (trade name Kengreal in the US and Kengrexal in Europe), and pharmaceutically acceptable salts or prodrugs thereof.
  • Ilb/IIIa antagonists e.g., tirofiban, eptifibatide, and abciximab
  • Clopidogrel acts by irreversibly inhibiting the P2Yn subtype of ADP receptor, which is important in activation of platelets and eventual cross-linking by the protein fibrin.
  • Cangrelor is a P2Yi2 inhibitor for intravenous application.
  • antiplatelet agents also include, prasugrel (Effient), Dipyridamole, dipyridamole/aspirin (Aggrenox), and ticlodipine (Ticlid).
  • PT prothrombin time
  • aPTT activated partial thromboplastin time
  • TT thrombin time
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • fucosylated non-galactosylated di-antenary oligosaccharides for Fe oligosaccharides.
  • 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.
  • 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.
  • 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).
  • the IgG S30A variant was expressed for analysis using standard transient expression techniques as described below.
  • single gene vectors SGVs
  • GS Xceed vectors Longza Biologies, Slough, UK
  • pXC IgG4pro ⁇ for the heavy chain constant domain encoding region and pXC Kappa for light chain constant domain encoding region
  • 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.
  • a single bacterial colony was picked into 15 ml Luria Bertani (LB) medium (LB Broth, Sigma-Aldrich, L7275) containing 50 ⁇ /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 ⁇ /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).
  • 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 10 5 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
  • Cells were electroporated at 300 V, 900 ⁇ F for the Gene Pulse XCell system and 300 V, 1300 ⁇ F 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% CO 2 , 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).
  • 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.
  • 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).
  • 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.
  • IgG S30A was found to be equivalent or more potent than IgG.
  • Table 5 shows IgG and IgG S30A binding affinities to thrombin using BiacoreTM 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).
  • Table 6 shows that both IgG S30A and IgG have equivalent solubility and both are soluble to >100 mg/ml concentration, with little reduction in solubility (and no aggregate formation) on storage.
  • Figure 24 shows the potency of IgG and IgG S30A in an ex vivo activated partial thromboplastin time (APTT) coagulation assay.
  • IgG S30A is equivalent or more potent than IgG.
  • 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.
  • 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.
  • 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.
  • Figure 28 shows that both IgG S30A and IgG are equivalent in the rat venous thrombosis model using ferric chloride (FeCl 3 ; see experimental section 2 above) at 2.5% concentration, with both IgG S3 OA and IgG causing total prevention of thrombus formation.
  • ferric chloride FeCl 3 ; see experimental section 2 above
  • Figure 29 shows that both IgG S30A and IgG are equivalent in the rat venous thrombosis model using ferric chloride (FeCl 3 ) at 5% concentration, with both IgG S3 OA and IgG causing similar reduction of thrombus formation.
  • the IgG S30A variant thus may be preferable from a manufacturing and production perspective for reasons described above.
  • Specific anti-exosite 1 antibody molecules disclosed herein include the following:
  • IgG synthetic anti-exosite 1 IgG antibody
  • IgG S30A synthetic anti-exosite 1 IgG S3 OA variant antibody
  • 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.
  • 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
  • the IgG S30A variant was expressed transiently using standard techniques for the purposes of analysing the variant.
  • double gene vector was constructed using previously established single gene vectors (see experimental section 6 above) in Lonza's GS Xceed vectors (pXC IgG4pro ⁇ 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.
  • Single gene vectors 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 ⁇ and incubated at 37°C for 2 hours. 4.0 ⁇ 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 ⁇ Lonza Simply Load Tandem DNA ladder was used as a reference ladder. The agarose gel was imaged using BioSpectrum Imaging System (IVP).
  • IVP BioSpectrum Imaging System
  • Vector DNA was isolated using the QIAGEN Plasmid Plus Gigaprep system (QIAGEN, 12991) and quantified using a Nanodrop 1000 spectrophotometer (Thermo- Scientific).
  • 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% CO 2 , 85% humidity, 140 rpm. Cells were routinely sub-cultured every 3-4 days, seeding at 2 x 10 5 cells/ml and were propagated in order to have sufficient cells available for transfection. Cells were discarded by passage 20.
  • 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 ⁇ g/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 10 7 cells/ml. 100 ⁇ linearized DNA at a concentration of 400 ⁇ g/ml was aliquoted into a 0.4 cm gap electroporation cuvette and 700 ⁇ cell suspension added.
  • the transfectant cultures were suitable to process.
  • Cells were seeded at 0.2x 10 6 cells/ml in a final volume of 100 ml in CD-CHO medium supplemented with 50 ⁇ 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% CO 2 , 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.
  • 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
  • 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.
  • 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).
  • 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
  • the clarified cell culture supernatant was purified using one-step Protein A chromatography.
  • SEQ ID NO: 14 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.
  • SEQ ID NO: 15 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.
  • SEQ ID NO: 17 includes the alanine (underlined) for serine substitution that corresponds the S30A substitution in SEQ ID NO:6.
  • Table 3 Effect of saturating concentration of anti-exosite 1 IgA (Fab) on thrombin inhibition by antithrombin (AT) in the absence and presence of 1 nM heparin (Hep).
  • Fab anti-exosite 1 IgA
  • Table 5 Binding affinities of IgG and IgG S30A to thrombin using BiacoreTM 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").
  • Coagulation cascade activation leading to fibrin formation is central to thrombosis.
  • Anticoagulant agents are of proven benefit in many thrombotic and/or embolic disorders but despite improvements, treatment-related bleeding continues to be a major concern and event rates remain unacceptably high [1-3]. All the currently available agents act to either prevent thrombin generation (e.g. vitamin K antagonists, factor Xa inhibitors, low molecular weight heparin) or block the catalytic site of the protease directly (e.g. dabigatran, bivalirudin).
  • 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 [4] .
  • JNJ- 64179375 has a heavy chain (HC) comprising SEQ ID NO: 14 and a light chain (LC) comprising 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).
  • HCDR1 heavy chain
  • VH variable heavy chain
  • VL variable light chain
  • the JNJ-64179375 sequences include an S30A substitution in the LC to remove a glycosylation site, a serine 228 to proline substitution (S228P, as numbered according to the EU numbering system) in the HC to stabilize the hinge region [46-47], and the C-terminal lysine was removed from the HC to eliminate heterogeneity.
  • Exosite 1 together with exosite 2, regulate thrombin enzymatic activity by providing initial binding sites for substrates, inhibitors, or co-factors, and by allosteric modification or steric hindrance of the active site [5,7].
  • Exosite 1 is predominantly the fibrinogen Aa recognition site, while exosite 2 binds to heparin and glycoprotein (GP) Iba.
  • JNJ-64179375 therefore acts as an anticoagulant by preventing binding of fibrinogen Aa whilst leaving the catalytic activity of the protease intact.
  • the mechanism of action is distinct from the current anticoagulants and by avoiding inhibiting all thrombin activity has the potential for a wider therapeutic window in terms of antithrombotic efficacy and haemorrhagic risk.
  • the mechanism of action is distinct from currently available direct thrombin inhibitors that block the active site only (eg, dabigatran, argatroban) or that block both the active site and exosite 1 (eg, bivalirudin, hirudin) (Figure X).
  • the mechanism of action is distinct 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).
  • Thrombus formation was assessed using the Badimon chamber perfusion model as previously described [8,9] .
  • a pump was used to draw native
  • Platelet p-selectin expression and platelet-monocyte aggregates are sensitive markers of in vivo platelet activation [12-14].
  • Blood (2.7 mL) was collected immediately distal to the final perfusion chamber into tubes containing 0.3 mL of 3.8% sodium citrate and Pefabloc FG (final concentration 1.5 mg/mL; Quadratech Diagnostics, Surrey, UK).
  • porcine strips with thrombus attached were removed and fixed in 4 % paraformaldehyde for 72 hours at 4 ° C prior to being prepared for histological analysis.
  • the mean cross-sectional area gives a reliable reflection of total thrombus formation [15].
  • the proximal and distal 1 mm of the exposed substrate were discarded and the remainder cut into eight segments. Segments were embedded in paraffin wax and 4- ⁇ m sections prepared.
  • a semi-automated slide scanner (Axioscan Z 1 ; Zeiss, Jena, Germany) and image analysis software (Definiens, Kunststoff, Germany) were used by a blinded operator to quantify thrombus area and composition. Digital images of each section were acquired at x20 magnification. High-resolution classifiers based on colour were established to detect total thrombus area, fibrin-rich thrombus area and platelet-rich thrombus area.
  • JNJ-64179375 caused dose-dependent prolongation of all measured blood coagulation markers, with thrombin time the most sensitive to the anticoagulant effect (Table 7). Pearson's correlation coefficient between plasma concentrations of JNJ- 64179375 and coagulation assays was 0.98 for prothrombin time, 0.87 for activated partial thromboplastin time, and 0.91 for thrombin time (p ⁇ 0.001 for all; Figure 31). Effect of JNJ-64179375 on platelet activation
  • JNJ-64179375 did not significantly reduce platelet-rich thrombus formation at either shear rate ( Figure 32).
  • JNJ-64179375 caused concentration-dependent prolongation of coagulation time and selective inhibition of thrombin-mediated platelet activation that led to reductions in low and high shear ex vivo thrombus formation driven by a decrease in fibrin-rich thrombus deposition.
  • the results suggest that JNJ-64179375 has a favourable anticoagulant and antithrombotic profile. Discussion
  • thromboplastin time may provide a useful assay to measure exosite 1 inhibition and JNJ- 64179375 activity.
  • thrombin exosite 1 inhibition differs from the currently available anticoagulants in that it prevents fibrinogen binding while leaving the active site and exosite 2-mediated substrate recognition intact. This results in a more selective inhibition of thrombin activity that may in turn avoid interfering with pathways primary involved in haemostasis.
  • JNJ-64179375 produces similar reductions in ex vivo thrombosis formation to the clinically approved anticoagulant edoxaban and indicate thrombin exosite 1 inhibition has a high probability of in vivo antithrombotic efficacy.
  • Thrombin activates platelets through binding to platelet surface glycoprotein (GP) Iba and cleavage of protease-activated receptors 1 (PARI) and 4 (PAR4) [29].
  • Exosite 1 interacts with the exodomain of PARI to facilitate efficient receptor cleavage [30] whereas PAR4 activation and GPIb binding are dependent almost entirely on the active site and exosite 2 respectively [18,31].
  • JNJ-64179375 demonstrated dose- dependent and selective inhibition of thrombin stimulated platelet activation, but was not associated with a reduction in platelet-rich thrombus formation.
  • exosite 1 has a role in platelet activation but appears to be minimally involved in pathways associated with irreversible platelet aggregation and incorporation into the developing thrombus. This is consistent with previous studies demonstrating exosite 1 inhibition only weakly inhibits thrombin- induced platelet aggregation [32]. Combination therapy with an anticoagulant and antiplatelet is an increasingly encountered and residual cause for dilemma in clinical practice because of the high bleeding risk and resultant narrow therapeutic window
  • Exosite 1 inhibition may be especially useful in this setting by providing robust inhibition of fibrin-rich thrombus formation but avoiding overly interfering with thrombin-mediated platelet activities.
  • porcine aorta includes collagen type I fibres and closely resembles that of human blood vessels, it is likely not to contain tissue factor [38-40] .
  • Tissue factor (TF) activates the coagulation cascade and is an important contributor to thrombogenicity [41,42].
  • JNJ-64179375 prolongs coagulation and substantially reduces ex vivo thrombus formation at both low and high shear rates.
  • JNJ-64179375 has a more selective effect on thrombin-mediated activity than existing agents with an emphasis on inhibiting fibrinogen Aa cleavage.
  • JNJ-64179375 represents a promising novel class of anticoagulant with the potential for a wider therapeutic window (i.e., therapeutic index) in terms of antithrombotic efficacy and bleeding risk.
  • JNJ-64179375 may preferentially inhibit fibrin-rich venous thrombosis with relative preservation of platelet-mediated hemostasis.
  • This specificity of JNJ-64179375 could provide benefits by way of improving therapeutic index for treatment with JNJ-64179375 and allowing for combinations with antiplatelet agents or treating patient populations already taking antiplatelet agents.
  • the tests included Thrombin Time (TT), activated Partial Thrombin Time (aPTT), Prothrombin Time (PT), and Ecarin Clotting Time (ECT).
  • TT Thrombin Time
  • aPTT activated Partial Thrombin Time
  • PT Prothrombin Time
  • ECT Ecarin Clotting Time
  • the tests additionally included platelet aggregation tests with PRP.
  • Additional animal models included a rat tail transection model.
  • the rat AV-shunt model is done under anesthesia.
  • the left jugular vein and right carotid artery are cannulated with 8cm long PE-100 tubing.
  • a baseline blood sample (lml) is collected and then compounds are administered either as an intravenous bolus or 15min infusion.
  • the shunt is assembled by connecting the jugular vein and carotid artery cannulae with a 6cm long tubing containing a 6cm 2-0 silk surgical thread.
  • the 6cm long connection is the shunt and the silk thread in the shunt acts as a foreign body to activate the intrinsic cascade to cause a blood clot (thrombus).
  • Blood is allowed to flow through the shunt forl5 min. At the end of 15 min, the external tubing with the thread and blood clot are removed and the blood clot is weighed. After removing the thread, additional blood samples (2x4.5ml) are collected for subsequent testing and the animal is euthanized.
  • rat arterial FeCb model For the rat arterial FeCb model, the right carotid artery and left jugular vein are isolated and jugular vein is cannulated.
  • An ultrasonic flow probe (transonic 1RB) is placed around the carotid artery and blood flow is recorded via a Transonic Flow Meter connected to a Powerlab recording system.
  • Test compounds or vehicle are administered via the jugular catheter either as an intravenous (IV) bolus or infusion.
  • Two pieces of filter paper, 3 mm in diameter soaked in FeCb (10% wt/vol) are applied to the surface and underneath of the carotid artery for lOmin. Flow is then monitored until complete occlusion of the artery, or after a period of 60 min. Terminal blood samples are subsequently collected to measure drug levels and to assess various clotting assays and platelet function tests.
  • Rat blood was collected in a BD tube with 3.2% sodium citrate and spun down @ 125g for 13mins at room temperature. Supernatant was collected (platelet-rich plasma, PRP). Remaining fraction was spun at 2500g for 15mins at room temperature. Supernatant was collected to obtain platelet-poor plasma (PPP). The number of platelets in PRP were counted, adjusted to approximately 600K cell/ ⁇ with PPP, and then rested at room temperature before the experiment. Optical Platelet Aggregation Assay
  • the aggregometer machine was allowed to warm up to 37°C. 250ul of PRP was added to the test cuvette with stir bars. A cuvette with PPP was placed into the background chamber and the test cuvette with PRP was placed into the test chamber. Baseline was calibrated (from 0% to 100%) and the agonist ADP was added into the PRP to initiate platelet aggregation. The test process was allowed to continue for at least 5 min to achieve to maximal aggregation.
  • JNJ-64179375, dabigatran, and apixaban all maximally inhibited thrombus formation in dose dependent manner in the rat AV-shunt model of venous thrombosis.
  • Significant inhibition of thrombus weights of 41.15%, 44.25% and 57.23% were observed at 0.3 mg/kg, IV, 0.1 mg/kg, IV and 1 mg/kg, IV with JNJ-64179375, dabigatran, and apixaban, respectively (Figure 35).
  • Dabigatran the proportion of total number that not occluded in each group were: Control (0/6), Vehicle (0/6), Dabigatran O. lmpk (0/6), Dabigatran 0.3mpk (2/6), Dabigatran lmpk (4/6).
  • Heparin the proportion of total number that not occluded in each group were: Control (0/6), Vehicle (0/6), Heparin 30U/kg (0/6), Heparin lOOU/kg (3/6), Heparin 300U/kg (5/6).
  • the observed inhibition of thromb in-induced platelet aggregation was about 50%, >95% and >95% at 1, 3 and 10 mg/kg, IV with JNJ-64179375, respectively.
  • arterial thrombosis only observed at 10 mg/kg.
  • this profile of JNJ-64179375 may make it amenable to more predictive dosing in combinations with antiplatelet therapy in indications such as acute coronary syndrome (ACS), coronary artery disease (CAD), peripheral artery disease (PAD) and in certain patient populations, including in patient populations that are already taking one or more antiplatelet agents.
  • ACS acute coronary syndrome
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • the rat arterial FeCb model was used to test double and triple combinations of JNJ-64179375 with different representative antiplatelet agents, e.g., clopidogrel and aspirin.
  • Clopidogrel and aspirin were dosed orally (at the dose indicated) once daily for 3 days. 2 hrs after dosing on day 3 the animals were anesthetized and thrombosis was induced. JNJ-64179375 was administered as an IV bolus once the animals were anesthetized.
  • Measured values for the rat arterial FeCb model included: mean blood flow, area under the curve (AUC), and Time to Occlusion (TTO).
  • Ex-vivo assessments include:
  • TT Thrombin time
  • aPTT Activated partial thrombin time
  • PT Prothrombin time
  • ECT Ecarin clotting time
  • TXB2 thromboxane B2
  • Clopidogrel sold as the brandname Plavix among others, is a medication that is used to reduce the risk of heart disease and stroke in those at high risk. Clopidogrel acts by irreversibly inhibiting the P2Yn subtype of ADP receptor, which is important in activation of platelets and eventual cross-linking by the protein fibrin. [48] Clopidogrel is also used together with aspirin in heart attacks and following the placement of a coronary artery stent (dual antiplatelet therapy).
  • Aspirin can cause several different effects in the body, including, for example, reduction of inflammation, analgesia, reduction of fever, and the inhibition of clotting. Aspirin inhibits clotting by irreversibly blocking the formation of thromboxane A2 in platelets, which is responsible for the aggregation of platelets that form blood clots. This antiplatelet property makes aspirin useful for reducing the incidence of heart attacks.
  • thrombin RNA (2009), vol. 15, pp. 2105-2111.
  • Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II. J Biol Chem 1999;274:6226-33.
  • Michelson AD Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: methods and clinical applications. Blood Coagul Fibrinolysis 1994;5: 121-31.
  • PAI-1 plasminogen activator inhibitor- 1
  • PAI-749 in clinical models of fibrinolysis. J Thromb Haemost 2010;8: 1333-9. doi: 10.1111/j.1538- 7836.2010.03872.x
  • EAPCI Percutaneous Cardiovascular Interventions
  • ACCA European Association of Acute Cardiac Care
  • HRS Heart Rhythm Society
  • AHRS Asia-Pacific Heart Rhythm Society
  • thromboprophylaxis after knee replacement (ADVANCE-2): a randomised double-blind trial. Lancet. 2010;375:807-815.

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Abstract

La présente invention concerne des anticorps anti-thrombine isolés qui reconnaissent l'épitope de l'exosite-1 de la thrombine et inhibent sélectivement la thrombine sans provoquer de saignement. Ces molécules d'anticorps anti-thrombine peuvent être utiles pour traiter ou inhiber des troubles thrombotiques et/ou emboliques et d'autres états pathologiques médiés par la thrombine. En particulier, la présente invention concerne l'utilisation des molécules d'anticorps anti-thrombine en combinaison avec un ou plusieurs agents antiagrégants plaquettaires.
PCT/IB2018/056198 2017-08-16 2018-08-16 Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires Ceased WO2019035055A1 (fr)

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