Tissue factor compositions and methods

Abstract

Tissue Factor (natural or recombinant truncated) can be incorporated into stable, soluble nanoscale particles so that activity is maintained. These particles can be used as a reagent in prothrombin clotting time assays or they can be used in therapeutic compositions for use in humans or animals. Therapeutic settings can include supplementation in the case of a genetic deficiency, uncontrolled bleeding, surgical incisions or seepage, thrombocytopenia, soft tissue trauma or other trauma, to effect tumor regression or to inhibit tumor growth.

1 . A method of inhibiting tumor growth in a human or animal patient, said method comprising the step of administering a composition comprising nanoscale particles comprising tissue factor or recombinant tissue factor, a membrane scaffold protein and a phospholipid, wherein said phospholipid comprises a net negatively charged phospholipid, in an amount effective to cause clotting in a tumor, whereby tumor growth is inhibited. 
     
     
         2 . The method of  claim 1 , wherein the net negatively charged phospholipid is from 1 to 50% of the phospholipid on a mole percent basis. 
     
     
         3 . The method of  claim 2 , wherein the nanoscale particles are embedded in a slow release material. 
     
     
         4 . The method of  claim 2 , wherein the nanoscale particles comprise at least one targeting molecule specific for the tumor. 
     
     
         5 . The method of  claim 3 , wherein the targeting molecule is a lectin, single chain antibody, or an antigen-binding fragment of an antibody. 
     
     
         6 . The method of  claim 1 , wherein the tissue factor is a recombinant tissue factor consisting of amino acids 23 to 277 of SEQ ID NO:95. 
     
     
         7 . A composition comprising nanoscale particles comprising tissue factor, a membrane scaffold protein and at least one net negatively charged phospholipid, wherein said particles are bound to a solid support. 
     
     
         8 . The composition of  claim 7 , wherein said particles are noncovalently bound to the solid support. 
     
     
         9 . The composition of  claim 7 , wherein said particles are covalently bound to the solid support. 
     
     
         10 . The composition of  claim 7 , wherein the solid support is a silicon chip, a cellulosic material, a polymeric material, a collagen-containing material, a chitin-derived material, a chitin-containing material, a chitosan-containing material, or material containing a chitosan derivative. 
     
     
         11 . The composition of  claim 7 , wherein said tissue factor is a recombinant tissue factor consisting essentially of amino acids 23 to 277 of SEQ ID NO:95. 
     
     
         12 . The composition of  claim 7 , wherein the at least one phospholipid is phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, dipalmitoyl-phosphatidylcholine, dimyristoyl phosphatidyl choline, 1-palmitoyl-2-oleoyl-phosphatidyl choline, 1-palmitoyl-2-oleoyl-phosphatidyl serine, 1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine, dihexanoyl phosphatidyl choline, dipalmitoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl inositol, dimyristoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl inositol, dihexanoyl phosphatidyl ethanolamine, dihexanoyl phosphatidyl inositol, 1-palmitoyl-2-oleoyl-phosphatidyl ethanolamine or 1-palmitoyl-2-oleoyl-phosphatidyl inositol. 
     
     
         13 . The composition of  claim 12 , wherein the phospholipid comprises a net negatively charged phospholipid in a mol percent of from 1 to 50% and a net-neutral phospholipid in a mol percent from 50 to 99%. 
     
     
         14 . The composition of  claim 13 , wherein the net negatively charged phospholipid is phosphatidylserine and the net neutral phospholipid is phosphatidylcholine. 
     
     
         15 . The composition of  claim 14 , wherein said phospholipid comprises phosphatidylserine and phosphatidylcholine, and wherein the mol percent of phosphatidylserine is from 5 to 40%. 
     
     
         16 . The composition of  claim 15 , wherein said phospholipid comprises phosphatidylserine and phosphatidylcholine, and wherein the mol percent of phosphatidylserine is from 10 to 30%. 
     
     
         17 . The composition of  claim 16 , wherein said phospholipid comprises phosphatidylserine and phosphatidylcholine, and wherein the mol percent of phosphatidylserine is 20%. 
     
     
         18 . The composition of  claim 17 , wherein the mol percent of phosphatidylcholine is 80%. 
     
     
         19 . The composition of  claim 7 , wherein the mol percent of phosphatidylcholine is 40% and the phospholipid further comprises 40 mol % phosphatidylethanolamine. 
     
     
         20 . The composition of  claim 11 , wherein said membrane scaffold protein comprises an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, amino acids 13-414 of SEQ ID NO:8, SEQ ID NO:10, amino acids 13-422 of SEQ ID NO:10, SEQ ID NO:12, amino acids 13-168 of SEQ ID NO:12, SEQ ID NO:14, amino acids 13-168 of SEQ ID NO:14, SEQ ID NO:16, amino acids 13-201 of SEQ ID NO:16, SEQ ID NO:17, amino acids 13-201 of SEQ ID NO:17, SEQ ID NO:18, amino acids 13-392 of SEQ ID NO:18, SEQ ID NO:50, amino acids 13-234 of SEQ ID NO:50, SEQ ID NO:51, amino acids 13-256 of SEQ ID NO:51, SEQ ID NO:52, amino acids 13-278 of SEQ ID NO:52, SEQ ID NO:53, amino acids 24-223 of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, amino acids 24-212 of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, amino acids 24-201 of SEQ ID NO:57, SEQ ID NO:58, amino acids 13-190 of SEQ ID NO:58, SEQ ID NO:59, amino acids 13-201 of SEQ ID NO:59, SEQ ID NO:60, amino acids 13-190 of SEQ ID NO:60, SEQ ID NO:61, amino acids 24-201 of SEQ ID NO:61, SEQ ID NO:62, amino acids 24-190 of SEQ ID NO:62, SEQ ID NO:63, amino acids 24-179 of SEQ ID NO:63, SEQ ID NO:64, amino acids 24-289 of SEQ ID NO:64, SEQ ID NO:65, amino acids 24-289 of SEQ ID NO:64, SEQ ID NO:65, amino acids 24-278 of SEQ ID NO:65, SEQ ID NO:66, amino acids 24-423 of SEQ ID NO:66, SEQ ID NO:67, amino acids 24-199 of SEQ ID NO:67, SEQ ID NO:68, amino acids 24-401 of SEQ ID NO:68, SEQ ID NO:69, amino acids 24-392 of SEQ ID NO:69, SEQ ID NO:81, amino acids 24-397 of SEQ ID NO:81, SEQ ID NO:83, amino acids 24-383 of SEQ ID NO:83, SEQ ID NO:85, amino acids 24-379 of SEQ ID NO:85, SEQ ID NO:87, amino acids 24-381 of SEQ ID NO:87, SEQ ID NO:89, amino acids 25-212 of SEQ ID NO:89, SEQ ID NO:91, amino acids 25-212 of SEQ ID NO:91, SEQ ID NO:93 and amino acids 13-212 of SEQ ID 93. 
     
     
         21 . The composition of  claim 11 , further comprising a targeting agent which specifically binds to a surface of a cell to which the tissue factor is delivered. 
     
     
         22 . The composition of  claim 21 , wherein said targeting agent is a lectin, single chain antibody or an antigen-binding fragment of an antibody. 
     
     
         23 . The composition of  claim 21 , wherein the targeting agent is covalently linked to said membrane scaffold protein. 
     
     
         24 . A nanoscale particle comprising at least one net-negatively charged phospholipid, a membrane scaffold protein and tissue factor. 
     
     
         25 . The nanoscale particle of  claim 24 , wherein a targeting agent is noncovalently bound to said particle. 
     
     
         26 . The nanoscale particle of  claim 24 , wherein said particle is covalently bound to a solid support. 
     
     
         27 . The nanoscale particle of  claim 24 , wherein said solid support is a collagen sponge, a cellulosic material, a chitin-derived material chitin-containing material, a chitosan-containing material, material containing a chitosan derivative or a cellulose-containing material. 
     
     
         28 . A kit for performing a prothrombin clotting time assay, said kit comprising the composition of  claim 11 . 
     
     
         29 . A method for performing a prothrombin clotting time assay in blood or plasma sample, said method comprises the step of adding the composition of  claim 11  to a blood or plasma sample.