US2021230570A1PendingUtilityA1

Modified factor ix polypeptides and uses thereof

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Assignee: CATALYST BIOSCIENCES INCPriority: Nov 3, 2010Filed: Mar 16, 2021Published: Jul 29, 2021
Est. expiryNov 3, 2030(~4.3 yrs left)· nominal 20-yr term from priority
Inventors:Edwin L. MadisonChristopher D. ThanosGrant E. Blouse
A61K 38/4846C12Y 304/21022A61P 7/04C12N 15/01C12N 9/644A61K 38/36A61K 48/00C07K 14/745
70
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Claims

Abstract

Modified Factor IX (FIX) polypeptides and uses thereof are provided. Nucleic acid molecules encoding modified factor IX (FIX) polypeptides and uses thereof also are provided. Such modified FIX polypeptides include FIXa and other forms of FIX. Among the modified FIX polypeptides provided are those that have altered activities, typically altered procoagulant activity, including increased procoagulant activities. Hence, such modified polypeptides are therapeutics.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A modified Factor IX (FIX) polypeptide, comprising one or more amino acid modification(s) in an unmodified FIX polypeptide, wherein:
 an amino acid modification corresponds to R318Y, R318E, R318F, or R318W, with reference to the sequence of amino acids set forth in SEQ ID NO:3;   corresponding amino acid residues are identified by alignment of the unmodified FIX polypeptide with the FIX polypeptide of SEQ ID NO:3;   the unmodified FIX polypeptide comprises a sequence of amino acids set forth in any of SEQ ID NOs: 2, 3, 20, or 325, or a sequence of amino acids having at least 95% sequence identity to the FIX polypeptide sequence set forth in any of SEQ ID NOs: 2, 3, 20, or 325, or a catalytically active fragment thereof; and   modifications are amino acid replacements, additions, or deletions, or any combination thereof.   
     
     
         2 . The modified FIX polypeptide of  claim 1 , that, when in active form (FIXa), exhibits increased resistance to antithrombin III and/or heparin, and/or one or both of increased catalytic activity and increased procoagulant activity, compared to the active form of the unmodified FIX polypeptide that does not contain the modification(s). 
     
     
         3 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide comprises the sequence of amino acids set forth in any of SEQ ID NOs: 2, 3, 20, or 325, or is unmodified activated FIX (FIXa), which consists of a light chain that consists of residues 1-14S, and a heavy chain that consists of residues 181-41S, of SEQ ID NO:3. 
     
     
         4 . The modified FIX polypeptide of  claim 1 , comprising the amino acid replacement corresponding to R318Y. 
     
     
         5 . The modified FIX polypeptide of  claim 1 , further comprising the amino acid replacements corresponding to replacements T343R, T343E or T343D. 
     
     
         6 . The modified FIX polypeptide of  claim 1 , further comprising an amino acid replacement at a residue corresponding to R338. 
     
     
         7 . The modified FIX polypeptide of  claim 6 , wherein the replacement at the residue corresponding to R338 corresponds to an amino acid replacement R338D or R338E. 
     
     
         8 . The modified FIX polypeptide of  claim 7  that comprises the amino acid replacement R338E. 
     
     
         9 . The modified FIX polypeptide of  claim 6 , wherein the replacement at the residue corresponding to R338 is R338L. 
     
     
         10 . The modified FIX polypeptide of  claim 1 , comprising the amino acid replacements corresponding to R318Y/R338E. 
     
     
         11 . The modified FIX polypeptide of  claim 7 , further comprising an amino acid replacement corresponding to T343R. 
     
     
         12 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:3 or SEQ ID NO:20. 
     
     
         13 . The modified FIX polypeptide of  claim 1 , comprising amino acid replacements corresponding to R318Y/T343R. 
     
     
         14 . The modified FIX polypeptide of  claim 1 , further comprising an amino acid replacement at the residue corresponding to E410, wherein the replacement is N or S. 
     
     
         15 . The modified FIX polypeptide of  claim 14  that comprises amino acid replacements corresponding to R318Y/E410N. 
     
     
         16 . The modified FIX polypeptide of  claim 1  that comprises an amino acid replacement corresponding to R318Y and one or more amino acid replacements at an amino acid residue corresponding to a residue selected from among 338, 343, 403, and 410. 
     
     
         17 . The modified FIX polypeptide of  claim 16 , comprising an amino acid replacement corresponding to R318Y, and one or more replacements selected from among R338E, T343R, R403E, and E410N. 
     
     
         18 . The modified FIX polypeptide of  claim 1  that comprises amino acid replacements corresponding to replacements selected from among R318Y/R338E/R403E/E410N, Y155F/K247N/N249S/R318Y/R338E, R318Y/T343R/E410N, Y155F/R318Y/R338E/R403E, R318Y/R338E/R403E, R318Y/R338E/E410N, K228N/R318Y/E410N, R318Y/R403E/E410N, D203N/F205T/R318Y/E410N, A103N/N105S/R318Y/R338E/R403E/E410N, D104N/K106S/R318Y/R338E/R403E/E410N, K228N/R318Y/R338E/R403E/E410N, I251S/R318Y/R338E/R403E/E410N, D104N/K106S/I251S/R318Y/R338E/R403E/E410N, D104N/K106S/R318Y/R338E/E410N, I251S/R318Y/E410N/R338E, D104N/K106S/I251S/R318Y/R338E/E410N, A103N/N105S/K247N/N249S/R318Y/R338E/R403E/E410N, D104N/K106S/K247N/N249S/R318Y/R338E/R403E/E410N, K228N/K247N/N249S/R318Y/R338E/R403E/E410N, A103N/N105S/Y155F/R318Y/R338E/R403E/E410N, D104N/K106S/Y155F/R318Y/R338E/R403E/E410N, Y155F/K228N/R318Y/R338E/R403E/E410N, Y155F/I251S/R318Y/R338E/R403E/E410N, K247N/N249S/R318Y/R338E/R403E/E410N, Y155F/R318Y/R338E/R403E/E410N, K247N/N249S/R318Y/R338E/E410N, Y155F/R318Y/R338E/E410N, Y155F/K247N/N249S/R318Y/R338E/E410N, R318Y/R338E/R403E/E410S, R318Y/R338E/R403E/E410N/T412V, R318Y/R338E/R403E/E410N/T412A, R318Y/R338E/R403E/T412A, R318Y/R338E/E410S, R318Y/R338E/T412A, R318Y/R338E/E410N/T412V, D85N/K228N/R318Y/R338E/R403E/E410N, N260S/R318Y/R338E/R403E/E410N, R318Y/R338E/N346D/R403E/E410N, Y155F/R318Y/R338E/N346D/R403E/E410N, K247N/N249S/N260S/R318Y/R338E/R403E/E410N, D104N/K106S/N260S/R318Y/R338E/R403E/E410N, Y155F/N260S/R318Y/R338E/R403E/E410N, D104N/K106S/Y155F/K247N/N249S/R318Y/R338E/R403E/E410N, D104N/K106S/K228N/K247N/N249S/R318Y/R338E/R403E/E410N, Y155F/K228N/K247N/N249S/R318Y/R338E/R403E/E410N, Y155F/K247N/N249S/N260S/R318Y/R338E/R403E/E410N, R318Y/R338E/Y345A/R403E/E410N, K228N/I251S/R318Y/R338E/R403E/E410N, R318Y/R338E/Y345A/N346D/R403E/E410N, Y155F/K247N/N249S/R318Y/R338E/R403E, K247N/N249S/R318Y/R338E/R403E, Y155F/K247N/N249S/R318Y/R403E/E410N, K247N/N249S/R318Y/R403E/E410N, R318Y/T343R/R403E/E410N, Y155F/K228N/I251S/R318Y/R338E/R403E/E410N, Y155F/K247N/N249S/R318Y/R403E, Y155F/K247N/N249S/R318Y/E410N, Y155F/K247N/N249S/R318Y/T343R/R403E/E410N, K247N/N249S/R318Y/T343R/R403E/E410N, Y155F/K247N/N249S/R318Y/T343R/R403E, K247N/N249S/R318Y/T343R/R403E, Y155F/K247N/N249S/R318Y/T343R/E410N, K247N/N249S/R318Y/T343R/E410N, Y155F/R318Y/T343R/R403E, Y155F/R318Y/T343R/E410N, Y155F/K247N/N249S/R318Y/T343R, Y155F/K247N/N249S/R318Y/R338E/R403E/E410N, K228N/K247N/N249S/R318Y/T343R/R403E/E410N, and Y155F/R318Y/R403E/E410N. 
     
     
         19 . The modified FIX polypeptide of  claim 1 , comprising amino acid replacements corresponding to R318Y/R338E/R403E/E410N, Y155F/K247N/N249S/R318Y/R338E, R318Y/T343R/E410N, or Y155F/R318Y/R338E/R403E. 
     
     
         20 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide is a mature FIX polypeptide of SEQ ID NO:3 or SEQ ID NO:20. 
     
     
         21 . The modified FIX polypeptide of  claim 1  that is an activated FIX (FIXa) polypeptide. 
     
     
         22 . The modified FIX polypeptide of  claim 1 , comprising a sequence of amino acids set forth in any of SEQ ID NOs: 121, 122, 153, 156, 161, 166, 169-173, 188, 219-226, 232-244, 249-258, 260, 264-267, 326-331, 333, 334, 336-342, 345-350, 353-360, 366, 368, 369, 372-375, 378-381, 383-386, 393-395, 397, 398, 403-408, 410, 411, 413, and 414, or an activated form thereof. 
     
     
         23 . The modified FIX polypeptide of  claim 1  that is a mature polypeptide. 
     
     
         24 . The modified FIX polypeptide of  claim 1  that is a two-chain polypeptide. 
     
     
         25 . The modified FIX polypeptide of  claim 1  that is a single-chain polypeptide. 
     
     
         26 . The modified FIX polypeptide of  claim 1  that is active or activated. 
     
     
         27 . The modified FIX polypeptide of  claim 1  that comprises one or more chemical modification(s) or post-translational modification(s). 
     
     
         28 . The modified FIX polypeptide of  claim 27 , wherein the modification is selected from among glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, albumination, and conjugation to a polyethylene glycol (PEG) moiety. 
     
     
         29 . The modified FIX polypeptide of  claim 1 , comprising up to 10 amino acid modifications, compared to the unmodified FIX polypeptide of SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         30 . The modified FIX polypeptide of  claim 1 , comprising modifications that introduce and/or eliminate one or more glycosylation sites compared to the unmodified FIX polypeptide of SEQ ID NO: 2, 3, 20, or 325, or compared to the unmodified FIXa polypeptide that consists of a light chain that consists of residues 1-14S, and a heavy chain that consists of residues 181-41S, of SEQ ID NO:3. 
     
     
         31 . The modified FIX polypeptide of  claim 1 , comprising the amino acid replacements R318Y/R338E/T343R, wherein:
 the unmodified FIX polypeptide consists of a sequence of amino acids set forth in any of SEQ ID NOs: 2, 3, 20, or 325; or   the unmodified FIX is a FIXa that consists of a light chain that consists of residues 1-14S, and a heavy chain that consists of residues 181-41S, of SEQ ID NO:3.   
     
     
         32 . A nucleic acid molecule, comprising a sequence of nucleotides encoding the modified FIX polypeptide of  claim 1 . 
     
     
         33 . A vector, comprising the nucleic acid molecule of  claim 30 . 
     
     
         34 . The vector of  claim 33 , wherein the vector is a prokaryotic vector, a viral vector, or a eukaryotic vector. 
     
     
         35 . The vector of  claim 33 , wherein the vector is a viral vector selected from among an adenovirus, an adeno-associated virus, a retrovirus, a herpes virus, a lentivirus, a poxvirus, and a cytomegalovirus. 
     
     
         36 . A cell, comprising the vector of  claim 33 . 
     
     
         37 . The cell of  claim 36 , that is a eukaryotic cell. 
     
     
         38 . The cell of  claim 37 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         39 . A pharmaceutical composition, comprising the modified FIX polypeptide of  claim 1  in a pharmaceutically acceptable vehicle. 
     
     
         40 . A method of treatment of a disease or condition, comprising administering the modified FIX polypeptide of  claim 1 , wherein the disease or condition to be treated is selected from among blood coagulation disorders, hematologic disorders, hemorrhagic disorders, hemophilias, and bleeding disorders. 
     
     
         41 . A method of treatment of a disease or condition, comprising administering the nucleic acid molecule of  claim 32 , or a vector comprising the nucleic acid molecule, wherein the disease or condition to be treated is selected from among blood coagulation disorders, hematologic disorders, hemorrhagic disorders, hemophilias, and bleeding disorders. 
     
     
         42 . The method of  claim 40 , wherein the disease or condition is a hemophilia. 
     
     
         43 . The method of  claim 41 , wherein the disease or condition is a hemophilia.

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