Thrombin-Binding Antibody Molecules And Uses Thereof

Abstract

This invention relates to isolated antibodies which recognize the exosite 1 epitope of thrombin and selectively inhibit thrombin without promoting bleeding. These antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other conditions mediated by thrombin.

What is claimed: 
     
         1 . A lyophilized antibody molecule that specifically binds to the exosite 1 epitope of thrombin, the antibody comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, and a VL domain comprising the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution at Ser30. 
     
     
         2 . A method for treating or inhibiting a thrombin-mediated thrombosis or embolism associated with surgery, the method comprising administering to a patient in need thereof an antibody molecule that specifically binds to the exosite 1 epitope of thrombin and comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, and a VL domain comprising the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution at Ser30. 
     
     
         3 . A method for inhibiting blood coagulation of a blood sample in vitro or ex vivo, the method comprising contacting the blood sample with an antibody molecule that specifically binds to the exosite 1 epitope of thrombin and comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, and a VL domain comprising the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution at Ser30. 
     
     
         4 . An isolated or recombinant nucleic acid molecule encoding an antibody molecule that specifically binds to the exosite 1 epitope of thrombin, the antibody comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, and a VL domain comprising the amino acid sequence of SEQ ID NO: 6 with an amino acid substitution at Ser30. 
     
     
         5 . The nucleic acid molecule according to  claim 4 , wherein the antibody molecule comprises an IgG constant region. 
     
     
         6 . The nucleic acid molecule according to  claim 5 , wherein the IgG constant region is an IgG1 or IgG4 constant region. 
     
     
         7 . The nucleic acid molecule according to  claim 4 , wherein the antibody molecule is a monoclonal antibody. 
     
     
         8 . A vector comprising the nucleic acid molecule of  claim 4 . 
     
     
         9 . A recombinant cell that expresses the nucleic acid molecule of  claim 4 . 
     
     
         10 . A method of producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin, comprising culturing the recombinant cell according to  claim 9  under conditions such that the antibody molecule is expressed. 
     
     
         11 . The method according to  claim 10 , further comprising recovering the antibody molecule. 
     
     
         12 . A method of producing an antibody antigen-binding domain for the exosite 1 epitope of thrombin, the method comprising:
 (i) providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein the parent VH domain HCDR1, HCDR2, and HCDR3 have the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, a VH domain which is an amino acid sequence of the parent VH domain,   (ii) optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and   (iii) testing the 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.   
     
     
         13 . A method for producing an antibody molecule that specifically binds to the exosite 1 epitope of thrombin, the method comprising:
 (i) providing a starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise an HCDR1, HCDR2, and/or HCDR3 to be replaced or lack an HCDR1, HCDR2, and/or HCDR3 encoding region;   (ii) combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1, HCDR2, and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, such that the 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;   (iii) expressing the nucleic acids of the product repertoire to produce product VH domains;   (iv) optionally combining the product VH domains with one or more VL domains;   (v) selecting an antibody molecule that binds the exosite 1 epitope of thrombin, which antibody molecule comprises a product VH domain and optionally a VL domain; and   (vi) recovering the antibody molecule or nucleic acid encoding it.