Coagulation Factor-Targeting to TLT-1 on Activated Platelets

Abstract

The current invention relates to: procoagulant proteins which may, for example, be fusion proteins or chemical conjugates; methods of producing said procoagulant proteins; polynucleotides that encode said fusion proteins and cells that expresses them. Furthermore, the current invention relates to procoagulant 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 the procoagulant proteins of the current invention.

1 .- 17 . (canceled) 
     
     
         18 . A procoagulant protein comprising (i) a monoclonal antibody or fragment thereof that is capable of binding to TREM-like transcript 1 (TLT-1) and (ii) a human wild-type Factor VIIa comprising the amino acid sequence of SEQ ID NO:157 or a variant thereof, wherein the variant is at least 99% identical to SEQ ID NO:157. 
     
     
         19 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is conjugated to the human wild-type Factor VIIa or variant thereof via glycosylation. 
     
     
         20 . The procoagulant protein according to  claim 18 , wherein the procoagulant protein is a fusion protein between the monoclonal antibody or fragment thereof and the human wild-type Factor VIIa or variant thereof. 
     
     
         21 . The procoagulant protein according to  claim 18 , wherein the fragment is selected from the group consisting of a Fab fragment, a F(ab′) 2  fragment, a Fab′ fragment, a Fd fragment, a Fv fragment, a ScFv fragment, a dAb fragment, and an isolated complimentarity determining region. 
     
     
         22 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody is a human monoclonal antibody. 
     
     
         23 . The procoagulant protein according to  claim 21 , wherein the Fab fragment is humanized. 
     
     
         24 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:5. 
     
     
         25 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:6. 
     
     
         26 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:7. 
     
     
         27 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:8. 
     
     
         28 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to an epitope on TLT-1 comprising residues 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. 
     
     
         29 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to an epitope on TLT-1 comprising residues 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, S100, and L101 of SEQ ID NO: 5. 
     
     
         30 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to an epitope on TLT-1 comprising residues 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. 
     
     
         31 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to an epitope on the stalk of TLT-1 comprising residues E5, T6, H7, K8, 19, G10, S11, L12, A13, E14, N15, A16, F17, S18, D19, and P20 of SEQ ID NO: 7. 
     
     
         32 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof is capable of binding to an epitope on the stalk of TLT-1 comprising residues K8, 19, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, and A21 of SEQ ID NO: 7. 
     
     
         33 . The procoagulant protein according to  claim 18 , wherein the monoclonal antibody or fragment thereof competes for binding to TLT-1 with a monoclonal antibody comprising SEQ ID NO:32 and SEQ ID NO:33. 
     
     
         34 . A procoagulant protein comprising (i) a humanized Fab fragment that is capable of binding to TLT-1 and (ii) a human wild-type Factor VIIa comprising the amino acid sequence of SEQ ID NO:157 or a variant thereof, wherein the humanized Fab fragment is conjugated to the human wild-type Factor VIIa or variant thereof via glycosylation, and wherein the variant is at least 99% identical to SEQ ID NO:157. 
     
     
         35 . The procoagulant protein according to  claim 34 , wherein the procoagulant protein comprises (i) a humanized Fab fragment that is capable of binding to TLT-1 and (ii) a human wild-type Factor VIIa comprising the amino acid sequence of SEQ ID NO:157. 
     
     
         36 . The procoagulant protein according to  claim 34 , wherein the humanized Fab fragment is capable of binding to a portion of TLT-1 selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. 
     
     
         37 . The procoagulant protein according to  claim 36 , wherein the humanized Fab fragment is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:7. 
     
     
         38 . The procoagulant protein according to  claim 36 , wherein the humanized Fab fragment is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:8. 
     
     
         39 . The procoagulant protein according to  claim 34 , wherein the humanized Fab fragment is capable of binding to an epitope on TLT-1 comprising residues 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. 
     
     
         40 . The procoagulant protein according to  claim 34 , wherein the humanized Fab fragment is capable of binding to an epitope on TLT-1 comprising residues 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, S100, and L101 of SEQ ID NO: 5. 
     
     
         41 . The procoagulant protein according to  claim 34 , wherein the humanized Fab fragment is capable of binding to an epitope on TLT-1 comprising residues 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. 
     
     
         42 . The procoagulant protein according to  claim 34 , wherein the humanized Fab fragment is capable of binding to an epitope on the stalk of TLT-1 comprising residues E5, T6, H7, K8, 19, G10, S11, L12, A13, E14, N15, A16, F17, S18, D19, and P20 of SEQ ID NO: 7. 
     
     
         43 . The procoagulant protein according to  claim 34 , wherein the humanized Fab fragment is capable of binding to an epitope on the stalk of TLT-1 comprising residues K8, 19, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, and A21 of SEQ ID NO: 7. 
     
     
         44 . A method for treating haemophilia A, comprising administering to a subject in need thereof an effective amount of a procoagulant protein comprising (i) a monoclonal antibody or fragment thereof that is capable of binding to TREM-like transcript 1 (TLT-1) and (ii) a human wild-type Factor VIIa comprising the amino acid sequence of SEQ ID NO:157 or a variant thereof, wherein the variant is at least 99% identical to SEQ ID NO:157. 
     
     
         45 . The method according to  claim 44 , wherein the monoclonal antibody or fragment thereof is conjugated to the human wild-type Factor VIIa or variant thereof via glycosylation. 
     
     
         46 . The method according to  claim 44 , wherein the procoagulant protein comprises a fusion protein between the monoclonal antibody or fragment thereof and the human wild-type Factor VIIa or variant thereof. 
     
     
         47 . The method according to  claim 44 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:5. 
     
     
         48 . The method according to  claim 44 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:6. 
     
     
         49 . The method according to  claim 44 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:7. 
     
     
         50 . The method according to  claim 44 , wherein the monoclonal antibody or fragment thereof is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:8. 
     
     
         51 . The method according to  claim 44 , wherein the monoclonal antibody is a human monoclonal antibody 
     
     
         52 . The method according to  claim 44 , wherein the fragment is a humanized Fab fragment. 
     
     
         53 . A method for treating haemophilia A, comprising administering to a subject in need thereof an effective amount of a procoagulant protein comprising (i) a humanized Fab fragment that is capable of binding to TREM-like transcript 1 (TLT-1) and (ii) a human wild-type Factor VIIa comprising the amino acid sequence of SEQ ID NO:157 or a variant thereof, wherein the humanized Fab fragment is conjugated to the human wild-type Factor VIIa via glycosylation, and wherein the variant is at least 99% identical to SEQ ID NO:157. 
     
     
         54 . The method according to  claim 53 , wherein the procoagulant protein comprises (i) a humanized Fab fragment that is capable of binding to TLT-1 and (ii) a human wild-type Factor VIIa comprising the amino acid sequence of SEQ ID NO:157. 
     
     
         55 . The method according to  claim 53 , wherein the humanized Fab fragment is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:7. 
     
     
         56 . The method according to  claim 53 , wherein the humanized Fab fragment is capable of binding to a portion of TLT-1 consisting of SEQ ID NO:8. 
     
     
         57 . The method according to  claim 53 , wherein the humanized Fab fragment is capable of binding to an epitope on the stalk of TLT-1 comprising residues E5, T6, H7, K8, 19, G10, S11, L12, A13, E14, N15, A16, F17, S18, D19, and P20 of SEQ ID NO: 7. 
     
     
         58 . The method according to  claim 53 , wherein the humanized Fab fragment is capable of binding to an epitope on the stalk of TLT-1 comprising residues K8, I9, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, and A21 of SEQ ID NO: 7.