Targeting tissue factor to activated platelets

Abstract

The current invention relates to procoagulant fusion proteins, polynucleotides that encode said fusion proteins and cells that expresses said fusion proteins. Furthermore, the current invention relates to fusion proteins for use as a medicament. Individuals that have a coagulopathy, such as haemophilia A and B with or without inhibitors, may be treated with fusions proteins of the current invention.

1 . A fusion protein comprising (i) at least one tissue factor polypeptide, or biologically functional variant(s) or fragment(s) thereof, which is/are covalently attached to (ii) a ligand that is capable of binding (iii) a receptor, and/or a fragment or variant thereof, wherein the receptor is only expressed on the surface of activated platelets. 
     
     
         2 . The fusion protein according to  claim 1 , wherein (iii) is TLT-1 or a fragment or variant thereof. 
     
     
         3 . The fusion protein according to  claim 1 , wherein (ii) is a monoclonal antibody or a fragment thereof. 
     
     
         4 . The fusion protein according to  claim 3 , wherein the epitope of (ii) comprises one or more residues selected from the group consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26, K27, A28, L63, G64, G65, G66, L67, L68, G89, A90, R91, G92, P93, Q94, I95 and L96 of SEQ ID NO: 5. 
     
     
         5 . The fusion protein according to  claim 3 , wherein the epitope of (ii) comprises one or more residues selected from the group consisting of L36, P37, E38, G39, C40, Q41, P42, L43, V44, S45, S46, A47, V73, T74, L75, Q76, E77, E78, D79, A80, G81, E82, Y83, G84, C85, M86, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S110 and L111 of SEQ ID NO: 5. 
     
     
         6 . The fusion protein according to  claim 3 , wherein the heavy chain of (ii) comprises:
 a CDR1 sequence of amino acids 50 to 54 (DYSMH) of SEQ ID NO: 43, wherein one of these amino acids may be substituted by a different amino acid; and/or   a CDR2 sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of SEQ ID NO: 43, wherein one, two, three or four of these amino acids may be substituted by a different amino acid; and/or   a CDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ ID NO: 43, wherein one, two or three of these amino acids may be substituted by a different amino acid.   
     
     
         7 . The fusion protein according to  claim 3 , wherein the epitope of (ii) comprises one or more residues selected from the group consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26, K27, A28, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 of SEQ ID NO: 5. 
     
     
         8 . The fusion protein according to  claim 3 , wherein the epitope of (ii) comprises one or more residues selected from the group consisting of K133, I134, G135, S136, L137, A138, N140, A141, F142, 5143, D144, P145 and A146 of SEQ ID NO: 4. 
     
     
         9 . The fusion protein according to  claim 3 , wherein the paratope of (ii) comprises one or more residues selected from the group consisting of H50, N52, Y56, H58, Y73, F79, S115, T116, V118 and Y120 of SEQ ID NO: 40 and residues V20, F45, R49, Y50, W51, E68, T75, N77, 5116, G117, V118 and T120 of SEQ ID NO: 39 
     
     
         10 . The fusion protein according to  claim 3 , wherein the heavy chain of (ii) comprises:
 a CDR1 sequence of amino acids 49 to 53 ( RYWMT ) of SEQ ID NO: 39, wherein one of these amino acids may be substituted by a different amino acid; and/or   a CDR2 sequence of amino acids 68 to 84 ( EINPDSSTINY T PSLKD ) of SEQ ID NO: 39, wherein one, two, three or four of these amino acids may be substituted by a different amino acid; and/or   a CDR3 sequence of amino acids 117 to 121 (G VFTS ) of SEQ ID NO: 39, wherein one, two or three of these amino acids may be substituted by a different amino acid.   
     
     
         11 . A fusion protein according to  claim 1  for use in the treatment of a coagulopathy. 
     
     
         12 . A monoclonal antibody, or fragment thereof, that is capable of binding TLT-1, or a fragment thereof, wherein the epitope of said monoclonal antibody comprises one or more residues selected from the group consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26, K27, A28, L63, G64, G65, G66, L67, L68, G89, A90, R91, G92, P93, Q94, I95 and L96 of SEQ ID NO: 5. 
     
     
         13 . A monoclonal antibody, or fragment thereof, that is capable of binding TLT-1, or a fragment thereof, wherein the epitope of said monoclonal antibody comprises one or more residues selected from the consisting of L36, P37, E38, G39, C40, Q41, P42, L43, V44, S45, S46, A47, V73, T74, L75, Q76, E77, E78, D79, A80, G81, E82, Y83, G84, C85, M86, R91, G92, P93, Q94, I95, L96, H97, R98, V99, 5110 and L111 of SEQ ID NO: 5. 
     
     
         14 . A monoclonal antibody, or fragment thereof, that is capable of binding TLT-1, or a fragment thereof, wherein the epitope of said monoclonal antibody comprises one or more residues selected from the group consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26, K27, A28, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 of SEQ ID NO: 5. 
     
     
         15 . A monoclonal antibody, or fragment thereof, that is capable of binding TLT-1, or a fragment thereof, wherein the epitope of said antibody comprises one or more residues selected from the group consisting of K133, I134, G135, 5136, L137, A138, N140, A141, F142, S143, D144, P145 and A146 of SEQ ID NO: 4. 
     
     
         16 . The fusion protein according to  claim 5 , wherein the heavy chain of (ii) comprises:
 a CDR1 sequence of amino acids 50 to 54 (DYSMH) of SEQ ID NO: 43, wherein one of these amino acids may be substituted by a different amino acid; and/or   a CDR2 sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of SEQ ID NO: 43, wherein one, two, three or four of these amino acids may be substituted by a different amino acid; and/or   a CDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ ID NO: 43, wherein one, two or three of these amino acids may be substituted by a different amino acid.   
     
     
         17 . The fusion protein according to  claim 8 , wherein the paratope of (ii) comprises one or more residues selected from the group consisting of H50, N52, Y56, H58, Y73, F79, S115, T116, V118 and Y120 of SEQ ID NO: 40 and residues V20, F45, R49, Y50, W51, E68, T75, N77, S116, G117, V118 and T120 of SEQ ID NO: 39. 
     
     
         18 . The fusion protein according to  claim 8 , wherein the heavy chain of (ii) comprises:
 a CDR1 sequence of amino acids 49 to 53 ( RYWMT ) of SEQ ID NO: 39, wherein one of these amino acids may be substituted by a different amino acid; and/or   a CDR2 sequence of amino acids 68 to 84 ( EINPDSSTINY T PSLKD ) of SEQ ID NO: 39, wherein one, two, three or four of these amino acids may be substituted by a different amino acid; and/or   a CDR3 sequence of amino acids 117 to 121 (G VFTS ) of SEQ ID NO: 39, wherein one, two or three of these amino acids may be substituted by a different amino acid.   
     
     
         19 . The fusion protein according to  claim 9 , wherein the heavy chain of (ii) comprises:
 a CDR1 sequence of amino acids 49 to 53 ( RYWMT ) of SEQ ID NO: 39, wherein one of these amino acids may be substituted by a different amino acid; and/or   a CDR2 sequence of amino acids 68 to 84 ( EINPDSSTINY T PSLKD ) of SEQ ID NO: 39, wherein one, two, three or four of these amino acids may be substituted by a different amino acid; and/or   a CDR3 sequence of amino acids 117 to 121 (G VFTS ) of SEQ ID NO: 39, wherein one, two or three of these amino acids may be substituted by a different amino acid.