Modified coagulation factor IX polypeptides and use thereof for treatment

Abstract

Provided are modified factor IX (FIX) polypeptides and methods of generating modified FIX polypeptides. Also provided are pharmaceutical compositions, including compositions formulation for oral administration, that contain the modified FIX polypeptides, and methods of treatment using modified FIX polypeptides.

What is claimed is: 
     
         1 . A modified factor IX (FIX) polypeptide, comprising an amino acid replacement that corresponds to one or more amino acid replacements selected from among E410N, K247N and K228N in the FIX polypeptide set forth in SEQ ID NO: 2 or 1035, wherein:
 corresponding residues are identified by alignment;   the modified FIX polypeptide comprises a sequence of amino acid residues that is at least 90% identical to a polypeptide with an amino acid sequence selected from among the sequences set forth in SEQ ID NOs: 2, 892, 893, 898-912 and 1035; and   the modified FIX polypeptide retains coagulant activity.   
     
     
         2 . The modified FIX polypeptide of  claim 1 , wherein the FIX polypeptide comprises an amino acid replacement at a position corresponding to an amino acid position selected from among Y1, S3, G4, K5, L6, E7, E8, F9, V10, G12, L14, E15, R16, E17, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, T38, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, K63, D64, D65, I66, Y69, W72, F75, G76, F77, E78, G79, E83, L84, V86, I90, K91, N92, G93, R94, E96, F98, K100, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, S123, A127, V128, P131, G133, V135, V137, S138, T140, S141, K142, L143, R145, A146, T148, V149, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, I168, F175, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, P189, G190, F192, P193, W194, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, E213, K214, W215, I216, V217, T218, A219, A220, V223, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, E245, R248, V250, I251, R252, I253, I254, P255, Y259, A261, A262, I263, K265, Y266, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, P287, I288, I290, A291, D292, K293, Y295, T296, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, G309, W310, G311, R312, V313, F314, K316, G317, R318, S319, A320, L321, V322, L323, Y325, L326, R327, V328, P329, L330, V331, D332, R333, A334, T335, L337, R338, S339, T340, K341, F342, T343, I344, Y345, M348, F349, A351, G352, F353, E355, G356, G357, R358, D359, S360, G363, D364, S365, G366, G367, P368, V370, V373, E374, G375, T376, S377, F378, L379, T380, G381, I382, I383, S384, W385, G386, E387, E388, A390, M391, K392, G393, K394, Y395, G396, I397, Y398, T399, K400, V401, S402, R403, Y404, V405, W407, I408, K409, E410, K411, T412, K413, and T415 of the FIX polypeptide set forth in SEQ ID NO: 2 or 1035. 
     
     
         3 . The modified FIX polypeptide of  claim 2 , wherein:
 the amino acid replacement corresponds to a replacement selected from among Y1H, Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L14I, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F251, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F32I, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, T38Q, T38H, T38N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y451, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L57I, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, Y69H, Y69I, W72S, W72H, F75I, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, E83Q, E83H, E83N, L84I, L84V, V86Q, V86H, V86N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, P131A, P131S, G133Q, G133H, G133N, V135Q, V135H, V135N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, R145H, R145Q, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, D152N, D152Q, V153Q, V153H, V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N, E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, I164H, I164N, L165I, L165V, L165Q, L165H, I168Q, I168H, I168N, F175I, F175V, F175H, F178I, F178V, F178H, T179Q, T179H, T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q, A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N, F192I, F192V, F192H, P193A, P193S, W194S, W194H, W194I, V196Q, V196H, V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S, G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q, A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N, I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, E245Q, E245H, E245N, R248H, R248Q, V250Q, V250H, V250N, I251Q, I251H, I251N, R252H, R252Q, I253Q, I253H, I253N, I254Q, I254H, I254N, P255A, P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, I270H, I270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N, Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, D292N, D292Q, K293N, K293Q, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q, V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H, R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q, V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q, V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N, D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, I344N, Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q, G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N, G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E38711, E387N, E388Q, E388H, E388N, A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N, K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N, S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N, W407S, W407H, I408Q, I408H, I408N, K409N, K409Q, E410Q, E410H, E410N, K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, T415Q, T415H, and T415N of the FIX polypeptide set forth in SEQ ID NO: 2 or 1035.   
     
     
         4 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide has a sequence of amino acids set forth in SEQ ID NO: 2. 
     
     
         5 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide has a sequence of amino acids set forth in SEQ ID NO: 1035. 
     
     
         6 . The modified FIX polypeptide of  claim 1 , comprising the sequence of amino acids set forth in one of SEQ ID NOs: 225, 242, 396, 1274, 1257 and 1428. 
     
     
         7 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide is a human polypeptide. 
     
     
         8 . The modified FIX polypeptide of  claim 1 , wherein the unmodified FIX polypeptide is a non-human polypeptide. 
     
     
         9 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide comprises 2, 3, 4, or 5 amino acid replacements compared to the unmodified polypeptide, wherein the unmodified polypeptide comprises the sequence of amino acids set forth in SEQ ID NO: 2 or 1035. 
     
     
         10 . The modified FIX polypeptide of  claim 1 , wherein the polypeptide is a naked polypeptide chain. 
     
     
         11 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, or conjugated to a polyethylene glycol (PEG) moiety. 
     
     
         12 . The modified FIX polypeptide of  claim 1 , comprising an amino acid modification that contributes to an altered property selected from among glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, PEGylation and a modification to decrease immunogenicity upon administration to a human. 
     
     
         13 . The modified FIX polypeptide of  claim 2 , wherein the amino acid replacement is selected from among natural amino acids, non-natural amino acids and a combination of natural and non-natural amino acids. 
     
     
         14 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide is a mature polypeptide. 
     
     
         15 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide is a precursor polypeptide. 
     
     
         16 . The modified FIX polypeptide of  claim 15 , wherein the modified FIX polypeptide comprises a signal peptide. 
     
     
         17 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide lacks a signal peptide. 
     
     
         18 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide comprises a propeptide. 
     
     
         19 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide lacks a propeptide. 
     
     
         20 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide is a zymogen. 
     
     
         21 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide is an activated FIX polypeptide. 
     
     
         22 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide is secreted. 
     
     
         23 . The modified FIX polypeptide of  claim 1 , wherein the modified FIX polypeptide comprises a heavy chain and/or a light chain. 
     
     
         24 . The modified FIX polypeptide of  claim 1 , comprising the sequence of amino acids set forth in SEQ ID NO: 242 or 1274, and optionally additional amino acid replacements. 
     
     
         25 . The modified FIX polypeptide of  claim 1 , comprising the sequence of amino acids set forth in SEQ ID NO: 225 or 1257, and optionally additional amino acid replacements. 
     
     
         26 . The modified FIX polypeptide of  claim 1 , comprising the sequence of amino acids set forth in SEQ ID NO: 396 or 1428, and optionally additional amino acid replacements. 
     
     
         27 . A pharmaceutical composition, comprising the modified FIX polypeptide of  claim 1 . 
     
     
         28 . The pharmaceutical composition of  claim 27 , wherein the pharmaceutical composition is formulated for local, systemic or topical administration. 
     
     
         29 . The pharmaceutical composition of  claim 28  that is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parenteral, intravenous, or intramuscular administration. 
     
     
         30 . The pharmaceutical composition of  claim 28  that is formulated for oral administration. 
     
     
         31 . The pharmaceutical composition of  claim 27 , wherein the modified FIX polypeptide is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, or conjugated to a polyethylene glycol (PEG) moiety. 
     
     
         32 . The pharmaceutical composition of  claim 27 , wherein the polypeptide comprises an amino acid modification that decreases immunogenicity upon administration to a human compared to the polypeptide that does not comprise the amino acid modification. 
     
     
         33 . The pharmaceutical composition of  claim 27 , wherein an amino acid modification comprises a substitution with a natural amino acids, a substitution with a non-natural amino acids or combinations thereof. 
     
     
         34 . The pharmaceutical composition of  claim 27  that is formulated for administration in a form selected from among a liquid, a pill, a tablet, a lozenge and a capsule. 
     
     
         35 . The pharmaceutical composition of  claim 34 , wherein the tablet or capsule is enterically coated. 
     
     
         36 . The pharmaceutical composition of  claim 27  that is formulated for controlled-release of the modified FIX polypeptide. 
     
     
         37 . The pharmaceutical composition of  claim 34 , wherein the pill, tablet, lozenge, or capsule delivers the modified FIX polypeptide to the mucosa of the mouth, throat, or gastrointestinal tract. 
     
     
         38 . A method, comprising treating a subject by administering the pharmaceutical composition of  claim 27 , wherein the subject has a disease or condition that is treated by administration of FIX. 
     
     
         39 . The method of  claim 38 , wherein the disease or condition is treated by administration of inactive FIX or active FIX. 
     
     
         40 . The method of  claim 38 , wherein the disease or condition to be treated is selected from among hemophilia, thrombotic disease, and hemorrhagic disease. 
     
     
         41 . The method of  claim 38 , wherein the condition to be treated is disseminated intravascular coagulation (DIC). 
     
     
         42 . The method of  claim 40 , wherein the disease or condition is hemophilia B. 
     
     
         43 . The method of  claim 40 , wherein the disease or conditions is hemophilia that is congenital. 
     
     
         44 . The method of  claim 40 , wherein the disease is hemophilia that is acquired. 
     
     
         45 . The method of  claim 40 , wherein disease or conditions is a thrombotic disease or hemorrhagic disease selected from among ischemia, stroke, atherosclerosis, antithrombin III deficiency and protein C deficiency. 
     
     
         46 . The modified FIX polypeptide of  claim 1 , wherein the mature form thereof comprises a sequence of amino acid residues that is at least 95% identical to a polypeptide with an amino acid sequence selected from among a mature form of a FIX polypeptide with the sequence set forth in any of SEQ ID NOs: 2, 892, 893, 898-912 and 1035.