Tissue factor protein variants

Abstract

The invention provides amino acid sequence variants of tissue factor protein. The tissue factor protein variants have a greater affinity for Factor VII/VIIa than wild-type counterparts. The invention also provides pharmaceutical compositions comprising the novel compositions as well as their use in diagnostic, therapeutic, and prophylactic methods.

What is claimed is:  
     
         1 . A tissue factor protein variant having an amino acid sequence derived from a mammalian tissue factor protein wherein at least one amino acid residue corresponding to a human amino acid residue selected from the group consisting of Asp54, Glu56, Glu130, Arg131, Leu133, Arg135 and Phe140 is substituted with another amino acid, the tissue factor protein variant having a greater affinity for FVII/FVIIa than the mammalian tissue factor protein from which it is derived.  
     
     
         2 . The tissue factor protein variant of  claim 1  wherein at least one amino acid residue selected from the group consisting of Asp54 and Glu56, and at least one amino acid selected from the group consisting of Glu130, Arg131, Leu133, Arg135 and Phe140 is substituted with another amino acid.  
     
     
         3 . The tissue factor protein variant of  claim 2  wherein the other amino acid residue for Asp54 is selected from the group consisting of Lys, Asn, Glu, Ala and Ser the other amino acid residue for Glu56 is selected from the group consisting of Asp, His, Gln and Trp, the other amino acid residue for Glu130 is selected from the group consisting of Asp, Ala, Ser and Gly, the other amino acid residue for Arg131 is selected from the group consisting of Gln, Ile, Pro, Ser, Leu, Lys, Thr and Met, the other amino acid residue for Leu133 is Ala, the other amino acid residue for Arg135 is selected from the group consisting of Trp, Gin, Leu, Tyr, Thr, and Ala and the other amino acid residue for Phe140 is selected from the group consisting of Asn, His, Val, Ala, Arg and Gly.  
     
     
         4 . The tissue factor protein variant of  claim 3  wherein the other amino acid residue for Asp 54 is Ser, the other amino acid for Glu 130 is selected from the group consisting of Asp, Gly and Ala, the other amino acid residue for Arg131 is Gln, the other amino acid residue for Arg135 is selected from the group consisting of Trp and Gln and the other amino acid for Phe140 is Asn.  
     
     
         5 . The tissue factor protein variant of  claim 4  wherein amino acid residues Asp54, Glu130, Arg131, Leu133, and Phe140 are substituted.  
     
     
         6 . The tissue factor protein variant of  claim 5  wherein the other amino acid residue for Glu130 is Asp.  
     
     
         7 . The tissue factor protein variant of  claim 1  further having at least one amino acid residue selected from the group consisting of Lys15 and Tyr185 subsituted with another amino acid residue.  
     
     
         8 . The tissue factor protein variant of  claim 7  having an amino acid substitution at Lys15 and Tyr185.  
     
     
         9 . The tissue factor protein variant of  claim 8  wherein the other amino acid residue for Lys15 and Tyr185 is Ala.  
     
     
         10 . The tissue factor protein variant of  claim 1  wherein the mammalian tissue factor protein is a human tissue factor protein.  
     
     
         11 . The tissue factor protein variant of  claim 10  wherein the human tissue factor protein is SEQ ID NO: 1.  
     
     
         12 . The tissue factor protein variant of  claim 10  wherein the tissue factor protein variant has a substitution of at least one other amino acid residue in the amino acid sequence of the human tissue factor protein.  
     
     
         13 . The tissue factor protein variant of  claim 12  wherein the tissue factor protein variant has a further amino acid substitution at an amino acid residue which contributes energetically to FVTI/FVIIa binding or which contributes to FVII/FVIIa cofactor activity.  
     
     
         14 . The tissue factor protein variant of  claim 13  wherein the amino acid residue required for FVII/FVIIa cofactor activity is selected from the group consisting of Asp44, Trp158, Ser163, Gly164, Lys165 and Lys166.  
     
     
         15 . The tissue factor protein variant of  claim 14  wherein the amino acid residue required for FVIIa cofactor activity is selected from the group consisting of Ser163 and Gly164.  
     
     
         16 . The tissue factor protein variant of  claim 15  wherein Ser163 and Gly164 are subsituted with Ala.  
     
     
         17 . The mutant tissue factor protein of  claim 14  wherein the human tissue factor protein is SEQ ID. NO: 2.  
     
     
         18 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the tissue factor protein variant of  claim 1 .  
     
     
         19 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the tissue factor protein variant of  claim 12 .  
     
     
         20 . A method for inhibiting human Factor VII/VIIa (FVII/FVIIa) procoagulant activity comprising contacting said FVII/FVIIa with the tissue factor protein variant of  claim 12  in an amount sufficient to inhibit the procoagulant activity of said FVII/FVIIa.  
     
     
         21 . A method for inhibiting human tissue factor-Factor VIIa (TF-FVIIa) procoagulant activity in a mammal comprising administering a therapeutically effective amount of the composition of  claim 19  to the mammal.  
     
     
         22 . The method of  claim 19  further comprising administrating the composition in combination with a thrombolytic agent.  
     
     
         23 . The method of  claim 19  further comprising administrating the composition in combination with an anticoagulant.  
     
     
         24 . A method of treating a mammal for which inhibiting Factor VIIa is indicated comprising administering a therapeutically effective amount of the composition of  claim 19  to the mammal.  
     
     
         25 . An isolated DNA molecule encoding the tissue factor protein variant of  claim 1 .  
     
     
         26 . The DNA molecule of  claim 25  further comprising an expression control sequence operably linked to the DNA molecule.  
     
     
         27 . An expression vector comprising the DNA molecule of  claim 26  wherein the control sequence is recognized by a host cell transformed with the vector.  
     
     
         28 . A host cell transformed with the vector of  claim 27 .  
     
     
         29 . A method for expressing the DNA molecule encoding a tissue factor protein variant comprising culturing the host cell of  claim 28  under conditions suitable for expression of the tissue factor protein variant.  
     
     
         30 . The method of  claim 29  further comprising recovering the tissue factor protein variant from the culture medium.