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Human coagulation factor vii polypeptidesRelated Patent Categories: Multicellular Living Organisms And Unmodified Parts Thereof And Related Processes, Method Of Using A Transgenic Nonhuman Animal To Manufacture A Protein Which Is Then To Be Isolated Or Extracted, The Protein Is Isolated Or Extracted From MilkHuman coagulation factor vii polypeptides description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080010693, Human coagulation factor vii polypeptides. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of Ser. No. 10/669,537 filed Sep. 24, 2003 and claims benefit of priority under 35 U.S.C. 119 of Danish application no. PA 2002 01423 filed Sep. 25, 2002 and U.S. provisional application No. 60/417,927 filed Oct. 11, 2002, the contents of which are fully incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to novel human coagulation Factor VIIa polypeptides having coagulant activity as well as polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment. BACKGROUND OF THE INVENTION [0003] Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually gives raise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation "cascade", are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself is an activated clotting factor). Coagulation factors that have undergone such a conversion are generally referred to as "active factors", and are designated by the addition of the letter "a" to the name of the coagulation factor (e.g. Factor VIIa). [0004] Initiation of the haemostatic process is mediated by the formation of a complex between tissue factor, exposed as a result of injury to the vessel wall, and Factor VIIa. This complex then converts Factors IX and X to their active forms. Factor Xa converts limited amounts of prothrombin to thrombin on the tissue factor-bearing cell. Thrombin activates platelets and Factors V and VII into Factors Va and VIIa, both cofactors in the further process leading to the full thrombin burst. This process includes generation of Factor Xa by Factor IXa (in complex with factor VIIIa) and occurs on the surface of activated platelets. Thrombin finally converts fibrinogen to fibrin resulting in formation of a fibrin clot. In recent years Factor VII and tissue factor have been found to be the main initiators of blood coagulation. [0005] Factor VII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive. Single-chain Factor VII may be converted to two-chain Factor VIIa by Factor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro. Factor Xa is believed to be the major physiological activator of Factor VII. Like several other plasma proteins involved in haemostasis, Factor VII is dependent on Vitamin K for its activity, which is required for the gamma-carboxylation of multiple glutamic acid residues that are clustered close to the amino terminus of the protein. These gamma-carboxylated glutamic acids are required for the metal ion-induced interaction of Factor VII with phospholipids. The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal Arg.sub.152-Ile.sub.153 peptide bond. In the presence of tissue factor, phospholipids and calcium ions, the two-chain Factor VIIa rapidly activates Factor X or Factor IX by limited proteolysis. [0006] It is often desirable to stimulate or improve the coagulation cascade in a subject. Factor VIIa has been used to control bleeding disorders that have several causes such as clotting factor deficiencies (e.g. haemophilia A and B or deficiency of coagulation Factors XI or VII) or clotting factor inhibitors. Factor VIIa has also been used to control excessive bleeding occurring in subjects with a normally functioning blood clotting cascade (no clotting factor deficiencies or inhibitors against any of the coagulation factors). Such bleeding may, for example, be caused by a defective platelet function, thrombocytopenia or von Willebrand's disease. Bleeding is also a major problem in connection with surgery and other forms of tissue damage. [0007] European Patent No. 200,421 (ZymoGenetics) relates to the nucleotide sequence encoding human Factor VII and the recombinant expression of Factor VII in mammalian cells. [0008] Dickinson et al. (Proc. Natl. Acad. Sci. USA (1996) 93, 14379-14384) relates to a Factor VII variant wherein Leu305 has been replaced by Ala (FVII(Ala305)). [0009] Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466, abstract 1474) relates to Factor VIIa variants wherein residues 316-320 are deleted or residues 311-322 are replaced with the corresponding residues from trypsin. [0010] International patent applications WO 01/83725 and WO 02/22776 relates to variants of Factor VIIa with increased activity. [0011] There is a need for variants of Factor VIIa having coagulant activity, variants with high activity that can be administered at relatively low doses, and variants which do not produce the undesirable side effects such as systemic activation of the coagulation system and bleeding, respectively, associated with conventional therapies. SUMMARY OF THE INVENTION [0012] The present invention provides Factor VII polypeptides comprising at least two substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions comprise: (i) a first substitution consisting of replacement of F374 with any other amino acid, and (ii) one or more substitutions consisting of replacement with any other amino acid of one or more amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. In some embodiments, both F374 and K157 are replaced with any other amino acid. In some embodiments, both F374 and K337 are replaced with any other amino acid. In some embodiments, both F374 and D334 are replaced with any other amino acid. In some embodiments, both F374 and S336 are replaced with any other amino acid. In some embodiments, both F374 and V158 are replaced with any other amino acid. In some embodiments, both F374 and E296 are replaced with any other amino acid. In some embodiments, both F374 and M298 are replaced with any other amino. In some embodiments, both F374 and L305 are replaced with any other amino acid. In some embodiments, both F374 and S314 are replaced with any other amino acid. [0013] The invention also provides Factor VII polypeptides in which, in addition to the above two substitutions, also comprise least one additional substitution at an amino acid residue in the protease domain, in which the additional residue has been replaced with any other amino acid. In some embodiments, at most 20 additional amino acids in the remaining positions in the protease domain have been replaced with any other amino acid. In different embodiments, the additional substitution is at a residue corresponding to a position selected from 159-170; 290-304; 306-312; or 330-339 of SEQ ID NO:1. In some embodiments, R304 has been replaced by Tyr, Phe, Leu, or Met. In some embodiments, M306 has been replaced by Asp or Asn. In some embodiments, D309 has been replaced by Ser or Thr. In some embodiments, wherein A274 has been replaced with any other amino acid, such as, e.g., Met, Leu, Lys, or Arg. [0014] The invention also encompasses Factor VII polypeptides with two substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of one amino acid selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with three substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of two amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with four substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of three amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with five substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of four amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with six substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of five amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with seven substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of six amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with eight substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of seven amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with nine substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of eight amino acids selected from the group consisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298. The invention also encompasses Factor VII polypeptides with ten substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein said substitutions are (i) replacement of F374 with any other amino acid and (ii) replacement with any other amino acid of the amino acids L305, S314, K157, K337, D334, S336, V158, E296, and M298. [0015] In some embodiments of the invention, K.sub.157 has been replaced by Gly, Val, Ser, Thr, Asn, Gln, Asp, or Glu. In some embodiments, K.sub.337 has been replaced by Ala, Gly, Val, Ser, Thr, Asn, Gln, Asp, or Glu. In some embodiments, D334 has been replaced by Gly or Glu. In some embodiments, S336 has been replaced by Gly or Glu. In some embodiments, V158 has been replaced by Ser, Thr, Asn, Gln, Asp, or Glu. In some embodiments, E296 has been replaced by Arg, Lys, Ile, Leu, or Val. In some embodiments, M298 has been replaced by Lys, Arg, Gln, or Asn. In some embodiments, L305 has been replaced by Val, Tyr, or lie. In some embodiments, S314 has been replaced by Gly, Lys, Gln, or Glu. [0016] In some embodiments, F374 has been replaced by Pro or Tyr. [0017] In some embodiments, the Factor VII polypeptide is human Factor VII. [0018] In some embodiments, the ratio between the activity of the Factor VII polypeptide of the invention and the activity of the native Factor VIIa polypeptide shown in SEQ ID NO:1 is at least about 1.25. In other embodiments, this ratio is at least about 2.0, preferably at least about 4.0. [0019] In one embodiment, the Factor VII polypeptide F374Y/L305V-FVII. In another embodiment, the Factor VII polypeptide is F374Y/L305V/S314E/K337A-FVII. In another embodiment, the Factor VII polypeptide is F374Y/L305V/S314E-FVII. In another embodiment, the Factor VII polypeptide is F374Y/L305V/K337A-FVII. [0020] The invention also encompasses polynucleotide constructs encoding the Factor VII polypeptides of the invention, vectors containing such constructs, and host cells comprising such constructs or vectors (which may be, but are not limited to, eukaryotic cells). In some embodiments, the host cells are mammalian cells, including, e.g., CHO cells, HEK cells and BHK cells. Continue reading about Human coagulation factor vii polypeptides... Full patent description for Human coagulation factor vii polypeptides Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Human coagulation factor vii polypeptides patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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