Modified coagulation factor IX polypeptides and use thereof for treatment

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in duplicate (labeled Copy #1 and Copy #2), the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned compact discs, created on Nov. 13, 2012, is identical, 6,982 kilobytes in size, and titled 4930Bseq.001.txt.

FIELD OF INVENTION

Modified coagulation factor IX (factor IX; FIX; F9) polypeptides are provided. The FIX polypeptides are modified to exhibit physical properties and activities that differ from unmodified and wild-type FIX polypeptides. Nucleic acid molecules encoding these polypeptides also are provided. Also provided are methods of treatment and diagnosis using the modified FIX polypeptides.

BACKGROUND

Effective delivery of therapeutic proteins for clinical use is a major challenge to pharmaceutical science. Once in the blood stream, these proteins are constantly eliminated from circulation within a short time by different physiological processes, involving metabolism as well as clearance using normal pathways for protein elimination, such as (glomerular) filtration in the kidneys or proteolysis in blood. Once in the luminal gastrointestinal tract, these proteins are constantly digested by luminal proteases. The latter can be a limiting process affecting the half-life of proteins used as therapeutic agents in per-oral administration and either intravenous or intramuscular injection. The problems associated with these routes of administration of proteins are known and various strategies have been used in attempts to solve them.

A protein family that has been the focus of clinical work and effort to improve its administration and bio-assimilation is the blood coagulation factor family, which includes procoagulation factors, such as factor IX (FIX) and factor VIII (FVIII). Deficiencies in the levels of functional coagulation pathway proteins can cause mild to severe bleeding disorders. For example, hemophilia A and B are inherited diseases characterized by deficiencies in FVIII and FIX polypeptides, respectively. The underlying cause of the deficiencies is frequently the result of mutations in FVIII and FIX genes, both of which are located on the X chromosome. Traditional therapy for hemophilias often involves intravenous administration of pooled plasma or semi-purified coagulation factors from normal individuals. These preparations can be contaminated by pathogenic agents or viruses, such as infectious prions, HIV, parvovirus, hepatitis A, and hepatitis C. Hence, there is an urgent need for therapeutic agents that do not require the use of human serum.

Recombinantly produced FIX polypeptides have been approved for procoagulation treatment of hemophilia. Also of therapeutic interest are FIX polypeptides that lack peptidase activity and can have anticoagulant activities useful in the treatment of thrombolytic diseases. Hence, FIX along with other coagulation factors are important therapeutic agents for procoagulant and anticoagulation therapies. Since naturally occurring variants can have undesirable side effects as well as the problems of administration, bioavailability, and short half-life, there is a need to improve properties of FIX for its use as a biotherapeutic agent. Therefore, among the objects herein, it is an object to provide modified FIX polypeptides that have improved therapeutic properties.

SUMMARY

Provided herein are modified Factor IX (FIX) polypeptides that exhibit increased protein stability compared to an unmodified FIX polypeptide. The modified FIX polypeptides provided herein are mutant variants of FIX that display improved protein stability. Modified FIX polypeptides provided herein exhibiting increased protein stability display increased protein half-life in vivo or in vitro compared to an unmodified FIX polypeptide. For example, modified FIX polypeptides provided herein exhibit increased stability in the bloodstream, following oral administration, and/or under storage conditions. Increased protein stability of a modified FIX provided herein can be manifested as increased resistance to digestion by proteases. Such increased stability includes stability as assessed by resistance to blood, intestinal or any other proteases.

Provided herein are modified FIX polypeptides containing one or more amino acid modifications corresponding to amino acid positions E36, R37, T39, E40, S61, S68, E70, P74, K80, D85, T87, S102, E119, K122, E125, P126, P129, R134, S136, T144, E147, P151, D166, T169, S171, T172, S174, D177, E224, T225, T241, E242, T244, K247, E294, T371, E372, and L414 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the one or more amino acid positions can include replacements of any of E36Q, E36H, E36N, R37H, R37Q, T39Q, T39H, T39N, E40Q, E40H, E40N, S61Q, S61H, S61N, S68Q, S68H, S68N, E70Q, E70H, E70N, P74A, P74S, K80N, K80Q, D85N, D85Q, T87Q, T87H, T87N, S102Q, S102H, S102N, K122N, K122Q, E125Q, E125H, E125N, P126A, P126S, P129A, P129S, R134H, R134Q, S136Q, S136H, S136N, S138Q, T144Q, T144H, T144N, E147Q, E147H, E147N, P151A, P151S, D166N, D166Q, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, D177N, D177Q, E224Q, E224H, E224N, T225Q, T225H, T225N, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, K247N, K247Q, E294Q, E294H, E294N, T371Q, T371H, T371N, E372Q, E372H, E372N, L414I, and L414V of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

Provided herein are modified FIX polypeptides containing one or more amino acid modifications corresponding to any of FIX polypeptides with modifications corresponding to Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9V, E15Q, E15N, R16H, R16Q, E17H, E17N, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24N, F25V, E26H, E26N, E27Q, E27N, R29H, E30Q, E30N, F321, F32V, T35Q, T35H, T35N, T38Q, T38H, T38N, F41I, K43N, K43Q, Y45I, D47Q, G48Q, G48H, G48N, D49N, E52Q, E52H, E52N, S53Q, S53N, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60N, K63N, K63Q, D64Q, D65N, D65Q, Y69I, F751, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, E83Q, E83H, E83N, L84I, L84V, K91N, K91Q, N92Q, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, D104N, D104Q, K106N, K106Q, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113N, Y115I, R116H, R116Q, L117I, L117V, E119Q, E119H, E119N, S123Q, S123N, A127Q, A127H, A127N, P131A, G133Q, G133H, G133N, S138N, T140Q, T140N, S141Q, S141H, S141N, K142N, K142Q, R145Q, A146Q, A146H, A146N, T148Q, T148N, D152N, D152Q, D154N, Y155I, S158H, S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, E162Q, E162H, E162N, T163Q, T163H, T163N, L165I, L165V, F175I, F175V, F178I, F178V, T179Q, T179H, T179N, E185Q, E185N, D186N, D186Q, K188N, K188Q, P189A, P189S, F192I, P193A, W194I, L198V, N199Q, G200Q, G200H, G200N, D203N, D203Q, F205I, G207Q, G207H, G207N, S209Q, S209H, S209N, E213Q, E213N, K214N, K214Q, T218Q, T218H, T218N, A219Q, A219N, A220Q, A220H, A220N, G226Q, G226H, G226N, K228N, K228Q, T230Q, T230H, T230N, E239Q, E239H, E239N, E240Q, E240N, E245Q, E245H, E245N, R248H, R252H, R252Q, P255A, P255S, Y259I, K265N, K265Q, Y266I, L272I, L272V, E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S, S283Q, S283H, S283N, Y284I, T286Q, T286H, T286N, P287A, P287S, D292N, D292Q, K293N, K293Q, Y295I, T296Q, T296H, T296N, F299I, K301N, K301Q, F302I, F302V, G303Q, G303N, S304Q, S304H, S304N, Y306I, S308Q, S308H, G309Q, G309H, G309N, G311Q, G311N, R312H, R312Q, F314V, K316N, K316Q, R318H, R318Q, L321I, L321V, Y325I, R327H, R327Q, P329A, P329S, D332N, D332Q, T335Q, T335H, T335N, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N, K341Q, F342I, F342V, T343Q, T343H, T343N, Y345I, F349V, G352Q, G352H, G352N, F353V, G363Q, G363H, G363N, D364Q, S365Q, S365H, G366Q, G366H, G366N, G367Q, G367H, G367N, P368A, E374Q, E374H, E374N, G375Q, G375N, T376Q, T376H, S377Q, S377H, S377N, F378I, L379V, T380Q, T380H, T380N, S384Q, S384H, G386Q, G386H, G386N, E387Q, E387N, M391V, K392N, K392Q, K394Q, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N, Y398I, T399Q, T399H, K400Q, S402Q, S402H, S402N, R403H, Y404I, K409N, K409Q, E410Q, E410N, K411N, K411 Q, T412Q, T412H, T412N, K413N, K413 Q, T415 Q, T415H, and T415N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In a particular example, the modifications in FIX polypeptide are any of T163N, T163H, I164N, I164Q, L165I, I168N, I168H, T169N, T169Q, T169H, S171N, S171Q, S171H, T172Q, T172H, S174N, S174Q, S174H, F175I, T179N, T179Q, T179H, D203Q, and D203N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In another example, the modifications in FIX polypeptide are any of D152Q, D154N, T163H, I164Q, I164H, D166Q, D166N, I168Q, I168H, T169H, S171N, S171Q, T172Q, S174Q, S174H, F175H, D177Q, D177N, T179N, T179Q, T179H, T179N, T179Q, T179H, V181Q, V182N, G184N, D186Q, D186N, A187Q, F192I, V197H, L198I, L198V, D203Q, and D203N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In yet another example, the modifications in FIX polypeptide are any of T163H, I164Q, I168H, I169H, S171N, S171Q, T172Q, S174Q, S174H, T179N, T179Q, T179H, D203Q, and D203N of the mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

Provided herein are modified FIX polypeptides containing two or more amino acid modifications corresponding to modifications at any two or more positions of 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, E36, R37, T38, T39, E40, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, S61, K63, D64, D65, I66, S68, Y69, E70, W72, P74, F75, G76, F77, E78, G79, K80, E83, L84, D85, V86, T87, I90, K91, N92, G93, R94, E96, F98, K100, S102, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, E119, K122, S123, E125, P126, A127, V128, P129, P131, G133, R134, V135, S136, V137, S138, T140, S141, K142, L143, T144, R145, A146, E147, T148, V149, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, S171, T172, S174, F175, D177, 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, E224, T225, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, T241, E242, T244, E245, K247, 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, E294, 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, T371, E372, 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, L414, and T415 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid amino acid replacements at any two or more of the amino acid positions can include replacements of any of 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, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, 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, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F75I, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, S102Q, S102H, S102N, 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, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, 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, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, 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, V19′7N, 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, E224Q, E224H, E224N, T225Q, T225H, T225N, 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, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, 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, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H, T296N, I298Q, I298H, I298N, F299I, F299V, L300I, L300V, K301N, K301Q, F302I, F302V, I303Q, G303H, I303N, S304Q, S304H, S304N, G305Q, G305H, I305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, W310S, W310H, G311Q, G311H, I311N, 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, T371Q, T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H, E374N, G375Q, I375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, F378I, F378V, L379I, L379V, T380Q, T380H, T380N, I381Q, I381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, W385S, W385H, G386Q, G386H, G386N, E387Q, E387H, 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, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

Provided herein are any of the above modified FIX polypeptides above that are further modified at one or more positions corresponding to amino acid positions 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, 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 mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the one or more further amino acid positions can include replacements of any of Y1H, Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L61, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F9I, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, 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, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, Y69H, Y691, W72S, W72H, F75I, F75V, G76Q, G76H, G76N, F77I, 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, F981, 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, E387H, 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 mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

Provided herein are modified FIX polypeptides containing an amino acid modification at a position corresponding to amino acid residue F 192 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. Provided herein is a modified FIX polypeptide in which the amino acid modification at position F192 is a replacement of phenylalanine (F) by isoleucine (I), valine (V), or histidine (H). In a particular example, the amino acid modification at position F192 is isoleucine (I). Provided herein are modified FIX polypeptides containing an amino acid modification at F 192 that also contains a further amino acid modification at another position. For example, such a modified FIX polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modifications. In one example, the further amino acid replacement is at positions corresponding to any of amino acid positions 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, 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 mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the further amino acid positions can include replacements of any of Y11, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F91, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, 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, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y691, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F751, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, S102Q, S102H, S102N, 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, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, 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, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, 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, 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, E224Q, E224H, E224N, T225Q, T225H, T225N, 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, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, 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, E294Q, E294H, E294N, 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, T371Q, T371H, T371N, E372Q, E372H, E372N, 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, E387H, 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, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

In some examples, exemplary amino acid modifications of a FIX polypeptide include amino acid modifications of any of T163H/F192I, I164Q/F192I, T169H/F192I, S171Q/F192I, D152N/D154N/F192I, S174Q/F192I, S174H/F192I, T179Q/F192I, T179H/F192I, T163N/I164N/F192I, D166Q/F192I, D166N/F192I, D186Q/F192I, D186N/F192I, T148A/D152N/D154N/F192I, F192I/D203Q, F192I/D203N, D152N/F192I, D152Q/F192I, T148A/D154N/F192I, D154N/F192I, D154Q/F192I, T163N/I164N, A187N/G190N, V196N/V197N, T140N/T144N, F175I/F178I, F192I/W194I, T148A/D152N, T148A/D152Q, T148A/D154N, T148A/D154Q, D152N/D154N, I168N/T169N/S171N/T172N/S174N, T179N/V181N/V182N/G183N/G184N, D152N/D154N/T148A, T163H/D166Q/F192I, T163H/D166N/F192I, T163H/D186Q/F192I, T163H/D186N/F192I, T163H/F192I/D203Q, T163H/F192I/D203N, T148A/D152N/D154N/T163H/F192I, T148A/D154N/T163H/F192I, I164Q/D166Q/F1921, I164Q/D166N/F1921, I164Q/D186Q/F192I, I164Q/D186N/F192I, I164Q/F192I/D203Q, I164Q/F192I/D203N, T148A/D152N/D154N/I164Q/F192I, T148A/D154N/I164Q/F192I, T163N/I164N/D166Q/F192I, T163N/I164N/D166N/F192I, T163N/I164N/D186Q/F192I, T163N/I164N/D186N/F192I, T163N/I164N/F192I/D203Q, T163N/I164N/F192I/D203N, T148A/D152N/D154N/T163N/I164N/F192I, T148A/D154N/T163N/I164N/F192I, D166Q/T169H/F192I, D166N/T169H/F192I, T169H/D186Q/F192I, T169H/D186N/F192I, T169H/F192I/D203Q, T169H/F192I/D203N, T148A/D152N/D154N/T169H/F192I, T148A/D154N/T169H/F192I, D166Q/S171Q/F192I, D166N/S171Q/F192I, S171Q/D186Q/F192I, S171Q/D186N/F192I, S171Q/F192I/D203Q, S171Q/F192I/D203N, T148A/D152N/D154N/S171Q/F192I, T148A/D154N/S171Q/F192I, D166Q/S174Q/F192I, D 166N/S174Q/F192I, S174Q/D186Q/F192I, S174Q/D186N/F192I, S174Q/F192I/D203Q, S174Q/F192I/D203N, T148A/D152N/D154N/S174Q/F192I, T148A/D154N/S174Q/F192I, D166Q/S174H/F192I, D166N/S174H/F192I, S174H/D186Q/F192I, S174H/D186N/F192I, S174H/F192I/D203Q, S174H/F192I/D203N, T148A/D152N/D154N/S174H/F192I, T148A/D154N/I174H/F192I, D166Q/T179Q/F192I, D166N/T179Q/F192I, T179Q/D186Q/F192I, T179Q/D186N/F192I, T179Q/F192I/D203Q, T179Q/F192I/D203N, T148A/D152N/D154N/T179Q/F192I, T148A/D154N/T179Q/F192I, D166Q/T179H/F192I, D166N/T179H/F192I, T179H/D186Q/F192I, T179H/D186N/F192I, T179H/F192I/D203Q, T179H/F192I/D203N, T148A/D152N/D154N/T179H/F192I, and T148A/D154N/T179H/F1921 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, a modified FIX polypeptide provided herein has a sequence of amino acids set forth in any of SEQ ID NOS: 917-1024 or 1927-2034. In particular examples, the modifications in FIX polypeptide are any of D186Q/F192I, T148A/D152N/D154N/F192I, T163H/D166Q/F192I, T163H/F192I/D203Q, T148A/D154N/T163H/F192I, I164Q/F192I/D203Q, I164Q/F192I/D203N, T148A/D154N/T163N/I164N/F192I, T169H/F192I/D203N, S174Q/F192I/D203Q, S174H/D186N/F192I, and T179Q/F192I/D203Q of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. In further examples, the modifications in FIX polypeptide are any of S174Q/F192I/D203Q, S174H/D186N/F192I, and T179Q/F192I/D203Q of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

The modified FIX polypeptides provided herein include precursor forms and mature forms, such as modification of a wild-type human FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 1 (precursor form) or SEQ ID NO: 2 or 1035 (mature forms). Mature forms of FIX polypeptides can include chimeric forms of FIX polypeptides such as set forth in SEQ ID NO: 1035, where homologous regions of coagulation factor family members, such as factor VII or factor X are inserted or replaced in the FIX amino acid sequence. Exemplary modified FIX polypeptides have a sequence of amino acids set forth in any of SEQ ID NOS: 3-891, 917-1034, or 1036-2044. It also is understood that amino acid modification of a FIX polypeptide can be in an allelic, species, or isoform variant of SEQ ID NO: 2 or 1035, where the allelic or species variant has 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide set forth in SEQ ID NO: 2 or 1035, excluding the modified positions. A modified FIX polypeptide can be a human polypeptide or a non-human polypeptide. Modified loci are identified with reference to the amino acid numbering of a known unmodified mature FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 2 or 1035. One of skill in the art can readily determine corresponding positions on a particular polypeptide, such as by alignment of unchanged residues. Furthermore, shortened or lengthened variants with insertions or deletions of amino acids, particularly at either terminus that retain an activity readily can be prepared and the loci for corresponding mutations identified.

In one example, provided herein is a modified structural homologue of a modified FIX as described herein containing one or more amino acid replacements in the structural homologue at positions corresponding to the 3-dimensional-structurally-similar positions within the 3-D structure of the modified FIX.

Modified FIX polypeptides provided herein include FIX polypeptides modified at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 positions as compared to an unmodified FIX polypeptide. The modified FIX polypeptides provided herein exhibit increased protein stability compared to the unmodified FIX polypeptide. Provided herein are FIX polypeptides in which increased protein stability of the modified FIX polypeptide is the result of modification to the primary sequence of the FIX polypeptide. The increased protein stability exhibited by a FIX polypeptide can be manifested as increased protease resistance. In some cases, increased protein stability of a FIX polypeptide can be due to increased resistance to proteolysis that can occur in serum, blood, saliva, digestive fluids, or in vitro when exposed to one or more proteases. The increased resistance to proteolysis is exhibited by the modified FIX when it is administered intravenously, orally, nasally, pulmonarily, or is present in the digestive tract. Such modified FIX polypeptides exhibit increased resistance to proteolysis by one or more proteases compared to the unmodified FIX. For example, the proteases can by any of pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA.

In some examples, a modified FIX polypeptide exhibits increased protein stability due to increased protease resistance to elastase. Exemplary of those modified FIX polypeptides with increased protease resistance to elastase are those with modifications corresponding to S3Q, S3H, S3N, G4Q, G4H, G4N, V10Q, V10H, V10N, G12Q, G12H, G12N, S24Q, S24H, S24N, A28Q, A28H, A28N, V31Q, V31H, V31N, T35Q, T35H, T35N, T38Q, T38H, T38N, T39Q, T39H, T39N, V46Q, V46H, V46N, G48Q, G48H, G48N, S53Q, S53H, S53N, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, 166Q, 166H, 166N, S68Q, S68H, S68N, G76Q, G76H, G76N, G79Q, G79H, G79N, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, G93Q, G93H, G93N, S102Q, S102H, S102N, A103Q, A103H, A103N, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, G114Q, G114H, G114N, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, G133Q, G133H, G133N, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, T144Q, T144H, T144N, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, V153Q, V153H, V153N, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, A161Q, A161H, A161N, T163Q, T163H, T163N, I164Q, I164H, I164N, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, T179Q, T179H, T179N, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, A187Q, A187H, A187N, G190Q, G190H, G190N, V196Q, V196H, V196N, V197Q, V197H, V197N, G200Q, G200H, G200N, V202Q, V202H, V202N, A204Q, A204H, A204N, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, I238Q, I238H, I238N, T241Q, T241H, T241N, T244Q, T244H, T244N, V250Q, V250H, V250N, I251Q, I251H, I251N, I253Q, I253H, I253N, I254Q, I254H, I254N, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, I270Q, I270H, I270N, A271Q, A271H, A271N, V280Q, V280H, V280N, S283Q, S283H, S283N, V285Q, V285H, V285N, T286Q, T286H, T286N, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, T296Q, T296H, T296N, I298Q, I298H, I298N, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, G311Q, G311H, G311N, V313Q, V313H, V313N, G317Q, G317H, G317N, S319Q, S319H, S319N, A320Q, A320H, A320N, V322Q, V322H, V322N, V328Q, V328H, V328N, V331Q, V331H, V331N, A334Q, A334H, A334N, T335Q, T335H, T335N, S339Q, S339H, S339N, T340Q, T340H, T340N, T343Q, T343H, T343N, I344Q, I344H, I344N, A351Q, A351H, A351N, G352Q, G352H, G352N, G356Q, G356H, G356N, G357Q, G357H, G357N, S360Q, S360H, S360N, G363Q, G363H, G363N, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, V370Q, V370H, V370N, T371Q, T371H, T371N, V373Q, V373H, V373N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, G386Q, G386H, G386N, A390Q, A390H, A390N, G393Q, G393H, G393N, G396Q, G396H, G396N, I397Q, I397H, I397N, T399Q, T399H, T399N, V401Q, V401H, V401N, S402Q, S402H, S402N, V405Q, V405H, V405N, I408Q, I408H, I408N, T412Q, T412H, T412N, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

A modified FIX polypeptide provided herein that contains one or more amino acid modifications and exhibits increased protein stability manifested as increased protease resistance to elastase also can contain any one or more further amino acid modification at amino acid positions corresponding to positions 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, 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 mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. For example, amino acid replacements at any one of the further amino acid positions can include replacements of any of 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, F321, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, T38Q, T38H, T38N, F411, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, 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, I66Q, I66H, I66N, Y69H, Y69I, W72S, W72H, F751, F75V, G76Q, G76H, G76N, F77I, 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, E387H, 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 mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

Modified FIX polypeptides provided herein can be naked polypeptides chains or can be post-translationally modified. Exemplary post-translational modifications include glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, or conjugation to albumin or a polyethylene glycol (PEG) moiety. The modified FIX polypeptides provided herein can be further modified at one or more amino acid positions, where the modification contributes to altered immunogenicity, glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, PEGylation or protease resistance of the modified FIX polypeptide. Exemplary amino acid modifications include replacement with natural amino acids, non-natural amino acids and combinations of natural and non-natural amino acids. Such modifications can increase the stability of the FIX polypeptide.

Provided herein are any of the above modified FIX polypeptides, further containing one or more pseudo-wild type mutations. In one embodiment, the pseudo-wild-type mutations include, but are not limited to, one or more of insertions, deletions or replacements of the amino acid residue(s) of the unmodified FIX polypeptide.

Typically, the modified FIX polypeptides provided herein exhibit increased protein stability compared to the unmodified FIX polypeptide and retain one or more activities of the unmodified FIX polypeptide. Provided herein are any of the above modified FIX polypeptides exhibiting increased activity compared to the unmodified FIX. Provided herein are any of the above modified FIX polypeptides exhibiting decreased activity compared to the unmodified FIX. Activity can be assessed, for example, by measuring peptidase activity or blood coagulation in vitro or in vivo. The results of such assays correlate with an in vivo activity and hence a biological activity. In some examples the modified FIX polypeptide promotes coagulation. In other examples, the modified FIX polypeptide inhibits coagulation.

In some examples, a modified FIX polypeptide containing a modification corresponding to position F192 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035, and that also contains a further amino acid modification, exhibits increased protein stability and retains one or more activities of the unmodified FIX polypeptide.

Provided herein are any of the above modified FIX polypeptides, in which the increased protein stability is manifested as an increased half-life in vivo or in vitro. In one example, the increased stability is manifested as an increased half-life when administered to a subject. In another example, the modified FIX has a half-life increased by at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the half-life of unmodified FIX. In yet another example, the modified FIX also has a half-life increased by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times and 1000 times, or more times when compared to the half-life of unmodified FIX.

Provided herein are any of the above modified FIX polypeptides that is a precursor polypeptide containing a signal peptide and a propeptide. In one example, the signal sequence is amino acids 1-28 and the propeptide is amino acids 29-46 of the sequence of amino acids set forth in SEQ ID NO: 1. Provided herein are any of the above modified FIX polypeptides that do not have a signal peptide and/or propeptide. Modified FIX polypeptides without a signal peptide and propeptide are mature FIX polypeptides. In some cases, the modified FIX polypeptides provided herein are secreted. Such a secreted polypeptide has a signal sequence and propeptide sequence that is processed prior to secretion. Such secreted FIX polypeptides can contain other post-translational modifications, such as for example, glycosylation, carboxylation, hydroxylation, sulfation, and/or phosphorylation.

Provide herein are modified FIX polypeptides that include a heavy chain and a light chain of the FIX polypeptide. In some examples, a modified FIX polypeptide can include a heavy chain and lack a light chain. In some other examples, a modified FIX polypeptide can include a light chain and lack a heavy chain.

Provided herein are libraries (collections) of modified FIX polypeptides containing two, three, four, five, 10, 50, 100, 500, 1000, 10³, 10⁴or more modified FIX polypeptides as described herein.

Provided herein are nucleic acid molecules containing a sequence of nucleotides encoding a modified FIX polypeptide as described herein. Provided herein are libraries (collections) of nucleic acid molecules comprising a plurality of the molecules as described herein.

Provided herein are vectors containing the nucleic acid molecules encoding modified FIX polypeptides as described herein. Exemplary vectors include, but are not limited to, a prokaryotic vector, a viral vector, or a eukaryotic vector. Exemplary viral vectors include adenovirus, adeno-associated-virus, retrovirus, herpes virus, lentivirus, poxvirus, and cytomegalovirus vectors. In some examples, the nucleic acid in the vector is operably linked to a promoter, such as a viral promoter or a eukaryotic promoter. Such promoters can be constitutive promoters or inducible promoters. Provided herein are libraries (collections) of such vectors containing two, three, four, five, 10, 50, 100, 500, 1000, 10³, 10⁴or more vectors containing the nucleic acid molecules encoding modified FIX polypeptides as described herein.

Provided herein are cells containing nucleic acid molecules encoding modified FIX polypeptides as described herein. Provided herein are cells containing vectors that contain the nucleic acid molecules encoding modified FIX polypeptides as described herein. Such cells can be prokaryotic or eukaryotic cells, and can express the modified FIX polypeptides.

Provided herein are methods for expressing a modified FIX polypeptide. Such methods can include the steps of: i) introducing a nucleic acid encoding a modified FIX polypeptide or a vector containing a nucleic acid encoding a modified FIX into a cell, and ii) culturing the cell under conditions in which the encoded modified FIX is expressed. In one embodiment, cells are eukaryotic cells or prokaryotic cells. In one embodiment, the modified FIX polypeptide is post-translationally modified. In another method for expression of a modified FIX polypeptide, a nucleic acid molecule encoding a modified FIX or a vector containing a nucleic acid encoding a modified FIX is introduced into a cell-free translation system, whereby the encoded modified FIX polypeptide is expressed. In such expression methods above, the method can further include detection of the modified FIX polypeptide.

Provided herein are non-human transgenic animals containing an exogenous nucleic acid encoding a modified FIX polypeptide. Exemplary non-human transgenic animals include pigs, goats, sheep, rabbits, rats, and cows. Provided herein are methods of expressing modified FIX polypeptides in non-human transgenic animals and detecting the expressed modified FIX polypeptides. Modified FIX polypeptides can be isolated from tissues or fluids of the non-human transgenic animals, such as in serum, milk, or eggs. Such expressed modified FIX polypeptides can be post-translationally modified. For example, the modified FIX polypeptides can be glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, or conjugated to a polyethylene glycol (PEG) moiety.

Provided herein are pharmaceutical compositions including any of the modified FIX polypeptides described herein. In some examples, the modified FIX polypeptides are any of amino acids set forth in SEQ ID NOS: 3-891, 917-1034, or 1036-2044.

In one example, the pharmaceutical compositions provided herein are formulated for local, systemic, or topical administration. For example the pharmaceutical compositions provided herein can be formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parenteral, intravenous, or intramuscular administration. In a particular example, the pharmaceutical compositions are formulated for oral administration.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide, where the FIX polypeptide is modified by replacement of one or more amino acids in its primary structure to be resistant to a protease and the composition is formulated for oral administration.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the modified FIX polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modifications. Provided herein are pharmaceutical compositions of a FIX polypeptide, in which only the primary sequence of the FIX polypeptide is modified, and the polypeptide exhibits increased protein stability. Such pharmaceutical compositions of a FIX polypeptide can include removal of proteolytic digestion sites.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which increased protein stability is manifested as increased resistance to proteolysis. In some instances, the modified FIX polypeptide in the pharmaceutical formulation exhibits increased protein stability in the gastrointestinal tract under conditions selected from exposure to saliva, exposure to proteases in the gastrointestinal tract and exposure to low pH conditions compared to an unmodified FIX polypeptide. In some instances, the increased resistance to proteolysis occurs in serum, blood, saliva, digestive fluids or in vitro when exposed to one or more proteases. Proteases include, but are not limited to one or more of a pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. In a particular example, that protease is elastase. In some instances, the modified FIX polypeptide in the pharmaceutical formulation exhibits increased protease resistance when it is administered orally or is present in the digestive tract.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide that are a FIX naked polypeptide or a post-translationally modified polypeptide, such as a FIX polypeptide that is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated albuminated, or conjugated to a polyethylene glycol (PEG) moiety.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide that further comprise one or more additional amino acid modifications that contribute to altered immunogenicity, glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, PEGylation or protease resistance of the modified FIX polypeptide. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which one or more additional amino acid modifications are selected from natural amino acids, non-natural amino acids or a combination of natural and non-natural amino acids. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which one or more additional amino acid modifications increase stability of the FIX polypeptide.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide that further comprise one or more pseudo-wild type mutations, including insertions, deletions, and replacements of the amino acid residue(s) of the unmodified FIX polypeptide.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide that exhibits increased protein stability compared to the unmodified FIX polypeptide and retains one or more activities of the unmodified FIX polypeptide. Provided herein are pharmaceutical compositions of a modified FIX polypeptide that promote coagulation. Provided herein are pharmaceutical compositions of a modified FIX polypeptide that inhibit coagulation.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide in which increased protein stability of the FIX polypeptide leads to increased protein half-life of the FIX polypeptide in vivo or in vitro. Increased protein stability can lead to increased protein half-life following administration to a subject. Protein half-life can be increased in an amount of at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the half-life of the unmodified FIX polypeptide. Alternatively, protein half-life can be increased in an amount of at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times or at least 1000 times or more compared to an unmodified protein. In one example, the modified FIX in the pharmaceutical composition exhibits increased protein half-life or bioavailability in the gastrointestinal tract.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide containing a composition that also contains a pharmaceutically acceptable carrier, diluent, or excipient. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the excipient is a binding agent, a filler, a lubricant, a disintegrant or a wetting agent. In a particular example, the excipient is anhydrous crystalline maltose or magnesium stearate. Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the additive is a suspending agent, an emulsifying agent, a non-aqueous vehicle, or a preservative.

Provided herein are pharmaceutical compositions of a modified FIX polypeptide, in which the pharmaceutical composition is formulated for administration in a form selected from among liquid, a pill, a tablet, a lozenge, and a capsule. In such pharmaceutical compositions, the modified FIX polypeptide is delivered to the gastrointestinal tract or the mucosa of the mouth, throat, or gastrointestinal tract. In some pharmaceutical compositions, the pill or tablet is chewable. In another example, the pill or tablet dissolves when exposed to saliva on the tongue or in the mouth. In other pharmaceutical compositions the capsule is in liquid form. Exemplary liquid pharmaceutical compositions include a solution, a syrup, or a suspension. In some pharmaceutical compositions, the tablet or capsule is enterically coated. In some pharmaceutical compositions, the modified FIX polypeptide is formulated for controlled-release.

Provided herein are pharmaceutical compositions prepared without the use of protease inhibitors, such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor, aprotinin and camostat. Provided herein are pharmaceutical compositions formulated without protective compounds.

Provided herein are pharmaceutical compositions of nucleic acid molecules encoding any of the modified FIX polypeptides described herein or a vector containing a nucleic acid molecule encoding any of the modified FIX polypeptides described herein and a pharmaceutically acceptable excipient.

Provided herein are methods of treating a subject exhibiting symptoms of or having FIX-mediated disease or condition by administering any of the pharmaceutical compositions described herein. For example, such pharmaceutical compositions can contain modified FIX polypeptides having a sequence of amino acids set forth in any one of SEQ ID NOS: 3-891, 917-1034, or 1036-2044. In some examples, the FIX-mediated disease or condition is treated by administration of an active FIX polypeptide. In other examples the FIX-mediated disease or condition is treated by administration of an inactive FIX polypeptide. Typically, treatment with the pharmaceutical composition ameliorates or alleviates the symptoms associated with the disease or condition. Provided herein are methods of monitoring the subject for changes in the symptoms associated with the FIX-mediated disease or condition.

In one example, the FIX-mediated disease or condition includes, but is not limited to hemophilia, thrombotic disease, disseminated intravascular coagulation (DIC), or hemorrhagic disease. In such an example where the disease to be treated is hemophilia, the disease or condition can be congenital or acquired. In another example where the disease to be treated is a thrombotic disease or hemorrhagic disease, the disease or condition can be, but is not limited to, ischemia, stroke, atherosclerosis, antithrombin III deficiency, or protein C deficiency. In cases where increased coagulation is desired (i.e. for hemophilia), modified FIX polypeptides that promote blood clotting are administered. In cases where decreased coagulation is desired (i.e. for thrombotic diseases), modified FIX polypeptides that inhibit blood clotting are administered.

Provided herein are methods of treating a subject exhibiting symptoms of or having a FIX-mediated disease or condition by administering modified FIX polypeptides with additional pro-coagulation factors, such as factor VIII, factor VII, vitamin K, pro-coagulants, platelet activators, vasoconstriction agents, plasminogen inhibitors, or fibrolytic inhibitors.

Provided herein are methods of treating a subject exhibiting symptoms of or having a FIX-mediated disease or condition by administering modified FIX polypeptides with additional anticoagulation factors, such as platelet inhibitors, vasodilators, fibrolytic activators, or other anticoagulants. Exemplary anticoagulants include heparin, coumarin, and hirudin. Exemplary platelet inhibitors include aspirin, naproxen, meclofenamic acid, ibuprofen, indomethacin, phenylbutazare, and ticlopidine. Exemplary fibrolytic activators include streptokinase, urokinase, and tissue plasminogen activator (t-PA).

Provided herein are articles of manufacture including, but not limited to, packaging material and a pharmaceutical composition of a modified FIX polypeptide described herein contained within the packaging material. In a particular embodiment, the pharmaceutical composition packaged within the article of manufacture is effective for treatment of a FIX-mediated disease or disorder, and the packaging material includes a label that indicates that the modified FIX is used for treatment of a FIX-mediated disease or disorder.

Provided herein are kits including a pharmaceutical composition of a modified FIX polypeptide as described herein, a device for administration of the modified FIX polypeptide and optionally instructions for administration.

Provided herein are methods for producing a modified FIX polypeptide, having an evolved predetermined property, wherein the evolved predetermined property is increased protein stability manifested as increased protease resistance. In such examples, the increased protein stability of the FIX polypeptide that is evolved is due to amino acid modifications, such that only the primary sequence of the polypeptide is modified to confer the property. In some examples, the methods include modifications of one or more additional amino acids that contribute to altered immunogenicity, glycosylation, carboxylation, hydroxylation, sulfation, phosphorylation, or PEGylation of the modified FIX polypeptide.

In one example, a method of increasing protein stability involves the step of introducing one or more amino acid modification that leads to the removal of proteolytic digestion sites such that the polypeptide exhibits increased protease resistance where the amino acid modifications are chosen from any one or more of 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, L141, L14V, E15Q, E15H, E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, 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, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F411, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, V46Q, V46H, V46N, D47N, D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N, P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F75I, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, I90Q, I90H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V10′7N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, 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, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, 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, F192IH, 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, E224Q, E224H, E224N, T225Q, T225H, T225N, 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, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, 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, E294Q, E294H, E294N, 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, T371Q, T371H, T371N, E372Q, E372H, E372N, 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, E387H, 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, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

In a particular example, a method of increasing protein stability involves the step of introducing one or more amino acid modification that leads to the removal of proteolytic digestion sites recognized by elastase such that the polypeptide exhibits increased protease resistance to elastase where the amino acid modifications are chosen from any one or more of S3Q, S3H, S3N, G4Q, G4H, G4N, V10Q, V10H, V10N, G12Q, G12H, G12N, S24Q, S24H, S24N, A28Q, A28H, A28N, V31Q, V31H, V31N, T35Q, T35H, T35N, T38Q, T38H, T38N, T39Q, T39H, T39N, V46Q, V46H, V46N, G48Q, G48H, G48N, S53Q, S53H, S53N, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, 166Q, 166H, 166N, S68Q, S68H, S68N, G76Q, G76H, G76N, G79Q, G79H, G79N, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, G93Q, G93H, G93N, S102Q, S102H, S102N, A103Q, A103H, A103N, V107Q, V107H, V10′7N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, G114Q, G114H, G114N, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, G133Q, G133H, G133N, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, T144Q, T144H, T144N, A146Q, A146H, A146N, T148Q, T148H, T148N, V149Q, V149H, V149N, V153Q, V153H, V153N, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, A161Q, A161H, A161N, T163Q, T163H, T163N, I164Q, I164H, I164N, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, T179Q, T179H, T179N, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, A187Q, A187H, A187N, G190Q, G190H, G190N, V196Q, V196H, V196N, V 197Q, V197H, V197N, G200Q, G200H, G200N, V202Q, V202H, V202N, A204Q, A204H, A204N, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V211H, V211N, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, I238Q, I238H, I238N, T241Q, T241H, T241N, T244Q, T244H, T244N, V250Q, V250H, V250N, I251Q, I251H, I251N, I253Q, I253H, I253N, I254Q, I254H, I254N, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, I270Q, I270H, I270N, A271Q, A271H, A271N, V280Q, V280H, V280N, S283Q, S283H, S283N, V285Q, V285H, V285N, T286Q, T286H, T286N, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, T296Q, T296H, T296N, I298Q, I298H, I298N, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, G311Q, G311H, G311N, V313Q, V313H, V313N, G317Q, G317H, G317N, S319Q, S319H, S319N, A320Q, A320H, A320N, V322Q, V322H, V322N, V328Q, V328H, V328N, V331Q, V331H, V331N, A334Q, A334H, A334N, T335Q, T335H, T335N, S339Q, S339H, S339N, T340Q, T340H, T340N, T343Q, T343H, T343N, I344Q, I344H, I344N, A351Q, A351H, A351N, G352Q, G352H, G352N, G356Q, G356H, G356N, G357Q, G357H, G357N, S360Q, S360H, S360N, G363Q, G363H, G363N, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, V370Q, V370H, V370N, T371Q, T371H, T371N, V373Q, V373H, V373N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, G386Q, G386H, G386N, A390Q, A390H, A390N, G393Q, G393H, G393N, G396Q, G396H, G396N, I397Q, I397H, I397N, T399Q, T399H, T399N, V401Q, V401H, V401N, S402Q, S402H, S402N, V405Q, V405H, V405N, I408Q, I408H, I408N, T412Q, T412H, T412N, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

DETAILED DESCRIPTION

Outline

A. Definitions

B. Hemostasis overview

- 1. Clot initiation
- 2. Signal Amplification and the clotting cascade
- 3. Termination of coagulation and fibrinolysis

C. Factor IX (FIX)

- 1. FIX structure and function
- 2. FIX polypeptides
- 3. FIX as a biopharmaceutical

D. Exemplary methods for modifying FIX

- 1. Non-Restricted Rational Mutagenesis One-Dimensional (1D)-Scanning
- 2. Two dimensional (2D) rational scanning (restricted rational mutagenesis)
  - a. Identifying in-silico HITS
  - b. Identifying replacing amino acids
  - c. Construction of mutant proteins and biological assays
- 3. Three dimensional (3D) scanning
  - a. Homology
  - b. 3D-scanning (structural homology) methods
- 4. Super-LEADs and additive directional mutagenesis (ADM)
- 5. Multi-overlapped primer extensions

E. Modified FIX polypeptides exhibiting increased protein stability

- 1. Protease resistance
  - a. Serine Proteases
  - b. Matrix Metalloproteinases
  - c. Increased resistance to proteolysis by removal of proteolytic sites
  - d. Modified FIX polypeptides exhibiting increased protease resistance
    - i. Modified FIX polypeptides exhibiting increased protease resistance to elastase only
  - e. Assessment of FIX variants with increased resistance to proteolysis
- 2. Super-LEADs
- 3. Other FIX modifications
  - a. Immunogenicity
  - b. Glycosylation
  - c. Additional modifications

F. Production of FIX polypeptides

- 1. Expression systems
  - a. Prokaryotic expression
  - b. Yeast
  - c. Insects and insect cells
  - d. Mammalian cells
  - e. Plants
- 2. Purification
- 3. Fusion proteins
- 4. Polypeptide modification
- 5. Nucleotide sequences

G. Assessing modified FIX polypeptide properties and activities

- 1. In vitro assays
- 2. Non-human animal models
- 3. Clinical Assays

H. Formulation/Packaging/Administration

- 1. Administration of modified FIX polypeptides
  - a. Oral administration
- 2. Administration of nucleic acids encoding modified FIX polypeptides (gene therapy)

I. Therapeutic Uses

- 1. Hemophilia
- 2. Thrombotic diseases and conditions
  - a. Arterial thrombosis
  - b. Venous thrombosis and thromboembolism
    - i. Ischemic stroke
- 3. Acquired coagulation disorders
  - a. Disseminated intravascular coagulation (DIC)
  - b. Bacterial infection and periodontitis
  - c. Liver disease
  - d. Warfarin and Heparin-induced bleeding
  - e. Intracerebral hemorrhage

J. Combination Therapies

K. Articles of manufacture and kits

L. EXAMPLES

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

As used herein, a “factor IX” polypeptide (abbreviated herein as FIX) refers to any factor IX polypeptide, including but not limited to, a recombinantly produced polypeptide, a synthetically produced polypeptide and a factor IX polypeptide extracted or isolated from cells or tissues including, but not limited to, liver and blood. Alternative names that are used interchangeably for factor IX include Factor 9, Christmas factor, plasma thromboplastin component (PTC), coagulation factor IX, and serum factor IX. Abbreviations for factor IX include FIX and F9. Factor IX includes related polypeptides from different species including, but not limited to animals of human and non-human origin. Human factor DC (hFIX) includes factor IX, allelic variant isoforms, synthetic molecules from nucleic acids, protein isolated from human tissue and cells, and modified forms thereof. Exemplary unmodified mature human factor IX polypeptides include, but are not limited to, unmodified and wild-type native factor IX polypeptide (such as the polypeptide containing a sequence set forth in SEQ ID NO: 2) and the unmodified and wild-type precursor factor IX polypeptide that includes a propeptide and/or a signal peptide (such as, the precursor FIX polypeptide that has the sequence set forth in SEQ ID NO: 1), and a polymorphic wild-type native factor IX polypeptide that has a mutation at T148A (SEQ ID NO: 892; or its precursor containing a sequence of amino acids set forth in SEQ ID NO: 893). One of skill in the art would recognize that the referenced positions of the mature factor IX polypeptide (SEQ ID NO: 2) differ by 46 amino acid residues when compared to the precursor FIX polypeptide SEQ ID NO: 1, which is the factor IX polypeptide containing the signal peptide and propeptide sequences Thus, the first amino acid residue of SEQ ID NO: 2 “corresponds to” the forty-seventh (47^th) amino acid residue of SEQ ID NO: 1. The term “factor IX” also encompasses the active form of the factor IX polypeptide, also called factor IXa (FIXa), containing FIX light chain (SEQ ID NO: 894) and FIX heavy chain (SEQ ID NO: 896) linked by a disulfide bond between residues 132C and 289C. FIXa is produced from a mature FIX polypeptide (SEQ ID NO: 2) by proteolytic cleavage after amino acid residues R145 and R180. Proteolytic cleavage can be carried out, for example, by activated factor XI (FXIa) or the tissue factor/activated factor VII complex. The FIX polypeptides provided herein can be further modified, such as by chemical modification or post-translational modification. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

Factor IX includes factor IX from any species, including human and non-human species. FIX polypeptides of non-human origin include, but are not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, and other primate factor IX polypeptides. Exemplary FIX polypeptides of non-human origin include, for example, chimpanzee (Pan troglodytes, SEQ ID NO: 904), rhesus macaque (Macaca mulatta, SEQ ID NO: 912), mouse (Mus musculus, SEQ ID NO: 903), rat (Rattus norvegicus, SEQ ID NO: 907), Guinea pig (Cavia porcellus, SEQ ID NO: 900), pig (Sus scrofa, SEQ ID NO: 905), dog (Canis familiaris, SEQ ID NO: 899), cat (Fells catus, SEQ ID NO: 902), rabbit (Oryctolagus cuniculus, SEQ ID NO: 906), chicken (Gallus gallus, SEQ ID NO: 901), cow (Bos Taurus, SEQ ID NO: 898), sheep (Ovis aries, SEQ ID NO: 908), frog (Xenopus tropicalis, SEQ ID NO: 909), zebrafish (Danio rerio, SEQ ID NO: 910), Japanese pufferfish (Takifugu rubripes, SEQ ID NO: 911). Exemplary non-human FIX sequences that include a signal sequence and propeptide sequence include SEQ ID NOS: 899, 901-904, 909-912. Exemplary mature non-human FIX sequences that do not include a signal sequence and propeptide sequence include SEQ ID NOS: 898 and 905. Exemplary fragments of non-human FIX sequences include SEQ ID NOS: 900 and 906-908.

Human and non-human FIX polypeptides include FIX polypeptides, allelic variant isoforms, tissue-specific isoforms and allelic variants thereof, synthetic molecules prepared by translation of nucleic acids, proteins isolated from human and non-human tissue and cells, chimeric FIX polypeptides and modified forms thereof. Human and non-human FIX also include fragments or portions of FIX that are of sufficient length or include appropriate regions to retain at least one activity of the full-length mature polypeptide. Human and non-human FIX polypeptides also can include FIX polypeptides that are of sufficient length to inhibit one or more activities of a full-length mature FIX polypeptide (such as acts as an anticoagulant).

As used herein, corresponding residues refer to residues compared among or between two polypeptides that are allelic or species variants or other isoforms. One of skill in the art can readily identify residues that correspond between or among such polypeptides. For example, by aligning the sequences of factor IX polypeptides, one of skill in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. For example, Y1 of SEQ ID NO: 2 (mature factor IX) corresponds to Y47 of SEQ ID NO: 1 (precursor factor IX with signal peptide and propeptide sequences). In other instances, corresponding regions can be identified. For example, the gla domain Y1 through V46 of SEQ ID NO: 2 (mature factor IX) corresponds to Y47 through V92 of SEQ ID NO: 1 (precursor factor 1× with signal peptide and propeptide sequences). One skilled in the art also can employ conserved amino acid residues as guides to find corresponding amino acid residues between and among human and non-human sequences. For example, residues R145 and R180 of SEQ ID NO: 2 (human) correspond to R191 and R226 of SEQ ID NO: 904 (chimpanzee) or R192 and R236 of SEQ ID NO: 903 (mouse).

As used herein, an “active portion or fragment of a factor IX polypeptide” refers to a portion of a human or non-human FIX polypeptide that includes at least one modification provided herein and exhibits an activity, such as one or more activities of a full-length FIX polypeptide or possesses another activity. Such activities include, but are not limited to peptidase activity, any coagulant (also referred to as procoagulant) activity, anticoagulant activity or other activity. Activity can be any percentage of activity (more or less) of the full-length polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity compared to the full polypeptide. Assays to determine function or activity of modified forms of FIX include those known to those of skill in the art, and exemplary assays are included herein. Assays include, for example, activated partial thromboplastin time (aPTT). In one such assay, coagulation activity is measured as the time required for formation of a fibrin clot. Activity also includes activities possessed by a fragment or modified form that are not possessed by the full length polypeptide or unmodified polypeptide.

As used herein, “mature factor IX” refers to a FIX polypeptide that lacks a signal sequence and a propeptide sequence. Typically, a signal sequence targets a protein for secretion via the endoplasmic reticulum (ER)-golgi pathway and is cleaved following insertion into the ER during translation. A propeptide sequence typically functions in post-translational modification of the protein and is cleaved prior to secretion of the protein from the cell. Thus, a mature FIX polypeptide is typically a secreted protein. In one example, a mature human FIX polypeptide is set forth in SEQ ID NO: 2. The amino acid sequence set forth in SEQ ID NO: 2 differs from that of the precursor polypeptide set forth in SEQ ID NO: 1 in that SEQ ID NO: 2 is lacking the signal sequence, which includes residues 1-21 of SEQ ID NO: 1, and the propeptide sequence, which includes residues 29-46 of SEQ ID NO: 1.

As used herein, “native factor IX” refers to a FIX polypeptide encoded by a naturally occurring FIX gene that is present in an organism in nature, including a human or other animal. Included among native FIX polypeptides are the encoded precursor polypeptide, fragments thereof, and processed forms thereof, such as a mature form lacking the signal peptide as well as any pre- or post-translationally processed or modified form thereof. For example, humans express FIX. Exemplary native human FIX sequences are set forth in SEQ ID NOS: 1 (precursor FIX with a signal peptide and propeptide), 2 (mature FIX lacking a signal peptide), 893 (precursor FIX T148A allelic variant), or 892 (mature FIX T148A allelic variant). Also included among native FIX polypeptides are those that are post-translationally modified, including, but not limited to, modification by glycosylation, carboxylation, hydroxylation, sulfation, and phosphorylation. Native FIX polypeptides also include those that have been activated by proteolytic cleavage, typically at R145 or R180 of SEQ ID NO: 2. Other animals produce native FIX, and include, but are not limited to, chimpanzees, mice, rats, pigs, dogs, cats, rabbits, chickens, cows, sheep, frogs, and fish. Exemplary native FIX sequences from other animals are provided in SEQ ID NOS: 898-912.

As used herein, “activated Factor IX” or “FIXa” refers to a FIX polypeptide that has been proteolytically cleaved to activate the peptidase activity of the FIX polypeptide. Typically, proteolytic cleavage occurs at two sites in the mature FIX polypeptide, after R145 and R180 (SEQ ID NO: 2). Cleavage is typically carried out by activated factor XI (FXIa) in the presence of vitamin K. The cleavage products, FIX heavy chain and FIX light chain, which are held together by disulfide bonds, form the active FIX enzyme.

As used herein, a “zymogen” refers to any compound, such as a polypeptide, that is an inactive precursor of an enzyme and requires some change, such as proteolysis of the polypeptide, to become active. For example, FIX polypeptides exist in the blood plasma as zymogens until activation of the coagulation cascade, whereby the FIX polypeptides are cleaved by activated FXI.

As used herein, “unmodified target protein,” “unmodified protein,” “unmodified polypeptide,” “unmodified coagulation factor,” “unmodified FIX” and grammatical variations thereof refer to a starting polypeptide that is selected for modification as provided herein. The starting target polypeptide can be a naturally-occurring, wild-type form of a polypeptide. In addition, the starting target polypeptide can be altered or mutated, such that it differs from a native wild type isoform but is nonetheless referred to herein as a starting unmodified target protein relative to the subsequently modified polypeptides produced herein. Thus, existing proteins known in the art that have been modified to have a desired increase or decrease in a particular activity or property compared to an unmodified reference protein can be selected and used as the starting unmodified target protein. For example, a protein that has been modified from its native form by one or more single amino acid changes and possesses either an increase or decrease in a desired property, such as reduced immunogenicity (see such as, US-2004-0254106) or a change in an amino acid residue or residues to alter glycosylation, can be a target protein, referred to herein as unmodified, for further modification of either the same or a different property. Exemplary modified FIX polypeptides known in the art include any FIX polypeptide described in, for example, U.S. Pat. Nos. 6,277,618, 6,315,995, and 6,531,298 and U.S. Patent Publication Nos. 2004-0102388, 2004-0110675, 2004-0254106, 2005-0100982, and 2006-0040856.

Existing proteins known in the art that previously have been modified to have a desired alteration, such as an increase or decrease, in a particular biological activity or property compared to an unmodified or reference protein can be selected and used as provided herein for identification of structurally homologous loci on other structurally homologous target proteins. For example, a protein that has been modified by one or more single amino acid changes and possesses either an increase or decrease in a desired property or activity, such as for example resistance to proteolysis, can be utilized with the methods provided herein to identify on structurally homologous target proteins, corresponding structurally homologous loci that can be replaced with suitable replacing amino acids and tested for either an increase or decrease in the desired activity.

As used herein, an “activity” or a “functional activity” of a FIX polypeptide refers to any activity exhibited by a factor IX polypeptide. Such activities will depend on the form of the FIX polypeptide. For example, active FIX polypeptide typically exhibits coagulant or procoagulant activity, whereas a zymogen or other inactive form of a FIX polypeptide typically has anticoagulant activity. Activities of a FIX polypeptide can be tested in vitro and/or in vivo and include, but are not limited to, coagulation activity, anticoagulation activity, enzymatic activity, and peptidase activity. Activity can be assessed in vitro or in vivo using recognized assays, for example, by measuring coagulation in vitro or in vivo. The results of such assays that indicate that a polypeptide exhibits an activity can be correlated to activity of the polypeptide in vivo, in which in vivo activity can be referred to as biological activity. Activity can be any level of percentage of activity of the polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more of activity compared to the full polypeptide. Assays to determine functionality or activity of modified forms of FIX are known to those of skill in the art. Exemplary assays to assess the functional activity of a FIX polypeptide include activated partial thromboplastin time (aPTT) assays, and are described in Example 3.

As used herein, “exhibits at least one activity” or “retains at least one activity” refers to the activity exhibited by a modified FIX polypeptide as compared to an unmodified FIX polypeptide of the same form and under the same conditions. For example, a modified FIX polypeptide that has been activated by cleavage into its heavy and light chain form is compared with an unmodified FIX polypeptide that also has been activated by cleavage into its heavy and light chain form, under the same experimental conditions, where the only difference between the two polypeptides is the modification under study. In another example, a modified FIX polypeptide in a zymogen form is compared with an unmodified FIX polypeptide in a zymogen form, under the same experimental conditions, where the only difference between the two polypeptides is the modification under study. Generally, a modified FIX polypeptide that retains an activity of an unmodified FIX polypeptide either improves or maintains the requisite biological activity (such as, coagulation activity) of an unmodified FIX polypeptide. In some instances, a modified FIX polypeptide can retain an activity that is increased compared to an unmodified FIX polypeptide. In some cases, a modified FIX polypeptide can retain an activity that is decreased compared to an unmodified FIX polypeptide. Activity of a modified FIX polypeptide can be any level of percentage of activity of the unmodified polypeptide, including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity compared to the unmodified polypeptide. For example, a modified FIX polypeptide retains at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% of the activity of the wild-type FIX polypeptide. In other embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times greater than unmodified FIX. Activity can be measured, for example, using assays such as those described in the Examples below.

As used herein, a “property” of a factor IX polypeptide refers to any property exhibited by a factor IX polypeptide. Such properties include, but are not limited to, protein stability, resistance to proteolysis, conformational stability, thermal tolerance, and tolerance to pH conditions. Changes in properties can alter an “activity” of the polypeptide.

As used herein, “protein stability” refers to increased protein-half-life under one or more conditions including, but not limited to, exposure to proteases, increased temperature, particular pH conditions and/or exposure to denaturing ingredients. Increased protein stability exhibited by a FIX polypeptide can be manifested as increased protease resistance, or increased conformational stability such as increased tolerance to temperature, pH, or tolerance to other denaturing ingredients. A modified polypeptide that exhibits increased protein stability in vitro or in vivo is, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more stable than an unmodified polypeptide. The protein stability of a polypeptide, for example, can be assessed in assays of protease resistance or conformational stability (i.e. resistance to temperature) to determine if an activity of the polypeptide is altered, such as is described in the Examples below. For example, the resistance of the modified FIX polypeptides compared to wild-type FIX against enzymatic cleavage by proteases (such as, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, and trypsin) can be empirically tested by treating the polypeptides with proteases over time and then testing the polypeptides for residual functional activity such as for example, coagulation activities.

As used herein, “serum stability” refers to protein stability in serum.

As used herein, “resistance to proteolysis” refers to any amount of decreased cleavage of polypeptide by a proteolytic agent, such as a protease such that the resulting polypeptide exhibits a changed therapeutic property, such as increase half-life, increased bioavailability, particularly upon oral administration or other such properties. With respect to FIX polypeptides for which therapeutic activity requires activation, the proteases to which resistance is increased do not include required to activate the polypeptide, such as a protease that cleaves a zymogen into its active form. For example, FIX and modified FIX polypeptides provided herein when present in an inactive zymogen form require cleavage by factor XI (FXIa) or the tissue factor/activated factor VII complex to generate active Factor IX. Hence, reference to a FIX polypeptide that exhibits resistance to proteolysis or increased resistance to proteolysis refers to a polypeptide that exhibits resistance to one or more proteases, including, but not limited to, proteases of the gastrointestinal tract, digestive system, bloodstream, and serum, but retains the ability to be cleaved by factor XI (FXIa) or the tissue factor/activated factor VII complex

Resistance to proteolysis can be achieved by modifying particular amino acid residues that are susceptible to cleavage by a particular protease to render them less susceptible to cleavage compared to cleavage by the same protease under the same conditions. A modified polypeptide that exhibits increased resistance to proteolysis exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more resistance to proteolysis than an unmodified polypeptide.

As used herein, a FIX polypeptide that is resistant to proteolysis or is resistant to protease cleavage or similar terminology, is a modified FIX polypeptide that exhibits increased resistance to a protease or proteases compared to a FIX polypeptide that does not include such modification. Modifications refer to changes in the primary amino acid sequence, unless noted otherwise.

As used herein, “conformational stability” refers to any amount of increased tolerance of a polypeptide to denaturation. This can be achieved by modifying particular amino acid residues that are susceptible to denaturation conditions to render them less susceptible to denaturation under the same conditions. Conformational stability can be determined by assessing the resistance or susceptibility of a polypeptide to denaturation conditions, such as resistance to temperature or pH. A modified polypeptide that exhibits increased conformational stability exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more resistance to denaturation than an unmodified polypeptide.

As used herein, “denaturation” refers to any noncovalent change in the structure of a protein. This change can alter the secondary, tertiary and/or quaternary structure of the polypeptide molecule. Denaturation of a polypeptide can occur by, for example but not limited to, exposure to chaotropic agents such as urea and guanidine hydrochloride, detergents, temperature, pH, and reagents which cleave disulfide bridges such as dithiothreitol or dithiothreitol.

As used herein, “thermal tolerance” refers to any temperature affected or dependent change in the stability of a protein. For example, a change, such as an increased thermal tolerance, can be reflected in a decreased amount of denaturation of a polypeptide after exposure to altered (particularly increased) temperatures compared to the unmodified protein under the same conditions. A modified polypeptide that exhibits increased thermal tolerance exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more stability at varied temperatures compared to the unmodified polypeptide. For example, a modified polypeptide can exhibit increased thermal tolerance in vivo when administered to a subject compared to an unmodified polypeptide, and thereby exhibit increased serum half-life.

As used herein, “EC₅₀” refers to the effective concentration of FIX necessary to give one-half of a maximum response. For purposes herein, the response measured is any activity of a FIX polypeptide, such as but not limited to, activity in a coagulation activity assay.

As used herein, “half-life” refers to the time required for a measured parameter, such the potency, activity and effective concentration of a polypeptide molecule to fall to half of its original level, such as half of its original potency, activity, or effective concentration at time zero. Thus, the parameter, such as potency, activity, or effective concentration of a polypeptide molecule is generally measured over time. For purposes herein, half-life can be measured in vitro or in vivo. For example, the half-life of FIX or a modified FIX polypeptide can be measured in vitro by assessing its activity (i.e. coagulation activity) following incubation over increasing time under certain conditions, such as for example, after exposure to proteases, or denaturing conditions such as temperature of pH. In another example, the half-life of FIX or a modified FIX polypeptide can be measured in vivo following administration (i.e. intravenous, subcutaneous, intraduodenal, oral) of the polypeptide to a human or other animal, followed by sampling of the blood over time to determine the remaining effective concentration and/or activity of the polypeptide in the blood sample.

As used herein, formulated for oral administration refers to a formulation for ingestion into the digestive system.

As used herein, mucosal delivery refers to delivery via the mucosa and, while mucosal administration can be into the mouth, its delivery does not involve the digestive system.

As used herein, “proteases,” “proteinases” or “peptidases” are interchangeably used to refer to enzymes that catalyze the hydrolysis of covalent peptidic bonds. Proteases include, for example, serine proteases and matrix metalloproteinases. Serine protease or serine endopeptidases constitute a class of peptidases, which are characterized by the presence of a serine residue in the active center of the enzyme. Serine proteases participate in a wide range of functions in the body, including blood clotting, inflammation as well as digestive enzymes in prokaryotes and eukaryotes. The mechanism of cleavage by “serine proteases,” is based on nucleophilic attack of a targeted peptidic bond by a serine. Cysteine, threonine, or water molecules associated with aspartate or metals also can play this role. Aligned side chains of serine, histidine, and aspartate form a catalytic triad common to most serine proteases. The active site of serine proteases is shaped as a cleft where the polypeptide substrate binds. Amino acid residues are labeled from N to C termini of a polypeptide substrate (Pi, . . . , P3, P2, P1, P1′, P2′, P3′, . . . , Pj). The respective binding sub-sites are labeled (Si, . . . , S3, S2, S1, S1′, S2′, S3′, . . . , Sj). The cleavage is catalyzed between P1 and P1′.

As used herein, a matrix metalloproteinases (MMP) refers to any of a family of metal-dependent, such as Zn²⁺-dependent, endopeptidases that degrade components of the extracellular matrix (ECM). MMPs include four classes: collagenases, stromelysin, membrane-type metalloproteinases, and gelatinases. Proteolytic activities of MMPs and plasminogen activators, and their inhibitors, are important for maintaining the integrity of the ECM. Cell-ECM interactions influence and mediate a wide range of processes including proliferation, differentiation, adhesion, and migration of a variety of cell types. MMPs also process a number of cell-surface cytokines, receptors and other soluble proteins and are involved in tissue remodeling processes such as wound healing, pregnancy and angiogenesis. Under physiological conditions in vivo, MMPs are synthesized as inactive precursors (zymogens) and are cleaved to produce an active form. Additionally, the enzymes are specifically regulated by endogenous inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs).

As used herein, “factor IX-mediated disease or disorder” refers to any disease or disorder in which treatment with a factor IX (or modified factor IX) ameliorates any symptom or manifestation of the disease or disorder. Exemplary factor IX-mediated diseases and disorders include, but are not limited to, hemorrhagic disorders, such as hemophilia or thrombotic disorders, such as thromboembolism. Accordingly, a disease or condition that is treated by administration of FIX includes any disease or condition for which factor IX (or modified factor IX) is employed for treatment, including, but not limited to, hemorrhagic disorders, such as hemophilia or thrombotic disorders, such as thromboembolism.

As used herein, “procoagulant” refers to any substance that promotes blood coagulation.

As used herein, “anticoagulant” refers to any substance that inhibits blood coagulation.

As used herein, “hemophilia” refers to a bleeding disorder caused by or involving a deficiency in blood clotting factors. Hemophilia can be the result, for example, of absence, reduced expression, or reduced function of a clotting factor. The most common type of hemophilia is hemophilia A, which results from a deficiency in factor VIII. The second most common type of hemophilia is hemophilia B, which results from a deficiency in factor IX. Another, more rare form of hemophilia is hemophilia C, which results from a deficiency in factor XI.

As used herein, “congenital hemophilia” refers to types of hemophilia that are inherited. Congenital hemophilia results from mutation, deletion, insertion, or other modification of a clotting factor gene in which the production of the clotting factor is absent, reduced, or non-functional. For example, hereditary mutations in clotting factor genes, such as factor VIII and factor IX result in the congenital hemophilias, Hemophilia A and B, respectively.

As used herein, a “hereditary mutation” is a gene alteration that is passed from parent to offspring.

As used herein, “acquired hemophilia” refers to hemophilia that results from clotting factor deficiencies caused by conditions such as liver disease, vitamin K deficiency, or coumadin (warfarin) or other anti-coagulant therapy.

As used herein, “hemostasis” refers to the stopping of bleeding or blood flow in an organ or body part. The term hemostasis can encompass the entire process of blood clotting to prevent blood loss following blood vessel injury to subsequent dissolution of the blot clot following tissue repair.

As used herein fibrinolysis refers to the dissolution of a blood clot.

As used herein, thrombosis refers to the formation of a clot, or thrombus, inside a blood vessel, obstructing the flow of blood. The vessel can be any vein or artery as, for example, in a deep vein thrombosis or a coronary artery thrombosis.

As used herein, a thromboembolism is a clot that breaks loose and travels through the bloodstream.

As used herein, “thrombotic disease or condition” or “thrombolytic disease or disorder” are used interchangeably to refer to a disease characterized by the overproduction of blot clots. The clots can often break off and travel through the blood stream, causing damage to tissues, such as the lungs (pulmonary embolism), brain (ischemic stroke, transient ischemic attack), heart (heart attack/myocardial infarction, unstable angina), skin (purpura fulminans), and adrenal gland (hemorrhage). The clots also can cause deep vein thrombosis, low blood pressure (hypotension), and salt loss.

As used herein, “treating” a subject with a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain static following treatment.

As used herein, treatment encompasses prophylaxis, therapy, and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. For example, treatment encompasses any pharmaceutical use of a modified factor IX and compositions provided herein.

As used herein, prevention refers to absolute prevention of a particular disease disorder. Since it generally is not possible to ascertain whether a disease or disorder never developed, prevention also includes reduction in the risk of developing or having a disease or disorder.

As used herein, a composition that does not include exogenously added proteases is a composition formulated without the addition of proteases, other than FIX or other active ingredient. Any additional proteases present in the composition would originate from the method of formulation.

As used herein, FIX is modified to be protease resistant by changes in primary structure. Among these are changes that do not result in changes in post-translational modification at that site.

As used herein, “therapeutically effective amount” or “therapeutically effective dose” refers to an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect.

As used herein, “subject” to be treated includes humans and human or non-human animals. Mammals include, primates, such as humans, chimpanzees, gorillas and monkeys; domesticated animals, such as dogs, horses, cats, pigs, goats, cows, and rodents, such as mice, rats, hamsters and gerbils.

As used herein, a patient is a human subject.

As used herein, “a directed evolution method” refers to methods that “adapt” either proteins, including natural proteins, synthetic proteins or protein domains to have changed proportions, such as the ability to act in different or existing natural or artificial chemical or biological environments and/or to elicit new functions and/or to increase or decrease a given activity, and/or to modulate a given feature. Exemplary directed evolution methods include, among others, rational directed evolution methods described in U.S. Published Application Nos. US 2003-0134351 A1 and US-2004-0132977-A1.

As used herein, “two dimensional rational mutagenesis scanning (2-D scanning)” refers to the processes provided herein in which two dimensions of a particular protein sequence are scanned: (1) one dimension is to identify specific amino acid residues along the protein sequence to replace with different amino acids, referred to as is-HIT target positions, and (2) the second dimension is the amino acid type selected for replacing the particular is-HIT target, referred to as the replacing amino acid.

As used herein, “in silico” refers to research and experiments performed using a computer. In silico methods include, but are not limited to, molecular modeling studies and biomolecular docking experiments.

As used herein, “is-HIT” refers to an in silico identified amino acid position along a target protein sequence that has been identified based on i) the particular protein properties to be evolved, ii) the protein's sequence of amino acids, and/or iii) the known properties of the individual amino acids. These is-HIT loci on the protein sequence are identified without use of experimental biological methods. For example, once the protein feature(s) to be optimized is (are) selected, diverse sources of information or previous knowledge (i.e., protein primary, secondary or tertiary structures, literature, patents) are exploited to determine those amino acid positions that are amenable to improved protein fitness by replacement with a different amino acid. This step uses protein analysis “in silico.” All possible candidate amino acid positions along a target protein's primary sequence that might be involved in the feature being evolved are referred to herein as “in silico HITs” (“is-HITs”). The collection (library), of all is-HITs identified during this step represents the first dimension (target residue position) of the two-dimensional scanning methods provided herein.

As used herein, “amenable to providing the evolved predetermined property or activity” in the context of identifying is-HITs refers to an amino acid position on a protein that is contemplated, based on in silico analysis, to possess properties or features that when replaced result in the desired activity being evolved. The phrase “amenable to providing the evolved predetermined property or activity” in the context of identifying replacement amino acids refers to a particular amino acid type that is contemplated, based on in silico analysis, to possess properties or features that when used to replace the original amino acid in the unmodified starting protein result in the evolution of a desired or preselected activity.

As used herein, “high-throughput screening” (HTS) refers to processes that test a large number of samples, such as samples of test proteins or cells containing nucleic acids encoding the proteins of interest to identify structures of interest or to identify test compounds that interact with the variant proteins or cells containing them. HTS operations are amenable to automation and are typically computerized to handle sample preparation, assay procedures, and the subsequent processing of large volumes of data.

As used herein, the term “restricted,” in the context of the identification of is-HIT amino acid positions along the amino acid residues in a protein selected for amino acid replacement and/or the identification of replacing amino acids, means that fewer than all amino acids on the protein-backbone are selected for amino acid replacement and/or fewer than all of the remaining 19 amino acids available to replace the original amino acid present in the unmodified starting protein are selected for replacement. In particular embodiments of the methods provided herein, the is-HIT amino acid positions are restricted such that fewer than all amino acids on the protein-backbone are selected for amino acid replacement. In other embodiments, the replacing amino acids are restricted such that fewer than all of the remaining 19 amino acids available to replace the native amino acid present in the unmodified starting protein are selected as replacing amino acids. In an exemplary embodiment, both of the scans to identify is-HIT amino acid positions and the replacing amino acids are restricted such that fewer than all amino acids on the protein-backbone are selected for amino acid replacement and fewer than all of the remaining 19 amino acids available to replace the native amino acid are selected for replacement.

As used herein, “candidate LEADs” are mutant proteins that are designed to have an alteration in property, activity or other attribute, typically a predetermined or preselected property, activity or other attribute, such as a, chemical, physical or biological property or activity in which such alteration is sought. The alteration can add, alter, remove or otherwise change a property, activity or other attribute of a polypeptide. In the methods herein, candidate LEADs are generally generated by systematically replacing is-HITS loci in a protein or a domain thereof with typically a restricted subset, or all, of the remaining 19 amino acids, such as obtained using PAM analysis. Candidate LEADs can be generated by other methods known to those of skill in the art tested by the high throughput methods herein.

As used herein, “LEADs” are “candidate LEADs” whose property, activity or other attribute has been changed, optimized, improved, added or eliminated. For purposes herein a “LEAD” typically has activity with respect to a property or activity of interest in an unmodified polypeptide that exhibits such activity or property that differs by at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more from the unmodified and/or wild type (native) protein. In certain embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times greater than the activity of the unmodified and/or wild type (native) protein. The desired alteration, which can be either an increase or a reduction in activity, depends upon the function or property of interest (such as, at least about or 10%, at least about or 20%, etc.). The LEADs can be further optimized by replacement of a plurality (2 or more) of “is-HIT” target positions on the same protein molecule to generate “super-LEADs.”

As used herein, the term “super-LEAD” refers to protein mutants (variants) obtained by adding the single mutations present in two or more of the LEAD molecules in a single protein molecule. Accordingly, in the context of the modified proteins provided herein, the phrase “proteins comprising one or more single amino acid replacements” encompasses addition of two or more of the mutations described herein for one respective protein. For example, the modified proteins provided herein containing one or more single amino acid replacements can have any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the amino acid replacements at the disclosed replacement positions. The collection of super-LEAD mutant molecules is generated, tested, and phenotypically characterized one-by-one in addressable arrays. Super-LEAD mutant molecules are molecules containing a variable number and type of LEAD mutation. Those molecules displaying further improved fitness for the particular feature being evolved, are referred to as super-LEADs. Super-LEADs can be generated by other methods known to those of skill in the art and tested by the high throughput methods herein. For purposes herein, a super-LEAD typically has activity with respect to the function of interest that differs from the altered activity (or the new activity or eliminated activity) of a LEAD by a desired amount, such as at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more from the LEAD mutant from which it is derived. As with LEADs, the change in the activity for super-LEADs is dependent upon the activity that is being “evolved.” The desired alteration, which can be either an increase or a reduction in activity, depends upon the function or property of interest. The desired alteration also can be an addition or elimination of a property or activity.

As used herein, the phrase “altered loci” refers to the is-HIT amino acid positions in the LEADs or super-LEADs that are replaced with different replacing amino acids resulting in the desired altered phenotype or activity.

As used herein, an “exposed residue” presents more than 15% of its surface exposed to the solvent.

As used herein, the phrase “structural homology” refers to the degree of coincidence in space between two or more protein backbones. Protein backbones that adopt the same protein structure, fold and show similarity upon three-dimensional structural superposition in space can be considered structurally homologous. Structural homology is not based on sequence homology, but rather on three-dimensional homology. Two amino acids in two different proteins said to be homologous based on structural homology between those proteins do not necessarily need to be in sequence-based homologous regions. For example, protein backbones that have a root mean squared (RMS) deviation of less than 3.5, 3.0, 2.5, 2.0, 1.7 or 1.5 angstroms at a given space position or defined region between each other can be considered to be structurally homologous in that region and are referred to herein as having a “high coincidence” between their backbones. It is contemplated herein that substantially equivalent (such as, “structurally related”) amino acid positions that are located on two or more different protein sequences that share a certain degree of structural homology have comparable functional tasks; also referred to herein as “structurally homologous loci.” These two amino acids then can be said to be “structurally similar” or “structurally related” with each other, even if their precise primary linear positions on the sequences of amino acids, when these sequences are aligned, do not match with each other. Amino acids that are “structurally related” can be far away from each other in the primary protein sequences, when these sequences are aligned following the rules of classical sequence homology.

As used herein, a “structural homolog” is a protein that is generated by structural homology. Exemplary FIX structural homologs include many other vitamin K dependent plasma proteins, including, for example, prothrombin, factor VII, factor X, and protein C.

As used herein, “variant,” “factor IX variant,” “modified factor IX polypeptides” and “modified factor IX proteins” refers to a FIX polypeptide that has one or more mutations compared to an unmodified factor IX polypeptide. The one or more mutations can be one or amino acid replacements, insertions or deletions and any combination thereof. Typically, a modified FIX polypeptide has one or more modifications in its primary sequence compared to an unmodified FIX polypeptide. For example, a modified FIX polypeptide provided herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations compared to an unmodified FIX polypeptide. Modifications that confer a property (such as, increased protein stability or increased protease resistance) by virtue of a change in a primary amino acid sequence do not require a change in post-translational modification of the modified polypeptide to confer the property. Any length polypeptide is contemplated as long as the resulting polypeptide exhibits at least one FIX activity associated with a native FIX polypeptide or inhibits an activity at least one FIX activity associated with a native FIX polypeptide (such as acts as an anticoagulant).

As used herein, a “single amino acid replacement” refers to the replacement of one amino acid by another amino acid. The replacement can be by a natural amino acid or non-natural amino acids. When one amino acid is replaced by another amino acid in a protein, the total number of amino acids in the protein is unchanged.

As used herein, the phrase “only one amino acid replacement occurs on each target protein” refers to the modification of a target protein, such that it differs from the unmodified form of the target protein by a single amino acid change. For example, in one embodiment, mutagenesis is performed by the replacement of a single amino acid residue at only one is-HIT target position on the protein backbone (such as, “one-by-one” in addressable arrays), such that each individual mutant generated is the single product of each single mutagenesis reaction. The single amino acid replacement mutagenesis reactions are repeated for each of the replacing amino acids selected at each of the is-HIT target positions. Thus, a plurality of mutant protein molecules are produced, whereby each mutant protein contains a single amino acid replacement at only one of the is-HIT target positions.

As used herein, the phrase “pseudo-wild type,” in the context of single or multiple amino acid replacements, are those amino acids that, while different from the original (such as, such as native) amino acid at a given amino acid position, can replace the native one at that position without introducing any measurable change in a particular protein activity. A population (library) of sets of nucleic acid molecules encoding a collection of mutant molecules is generated and phenotypically characterized such that proteins with sequences of amino acids different from the original amino acid, but that still elicit substantially the same level (i.e., at least about or 10%, 50%, 80%, 90%, 95%, 100%, depending upon the protein) and type of desired activity as the original protein are selected. A library, contains three, four, five, 10, 50, 100, 500, 1000, 10³, 10⁴or more modified FIX polypeptides.

As used herein, “in a position or positions corresponding to an amino acid position” of a protein, refers to amino acid positions that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference protein. For example, in a position corresponding to an amino acid position of human FIX set forth as SEQ ID NO: 2 can be determined empirically by aligning the sequence of amino acids set forth in SEQ ID NO: 2 with a particular FIX polypeptide of interest. Corresponding positions can be determined by such alignment by one of skill in the art using manual alignments or by using the numerous alignment programs available (for example, BLASTP). Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure. Recitation that amino acids of a polypeptide correspond to amino acids in a disclosed sequence refers to amino acids identified upon alignment of the polypeptide with the disclosed sequence to maximize identity or homology (where conserved amino acids are aligned) using a standard alignment algorithm, such as the GAP algorithm.

As used herein, “at a position corresponding to” refers to a position of interest (i.e., base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. The position of interest to the position in another reference protein can be in, for example, a precursor protein, an allelic variant, a heterologous protein, an amino acid sequence from the same protein of another species, etc. Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 95%, 96%, 97%, 98% or 99% or more. The position of interest is then given the number assigned in the reference nucleic acid molecule.

As used herein, the terms “homology” and “identity”” are used interchangeably, but homology for proteins can include conservative amino acid changes. In general to identify corresponding positions the sequences of amino acids are aligned so that the highest order match is obtained (see, such as: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

As use herein, “sequence identity” refers to the number of identical amino acids (or nucleotide bases) in a comparison between a test and a reference polypeptide or polynucleotide. Homologous polypeptides refer to a pre-determined number of identical or homologous amino acid residues. Homology includes conservative amino acid substitutions as well identical residues. Sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Homologous nucleic acid molecules refer to a pre-determined number of identical or homologous nucleotides. Homology includes substitutions that do not change the encoded amino acid (i.e., “silent substitutions”) as well identical residues. Substantially homologous nucleic acid molecules hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid or along at least about 70%, 80%, or 90% of the full-length nucleic acid molecule of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule. (For determination of homology of proteins, conservative amino acids can be aligned as well as identical amino acids; in this case, percentage of identity and percentage homology vary). Whether any two nucleic acid molecules have nucleotide sequences (or any two polypeptides have amino acid sequences) that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 2444 (other programs include the GCG program package (Devereux, J., et al. (1984) Nucleic Acids Research 12(I): 387), BLASTP, BLASTN, FASTA (Atschul, S. F., et al. (1990) J. Molec. Biol. 215:403; Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994), and Carillo et al. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (such as, Needleman et al. (1970) J. Mol. Biol. 48: 443, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2: 482. Briefly, a GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as described by Schwartz and Dayhoff, eds. (1979) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide. In one non-limiting example, “at least 90% identical to” refers to percent identities from 90 to 100% relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) of amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, such as, 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

As used herein, “corresponding structurally-related” positions on two or more polypeptides, such as FIX polypeptides and other coagulation factors, refer to those amino acid positions determined based upon structural homology to maximize tri-dimensional overlapping between or among polypeptides.

As used herein, the phrase “sequence-related proteins” refers to proteins that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% amino acid sequence identity or homology with each other.

As used herein, families of non-related proteins or “sequence-non-related proteins” refer to proteins having less than 50%, less than 40%, less than 30%, less than 20% amino acid identity, or homology with each other.

As used herein, it also is understood that the terms “substantially identical” or “similar” varies with the context as understood by those skilled in the relevant art.

As used herein, “a naked polypeptide chain” refers to a polypeptide that is not post-translationally modified or otherwise chemically modified, but contains only covalently linked amino acids.

As used herein, a polypeptide complex includes polypeptides produced by chemical modification or post-translational modification. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

As used herein, “output signal” refers to parameters that can be followed over time and, optionally, quantified. For example, when a recombinant protein is introduced into a cell, the cell containing the recombinant protein undergoes a number of changes. Any such change that can be monitored and used to assess the transformation or transfection is an output signal, and the cell is referred to as a reporter cell; the encoding nucleic acid is referred to as a reporter gene; and the construct that includes the encoding nucleic acid is a reporter construct. Output signals include, but are not limited to, enzyme activity, fluorescence, luminescence, amount of product produced, and other such signals. Output signals include expression of a gene or gene product, including heterologous genes (transgenes) inserted into the plasmid virus. Output signals are a function of time (“t”) and are related to the amount of protein used in the composition. For higher concentrations of protein, the output signal can be higher or lower. For any particular concentration, the output signal increases as a function of time until a plateau is reached. Output signals also can measure the interaction between cells, expressing heterologous genes and biological agents.

As used herein, a population of sets of nucleic acid molecules encoding a collection (library) of mutants refers to a collection of plasmids or other vehicles that carry (encode) the gene variants. Thus, individual plasmids or other individual vehicles carry individual gene variants. Each element (member) of the collection is physically separated from the others in an appropriate addressable array and has been generated as the single product of an independent mutagenesis reaction. When a collection (library) of such proteins is contemplated, it will be so-stated. A library contains three, four, five, 10, 50, 100, 500, 1000, 10³, 10⁴or more modified FIX polypeptides.

As used herein, a “reporter cell” is the cell that undergoes the change in response to a condition. For example, in response to exposure to a protein or a virus or to a change in its external or internal environment, the reporter cell “reports” (i.e., displays or exhibits the change).

As used herein, “reporter” or “reporter moiety” refers to any moiety that allows for the detection of a molecule of interest, such as a protein expressed by a cell. Reporter moieties include, but are not limited to, fluorescent proteins (such as, red, blue, and green fluorescent proteins), LacZ and other detectable proteins and gene products. For expression in cells, nucleic acids encoding the reporter moiety can be expressed as a fusion protein with a protein of interest or under the control of a promoter of interest.

As used herein, phenotype refers to the physical, physiological, or other manifestation of a genotype (a sequence of a gene). In methods herein, phenotypes that result from alteration of a genotype are assessed.

As used herein, culture medium is any medium suitable for supporting the viability, growth, and/or differentiation of mammalian cells ex vivo. Any such medium is known to those of skill in the art. Examples of culture medium include, but are not limited to, X-Vivo15 (BioWhittaker), RPMI 1640, DMEM, Ham=s F12, McCoys 5A and Medium 199. The medium can be supplemented with additional ingredients including serum, serum proteins, growth suppressing, and growth promoting substances, such as mitogenic monoclonal antibodies and selective agents for selecting genetically engineered or modified cells.

As used herein, the amino acids that occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide comprises two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids, and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain).

As used herein, the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (1972) Biochem. 11: 1726). Each naturally occurring L-amino acid is identified by the standard three letter code (or single letter code) or the standard three letter code (or single letter code) with the prefix “L-;” the prefix “D-” indicates that the stereoisomeric form of the amino acid is D.

As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. “NH₂” refers to the free amino group present at the amino terminus of a polypeptide. “COOH” refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in (1969) J. Biol. Chem., 243: 3552-3559, and adopted 37 C.F.R. □§§1.821-1.822, abbreviations for amino acid residues are shown in Table 1:

TABLE 1

Table of Correspondence

SYMBOL

1-Letter
3-Letter
AMINO ACID

Y
Tyr
tyrosine

G
Gly
glycine

F
Phe
phenylalanine

M
Met
methionine

A
Ala
alanine

S
Ser
serine

I
Ile
isoleucine

L
Leu
leucine

T
Thr
threonine

V
Val
valine

P
Pro
proline

K
Lys
lysine

H
His
histidine

Q
Gln
glutamine

E
Glu
glutamic acid

Z
Glx
Glu and/or Gln

W
Trp
tryptophan

R
Arg
arginine

D
Asp
aspartic acid

N
Asn
asparagine

B
Asx
Asn and/or Asp

C
Cys
cysteine

X
Xaa
Unknown or other

All amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH₂or to a carboxyl-terminal group such as COOH.

As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.

As used herein, the term “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary non-natural amino acids are described herein and are known to those of skill in the art.

As used herein, nucleic acids include DNA, RNA, and analogs thereof, including protein nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single- or double-stranded. When referring to probes or primers (optionally labeled with a detectable label, such as, a fluorescent or a radiolabel), single-stranded molecules are contemplated. Such molecules are typically of a length such that they are statistically unique of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 10, 15, 20, 25, or 30 contiguous nucleic acid bases of sequence complementary to, or identical to, a gene of interest. Probes and primers can be 5, 6, 7, 8, 9, 10, or more, 20 or more, 30 or more, 50 or more, 100, or more nucleic acids long.

As used herein, heterologous or foreign nucleic acid, such as DNA and RNA, are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it occurs or is found at a locus or loci in a genome that differs from that in which it occurs in nature. Heterologous nucleic acid includes nucleic acid not endogenous to the cell into which it is introduced, but that has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Heterologous DNA herein encompasses any DNA or RNA that one of skill in the art recognizes or considers as heterologous or foreign to the cell or locus in or at which it is expressed. Heterologous DNA and RNA also can encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes. Examples of heterologous nucleic acid include, but are not limited to, nucleic acid that encodes traceable marker proteins (such as, a protein that confers drug resistance), nucleic acid that encodes therapeutically effective substances (such as, anti-cancer agents), enzymes and hormones, and DNA that encodes other types of proteins (such as, antibodies). Hence, herein heterologous DNA or foreign DNA includes a DNA molecule not present in the exact orientation and position as the counterpart DNA molecule found in the genome. It also can refer to a DNA molecule from another organism or species (i.e., exogenous).

As used herein, “isolated with reference to a nucleic acid molecule or polypeptide or other biomolecule” means that the nucleic acid or polypeptide has separated from the genetic environment from which the polypeptide or nucleic acid were obtained. It also can mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated,” as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated. Also intended as an “isolated polypeptide” or an “isolated polynucleotide” are polypeptides or polynucleotides that have been partially or substantially purified from a recombinant host cell or from a native source. For example, a recombinantly produced version of a compound can be substantially purified by the one-step method described in Smith et al. (1988) Gene, 67:31-40. The terms isolated and purified can be used interchangeably.

Thus, by “isolated” it is meant that the nucleic acid is free of coding sequences of those genes that, in the naturally-occurring genome of the organism (if any), immediately flank the gene encoding the nucleic acid of interest. Isolated DNA can be single-stranded or double-stranded, and can be genomic DNA, cDNA, recombinant hybrid DNA or synthetic DNA. It can be identical to a starting DNA sequence or can differ from such sequence by the deletion, addition, or substitution of one or more nucleotides.

“Purified” preparations made from biological cells or hosts mean at least the purity of a cell extracts containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques, and the DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures can include, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, density gradient centrifugation, and electrophoresis.

A preparation of DNA or protein that is “substantially pure” or “isolated” refers to a preparation substantially free from naturally-occurring materials with which such DNA or protein is normally associated in nature and generally contains 5% or less of the other contaminants.

A cell extract that contains the DNA or protein of interest refers to a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest. The term “cell extract” is intended to include culture medium, especially spent culture medium from which the cells have been removed.

As used herein, “a targeting agent” refers to any molecule that can bind another target-molecule, such as an antibody, receptor, or ligand.

As used herein, “receptor” refers to a biologically active molecule that specifically binds to (or with) other molecules. The term “receptor protein” can be used to more specifically indicate the proteinaceous nature of a specific receptor.

As used herein, “recombinant” refers to any progeny formed as the result of genetic engineering.

As used herein, a “promoter region” refers to the portion of DNA of a gene that controls transcription of the DNA to which it is operatively linked. The promoter region includes specific sequences of DNA sufficient for RNA polymerase recognition, binding, and transcription initiation. This portion of the promoter region is referred to as the “promoter”. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. Promoters, depending upon the nature of the regulation, can be constitutive or regulated by cis acting or trans acting factors.

As used herein, the phrase “operatively linked” with reference to a nucleic acid molecule generally means the sequences or segments have been covalently joined into one piece of DNA, whether in single- or double-stranded form, whereby control or regulatory sequences on one segment control or permit expression or replication or other such control of other segments. The two segments are not necessarily contiguous. For gene expression, a DNA sequence and a regulatory sequence(s) are connected in such a way to control or permit gene expression when the appropriate molecular, such as, transcriptional activator proteins, are bound to the regulatory sequence(s).

As used herein, “production by recombinant means by using recombinant DNA methods” means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA, including cloning expression of genes and methods.

As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.

As used herein, a composition refers to any mixture of two or more products or compounds (such as, agents, modulators, regulators, etc.). It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous formulations or any combination thereof.

As used herein, a combination refers to any association between two or more items.

As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass pharmaceutical compositions of modified FIX polypeptides and/or nucleic acids as described herein contained in articles of packaging.

As used herein, a “kit” refers to a combination of modified FIX polypeptides or nucleic acid molecules as described herein provided in pharmaceutical compositions and another item for a purpose including, but not limited to, administration, diagnosis, and assessment of an activity or property of the polypeptides described herein. Kits, optionally, include instructions for use.

As used herein, “substantially identical to a product” means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.

As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. “Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of exemplary vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Exemplary vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked; such vectors typically include origins of replication. Vectors also can be designed for integration into host chromosomes. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Expression vectors are often in the form of “plasmids,” which refer generally to circular double-stranded DNA loops which, in their vector form are not bound to the chromosome. “Plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vectors. Other such forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.

As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

As used herein, “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof among a population. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation. Typically, allelic variants, have at least 80%, 90%, 95% or greater amino acid identity with a wild-type and/or predominant form from the same species.

As used herein, the terms “gene” or “recombinant gene” refer to a nucleic acid molecule containing an open reading frame and including at least one exon and, optionally, an intron-encoding sequence. A gene can be either RNA or DNA. Genes can include regions preceding and following the coding region (leader and trailer).

As used herein, “intron” refers to a DNA fragment that occurs in a gene, but that is spliced out during mRNA maturation.

As used herein, “nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1” refers to the nucleotide sequence of the complementary strand of a nucleic acid strand encoding a polypeptide that includes an amino acid sequence having the particular SEQ ID NO: 1. An exemplary nucleic acid sequence that encodes an amino acid sequence having the particular SEQ ID NO: 1 is set forth in SEQ ID NO: 897.

The term “complementary strand” is used herein interchangeably with the term “complement.” The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand. When referring to double-stranded nucleic acids, the complement of a nucleic acid encoding a polypeptide containing amino acid residues having a sequence set forth in a particular SEQ ID NO: 1 refers to the complementary strand of the strand encoding the amino acid sequence set forth in the particular SEQ ID NO: 1 or to any nucleic acid molecule containing the nucleotide sequence of the complementary strand of the particular nucleic acid sequence. When referring to a single-stranded nucleic acid molecule containing a nucleotide sequence, the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of the particular nucleic acid sequence.

As used herein, the term “coding sequence” refers to that portion of a gene that encodes a sequence of amino acids present in a protein.

As used herein, the term “sense strand” refers to that strand of a double-stranded nucleic acid molecule that has the sequence of the mRNA that encodes the sequence of amino acids encoded by the double-stranded nucleic acid molecule.

As used herein, the term “antisense strand” refers to that strand of a double-stranded nucleic acid molecule that is the complement of the sequence of the mRNA that encodes the sequence of amino acids encoded by the double-stranded nucleic acid molecule.

As used herein, an “array” refers to a collection of elements, such as nucleic acid molecules, containing three or more members. An addressable array is one in which the members of the array are identifiable, typically by position on a solid phase support or by virtue of an identifiable or detectable label, such as by color, fluorescence, electronic signal (i.e., RF, microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label. In certain embodiments, the members of the array are immobilized to discrete identifiable loci on the surface of a solid phase or directly or indirectly linked to or otherwise associated with the identifiable label, such as affixed to a microsphere or other particulate support (herein referred to as beads) and suspended in solution or spread out on a surface.

As used herein, a “support” (such as, a matrix support, a matrix, an insoluble support or solid support, etc.) refers to any solid or semisolid or insoluble support to which a molecule of interest (such as, a biological molecule, organic molecule or biospecific ligand) is linked or contacted. Such materials include any materials that are used as affinity matrices or supports for chemical and biological molecule syntheses and analyses, such as, but are not limited to: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, silicon, rubber, and other materials used as supports for solid phase syntheses, affinity separations and purifications, hybridization reactions, immunoassays and other such applications. The matrix herein can be particulate or can be in the form of a continuous surface, such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, or other such materials. When particulate, typically the particles have at least one dimension in the 5-10 mm range or smaller. Such particles, referred collectively herein as “beads,” are often, but not necessarily, spherical. Such reference, however, does not constrain the geometry of the matrix, which can be any shape, including random shapes, needles, fibers, and elongated. Roughly spherical “beads,” particularly microspheres that can be used in the liquid phase, also are contemplated. The “beads” can include additional components, such as magnetic or paramagnetic particles (see, for example, Dynabeads (Dynal, Oslo, Norway)) for separation using magnets as long as the additional components do not interfere with the methods and analyses herein.

As used herein, matrix or support particles refer to matrix materials that are in the form of discrete particles. The particles have any shape and dimensions, but typically have at least one dimension that is 100 mm or less, 50 mm or less, 10 mm or less, 1 mm or less, 100 μm or less, 50 μm or less and typically have a size that is 100 mm³or less, 50 mm³or less, 10 mm³or less, 1 mm³or less, 100 μm³or less and can be order of cubic microns. Such particles are collectively called “beads.”

B. HEMOSTASIS OVERVIEW

Provided herein are modified FIX polypeptides. These modified polypeptides have a variety of uses and applications. To appreciate the biological role of FIX polypeptides and some of their uses, an understanding of their role function in vivo is helpful. The following discussion provides such background.

The process by which the body responds to vascular injury involves a series of dependent signaling events that ultimately leads to the formation of a blot clot. Effective coagulation of blood is needed to prevent excessive blood loss at the site of injury. Once a blood vessel is injured or ruptured, vascular constriction, which limits blood flow to the area of the injury, accompanies the initial physiologic response. The rupture exposes highly thrombogenic subendothelial connective tissue that is composed of fibrillar collagen, which binds and activates platelets and stimulates platelet aggregation at the injury site. The activated platelets secrete factors which stimulate additional platelet activation, and other molecules, such as serotonin, phospholipids, and lipoproteins, which are important mediators of the clotting process. Fibrinogen, a component of the blood plasma also binds platelets and aids in clumping of the activated platelets. The activated platelets in turn undergo changes in cell shape to form a loose platelet plug. A clotting cascade of peptidases is simultaneously initiated that generates activated molecules such as thrombin (from cleavage of prothrombin), which further activates platelets, and fibrin (from cleavage of fibrinogen), which forms a cross-linked polymer around the platelet plug to stabilize the clot. During clot formation, coagulation factor inhibitors also circulate through the blood to prevent clot formation beyond the injury site.

Following repair of the injured tissue, the body must efficiently dissolve the clot in order to restore normal blood flow. Fibrinolysis is the process by which the clotting cascade is reduced and the clot is eliminated. Dissolution of the clot is mediated by the protein plasmin.

The processes of clot initiation, signal amplification, and fibrinolysis, and the factors involved are presented in further detail below.

1. Clot Initiation

Under normal conditions, vascular endothelium is resistant to clot formation. This resistance is supported by mechanisms that enhance vasodilation, inhibit platelet adhesion and activation, suppress coagulation, and promote fibrin cleavage. Vascular endothelial cells secrete molecules such as nitrous oxide (NO) and prostacylin, which inhibit platelet aggregation and dilate blood vessels. Release of these molecules activates soluble guanylate cyclases (sGC) and cGMP-dependent protein kinase I (cGKI) and increases cyclic guanosine monophosphate (cGMP) levels, which cause relaxation of the smooth muscle in the vessel wall. Furthermore, endothelial cells express cell-surface ADPases, such as CD39, which control platelet activation and aggregation by converting ADP released from platelets into adenine nucleotide platelet inhibitors. Other membrane proteins expressed on the surface of vascular endothelial cells include heparin sulfate proteoglycans that function as cofactors for antithrombin III, which inhibits thrombin and other coagulation factors. In addition, vascular endothelial cells secrete plasminogen to promote fibrinolysis, which lyses and clears clots.

Upon blood vessel injury, the damaged vessel wall exposes the subendothelial connective tissue to the circulating blood. In contrast to normal endothelial cells, this tissue is extremely thrombogenic. The layer is composed of proteins such as von Willebrand factor (vWF) and fibrillar collagen, which bind to circulating platelets via glycoprotein receptors expressed on the surface of the platelets. The platelet glycoprotein receptor complex GPIb-V-IX binds vWF, whereby vWF can bridge the platelets to the collagen fibrils. vWF is a multimeric glycoprotein, which contributes to its ability to aggregate platelets. vWF also functions to stabilize coagulation factors, such as factor VIII, and promotes their survival in the blood stream. Furthermore, the platelet integrin receptors α₂β₁and GPVI can bind to collagen directly. Binding of collagen to α₂β₁can facilitate binding to the lower affinity GPVI receptor. The collagen interactions with α₂β₁and GPVI contribute to platelet adherence to the site of injury and furthermore promote platelet activation through activation of intracellular signaling cascades.

2. Signal Amplification and the Clotting Cascade

During clot formation, positive feedback loops are activated that enhance the maturation of a clot. Following platelet activation, expression of the platelet surface integrin α_IIbβ₃is stimulated. α_IIbβ₃can bind to vWF and fibrinogen. Binding of GPIb-V-IX to vWF can enhance the affinity of α_IIbβ₃for vWF to promote platelet adhesion. Further, one fibrinogen molecule can bind to multiple α_IIbβ₃integrin molecules from different cells, thus, enhancing platelet aggregation.

Platelet activation stimulates the release of platelet alpha and dense granules. The released contents of the granules include factors, such as vWF and ADP, which further contribute to aggregation and platelet activation. ADP helps stimulate modification of the platelet membrane that allows binding of fibrinogen to α_IIbβ₃, and vWF contributes to adhesion and aggregation as discussed above.

In addition to ADP, activated platelets also secrete factors such as serotonin and thromboxane A₂(TXA₂) which also positively regulate platelet activation. Signal transduction cascades activated in response to thrombin binding control release of these factors. During platelet activation, thrombin binds to a G-protein coupled receptor on the surface of platelets. The receptor becomes stimulated, and the activated G-protein activates phospholipase C-γ 2 (PLCγ2), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate inositol triphosphate (IP₃) and diacylglycerol (DAG). IP₃stimulates release of intracellular calcium (Ca²⁺) stores from the endoplasmic reticulum by binding to IP₃receptors. The released calcium in combination with collagen binding to platelets leads to the activation of phospholipase A2 (PLA₂). PLA₂then hydrolyzes membrane phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), to generate arachidonic acid, which in turn stimulates the production and release of TXA₂.

The production of DAG by activated platelets stimulates protein kinase C, which then phosphorylates a platelet specific 47 kDa protein (p47). The phosphorylated p47 protein induces the release of the platelet granules as discussed above. Intracellular Ca²⁺ release also activates myosin light chain kinase (MLCK) that phosphorylates the light chain of myosin. The phosphorylated myosin then interacts with actin, bringing about a change in platelet morphology and motility, necessary for clot formation.

Platelet activation through collagen binding also elicits activation of a similar signaling cascade. When collagen binds to the GPVI integrin, it promotes clustering of GPVI with the Fc receptor γ-chain (FcγR). The clustering induces tyrosine phosphorylation of the FcγR by Src-family kinases Lyn and Fyn, which promote binding and activation of the tyrosine kinase Syk. Syk, in turn phosphorylates the transmembrane adapter protein LAT, which then assembles a signaling complex composed of proteins including phosphoinositide 3-kinase (PI3K), PLCγ2, adaptor proteins such as Gads, SLP-75, and SLAP-130, and the RhoGTP exchange factor Vav. PI3K can then regulate protein kinase B (PKB), phosphoinositide-dependent kinase (PDK1) and integrin-linked kinase (ILK), which can regulate integrin expression and signaling. PLCγ2 generates IP₃and DAG to mobilize calcium and activate PKC as described above.

For stable clot formation, cross-linked fibrin polymers must be formed surrounding the activated platelet plug. Fibrin formation is generated though activation of coagulation factor cascades which occurs simultaneously with platelet activation, following vessel injury. Two converging pathways can be activated, the intrinsic and extrinsic pathway. Key factors that participate in the intrinsic and extrinsic coagulation cascades are listed in Tables 2 and 3. The high-molecular weight kininogen, prekallikrein, and factors XII, XI, IX, and VIII participate in the intrinsic pathway, while tissue factor and the factor VII complex contribute to the extrinsic pathway. The two pathways converge at the activation of factor X (to generate FXa), through proteolytic action of the activated factor IX (FIXa)/factor VIII (FVIIIa) tenase complex (intrinsic) or by the activated factor VII (FVIIa)/tissue factor (TF) complex (extrinsic). Activated factor X (FXa), with the help of factor V, calcium, and platelet phospholipid, generates thrombin from prothrombin. In turn, thrombin cleaves fibrinogen to produce fibrin and also activates factor XIII (transglutamidase), which cross-links fibrin polymers to form the stable clot. The surface of activated platelets facilitates the formation of the activated coagulation factor complexes and thus participates in amplification of the cascade.

The primary pathway for initiation is the extrinsic pathway; the intrinsic pathway functions to amplify the production of activated factor X. Following vascular injury, tissue factor (TF) in the vessel wall is exposed and generated. TF is expressed by endothelial cells, subendothelial tissue, and monocytes. When the vessel wall is disrupted TF produced by the subendothelial layer is exposed. In the case of endothelial cell damage in the absence of a vessel wall lesion, contact is made between the blood and TF expressed on the damaged endothelial layer in the cell wall. vWF aids in the interaction of platelets with the damaged endothelium. TF binds to VII and in turn catalyzes the activation of factor X. The thrombin that is generated by the extrinsic pathway then contributes to activation of factor XI of the intrinsic cascade, leading to the activation of factor IX. Factor IX also can be activated directly by the TF/factor VII complex. The intrinsic cascade also can be activated via factor XII-mediated activation of factor XI when prekallikrein is converted to kallikrein in response to collagen contact with the vessel surface (called the contact pathway), though this pathway plays a minimal role in coagulation as compared to the amplification effects of thrombin. The contact pathway plays a more important role in fibrinolysis since factor XII and kallikrein can convert plasminogen into plasmin and bradykinin, which is a potent vasodilator and stimulator for tissue plasminogen activator (tPA).

3. Termination of Coagulation and Fibrinolysis

During coagulation constitutive and stimulated processes inhibit further clot formation. Antithrombin III and tissue factor pathway inhibitor (TFPI) work constitutively to inhibit factors in the coagulation cascade. Specifically, antithrombin III inhibits thrombin, FIXa, and FXa, where as TFPI inhibits FXa and FVIIa/TF complex. Protein C, which is stimulated via platelet activation, proteolytically inactivates FVa and FVIIIa. Further, another factor which contributes to coagulation inhibition is the integral membrane protein thrombomodulin, which is produced by vascular endothelial cells and binds thrombin. Complex formation inhibits thrombin procoagulant activities and also contributes to protein C activation.

Clearance of the fibrin clot is carried out by the serine protease plasmin, which can digest fibrin. Prior to activation of this enzyme, it circulates in the blood in the form of the proenzyme plasminogen. Plasminogen can bind to fibrin and fibrinogen, which allows it to be incorporated into the clot as it is formed. Cleavage of plasminogen into active plasmin is primarily carried out by activated tPA, which is secreted by endothelial cells following injury, through stimulation of tPA activity. Production of plasmin also can be initiated and enhanced by the actions of factor XII as discussed above.

TABLE 2

Coagulation Factors

Factor
Common Name
Pathway
Characteristic

I
Fibrinogen
Both
—

II
Prothrombin
Both
Contains N-terminal

gla segment

III
Tissue Factor
Extrinsic
—

IV
Calcium
Both
—

V
Proaccelerin, labile factor,
Both
Protein cofactor

Accelerator globulin

VI
Accelerin
—
(Redundant to factor V)

(Va)

VII
Proconvertin, serum prothrombin
Extrinsic
Endopeptidase with

conversion accelerator (SPCA)

gla residues

cothromboplastin

VIII
Antihemophiliac factor A,
Intrinsic
Protein cofactor

antihemophiliac globulin (AHG)

IX
Christmas factor, antihemophiliac
Intrinsic
Endopeptidase with

factor B, plasma thromboplastin

gla residues

component (PTC)

X
Stuart-prower factor
Both
Endopeptidase with

gla residues

XI
Plasma thromboplastin
Intrinsic
Endopeptidase

antecedent (PTA)

XII
Hageman factor
Intrinsic
Endopeptidase

XIII
Protransglutamidase, fibrin
Both
Transpeptidase

stabilizing factor (FSF),

fibrinoligase

*Table adapted from M. W. King (2006) med.unibs.it/~marchesi/blood.html

TABLE 3

Coagulation Factor Zymogens and Cofactors

Name of Factor
Activity

Zymogens of Serine Proteases

Factor XII
Binds exposed collagen at site of vessel

wall injury, activated by high-MW

kininogen and kallikrein

Factor XI
Activated by factor XIIa

Factor IX
Activated by factor XIa + Ca²⁺

Factor VII
Activated by thrombin + Ca²⁺

Factor X
Activated on platelet surface by tenase

complex;

Also activated by factor VIIa + tissue

factor + Ca²⁺

Factor II
Activated on platelet surface by

prothrombinase complex;

Cofactors

Factor VIII
Activated by thrombin; factor VIIa acts

as cofactor for factor IXa in activation of

factor X

Factor V
Activated by thrombin; factor Va acts as

cofactor for factor Xa in activation of

prothrombin

Factor III (Tissue factor)
Acts as cofactor for factor VII

Fibrinogen

Factor I
Cleaved by thrombin to form fibrin

Transglutaminase

Factor XIII
Activated by thrombin + Ca²⁺; promotes

covalent cross-linking of fibrin

Regulatory and other proteins

von Willebrand factor (vWF)
Acts as bridge between GPIb-V-IX

complex and collagen

Protein C
Activated by thrombin bound to

thrombomodulin; Ca degrades factors

VIIIa and Va

Protein S
Acts as cofactor of protein C

Thrombomodulin
Endothelial cell surface protein; binds

thrombin, which activates protein C

Antithrombin III
Coagulation inhibitor of thrombin, and

factors IXa, Xa, XIa, and XIIa.

*Table adapted from M. W. King (2006) med.unibs.it/~marchesi/blood.html

C. FACTOR IX (FIX)

Coagulation Factor IX (FIX) is a vitamin-K dependent serine protease produced in animals, including mammals, that plays a pivotal role in the blood clotting cascade that functions to seal injured blood vessels. FIX shows a high degree of homology to other vitamin-K dependent serine proteases, including factor VII, factor X, and protein C (Furie et al., 1988). FIX is synthesized in the liver as an inactive zymogen that is secreted into the blood stream. During the coagulation process, FIX is cleaved via the intrinsic pathway by active factor XIa to generate an active serine protease, FIXa. FIXa also is generated via the extrinsic pathway by factor VIIa/tissue factor (FVIIa/TF). FIXa binds to activated Factor VIII (FVIIIa) and catalyzes the conversion of factor X to its active form, factor Xa, which hydrolyzes prothrombin into active thrombin

Coagulation factors, including FIX, are used as therapeutic agents. Treatment with recombinant FIX is an established therapy. Recombinant FIX is used, for example, as a therapeutic for treatment of diseases such as hemophilia, in particular, hemophilia B. Patients receiving FIX are subject to frequent, repeated applications of the drug due to instability of FIX in the blood stream and under storage conditions. Hence, improved FIX stability (half-life) in serum or following oral administration and/or in in vitro applications can improve its activity and efficiency as a drug. Accordingly, provided herein are modified FIX polypeptides that display improved protein stability, such as by increased resistance to proteases, resulting in increased protein half-life. The modified polypeptides can possess increased stability in the bloodstream or following oral administration (in vivo) and/or under storage conditions (in vitro).

1. Factor IX Structure and Function

The human coagulation FIX gene is located on chromosome X at Xq27.1 and contains 8 exons of varying length from 25 base pairs to about 2000 base pairs. The mRNA for FIX is about 3 kilobases in length, comprising 205 bases of 5′ UTR (untranslated region), 1386 bases encoding the FIX polypeptide, and 1392 bases of 3′ UTR. The FIX mRNA encodes for a 461 amino acid precursor FIX polypeptide. The nucleotide coding sequence and the encoded precursor polypeptide are provided in SEQ ID NOS: 897 and 1, respectively.

The precursor FIX polypeptide comprises the following segments and domains: a hydrophobic signal peptide (aa 1-28 of SEQ ID NO: 1), a propeptide (aa 29-46 of SEQ ID NO: 1), a gla domain (aa 47-92 of SEQ ID NO: 1), a type B epidermal growth factor domain (EGF-like 1, aa 93-129 of SEQ ID NO: 1), a type A epidermal growth factor domain (EGF-like 2, aa 130-171 of SEQ ID NO: 1), an activation peptide (aa 192-226 of SEQ ID NO: 1), and a serine protease domain (aa 227-461 of SEQ ID NO: 1). The mature form of the FIX polypeptide lacks the signal peptide and propeptide sequences (SEQ ID NO: 2). In the mature form of the FIX polypeptide the corresponding amino acid positions for the above mentioned domains are as follows: gla domain (aa 1-46 of SEQ ID NO: 2), EGF-like 1 (aa 47-83 of SEQ ID NO: 2), EGF-like 2 (aa 84-125 of SEQ ID NO: 2), activation peptide (aa 146-180 of SEQ ID NO: 2), and serine protease domain (aa 181-415 of SEQ ID NO: 2). Amino acid positions for FIX polypeptides provided herein are given with respect to the mature form of the FIX polypeptide (SEQ ID NO: 2), unless otherwise noted.

The signal peptide at the N-terminus of the precursor FIX polypeptide (SEQ IS NO: 1) is 28 amino acids in length and functions to target the FIX polypeptide to the cellular secretory pathway by insertion into the endoplasmic reticulum (ER) during translation. The signal sequence is cleaved by a signal peptidase within the ER lumen. Once inside the ER lumen, the FIX polypeptide is post-translationally modified by N-linked and O-linked glycosylation, β-hydroxylation, and γ-carboxylation. β-hydroxylation and γ-carboxylation are completed in the ER, while glycosylation commences in the ER and finishes in the Golgi complex.

The propeptide, which is 18 amino acids in length, provides a binding site for a vitamin K-dependent carboxylase. The recognition element for the carboxylase is located in the N-terminal portion of the propeptide, which forms a 10 residue amphipathic α-helix. After binding, the carboxylase γ-carboxylates 12 glutamic acid residues within the gla domain of the FIX polypeptide, producing γ-carboxyglutamyl residues at positions E53, E54, E61, E63, E66, E67, E72, E73, E76, E79, E82, and E86 relative to the FIX precursor amino acid sequence set forth in SEQ ID NO: 1. These positions correspond to positions E7, E8, E15, E17, E20, E21, E26, E27, E30, E33, E36, and E40 relative to the mature FIX polypeptide set forth in SEQ ID NO: 2. The γ-carboxyglutamyl modifications aid in the correct folding of the mature FIX polypeptide and in the ability to FIX to bind calcium (Ca²⁺) and phospholipids. γ-Carboxyglutamyl residues located at amino acid positions 17, 21, and 27, relative to the mature FIX polypeptide (SEQ ID NO: 2), form ion pairs with the amino terminal alanine in prothrombin, which stabilize the structure of the gla domain and promote interaction with phospholipid membranes (Soriano-Garcia et al. (1992) Biochemistry 31:2554-2566). The C-terminal end of the gla domain also contains a conserved hydrophobic domain containing a Phe-Trp-X-X-Tyr motif, located at amino acid positions 41-45 relative to the mature FIX polypeptide (SEQ ID NO: 2), which is required for Ca²⁺ dependent changes in protein structure and for formation of a membrane binding site with the gla domain. In addition, proper folding of the gla domain enables binding of cofactor FVIIIa to FIXa, in order to form the complex which catalyzes the conversion of factor X into its active form, FXa. With respect to the other post-translational modifications of FIX, β-hydroxylation occurs at D64 to form β-hydroxyaspartic acid, N-linked glycosylation at N157 and N167, O-linked glycosylation at S53, S61, T159, T169, T172, and T179; sulfation at Y155; and phosphorylation of S68 and S158, relative to the mature FIX polypeptide, SEQ ID NO: 2 (Furie et al. (1988) Cell 53:505-518 and Kaufman et al. (1998) Thromb. Haemost 79:1068-1079).

After post-translation modification and prior to secretion, the propeptide is cleaved by a propeptidase to generate the mature FIX polypeptide, which is 415 amino acids in length (SEQ ID NO: 2). The recognition element for binding of the propeptidase is proposed to be located on the carboxyl side of the propeptide, closer to the site of cleavage. PACE/furin is a subtilisin-like calcium-dependent serine propeptidase localized to the trans Golgi compartment that has the ability to cleave the FIX propeptide into its mature form (Wasley et al. (1993) J Biol Chem. 268(12):8458-65). The name PACE is an acronym for Paired basic Amino acid Cleaving Enzyme, which describes the characteristic of the enzyme to cleave after a pair of basic amino acids, namely K45-R46 in FIX (positions relative to precursor FIX polypeptide as set forth in SEQ ID NO: 1; K-2 and R-1 relative to the mature FIX polypeptide set forth in SEQ ID NO: 2). The consensus motif for PACE cleavage is Arg-X-(Lys/Arg)-Arg. PACE/furin also functions to process the propeptides of other proteins, such as pro-von Willebrand factor, pro-nerve growth factor proalbumin, and complement pro-C3. PACE/furin is ubiquitously expressed and is likely to process additional proteins, including growth factors, receptors, and other coagulation factors. Other members of the PACE family of peptidases include PC1/3, PC2, PACE4, PC4, PC5/6 and PC7/SPC7/LPC/PC8, though functional and expression data suggest that PACE/furin is most likely the FIX propeptidase in vivo. Mutation of the tyrosine residue at position 1 of the mature FIX polypeptide (Y47 in the precursor FIX polypeptide) has been shown to increase the efficiency of cleavage of the propeptide sequence (Meulian et al. (1990) Protein Eng. 3(7):629-33).

The first EGF-like domain (EGF-like 1, EGF 1) is a type B EGF domain and contains the β-hydroxylation site at D64. β-Hydroxylation aids in the binding of additional Ca²⁺, which promotes binding of cofactor VIII to the domain. Two O-linked glycosylation sites are located within this domain at S53 and S61 (relative to the mature FIX polypeptide, SEQ ID NO: 2).

The second EGF-like domain (EGF-like 2, EGF2) is a type A EGF domain, which lacks β-hydroxylated residues compared to the type B domain. The EGF2 domain is responsible for binding to platelets and for assembly of the factor X activating complex (Wilkinson et al. (2002) J. Biol. Chem., 277(8):5734-5741). Formation of this complex on the surface of activated platelets promotes efficient hydrolysis of factor X.

The activation peptide contains the two cleavage sites, R145-A146 and R180-V181 (relative to mature FIX polypeptide, SEQ ID NO: 2) which are hydrolyzed by FXIa in the presence of Ca²⁺, or by FVIIa in the presence of TF, phospholipid, and Ca²⁺ to generate the activated form, FIXa. The activation peptide also contains two of the N-linked glycosylation sites at N157 and N167 and four of the O-linked glycosylation sites at T159, T169, T172, and T179. Upon cleavage, the activation peptide (SEQ ID NO: 895) is excised from the polypeptide, leaving the activated FIX polypeptide, containing of a light chain and a heavy chain that are attached via cysteine disulfide bonds. The light chain includes the amino terminal portion of the FIX polypeptide, comprising the gla, EGF1, and EGF2 domains (SEQ ID NO: 894). The heavy chain comprises the FIX serine protease domain (SEQ ID NO: 896).

The serine protease domain is a catalytic domain that is homologous to known serine proteases, such as chymotrypsin, and contains an analogous catalytic core of residues at H221, D269, and S365 (relative to the mature FIX polypeptide, SEQ ID NO: 2). The protease domain catalyzes the hydrolysis of factor X in the presence of Ca²⁺, phospholipids, and FVIIIa by cleaving the R53-I53 bond of mature factor X.

2. Factor IX Polypeptides

Factor IX (FIX) polypeptides are heterogeneous polypeptides, are made of varying amino acid sequence lengths and include, but are not limited to, recombinantly produced polypeptide, synthetically produced polypeptide and FIX extracted from cells as tissues such as, for example, liver. FIX includes related polypeptides from different species including, but not limited to, animals of human and non-human origin, allelic variant isoforms, species isoforms, synthetic molecules from nucleic acids, protein isolated from human tissue and cells, and modified forms thereof. Exemplary unmodified mature FIX polypeptides include, but are not limited to, unmodified and wild-type native FIX polypeptide (such as the polypeptide containing a sequence set forth in SEQ ID NO: 2), the unmodified and wild-type precursor FIX polypeptide that includes a signal peptide and a propeptide (such as the polypeptide that has the sequence set forth in SEQ ID NO: 1), a polymorphic wild-type variant of native factor IX polypeptide that has alanine at amino acid position T148 (SEQ ID NO: 892; or its precursor containing a sequence of amino acids set forth in SEQ ID NO: 893), and chimeric variants of FIX polypeptides such as set forth in SEQ ID NO: 1035. Unmodified FIX polypeptides also include FIX polypeptides that are modified by the cellular machinery and include, for example, glycosylation, carboxylation, β-hydroxylation, sulfation, and phosphorylation.

Based on alignment of FIX with other coagulation factor family members, such as factor VII (FVII) or factor X (FX), homologous domains among the family members are readily identified. Chimeric variants of FIX polypeptides can be constructed where one or more amino acids or entire domains are replaced in the FIX amino acid sequence using the amino acid sequence of the corresponding family member. Such chimeric proteins can be used as the unmodified FIX polypeptide for the methods described herein. For example, amino acid residues of the gla domain can be replaced with residues from the analogous FVII or FX gla domains (Lin et al. (1990) J. Biol. Chem. 265(1):144-150 and Cheung et al. (1991) J. Biol. Chem. 266(14):8797-880). A chimeric FIX protein can then be further modified by amino acid replacement for increased protein stability using any the methods described herein. Exemplary modified chimeric FIX polypeptides have amino acid sequences set forth in SEQ ID NOS: 1036-2044.

FIX is functionally active following cleavage by FXIa in the presence of Ca²⁺ or by FVIIa in the presence of TF, phospholipid, and Ca²⁺. Hydrolysis of mature FIX polypeptides generates heavy and light chains of FIX that are connected by disulfide bridges. FIX polypeptides therefore also include FIX heavy chain and FIX light chain polypeptides (SEQ ID NOS: 896 and 894, respectively) and combinations thereof.

The full structure of coagulation FIX is formed from six FIX molecules. Modified polypeptides can be stabilized by intra- (within FIX polypeptide) and inter- (between heavy and light chains of FIX polypeptide or between subunits) subunit interactions. Amino acids within the helices of each FIX polypeptide chain can interact with each other, thereby contributing to protein stability within a FIX polypeptide (i.e., “intrastability”). For example, cysteine residues form disulfide bonds between the FIX heavy and light polypeptide chains. In addition, interhelical contacts occur between helices of different subunits thereby contributing to protein stability between the monomers (i.e., “interstability”). For example, amino acid residues in helices on monomer A can interact with amino acid residues in helices on monomer B.

Contemplated herein are modified FIX polypeptides that differ from unmodified or wild-type FIX polypeptides with respect to a property or an activity. Modified FIX polypeptides provided herein can have increased stability, as manifested as increased serum half-life or increased resistance to proteases as compared to unmodified FIX polypeptides.

Generally, FIX is produced as a larger polypeptide (461 aa, SEQ ID NO: 1, for example) that is matured to a smaller polypeptide upon cleavage of the signal sequence (amino acids 1-28 of SEQ ID NO: 1, for example). FIX polypeptides are typically about 433 amino acids in length after cleavage of the signal sequence, and typically about 415 amino acids in length following further cleavage of the propeptide sequence, resulting in a mature FIX polypeptide (SEQ ID NO: 2, for example).

FIX is a secreted, glycosylated protein, including O- and N-linked glycosylation sites. The extent of glycosylation of the FIX polypeptides provided herein can contribute to the stability of the protein and to protease resistance. Glycosylation of FIX polypeptides provided herein also can contribute to the amount of protein expression, secretion, and potency of the FIX polypeptide. FIX polypeptides provided herein can be γ-carboxylated, β-hydroxylated, tyrosine sulfated, and/or phosphorylated.

FIX polypeptides from different species share conserved sequences of amino acids (see, for example, mouse, rat, guinea pig, cow, sheep, dog, cat, chicken, pig, rabbit, fish, and chimpanzee FIX polypeptides). Such sequences can be used for modification using the methods described herein.

Active and inactive forms of FIX polypeptides are known in the art. Active peptidase forms of FIX can be used in the treatment of hemophilias, where FIX polypeptides can function to promote blood coagulation. FIX polypeptides that have an inactive protease domain can be used in the treatment of thrombotic diseases by inhibiting blood coagulation via interference of the coagulation cascade.

Modes of administration include, but are not limited to, FIX polypeptide injection or the nucleic acid molecules encoding modified FIX polypeptides provided herein and also can include additional treatments administered similarly or through other routes of administration, including oral administration and inhalation, stem cell engraftment, and systemic administration of viral vectors encoding a FIX polypeptide. FIX also can be targeted for delivery into specific cell types. For example, adenoviral vectors encoding FIX polypeptides can be used for stable expression in nondividing cells, such as liver cells, and skeletal muscle cells (Arruda et al. (2001) Blood 97(1): 130-138 and Yao et al. (1992) PNAS 89: 3357-3361). In another example, viral or nonviral vectors encoding FIX polypeptides can be transduced into isolated cells for subsequent delivery. Additional cell types for expression and delivery of FIX are known in the art and include but are not limited to, fibroblasts and endothelial cells (Palmer et al. (1989) Blood 73: 438-445; Yao et al. (1991) PNAS 88: 8101-8105).

3. FIX as a Pharmaceutical

Activities induced by FIX include, but are not limited to, blood coagulation. Administration of FIX has been shown to promote blood clotting in patients suffering from hemophilia, particularly hemophilia B. In man, treatment with doses of FIX have been found to be safe and well-tolerated.

FIX can be administered orally, systemically, buccally, transdermally, intravenously, intramuscularly and subcutaneously and, typically, multiple administrations are used in treatment regimens. The formulations are typically stored in refrigerated (2-8° C.) conditions to ensure retention of activity. Hence, improved FIX stability (half-life) in administered conditions (in vivo), such as stability in serum, and in vitro (such as, during production, purification and storage conditions) can improve its utility and efficiency as a drug.

Provided herein are variants of the FIX polypeptide that display improved stability as assessed by resistance to proteases (blood, intestinal, etc) and/or increased thermal tolerance and/or pH conditions, wherein the mutant variants exhibit increased protein half-life. The mutant variants that exhibit improved stability possess increased stability in administration conditions such as in the bloodstream, gastrointestinal tract, under low pH conditions (such as, the stomach), mouth, throat, and/or under storage conditions.

D. EXEMPLARY METHODS FOR MODIFYING FIX

Provided herein are methods for increasing stability and half-life of a FIX polypeptide by increasing resistance to proteolysis. Provided herein are methods of modifying FIX polypeptides to increase resistance to proteolysis by proteases (blood, serum, gastrointestinal, etc.), whereby the modified polypeptide exhibits increased half-life in vitro and/or in vivo. Provided herein are modified FIX polypeptides in which the primary amino acid sequence is modified to confer increased protein stability. Among the amino acid modifications provided herein are such modifications including replacement of amino acids in the primary sequence of the FIX polypeptide in order to decrease proteolytic cleavage of the FIX polypeptide. Further modifications of the modified FIX polypeptide can be included, such as, but not limited to, addition of carbohydrate, phosphate, sulfur, hydroxyl, carboxyl, and polyethylene glycol (PEG) moieties. Thus, the modified FIX polypeptides provided herein can be further modified, for example, by glycosylation, phosphorylation, sulfation, hydroxylation, carboxylation, and/or PEGylation. Such modifications can be performed in vivo or in vitro.

Provided herein are methods of modifying polypeptides to increase resistance to proteolysis by proteases and contacting proteolytic enzymes with peptide inhibitors, thereby inhibiting activity of the proteases. Also provided herein are the modified polypeptides generated by said methods. Provided herein are FIX polypeptides that display improved stability as assessed by resistance to proteases; the modified FIX polypeptides exhibiting these properties possess, thereby, increased protein half-life in vitro or in vivo.

The modified FIX polypeptides (also referred to herein as variants) are more stable compared to unmodified FIX polypeptides. Increasing stability (i.e., the half-life of proteins in vivo) can result in a decrease in the frequency of injections needed to maintain a sufficient drug level in serum, thus leading to: i) higher comfort to, and acceptance by, treated subjects, particularly human subjects, ii) lower doses necessary to achieve comparable biological effects, and iii) as a consequence, an attenuation of the (dose-dependent) secondary effects.

Increased stability of FIX can be achieved, for example, by destruction of protease target residues or sequences and/or by modification of residues that contribute to conformational stability and are susceptible to denaturation by temperature, pH, or other denaturing agent. Modification of FIX to increase stability can be accomplished while keeping activity unchanged compared to the unmodified or wild-type FIX. Alternatively, modification of FIX stability can be accomplished while increasing activity compared to the unmodified or wild-type FIX. Any methods known in the art can be used to create modified FIX polypeptides. In the methods described herein, modifications are chosen using the method of 2D-scanning mutagenesis as described, for example, in PCT published applications WO 2004/022747 and WO 2004/022593.

In principle, there are several general approaches described for protein-directed evolution based on mutagenesis. Any of these, alone or in combination can be used to modify a FIX polypeptide to achieve increased stability and/or resistance to proteolysis. Such methods include random mutagenesis, where the amino acids in the starting protein sequence are replaced by all (or a group) of the 20 amino acids either in single or multiple replacements at different amino acid positions are generated on the same molecule, at the same time. Another method, restricted random mutagenesis, introduces either all of the 20 amino acids or DNA-biased residues. The bias is based on the sequence of the DNA and not on that of the protein in a stochastic or semi-stochastic manner, respectively, within restricted or predefined regions of the protein known in advance to be involved in the activity being “evolved.” Additionally, methods of rational mutagenesis including 1D-scanning, 2D-scanning, and 3D-scanning can be used alone or in combination to construct modified FIX variants.

1. Non-Restricted Rational Mutagenesis One-Dimensional (1D)-Scanning

Rational mutagenesis, also termed 1D-scanning, is a two-step process, and is described in co-pending U.S. application Ser. No. 10/022,249 (U.S. Publication No. 2003/0134351-A1). 1D-scanning can be used to modify FIX polypeptides and, additionally, to identify positions for further modification by other methods such as 2D- and 3D-scanning. Briefly, in the first step, full-length amino acid scanning is performed where all and each amino acid in the starting FIX polypeptide sequence (for example SEQ ID NO: 2) is replaced by a designated reference amino acid (such as, alanine). Only a single amino acid is replaced on each protein molecule at a time. A collection of protein molecules having a single amino acid replacement is generated such that molecules differ from each other by the amino acid position at which the replacement has taken place. Mutant DNA molecules are designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and such that each one is the single product of an independent mutagenesis reaction. Mutant protein molecules derived from the collection of mutant nucleic acid molecules also are physically separated from each other, such as by formatting in addressable arrays. Activity assessment on each protein molecule allows for the identification of those amino acid positions that result in a drop in activity when replaced, thus indicating the involvement of that particular amino acid position in the protein's biological activity and/or conformation that leads to fitness of the particular feature being evolved. Those amino acid positions are referred to as HITs.

At the second step, a new collection of molecules is generated such that each molecule differs from each of the others by the amino acid present at the individual HIT positions identified in step 1. All 20 amino acids (19 remaining) are introduced at each of the HIT positions identified in step 1; while each individual molecule contains, in principle, one and only one amino acid replacement. Mutant DNA molecules are designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and such that each one is the single product of an independent mutagenesis reaction. Mutant protein molecules derived from the collection of mutant DNA molecules also are physically separated from each other, such as by formatting in addressable arrays. Activity assessment then is individually performed on each individual mutant molecule. The newly generated mutants that lead to a desired alteration (such as an improvement) in a protein activity are referred to as LEADs. This method permits an indirect search for property or activity alteration, such as improved stability and/or improved resistance to proteases or thermal conditions based on one rational amino acid replacement and sequence change at a single amino acid position at a time, in search of a new, unpredicted amino acid sequence at some unpredicted regions along a protein to produce a protein that exhibits a desired activity or altered activity, such as better performance than the starting protein.

In this approach, neither the amino acid position nor the replacing amino acid type are restricted. Full length protein scanning is performed during the first step to identify HIT positions, and then all 20 amino acids are tested at each of the HIT positions, to identify LEAD sequences; while, as a starting point, only one amino acid at a time is replaced on each molecule. The selection of the target region (HITs and surrounding amino acids) for the second step is based upon experimental data on activity obtained in the first step. Thus, no prior knowledge of protein structure and/or function is necessary. Using this approach, LEAD sequences have been found on proteins that are located at regions of the protein not previously known to be involved in the particular biological activity being modified; thus emphasizing the power of this approach to discover unpredictable regions (HITs) as targets for fitness improvement.

2. Two Dimensional (2D) Rational Scanning (Restricted Rational Mutagenesis)

The 2D-scanning (or restricted rational mutagenesis) methods for protein rational evolution (see, co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published International applications WO 2004/022593 and WO 2004/022747) are based on scanning over two dimensions. The first dimension is the amino acid position along the protein sequence, in order to identify is-HIT target positions. The second dimension is scanning the amino acid type selected for replacing a particular is-HIT amino acid position. An advantage of the 2D-scanning methods provided herein is that at least one, and typically the amino acid position and/or the replacing amino acid, can be restricted such that fewer than all amino acids on the protein-backbone are selected for amino acid replacement; and/or fewer than all of the remaining 19 amino acids available to replace an original, such as native, amino acid are selected for replacement.

In particular embodiments, based on i) the particular protein properties to be evolved (such as, resistance to proteolysis), ii) sequence of amino acids of the protein, and iii) the known properties of the individual amino acids, a number of target positions along the protein sequence are selected, in silico, as “is-HIT target positions.” This number of is-HIT target positions is as large as reasonably possible such that all reasonably possible target positions for the particular feature being evolved are included. In particular, embodiments where a restricted number of is-HIT target positions are selected for replacement, the amino acids selected to replace the is-HIT target positions on the particular protein being optimized can be either all of the remaining 19 amino acids or, more frequently, a more restricted group comprising selected amino acids that are contemplated to have the desired effect on protein activity. In another embodiment, so long as a restricted number of replacing amino acids are used, all of the amino acid positions along the protein backbone can be selected as is-HIT target positions for amino acid replacement. Mutagenesis then is performed by the replacement of single amino acid residues at specific is-HIT target positions on the protein backbone (such as, “one-by-one,” such as in addressable arrays), such that each individual mutant generated is the single product of each single mutagenesis reaction. Mutant DNA molecules are designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and that each one is the single product of an independent mutagenesis reaction. Mutant protein molecules derived from the collection of mutant DNA molecules also are physically separated from each other, such as by formatting in addressable arrays. Thus, a plurality of mutant protein molecules is produced. Each mutant protein contains a single amino acid replacement at only one of the is-HIT target positions. Activity assessment is then individually performed on each individual protein mutant molecule, following protein expression and measurement of the appropriate activity. An example of practice of this method is shown in the Examples in which mutant FIX molecules are produced.

The newly generated proteins that lead to altered, typically improved, target protein activity are referred to as LEADs. This method relies on an indirect search for protein improvement for a particular activity (such as increased resistance to proteolysis), based on amino acid replacement and sequence change at single or, in another embodiment, a limited number of amino acid positions at a time. As a result, optimized proteins, which have modified sequences of amino acids at some regions along the protein that perform better (at a particular target activity or other property) than or different from the starting protein, are identified and isolated.

2D-scanning on FIX was used to generate variants improved in protein stability, including improved resistance to proteolysis. To effect such modifications, amino acid positions were selected using in silico analysis of FIX.

a. Identifying in-Silico HITs

The 2D-scanning method for directed evolution of proteins includes identifying and selecting (using in silico analysis) specific amino acids and amino acid positions (referred to herein as is-HITs) along the protein sequence that are contemplated to be directly or indirectly involved in the feature being evolved. As noted, the 2D-scanning methods provided include the following two steps. The first step is an in silico search of a target sequence of amino acids of the protein to identify all possible amino acid positions that can be targets for the activity being evolved. This is effected, for example, by assessing the effect of amino acid residues on the property or properties to be altered on the protein, using any known standard software. The particulars of the in silico analysis is a function of the property to be modified.

Once identified, these amino acid positions or target sequences are referred to as “is-HITs” (in silico HITs). In silico HITs are defined as those amino acid positions (or target positions) that potentially are involved in the “evolving” feature, such as increased resistance to proteolysis. The discrimination of the is-HITs among all the amino acid positions in a protein sequence can be made based on the amino acid type at each position in addition to the information on the protein secondary or tertiary structure. In silico HITs constitute a collection of mutant molecules such that all possible amino acids, amino acid positions or target sequences potentially involved in the evolving feature are represented. No strong theoretical discrimination among amino acids or amino acid positions is made at this stage. In silico HIT positions are spread over the full length of the protein sequence. Single or a limited number of is-HIT amino acids are replaced at a time on the target FIX polypeptide.

A variety of parameters can be analyzed to determine whether or not a particular amino acid on a protein might be involved in the evolving feature, typically a limited number of initial premises (typically no more than 2) are used to determine the in silico HITs. For example, as described herein, to increase the stability of FIX, the first condition is the nature of the amino acids linked to stability of the molecule such as its participation in directing proteolytic cleavage. A second premise, for example, can be related to the specific position of those amino acids along the protein structure.

During the first step of identification of is-HITs according to the methods provided herein, each individual amino acid along the protein sequence is considered individually to assess whether it is a candidate for is-HIT. This search is done one-by-one and the decision on whether the amino acid is considered to be a candidate for a is-HIT is based on (1) the amino acid type; (2) the position in the protein and protein structure if known; and (3) the predicted interaction between that amino acid and its neighbors in sequence and space.

Is-HITs were identified for a number of properties of FIX that contribute to protein stability, such as removal/modification of protease sensitive sites. Such modifications contribute to protein stability and thereby, to increasing the half-life of a FIX polypeptide in vitro, in vivo or ex vivo.

b. Identifying Replacing Amino Acids

Once the is-HITs target positions are selected, the next step is identifying those amino acids that will replace the original, such as native, amino acid at each is-HIT position to alter the activity level for the particular feature being evolved. The set of replacing amino acids to be used to replace the original, such as native, amino acid at each is-HIT position can be different and specific for the particular is-HIT position. The choice of the replacing amino acids takes into account the need to preserve the physicochemical properties such as hydrophobicity, charge and polarity of essential (such as, catalytic, binding, etc.) residues and alter some other property of the protein (such as, protein stability). The number of replacing amino acids of the remaining 19 non-native (or non-original) amino acids that can be used to replace a particular is-HIT target position ranges from 1 up to about 19, and anywhere in between, depending on the properties for the particular modification.

Numerous methods of selecting replacing amino acids (also referred to herein as “replacement amino acids”) are well known in the art. Protein chemists determined that certain amino acid substitutions commonly occur in related proteins from different species. As the protein still functions with these substitutions, the substituted amino acids are compatible with protein structure and function. Often, these substitutions are to a chemically similar amino acid, but other types of changes, although relatively rare, also can occur.

Knowing the types of changes that are most and least common in a large number of proteins can assist with predicting alignments and amino acid substitutions for any set of protein sequences. Amino acid substitution matrices are used for this purpose. A number of matrices are available. A detailed presentation of such matrices can be found in the co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published International applications WO 2004/022593 and WO 2004/022747, each of which is incorporated herein in their entirety herein. Such matrices also are known and available in the art, for example in the reference listed below.

In amino acid substitution matrices, amino acids are listed horizontally and vertically, and each matrix position is filled with a score that reflects how often one amino acid would have been paired with the other in an alignment of related protein sequences. The probability of changing amino acid “A” into amino acid “B” is assumed to be identical to the reverse probability of changing “B” into “A”. This assumption is made because, for any two sequences, the ancestor amino acid in the phylogenetic tree is usually not known. Additionally, the likelihood of replacement should depend on the product of the frequency of occurrence of the two amino acids and on their chemical and physical similarities. A prediction of this model is that amino acid frequencies will not change over evolutionary time (Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3): 345-352, 1978). Several exemplary amino acid substitution matrices, including, but not limited to block substitution matrix (BLOSUM) (Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 10915-10919), Jones et al. (1992) Comput. Appl. Biosci., 8: 275-282, Gonnet et al. (1992) Science, 256: 1433-1445, Fitch (1966) J. Mol. Evol., 16(1): 9-16, Feng et al. (1985) J. Mol. Evol., 21: 112-125, McLachlan (1971) J. Mol. Biol., 61: 409-424, Grantham (1974) Science, 185: 862-864, Miyata (1979) J. Mol. Evol., 12: 219-236, Rao (1987) J. Pept. Protein Res., 29: 276-281, Risler (1988) J. Mol. Biol., 204: 1019-1029, Johnson et al. (1993) J. Mol. Biol., 233: 716-738, and Point Accepted Mutation (PAM) (Dayhoff et al. (1978) Atlas of Protein Sequence and Structure 5: 345-352.

Dayhoff and coworkers developed a model of protein evolution that resulted in the development of a set of widely used replacement matrices (Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5(3):345-352) termed percent accepted mutation matrices (PAM). In deriving these matrices, each change in the current amino acid at a particular site is assumed to be independent of previous mutational events at that site. Thus, the probability of change of any amino acid A to amino acid B is the same, regardless of the previous changes at that site and also regardless of the position of amino acid A in a protein sequence.

In the Dayhoff approach, replacement rates are derived from alignments of protein sequences that are at least 85% identical; this constraint ensures that the likelihood of a particular mutation being the result of a set of successive mutations is low. Because these changes are observed in closely related proteins, they represent amino acid substitutions that do not significantly change the function of the protein. Hence, they are called “accepted mutations,” as defined as amino acid changes that are accepted by natural selection.

The outcome of the two steps set forth above, which is performed in silico is that: (1) the amino acid positions that are the target for mutagenesis are identified (referred to as is-HITs); and (2) the replacing amino acids for the original, such as native, amino acids at the is-HITs are identified, to provide a collection of candidate LEAD mutant molecules that are expected to perform differently from the native molecule. These are assayed for a desired optimized (or improved or altered) activity.

c. Construction of Modified Proteins and Biological Assays

Once is-HITs are selected as set forth above, replacing amino acids are introduced. Mutant proteins typically are prepared using recombinant DNA methods and assessed in appropriate biological assays for the particular activity (feature) optimized. An exemplary method of preparing the mutant proteins is by mutagenesis of the original, such as native, gene using methods well known in the art. Mutant molecules are generated one-by-one, such as in addressable arrays, such that each individual mutant generated is the single product of each single and independent mutagenesis reaction. Individual mutagenesis reactions are conducted separately, such as in addressable arrays where they are physically separated from each other. Once a population of sets of nucleic acid molecules encoding the respective mutant proteins is prepared, each is separately introduced one-by-one into appropriate cells for the production of the corresponding mutant proteins. This also can be performed, for example, in addressable arrays where each set of nucleic acid molecules encoding a respective mutant protein is introduced into cells confined to a discrete location, such as in a well of a multi-well microtiter plate. Each individual mutant protein is individually phenotypically characterized and performance is quantitatively assessed using assays appropriate for the feature being optimized (i.e., feature being evolved). Again, this step can be performed in addressable arrays. Those mutants displaying a desired increased or decreased performance compared to the original, such as native molecules are identified and designated LEADs. From the beginning of the process of generating the mutant DNA molecules up through the readout and analysis of the performance results, each candidate LEAD mutant is generated, produced and analyzed individually, such as from its own address in an addressable array. The process is amenable to automation.

3. Three Dimensional (3D) Scanning

3D scanning, as described in co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published PCT applications WO 2004/022747 and WO 2004/022593, is an additional method of rational evolution of proteins based on the identification of potential target sites for mutagenesis (is-HITs). The method uses comparison of patterns of protein backbone folding between structurally related proteins, irrespective of the underlying sequences of the compared proteins. Once the structurally related amino acid positions are identified on the protein of interest, then suitable amino acid replacement criteria, such as PAM analysis, can be employed to identify candidate LEADs for construction and screening.

For example, analysis of “structural homology” between and among a number of related vitamin K-dependent proteases can be used to identify on various members of the vitamin K-dependent protease family, those amino acid positions and residues that are structurally similar or structurally related. For example, 3D scanning can be used to identify amino acid positions on FIX that are structurally similar or structurally related to those found in factor VII mutants, for example, that have been modified for improved stability. Exemplary vitamin K-dependent proteases include, but are not limited to, prothrombin, factor VII, factor X and protein C.

Using the 3D-scanning methods described herein, once one protein within a family of proteins (such as, FIX within the vitamin-K dependent protease family) is modified using the methods provided herein for generating LEAD mutants, is-HITs can be identified for other or all proteins within a particular family by identifying the corresponding amino acid positions therein using structural homology analysis (based upon comparisons of the 3D structures of the family members with original protein to identify corresponding residues for replacement) as described hereinafter. The is-HITs for the family members identified in this manner then can be subjected to the next step of identifying replacing amino acids and further assayed to obtain LEADs or super-LEADs as described herein. Similarly, information from 2D-scanning performed on other vitamin K-dependent proteases such as prothrombin, factor VII, factor X or protein C, for example, can be used to optimize FIX polypeptides.

This method can be applied to any desired phenotype using any protein, such as a vitamin K-dependent protease, as the starting material to which an evolution procedure, such as the rational directed evolution procedure of U.S. Published Application No. US 2003-0134351 A1 or the 2-dimensional scanning method described herein. The structurally corresponding residues are then altered on members of the family to produce additional vitamin K-dependent proteases with similar phenotypic alterations.

a. Homology

Typically, homology between proteins is compared at the level of their amino acid sequences, based on the percent or level of coincidence of individual amino acids, amino acid per amino acid, when sequences are aligned starting from a reference, generally the residue encoded by the start codon. For example, two proteins are said to be “homologous” or to bear some degree of homology whenever their respective amino acid sequences show a certain degree of matching upon alignment comparison. Comparative molecular biology is primarily based on this approach. From the degree of homology or coincidence between amino acid sequences, conclusions can be made on the evolutionary distance between or among two or more protein sequences and biological systems.

The concept of “convergent evolution” is applied to describe the phenomena by which phylogenetically-unrelated organisms or biological systems have evolved to share features related to their anatomy, physiology and structure as a response to common forces, constraints and evolutionary demands from the surrounding environment and living organisms. Alternatively, “divergent evolution,” is applied to describe the phenomena by which strongly phylogenetically related organisms or biological systems have evolved to diverge from identity or similarity as a response to divergent forces, constraints and evolutionary demands from the surrounding environment and living organisms.

In the typical traditional analysis of homologous proteins there are two conceptual biases corresponding to: i) “convergent evolution,” and ii) “divergent evolution.” Whenever the aligned amino acid sequences of two proteins do not match well with each other, these proteins are considered “not related” or “less related” with each other and have different phylogenetic origins. There is no (or low) homology between these proteins and their respective genes are not homologous (or show little homology). If these two “non-homologous” proteins under study share some common functional features (such as, interaction with other specific molecules, activity), they are determined to have arisen by “convergent evolution,” i.e., by evolution of their non-homologous amino acid sequences, in such a way that they end up generating functionally “related” structures.

On the other hand, whenever the aligned amino acid sequences of two proteins do match with each other to a certain degree, these proteins are considered to be “related” and to share a common phylogenetic origin. A given degree of homology is assigned between these two proteins and their respective genes likewise share a corresponding degree of homology. During the evolution of their initial highly homologous amino acid sequence, enough changes can be accumulated in such a way that they end up generating “less-related” sequences and less related function. The divergence from perfect matching between these two “homologous” proteins under study is said come from “divergent evolution.”

b. 3D-Scanning (Structural Homology) Methods

Structural homology refers to homology between the topology and three-dimensional structure of two proteins. Structural homology is not necessarily related to “convergent evolution” or to “divergent evolution,” nor is it related to the underlying amino acid sequence. Rather, structural homology is likely driven (through natural evolution) by the need of a protein to fit specific conformational demands imposed by its environment. Particular structurally homologous “spots” or “loci” would not be allowed to structurally diverge from the original structure, even when its own underlying sequence does diverge. This structural homology is exploited herein to identify loci for mutation.

Within the amino acid sequence of a protein resides the appropriate biochemical and structural signals to achieve a specific spatial folding in either an independent or a chaperon-assisted manner. Indeed, this specific spatial folding ultimately determines protein traits and activity. Proteins interact with other proteins and molecules in general through their specific topologies and spatial conformations. In principle, these interactions are not based solely on the precise amino acid sequence underlying the involved topology or conformation. If protein traits, activity (behavior and phenotypes) and interactions rely on protein topology and conformation, then evolutionary forces and constraints acting on proteins can be expected to act on topology and conformation. Proteins sharing similar functions will share comparable characteristics in their topology and conformation, despite the underlying amino acid sequences that create those topologies and conformations.

4. Super-LEADs and Additive Directional Mutagenesis (ADM)

Modification of FIX polypeptides also can include combining two or more mutations. For example, Additive Directional Mutagenesis (ADM) can be used to assemble on a single mutant protein multiple mutations present on the individual LEAD molecules, so as to generate super-LEAD mutant proteins (see co-pending U.S. Published Application Nos. US 2005-0202438 A1 and US-2004-0132977-A1 and published PCT applications WO 2004/022747 and WO 2004/022593). ADM is a repetitive multi-step process where at each step after the creation of the first LEAD mutant protein a new LEAD mutation is added onto the previous LEAD mutant protein to create successive super-LEAD mutant proteins. ADM is not based on genetic recombination mechanisms, nor on shuffling methodologies; instead it is a simple one-mutation-at-a-time process, repeated as many times as necessary until the total number of desired mutations is introduced on the same molecule. To avoid the exponentially increasing number of all possible combinations that can be generated by putting together on the same molecule a given number of single mutations, a method is provided herein that, although it does not cover all the combinatorial possible space, still captures a big part of the combinatorial potential. “Combinatorial” is used herein in its mathematical meaning (i.e., subsets of a group of elements, containing some of the elements in any possible order) and not in the molecular biological or directed evolution meaning (i.e., generating pools, or mixtures, or collections of molecules by randomly mixing their constitutive elements).

A population of sets of nucleic acid molecules encoding a collection of new super-LEAD mutant molecules is generated tested and phenotypically characterized one-by-one in addressable arrays. Super-LEAD mutant molecules are such that each molecule contains a variable number and type of LEAD mutations. Those molecules displaying further improved fitness for the particular feature being evolved are referred to as super-LEADs. Super-LEADs can be generated by other methods known to those of skill in the art and tested by the high throughput methods herein. For purposes herein a super-LEAD typically has activity with respect to the function or biological activity of interest that differs from the improved activity of a LEAD by a desired amount, such as at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more from at least one of the LEAD mutants from which it is derived. In yet other embodiments, the change in activity is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more greater than at least one of the LEAD molecules from which it is derived. As with LEADs, the change in the activity for super-LEADs is dependent upon the property that is being “evolved.” The desired alteration, which can be either an increase or a reduction in a feature or property, will depend upon the function or property of interest.

In one embodiment, the ADM method employs a number of repetitive steps, such that at each step a new mutation is added on a given molecule. Although numerous different ways are possible for combining each LEAD mutation onto a super-LEAD protein, an exemplary way the new mutations (such as, mutation 1 (m1), mutation 2 (m2), mutation 3 (m3), mutation 4 (m4), mutation 5 (m5), mutation n (mn)) can be added corresponds to the following diagram:

m1+m2

m1+m2+m3

m1+m2+m3+m4

m1+m2+m3+m4+m5

m1+m2+m3+m4+m5+ . . . +nm

m1+m2+m4

m1+m2+m4+m5

m1+m2+m4+m5+ . . . +nm

m1+m2+m5

m1+m2+m5+ . . . +nm

m2+m3

m2+m3+m4

m2+m3+m4+m5

m2+m3+m4+m5+ . . . +nm

m2+m4

m2+m4+m5

m2+m4+m5+ . . . +nm

m2+m5

m2+m5+ . . . +nm

. . . , etc. . . .

5. Multi-Overlapped Primer Extensions

Another method that can be employed to generate combinations of two or more mutations is using oligonucleotide-mediated mutagenesis referred to as “multi overlapped primer extensions”. This method can be used for the rational combination of mutant LEADs to form super-LEADS. This method allows the simultaneous introduction of several mutations throughout a small protein or protein-region of known sequence. Overlapping oligonucleotides of typically around 70 bases in length (since longer oligonucleotides lead to increased error) are designed from the DNA sequence (gene) encoding the mutant LEAD proteins in such a way that they overlap with each other on a region of typically around 20 bases. Although typically about 70 bases are used to create the overlapping oligonucleotides, the length of additional overlapping oligonucleotides for use can range from about 30 bases up to about 100 bases. Likewise, although typically the overlapping region of the overlapping oligonucleotides is about 20 bases, the length of other overlapping regions for use herein can range from about 5 bases up to about 40 bases. These overlapping oligonucleotides (including or not point mutations) act as template and primers in a first step of PCR (using a proofreading polymerase, such as, Pfu DNA polymerase, to avoid unexpected mutations) to create small amounts of full-length gene. The full-length gene resulting from the first PCR is then selectively amplified in a second step of PCR using flanking primers, each one tagged with a restriction site in order to facilitate subsequent cloning. One multi overlapped extension process yields a full-length (multi-mutated) nucleic acid molecule encoding a candidate super-LEAD protein having multiple mutations therein derived from LEAD mutant proteins.

E. MODIFIED FIX POLYPEPTIDES EXHIBITING INCREASED PROTEIN STABILITY, PARTICULARLY MODIFICATIONS THAT INCREASE RESISTANCE TO PROTEASES

Provided herein are modified FIX polypeptides (also referred to herein as variants of FIX) that display improved protein stability (i.e. increased protease resistance, or increased conformational stability that, for example, renders a polypeptide more resistant to denaturation by temperature or pH changes). A FIX polypeptide provided herein exhibiting increased protein stability can lead to an increased half-life of the polypeptide in vitro (such as, during production, purification and storage) or in vivo (such as, after administration to a subject). For example, increased half-life can occur following administration of the polypeptide to a subject, such as a human subject. The increased half-life of the modified FIX polypeptide can be increased by an amount that is at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the half-life of the unmodified FIX polypeptide. In some examples, the increased half-life of the modified FIX polypeptide can be increased by an amount that is at least 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more times when compared to the half-life of the unmodified FIX polypeptide. Hence, the modified FIX polypeptides provided herein offer FIX with advantages including a decrease in the frequency of injections needed to maintain a sufficient drug level in serum, thus leading to, for example, higher comfort and acceptance by subjects, lower doses necessary to achieve comparable biological effects and attenuation of secondary effects.

Provided herein are modified FIX polypeptides containing modifications that alter any one or more of the properties of FIX that contribute to increased protein stability, such as increased protease resistance, and any combinations of the modifications thereof. Increased protein stability can be accomplished by amino acid replacement, such that resistance to proteases by amino acid replacements can be achieved by direct destruction of the protease target residue or sequence. Generally, modified FIX polypeptides retain one or more activities of an unmodified FIX polypeptide. For example, the modified FIX polypeptides provided herein exhibit at least one activity that is substantially unchanged (less than 1%, 5% or 10% changed) compared to the unmodified or wild-type FIX. In other examples, the activity of a modified FIX polypeptide is increased or is decreased as compared to an unmodified FIX polypeptide. In another embodiment, the modified FIX polypeptides provided herein can inhibit an activity of the unmodified and/or wild-type native FIX polypeptide. Activity includes, for example, but not limited to blood coagulation, platelet binding, cofactor binding and protease activity. Activity can be assessed in vitro or in vivo and can be compared to the unmodified FIX polypeptide, such as for example, the mature, wild-type native FIX polypeptide (SEQ ID NO: 2), the wild-type precursor FIX polypeptide (SEQ ID NO: 1), or any other FIX polypeptide known to one of skill in the art that is used as the starting material.

Modified FIX polypeptides provided herein can be modified at one or more amino acid positions corresponding to amino acid positions of a mature FIX polypeptide, for example, a mature FIX polypeptide having an amino acid sequence set forth in SEQ ID NO: 2. FIX polypeptides can be modified compared to a mature or precursor FIX polypeptide having an amino acid sequence set forth in SEQ ID NO: 2 or 1, respectively. Modified FIX polypeptides provided herein include human FIX (hFIX) variants. An exemplary hFIX variant occurs at amino acid position 148 of the mature hFIX polypeptide, wherein the amino acid is Alanine or Threonine. The hFIX polypeptide can be of any human tissue or cell-type origin. Modified FIX polypeptides provided herein also include variants of FIX of non-human origin. Such alignments and selection of positions can be performed with any FIX polypeptide by aligning it with hFIX and selecting corresponding positions for modification. For example, modified FIX polypeptides can be variants of a non-human FIX, including, but not limited to, mouse, rat, guinea pig, cow, sheep, dog, cat, chicken, pig, rabbit, fish, and chimpanzee FIX. Exemplary unmodified non-human FIX polypeptides have amino acid sequences set forth in SEQ ID NOS: 898-912. Modified FIX polypeptides also include polypeptides that are hybrids of different FIX polypeptides and also synthetic FIX polypeptides prepared recombinantly or synthesized or constructed by other methods known in the art based upon known polypeptides.

Typically, modifications include replacement (substitution), addition, deletion or a combination thereof, of amino acid residues as described herein. Modified FIX polypeptides include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. Generally, the modification results in increased stability without losing at least one activity, such as coagulant activity (L e. retains at least one activity as defined herein) of an unmodified FIX polypeptide. A modified FIX exhibiting increased protein stability containing a single amino acid change at an is-HIT position as compared to an unmodified FIX is called a LEAD. FIX polypeptide candidate LEAD polypeptides can include amino acid replacement or replacements at any one or more of the is-HIT positions selected using methods described herein or known in the art, such as obtained using PAM analysis. Exemplary amino acid modifications corresponding to amino acid positions of a mature FIX polypeptide that can contribute to an increase in protein stability with respect to protease resistance are set forth in Table 5. In Table 5 below, the sequence identifier (SEQ ID NO.) is in parenthesis next to each substitution.

Also among the variants provided herein are modified FIX polypeptides with two or more modifications compared to native or wild-type FIX. Modified FIX polypeptides include those with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. The two or more modifications can include two or more modifications of the same property, such as, two modifications that modify resistance to proteases (blood, intestinal, etc.). In another embodiment, the two or more modifications include combinations of properties that each contribute to FIX stability. For example, a FIX variant can include one or more modifications that remove a protease sensitive site and one or more modifications that alter FIX conformational stability. FIX variants carrying replacements at more than one is-HIT sites and displaying improved stability are called super-LEADs. A FIX super-LEAD can for example, contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acid changes compared to wild-type or unmodified FIX. In one example, a modified FIX polypeptide candidate super-LEAD can contain two or more amino acid modifications set forth in Table 5.

1. Protease Resistance

The delivery of stable peptide and protein drugs to patients is a major challenge for the pharmaceutical industry. These types of drugs in the human body are constantly eliminated or taken out of circulation by different physiological processes including internalization, glomerular filtration and proteolysis. The latter is often the limiting process affecting the half-life of proteins used as therapeutic agents in per-oral administration and either intravenous or intramuscular injections. To solve this problem, therapeutic proteins that increase protein stability manifested as an increased resistance to digestion by proteases are provided. Among modifications for therapeutic proteins are those that increase protection against protease digestion without destroying or completely eliminating a therapeutic use or the therapeutic activity. Such changes result in longer-lasting therapeutic proteins, and, also permit oral administration of therapeutic proteins.

FIX polypeptides provided herein are modified to increase resistance to proteolysis compared to in the absence of the modification. The resulting FIX polypeptides exhibit increased the half-life of the modified FIX polypeptide in vitro (such as, for production, processing, storage and assays.) and/or in vivo (such as, serum stability) and/or other properties, such as increased bioavailability upon oral administration. The modifications that alter susceptibility to cleavage by a proteases, unless specifically desired, do not alter activation cleavage sites for conversion of the zymogen into an activated form.

Proteases, proteinases or peptidases catalyze the hydrolysis of covalent peptidic bonds. Modified FIX polypeptides provided herein exhibit increased resistance to proteolysis by proteases, including those that occur, for example, in body fluids and tissues, such as those that include, but are not limited to, saliva, blood, serum, intestinal, stomach, blood, cell lysates, cells and others. These include proteases of all types, such as, for example, serine proteases and matrix metalloproteinases.

Modifications of FIX polypeptides result in resistance to one or more proteases that include, but are not limited to, pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA.

FIX polypeptides are cleaved by proteases and their activity reduced or inactivated. For example, human neutrophil elastase cleaves and inactivate FIX polypeptides after amino acid residues, such as T140, T144, I164, T172, and V181 (see, e.g., Takaki et al. (1983) J. Clin. Invest. 72: 1706-1715 and Samis et al. (1998) Blood 92(4): 1287-1296). In another example, thrombin cleaves and inactivates FIX polypeptides after amino acid residues, such as R318 and R327 (see, e.g., Kisiel et al. Blood 66(6): 1302-1308 (1985). Plasmin cleaves and inactivates FIX polypeptides after amino acid residues (Samis et al. (2000) Blood 95(3) 943-951). As shown herein, modifications of FIX polypeptides at sites, such as such as K43, R145, R180, K316, and R318 increase resistance of the FIX polypeptides to hydrolysis a protease. In a non-limiting example, modification of T140, T144, I164, T172 and/or V181 or other sites of a mature FIX polypeptide is shown herein to increase resistance to elastase.

Modified FIX polypeptides provided herein exhibit increased resistance to proteolysis, particularly by enzymes present in serum, blood, the gut, the mouth and other body fluids. Such increase in resistance can manifested as increased half-life of the FIX polypeptide by an amount that is at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the unmodified or wild-type FIX polypeptide in either in vivo (such as in human blood, human serum, saliva, digestive fluid and the intestinal tract,) and/or an in vitro mixture containing one or more proteases. Increased resistance also is manifested by increased bioavailability upon oral administration. As described herein, therapeutic proteins are not generally amenable to oral administration by virtue of the activity of digestive and/or blood and serum proteases. The modified FIX polypeptides provided herein can be administered orally. Hence, as described herein, provided are oral compositions containing a modified FIX polypeptide. The oral compositions do not require additional protease inhibitors nor any additional modifications of the FIX polypeptides. Additional modifications of the FIX polypeptides are optional.

Typically, the half-life in vitro or in vivo of the modified FIX polypeptides provided herein is increased by an amount selected from at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more when compared to the half-life of unmodified or wild-type FIX in either blood, serum, or in an in vitro preparation or an in vitro mixture containing one or more proteases.

Typically, the modified FIX polypeptides provided herein exhibit at least one activity that is substantially unchanged (less than 1%, 5% or 10% changed) compared to the unmodified or wild-type FIX. In some examples, the activity is increased compared to the unmodified FIX. In other examples, the activity is decreased compared to the unmodified FIX polypeptide. The activity is retained at level so that the FIX polypeptide can be used therapeutically for the desired purpose. Activity includes, for example, blood coagulation activity, and can be compared to the unmodified polypeptide, such as for example, the mature, wild-type native FIX polypeptide (SEQ ID NO: 2), the wild-type precursor FIX polypeptide (SEQ ID NO: 1), or any other FIX polypeptide used as the starting material. In another embodiment the modified FIX polypeptide can inhibit one or more activities of an unmodified FIX polypeptide and/or act as an anticoagulant.

The FIX polypeptides can be modified to have resistance to a variety of proteases. These include, for example, members of the serine protease family and also metalloproteinases.

a. Serine Proteases

Serine proteases participate in a range of functions in the body, including blood clotting, inflammation as well as digestive enzymes in prokaryotes and eukaryotes. Serine proteases are sequence specific. While cascades of protease activations control blood clotting and complement, other proteases are involved in signaling pathways, enzyme activation and degradative functions in different cellular or extracellular compartments.

Serine proteases include, but are not limited, to chymotrypsin, trypsin, elastase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. Chymotrypsin, trypsin and elastase are synthesized by the pancreatic acinar cells, secreted in the small intestine and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are similar in structure, as shown through their X-ray structures. Each of these digestive serine proteases targets different regions of the polypeptide chain, based upon the amino acid residues and side chains surrounding the site of cleavage. The active site of serine proteases is shaped as a cleft where the polypeptide substrate binds. Amino acid residues are labeled from N to C term of the polypeptide substrate (Pi, . . . , P3, P2, P1, P1′, P2′, P3′, . . . , Pj) and their respective binding sub-sites (Si, . . . , S3, S2, S1, S1′, S2′, S3′, . . . , Sj). The cleavage is catalyzed between P1 and P1′. Chymotrypsin hydrolyzes peptide bonds flanked with bulky hydrophobic amino acid residues. Particular residues include phenylalanine, tryptophan and tyrosine, which fit into a snug hydrophobic pocket. Trypsin hydrolyzes peptide bonds flanked with positively charged amino acid residues. Instead of having the hydrophobic pocket of the chymotrypsin, trypsin possesses an aspartic acid residue at the back of the pocket, which can interact with positively charged residues such as arginine and lysine. Elastase hydrolyzes peptide bonds flanked with small neutral amino acid residues, such as alanine, glycine and valine. In contrast to trypsin and chymotrypsin, elastase contains a pocket that is lined with valine and threonine, rendering it a mere depression, which can accommodate the smaller amino acid residues. Serine proteases are ubiquitous in prokaryotes and eukaryotes and serve important and diverse biological functions such as hemostasis, fibrinolysis, complement formation and the digestion of dietary proteins. Coagulation factors, such as FIX, FVII and FX possess serine protease domains that hydrolyze specific members of the coagulation cascade.

Elastases that belong to the serine protease family display extensive sequence homology to other known serine proteases, including trypsin and chymotrypsin. Serine elastases preferentially cleave polypeptides adjacent to aliphatic amino acids residues, typically alanine, valine and methionine, and to a lesser extent, leucine and isoleucine. Humans have six elastase genes which encode the structurally similar proteins, elastase 1 (ELA-1, also known as pancreatic elastase, PE), elastase 2 (neutrophil elastase, NE, also known as PMN elastase, bone marrow serine protease, medullasin, human leukocyte elastase, HLE), elastase 2A (ELA-2A), elastase 2B (ELA-2B), elastase 3A (ELA-3A, elastase IIIA, Protease E), and elastase-3B (ELA-3B, elastase IIIB, protease E). Other serine proteases with elastase activity include, but are not limited to, proteinase-3 (PR-3), endogenous vascular elastase (EVE), and endothelial cell elastase (ECE).

Neutrophil primary azurophil granules carry NE (ELA-2) and PR-3, which are released upon neutrophil activation. NE is involved in degradation of the extracellular matrix and (ECM), including degradation of elastin, cartilage proteoglycans, collagens, and fibronectin, and digestion of material taken into the cell by phagocytosis. NE also helps in degradation of proteins, such as immunoglobulins and surfactant apoproteins. NE preferentially cleaves Val-X bonds and to a lesser extent Ala-X bonds. Abnormal or excessive release of NE has been linked to defects in connective tissue turnover, arthritis and inflammation. Like NE, PR-3 also functions to activate proenzymes, such as metalloproteinases, and cytokines, such as TNF-α, IL-1β, and interleukin-8 (IL-8).

Pancreatic elastase (ELA-1) preferentially cleaves Ala-X bonds and is expressed primarily in skin keratinocytes. Expression of ELA is not normally found in the adult pancreas though it is often expressed in and used as a marker for pancreatic cancers. Elastase activity of the normal pancreas is attributable to ELA-2A and ELA-2B. ELA-2A and ELA-2B preferentially cleaves Leu-X, Met-X and Phe-X bonds.

Some pathological conditions are result at least in part from an imbalance between the elastases and their endogenous inhibitors. Uncontrolled proteolytic degradation by neutrophil elastases, especially ELA-2 has been implicated in a number of pathological conditions like pulmonary emphysema, acute respiratory distress syndrome, septic shock, multiple organ failure, rheumatoid arthritis and cystic fibrosis.

High concentrations of elastases can be found in the gastrointestinal tract and blood stream. Hence, effective therapeutics to be administered via these routes can be achieved through modification of elastase cleavage sites. As mentioned above, FIX is known to be cleaved by human neutrophil elastase (HNE) at amino acid positions T140, T144, I164, T172 and V181 of a mature FIX polypeptide. Cleavage of FIX by HNE generates a FIX polypeptide without coagulant activity. Modification of FIX polypeptides at one or more elastase cleavage sites can increase resistance of FIX polypeptides to proteolytic degradation and thus, improve the therapeutic properties of the FIX polypeptide.

Increased levels of elastase are associated with coagulation disease states, such as disseminated intravascular coagulation (DIC), wherein there is a depletion of active coagulation factors. The decrease in pool of coagulation factors can result in excessive bleeding at sites of tissue damage due to the deficiency in clotting factors needed at the injury location. Modification of FIX polypeptides at elastase cleavage sites, such as the modified FIX polypeptides provided herein, can increase the resistance of FIX polypeptides to elevated levels of elastase in such disease states and thus, improve the therapeutic effectiveness of FIX polypeptides.

b. Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are a family of Zn²⁺- and calcium-dependent endopeptidases that degrade components of the extracellular matrix (ECM). In addition, MMPs also can process a number of cell-surface cytokines, receptors and other soluble proteins. They are involved in normal tissue remodeling processes such as wound healing, pregnancy and angiogenesis. Under physiological conditions, MMPs are made as inactive precursors (zymogens) and are processed to their active form. Additionally, the enzymes are specifically regulated by endogenous inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs). The proteolytic activity of MMPs acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. The excess synthesis and production of these proteins lead to accelerated degradation of the ECM which is associated with a variety of diseases and conditions such as, for example, bone homeostasis, arthritis, cancer, multiple sclerosis and rheumatoid arthritis. In the context of neuroinflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system (Leppert et al. (2001) Brain Res. Rev. 36(2-3): 249-57; Borkakoti et al. (1998) Prog. Biophys. Mol. Biol. 70(1): 73-94).

Members of the MMP family include collagenases, gelatinases, stromelysins, matrilysin and membrane-bound MMPs. Most MMPs are secreted in the inactive proenzyme form. The secreted proenzyme MMPs can be activated by several proinflammatory agents such as oxidants, proteinases including elastase, plasmin, and trypsin, and other MMPs (Cuzner and Opdenakker (1999) J. Neuroimmunol. 94(1-2): 1-14). In tissues, physiological MMP activators include tissue or plasma proteinases or opportunistic bacterial proteinases. For example, the plasminogen activator/plasmin system, including ubiquitous plasminogen by urokinase (u-Pa) and tissue-type plasminogen activator (t-Pa), is an important activator of pro-MMP in pathological situations. MMP activity can be inhibited by tissue inhibitors of metalloproteinases (TIMPs), by serine proteinase inhibitors (serpins), and by nonspecific proteinase inhibitors, such as α2-macroglobulin. TIMPs inhibit the MMP activity through noncovalent binding of the active zinc-binding sites of MMPs. Proteolytic activities of MMPs and plasminogen activators, and their inhibitors, are important in maintaining the integrity of the ECM as cell-ECM interactions influence and mediate a wide range of processes including proliferation, differentiation, adhesion and migration of a variety of cell types. Excessive production of matrix metalloproteinases has been implicated in tissue damage and wound healing, inflammatory disorders, proliferative disorders and autoimmune diseases (St-Pierre et al. (2003) Curr. Drug Targets Inflamm. Allergy 2(3): 206-215; Opdenakker (1997) G. Verh. K. Acad. Geneeskd. Belg. 59(6): 489-514).

c. Increased Resistance to Proteolysis by Removal of Proteolytic Sites

As exemplified herein, the 2D-scanning methodology was used to identify the amino acids of hFIX that lead to an increase in stability when challenged with proteases (such as blood and/or intestinal proteins), blood lysate and/or serum. Increasing protein stability to proteases (such as blood, lysate and intestinal serum), provides a longer in vivo half-life for the particular protein molecules. This can reduce the frequency of administered doses and/or the total amount administered.

A first step in the design of hFIX mutants provided resistant to proteolysis included identifying sites vulnerable to proteolysis along the protein sequence. Based on a list of selected blood, intestinal or any other type of proteases considered (Table 4), the complete list of all amino acids and sequences of amino acids in hFIX that can be targeted by those proteases was first determined in silico. The protease targets (amino acids or sequences of amino acids along the hFIX polypeptide) are named in silico HITs (is-HITs).

The second step in the design of hFIX mutants that are resistant to proteolysis included identifying the appropriate replacing amino acids such that if they replaced the natural amino acids in hFIX at is-HITs, the protein (i) becomes resistant to proteolysis; and (ii) elicits a level of activity at least comparable to the wild-type hFIX polypeptide. In another embodiment the protein (i) becomes resistant to proteolysis; and (ii) inhibits an activity of the wild-type hFIX polypeptide and/or acts as an anticoagulant. The choice of the replacing amino acids included consideration of the broad target specificity of certain proteases and the need to preserve the physicochemical properties such as hydrophobicity, charge and polarity of essential (such as, catalytic, binding, etc.) residues in hFIX.

“Point Accepted Mutation” (PAM; Dayhoff et al., 1978) can be used as part of the 2D scanning approach. PAM values, originally developed to produce alignments between protein sequences, are available in the form of probability matrices that reflect an evolutionary distance between amino acids. Conservative substitutions of a residue in a reference sequence are those substitutions that are physically and functionally similar to the corresponding reference residues, i.e., that have a similar size, shape, electric charge, and/or chemical properties, including the ability to form covalent or hydrogen bonds and other such interactions. Conservative substitutions show the highest scores fitting with the PAM matrix criteria in the form of accepted point mutations. The PAM250 matrix is used in the frame of 2D-scanning to identify candidate replacing amino acids for the is-HITs in order to generate conservative mutations without affecting protein function. At least two amino acids with the highest values in PAM250 matrix corresponding to conservative substitutions or accepted point mutations were chosen for replacement at each is-HIT. The replacement of amino acids by cysteine residues is explicitly avoided since this change can lead to the formation of intermolecular disulfide bonds.

Briefly, using the algorithm PROTEOL (on-line at infobiogen.fr and at bioinfo.hku.hk/services/analyseq/cgi-bin/proteol_in.pl), a list of residues along the mature hFIX polypeptide of 415 amino acids (SEQ ID NO: 2), which can be recognized as substrate for proteases (blood, intestinal, etc.) in Table 4 was established. The algorithm generates a proteolytic digestion map based on a list of proteases, the proteolytic specificity of the proteases, and the polypeptide amino acid sequence that is entered. Table 4 shows the in silico identification of amino acid positions that are targets for proteolysis using selected proteases and chemical treatment.

TABLE 4

Amino Acid
Protease or Chemical

Abbreviation
Position
Treatment

AspN
D
Endoproteinase Asp-N

Chymo
(F, W, Y, M, L)~P
Chymotrypsin

Clos
R
Clostripain

CnBr
M
Cyanogen Bromide

IBzO
W
IodosoBenzoate

Myxo
K
Myxobacter

NH₂OH
N G
Hydroxylamine

pH2.5
D P
pH 2.5

ProEn
P
Proline Endopeptidase

Staph
E
Staphylococcal Protease

Tryp
(K, R)~P
Trypsin

TrypK
K~P
Trypsin (Arg blocked)

TrypR
R~P
Trypsin (Lys blocked)

Is-HITS were identified and LEADS created for higher resistance to proteolysis of hFIX. The native amino acids at each of the is-HIT positions and replacing amino acids for increased resistance to proteolysis can include, but are not limited to replacing any of Y, A, L, S, T, I, V, F, Q and M by any of E, D, K, R, N, Q, S and T. Is-HITS and LEADs can include modifications at regions susceptible to proteolysis.

d. Modified FIX Polypeptides Exhibiting Increased Protease Resistance

Using such methods as outlined above, modified FIX proteins were generated. Sites, designated is-HIT positions, were identified and modified proteins prepared. The resulting proteins exhibited increased protease resistance. This was manifested as, for example, increased the methods described herein, the following is-HIT positions were identified to eliminate protease sensitive sites and increase protein stability of a FIX polypeptide. In addition, the modified FIX proteins can be administered orally and deliver therapeutically effective dosages to the blood.

Among the modified FIX polypeptides provided herein are those that included modifications at one or more loci. Typically modified FIX polypeptides include one, two, three, four or five such modifications. In addition, the background polypeptide that is modified can include other modifications known to alter desired properties, such as modification of sites to decrease immunogenicity and/or to increase glycosylation. The polypeptides can include further modifications, such as pegylation.

Provided herein are modified FIX polypeptides that include modifications of the amino acids at positions selected from among one or more of the following: 1, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 52, 53, 55, 57, 58, 59, 60, 61, 63, 64, 65, 66, 68, 69, 70, 72, 74, 75, 76, 77, 78, 79, 80, 82, 83, 84, 85, 86, 87, 90, 91, 92, 93, 94, 96, 98, 100, 101, 102, 103, 104, 106, 107, 108, 110, 112, 113, 114, 115, 116, 117, 118, 119, 122, 123, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 168, 169, 171, 172, 174, 175, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217, 218, 219, 220, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 238, 239, 240, 241, 242, 244, 245, 247, 248, 250, 251, 252, 253, 254, 255, 259, 261, 262, 263, 265, 266, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 283, 284, 285, 286, 287, 288, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 341, 342, 343, 344, 345, 348, 349, 351, 352, 353, 355, 356, 357, 358, 359, 360, 363, 364, 365, 366, 367, 368, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 407, 408, 409, 410, 411, 412, 413, 414, and 415. These positions (loci) are relative to the mature FIX polypeptide whose sequence is set forth in SEQ ID NO.: 2 or 1035 or corresponding loci in an allelic or species variant and/or in a background polypeptide (starting FIX polypeptide) including other modifications. The FIX polypeptides include precursor forms and mature forms, zymogens and activated forms.

In particular examples, amino acid replacement or replacements can be at any one or more positions corresponding to any of the following positions: 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, E36, R37, T38, T39, E40, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, S61, K63, D64, D65, I66, S68, Y69, E70, W72, P74, F75, G76, F77, E78, G79, K80, E83, L84, D85, V86, T87, I90, K91, N92, G93, R94, E96, F98, K100, S102, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, E119, K122, S123, E125, P126, A127, V128, P129, P131, G133, R134, V135, S136, V137, S138, T140, 5141, K142, L143, T144, R145, A146, E147, T148, V149, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, S171, T172, S174, F175, D177, 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, E224, T225, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, T241, E242, T244, E245, K247, 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, E294, 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, T371, E372, 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, L414, and T415 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or in corresponding loci in an allelic or species variant and/or in a background polypeptide including other modifications.

In one embodiment, positions are typically replaced as follows: replacement of D with N or Q, replacement of E with H, Q or N, replacement of F with I or V, replacement of K with Q or N, replacement of L with I or V, replacement of M with I or V, replacement of N with Q or S, replacement of P with A or S, replacement of R with H or Q, replacement of W with H or S, replacement of Y with I or H, replacement of A, G, I, S, T, or V with Q, H, or N.

In one embodiment, positions corresponding to hFIX are selected (is-HITS) and amino acid replacements are made (LEADs) with increased resistance to proteolysis that include, but are not limited to replacements corresponding to those set forth in Table 5, where the replacements are made compared to the sequence of amino acids set forth in SEQ ID NO: 2. Table 5 provides non-limiting examples of amino acid replacements, corresponding to amino acid positions of a mature FIX polypeptide, that increase resistance to proteolysis and, thereby, protein stability. The modified FIX polypeptides include precursor and mature forms, zymogen and active forms, allelic and species variants and/or modifications in a background FIX polypeptide including other modifications.

In referencing such mutants, the first amino acid (one-letter abbreviation) corresponds to the amino acid that is replaced, the number corresponds to position in the hFIX polypeptide sequence with reference to SEQ ID NO: 2, and the second amino acid (one-letter abbreviation) corresponds to the amino acid selected that replaces the first amino acid at that position. In Table 5 below, the sequence identifier (SEQ ID NO.) is in parenthesis next to each substitution. The FIX polypeptides employed for modification can be any FIX polypeptide, including other mammalian FIX polypeptides. Corresponding positions, as assessed by appropriate alignment, are identified and modified as described herein.

TABLE 5

List of Human FIX Modifications to Increase Resistance to Proteolysis

Y1H (3)
Y1I (4)
K5N (5)
K5Q (6)
L6I (7)
L6V (8)
E7Q (9)

E7H (10)
E7N (11)
E8Q (12)
E8H (13)
E8N (14)
F9I (15)
F9V (16)

L14I (17)
L14V (18)
E15Q (19)
E15H (20)
E15N (21)
R16H (22)
R16Q (23)

E17Q (24)
E17H (25)
E17N (26)
M19I (27)
M19V (28)
E20Q (29)
E20H (30)

E20N (31)
E21Q (32)
E21H (33)
E21N (34)
K22N (35)
K22Q (36)
F25I (37)

F25V (38)
E26Q (39)
E26H (40)
E26N (41)
E27Q (42)
E27H (43)
E27N (44)

R29H (45)
R29Q (46)
E30Q (47)
E30H (48)
E30N (49)
F32I (50)
F32V (51)

E33Q (52)
E33H (53)
E33N (54)
E36Q (55)
E36H (56)
E36N (57)
R37H (58)

R37Q (59)
E40Q (60)
E40H (61)
E40N (62)
F41I (63)
F41V (64)
W42S (65)

W42H (66)
K43N (67)
K43Q (68)
Y45H (69)
Y45I (70)
D47N (71)
D47Q (72)

D49N (73)
D49Q (74)
E52Q (75)
E52H (76)
E52N (77)
P55A (78)
P55S (79)

L57I (80)
L57V (81)
N58Q (82)
N58S (83)
K63N (84)
K63Q (85)
D64N (86)

D64Q (87)
D65N (88)
D65Q (89)
Y69H (90)
Y69I (91)
E70Q (92)
E70H (93)

E70N (94)
W72S (95)
W72H (96)
P74A (97)
P74S (98)
F75I (99)
F75V (100)

F77I (101)
F77V (102)
E78Q (103)
E78H (104)
E78N (105)
K80N (106)
K80Q (107)

E83Q (108)
E83H (109)
E83N (110)
L84I (111)
L84V (112)
D85N (113)
D85Q (114)

K91N (115)
K91Q (116)
N92Q (117)
N92S (118)
R94H (119)
R94Q (120)
E96Q (121)

E96H (122)
E96N (123)
F98I (124)
F98V (125)
K100N (126)
K100Q (127)
D104N (128)

D104Q (129)
K106N (130)
K106Q (131)
E113Q (132)
E113H (133)
E113N (134)
Y115H (135)

Y115I (136)
R116H (137)
R116Q (138)
L117I (139)
L117V (140)
E119Q (141)
E119H (142)

E119N (143)
K122N (144)
K122Q (145)
E125Q (146)
E125H (147)
E125N (148)
P126A (149)

P126S (150)
P129A (151)
P129S (152)
P131A (153)
P131S (154)
R134H (155)
R134Q (156)

K142N (157)
K142Q (158)
L143I (159)
L143V (160)
R145H (161)
R145Q (162)
E147Q (163)

E147H (164)
E147N (165)
P151A (166)
P151S (167)
D152N (168)
D152Q (169)
D154N (170)

D154Q (171)
Y155H (172)
Y155I (173)
E160Q (174)
E160H (175)
E160N (176)
E162Q (177)

E162H (178)
E162N (179)
L165I (180)
L165V (181)
D166N (182)
D166Q (183)
F175I (184)

F175V (185)
D177N (186)
D177Q (187)
F178I (188)
F178V (189)
R180H (190)
R180Q (191)

E185Q (192)
E185H (193)
E185N (194)
D186N (195)
D186Q (196)
K188N (197)
K188Q (198)

P189A (199)
P189S (200)
P193A (201)
P193S (202)
W194S (203)
W194H (204)
L198I (205)

L198V (206)
N199Q (207)
N199S (208)
K201N (209)
K201Q (210)
D203N (211)
D203Q (212)

F205I (213)
F205V (214)
E213Q (215)
E213H (216)
E213N (217)
K214N (218)
K214Q (219)

W215S (220)
W215H (221)
E224Q (222)
E224H (223)
E224N (224)
K228N (225)
K228Q (226)

E235Q (227)
E235H (228)
E235N (229)
E239Q (230)
E239H (231)
E239N (232)
E240Q (233)

E240H (234)
E240N (235)
E242Q (236)
E242H (237)
E242N (238)
E245Q (239)
E245H (240)

E245N (241)
K247N (242)
K247Q (243)
R248H (244)
R248Q (245)
R252H (246)
R252Q (247)

P255A (248)
P255S (249)
Y259H (250)
Y259I (251)
K265N (252)
K265Q (253)
Y266H (254)

Y266I (255)
D269N (256)
D269Q (257)
L272I (258)
L272V (259)
L273I (260)
L273V (261)

E274Q (262)
E274H (263)
E274N (264)
L275I (265)
L275V (266)
D276N (267)
D276Q (268)

E277Q (269)
E277H (270)
E277N (271)
P278A (272)
P278S (273)
L279I (274)
L279V (275)

L281I (276)
L281V (277)
Y284H (278)
Y284I (279)
P287A (280)
P287S (281)
D292N (282)

D292Q (283)
K293N (284)
K293Q (285)
E294Q (286)
E294H (287)
E294N (288)
Y295H (289)

Y295I (290)
F299I (291)
F299V (292)
L300I (293)
L300V (294)
K301N (295)
K301Q (296)

F302I (297)
F302V (298)
Y306H (299)
Y306I (300)
W310S (301)
W310H (302)
R312H (303)

R312Q (304)
F314I (305)
F314V (306)
K316N (307)
K316Q (308)
R318H (309)
R318Q (310)

L321I (311)
L321V (312)
L323I (313)
L323V (314)
Y325H (315)
Y325I (316)
L326I (317)

L326V (318)
R327H (319)
R327Q (320)
P329A (321)
P329S (322)
L330I (323)
L330V (324)

D332N (325)
D332Q (326)
R333H (327)
R333Q (328)
L337I (329)
L337V (330)
R338H (331)

R338Q (332)
K341N (333)
K341Q (334)
F342I (335)
F342V (336)
Y345H (337)
Y345I (338)

M348I (339)
M348V (340)
F349I (341)
F349V (342)
F353I (343)
F353V (344)
E355Q (345)

E355H (346)
E355N (347)
R358H (348)
R358Q (349)
D359N (350)
D359Q (351)
D364N (352)

D364Q (353)
P368A (354)
P368S (355)
E372Q (356)
E372H (357)
E372N (358)
E374Q (359)

E374H (360)
E374N (361)
F378I (362)
F378V (363)
L379I (364)
L379V (365)
W385S (366)

W385H (367)
E387Q (368)
E387H (369)
E387N (370)
E388Q (371)
E388H (372)
E388N (373)

M391I (374)
M391V (375)
K392N (376)
K392Q (377)
K394N (378)
K394Q (379)
Y395H (380)

Y395I (381)
Y398H (382)
Y398I (383)
K400N (384)
K400Q (385)
R403H (386)
R403Q (387)

Y404H (388)
Y404I (389)
W407S (390)
W407H (391)
K409N (392)
K409Q (393)
E410Q (394)

E410H (395)
E410N (396)
K411N (397)
K411Q (398)
K413N (399)
K413Q (400)
L414I (401)

L414V (402)
S3N (403)
S3Q (404)
S3H (405)
G4N (406)
G4Q (407)
G4H (408)

V10N (409)
V10Q (410)
V10H (411)
G12N (412)
G12Q (413)
G12H (414)
S24N (415)

S24Q (416)
S24H (417)
A28N (418)
A28Q (419)
A28H (420)
V31N (421)
V31Q (422)

V31H (423)
T35N (424)
T35Q (425)
T35H (426)
T38N (427)
T38Q (428)
T38H (429)

T39N (430)
T39Q (431)
T39H (432)
V46N (433)
V46Q (434)
V46H (435)
G48N (436)

G48Q (437)
G48H (438)
S53N (439)
S53Q (440)
S53H (441)
G59N (442)
G59Q (443)

G59H (444)
G60N (445)
G60Q (446)
G60H (447)
S61N (448)
S61Q (449)
S61H (450)

I66N (451)
I66Q (452)
I66H (453)
S68N (454)
S68Q (455)
S68H (456)
G76N (457)

G76Q (458)
G76H (459)
G79N (460)
G79Q (461)
G79H (462)
V86N (463)
V86Q (464)

V86H (465)
T87N (466)
T87Q (467)
T87H (468)
I90N (469)
I90Q (470)
I90H (471)

G93N (472)
G93Q (473)
G93H (474)
S102N (475)
S102Q (476)
S102H (477)
A103N (478)

A103Q (479)
A103H (480)
V107N (481)
V107Q (482)
V107H (483)
V108N (484)
V108Q (485)

V108H (486)
S110N (487)
S110Q (488)
S110H (489)
T112N (490)
T112Q (491)
T112H (492)

G114N (493)
G114Q (494)
G114H (495)
A118N (496)
A118Q (497)
A118H (498)
S123N (499)

S123Q (500)
S123H (501)
A127N (502)
A127Q (503)
A127H (504)
V128N (505)
V128Q (506)

V128H (507)
G133N (508)
G133Q (509)
G133H (510)
V135N (511)
V135Q (512)
V135H (513)

S136N (514)
S136Q (515)
S136H (516)
V137N (517)
V137Q (518)
V137H (519)
S138N (520)

S138Q (521)
S138H (522)
T140N (523)
T140Q (524)
T140H (525)
S141N (526)
S141Q (527)

S141H (528)
T144N (529)
T144Q (530)
T144H (531)
A146N (532)
A146Q (533)
A146H (534)

T148N (535)
T148Q (536)
T148H (537)
V149N (538)
V149Q (539)
V149H (540)
V153N (541)

V153Q (542)
V153H (543)
V156N (544)
V156Q (545)
V156H (546)
S158N (547)
S158Q (548)

S158H (549)
T159N (550)
T159Q (551)
T159H (552)
A161N (553)
A161Q (554)
A161H (555)

T163N (556)
T163Q (557)
T163H (558)
I164N (559)
I164Q (560)
I164H (561)
I168N (562)

I168Q (563)
I168H (564)
T169N (565)
T169Q (566)
T169H (567)
S171N (568)
S171Q (569)

S171H (570)
T172N (571)
T172Q (572)
T172H (573)
S174N (574)
S174Q (575)
S174H (576)

T179N (577)
T179Q (578)
T179H (579)
V181N (580)
V181Q (581)
V181H (582)
V182N (583)

V182Q (584)
V182H (585)
G183N (586)
G183Q (587)
G183H (588)
G184N (589)
G184Q (590)

G184H (591)
A187N (592)
A187Q (593)
A187H (594)
G190N (595)
G190Q (596)
G190H (597)

V196N (598)
V196Q (599)
V196H (600)
V197N (601)
V197Q (602)
V197H (603)
G200N (604)

G200Q (605)
G200H (606)
V202N (607)
V202Q (608)
V202H (609)
A204N (610)
A204Q (611)

A204H (612)
G207N (613)
G207Q (614)
G207H (615)
G208N (616)
G208Q (617)
G208H (618)

S209N (619)
S209Q (620)
S209H (621)
I210N (622)
I210Q (623)
I210H (624)
V211N (625)

V211Q (626)
V211H (627)
I216N (628)
I216Q (629)
I216H (630)
V217N (631)
V217Q (632)

V217H (633)
T218N (634)
T218Q (635)
T218H (636)
A219N (637)
A219Q (638)
A219H (639)

A220N (640)
A220Q (641)
A220H (642)
V223N (643)
V223Q (644)
V223H (645)
T225N (646)

T225Q (647)
T225H (648)
G226N (649)
G226Q (650)
G226H (651)
V227N (652)
V227Q (653)

V227H (654)
I229N (655)
I229Q (656)
I229H (657)
T230N (658)
T230Q (659)
T230H (660)

V231N (661)
V231Q (662)
V231H (663)
V232N (664)
V232Q (665)
V232H (666)
A233N (667)

A233Q (668)
A233H (669)
G234N (670)
G234Q (671)
G234H (672)
I238N (673)
I238Q (674)

I238H (675)
T241N (676)
T241Q (677)
T241H (678)
T244N (679)
T244Q (680)
T244H (681)

V250N (682)
V250Q (683)
V250H (684)
I251N (685)
I251Q (686)
I251H (687)
I253N (688)

I253Q (689)
I253H (690)
I254N (691)
I254Q (692)
I254H (693)
A261N (694)
A261Q (695)

A261H (696)
A262N (697)
A262Q (698)
A262H (699)
I263N (700)
I263Q (701)
I263H (702)

I270N (703)
I270Q (704)
I270H (705)
A271N (706)
A271Q (707)
A271H (708)
V280N (709)

V280Q (710)
V280H (711)
S283N (712)
S283Q (713)
S283H (714)
V285N (715)
V285Q (716)

V285H (717)
T286N (718)
T286Q (719)
T286H (720)
I288N (721)
I288Q (722)
I288H (723)

I290N (724)
I290Q (725)
I290H (726)
A291N (727)
A291Q (728)
A291H (729)
T296N (730)

T296Q (731)
T296H (732)
I298N (733)
I298Q (734)
I298H (735)
G303N (736)
G303Q (737)

G303H (738)
S304N (739)
S304Q (740)
S304H (741)
G305N (742)
G305Q (743)
G305H (744)

V307N (745)
V307Q (746)
V307H (747)
S308N (748)
S308Q (749)
S308H (750)
G309N (751)

G309Q (752)
G309H (753)
G311N (754)
G311Q (755)
G311H (756)
V313N (757)
V313Q (758)

V313H (759)
G317N (760)
G317Q (761)
G317H (762)
S319N (763)
S319Q (764)
S319H (765)

A320N (766)
A320Q (767)
A320H (768)
V322N (769)
V322Q (770)
V322H (771)
V328N (772)

V328Q (773)
V328H (774)
V331N (775)
V331Q (776)
V331H (777)
A334N (778)
A334Q (779)

A334H (780)
T335N (781)
T335Q (782)
T335H (783)
S339N (784)
S339Q (785)
S339H (786)

T340N (787)
T340Q (788)
T340H (789)
T343N (790)
T343Q (791)
T343H (792)
I344N (793)

I344Q (794)
I344H (795)
A351N (796)
A351Q (797)
A351H (798)
G352N (799)
G352Q (800)

G352H (801)
G356N (802)
G356Q (803)
G356H (804)
G357N (805)
G357Q (806)
G357H (807)

S360N (808)
S360Q (809)
S360H (810)
G363N (811)
G363Q (812)
G363H (813)
S365N (814)

S365Q (815)
S365H (816)
G366N (817)
G366Q (818)
G366H (819)
G367N (820)
G367Q (821)

G367H (822)
V370N (823)
V370Q (824)
V370H (825)
T371N (826)
T371Q (827)
T371H (828)

V373N (829)
V373Q (830)
V373H (831)
G375N (832)
G375Q (833)
G375H (834)
T376N (835)

T376Q (836)
T376H (837)
S377N (838)
S377Q (839)
S377H (840)
T380N (841)
T380Q (842)

T380H (843)
G381N (844)
G381Q (845)
G381H (846)
I382N (847)
I382Q (848)
I382H (849)

I383N (850)
I383Q (851)
I383H (852)
S384N (853)
S384Q (854)
S384H (855)
G386N (856)

G386Q (857)
G386H (858)
A390N (859)
A390Q (860)
A390H (861)
G393N (862)
G393Q (863)

G393H (864)
G396N (865)
G396Q (866)
G396H (867)
I397N (868)
I397Q (869)
I397H (870)

T399N (871)
T399Q (872)
T399H (873)
V401N (874)
V401Q (875)
V401H (876)
S402N (877)

S402Q (878)
S402H (879)
V405N (880)
V405Q (881)
V405H (882)
I408N (883)
I408Q (884)

I408H (885)
T412N (886)
T412Q (887)
T412H (888)
T415N (889)
T415Q (890)
T415H (891)

L165Q (1025)
L165H (1026)
F175H (1027)
F178H (1028)
F192H (1029)
F192I (1030)
F192V (1031)

W194I (1032)
L198Q (1033)
L198H (1034)

A modified FIX polypeptide provided herein that exhibits increased protease resistance can contain one or more amino acid modifications corresponding to any one or more modifications of Y1H (i.e., replacement of Y by H at a position corresponding to amino acid position 1 of mature human FIX (such as, SEQ ID NO: 2)), Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L6I, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N, F91, 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, F25I, 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, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, F41I, F41V, W42S, W42H, K43N, K43Q, Y45H, Y45I, 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, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F75I, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q, S102Q, S102H, S102N, 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, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, 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, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, 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, F192IH, 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, E224Q, E224H, E224N, T225Q, T225H, T225N, 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, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, 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, E294Q, E294H, E294N, 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, T371Q, T371H, T371N, E372Q, E372H, E372N, 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, E387H, 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, L414I, L414V, T415Q, T415H, and T415N, where the modified polypeptide exhibits increased resistance to proteolysis. In some examples, the modifications are in an unmodified FIX polypeptide, such as a FIX having a sequence of amino acids set forth in SEQ ID NO: 2. Exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 3-891 or 1025-1034. In some examples, the modifications are in a FIX polypeptide, such as a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 892 or 1035. For example, exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 1036-1926 or 2035-2044.

Additionally, a modified FIX polypeptide as set forth above can contain a further modification compared to an unmodified FIX polypeptide. Generally, the resulting modified FIX polypeptide retains one or more activities of the unmodified FIX polypeptide. In some embodiments, the modified FIX polypeptide inhibits the activity of an unmodified or wild-type FIX polypeptide and/or acts as an anticoagulant.

i. Modified FIX Polypeptides Exhibiting Increased Protease Resistance to Elastase

Exemplary of modified FIX polypeptides exhibiting increased protease resistance are FIX polypeptides exhibiting increased resistance to elastase. Non-limiting modifications in a FIX polypeptide that confer increased resistance to elastase have been rationally determined herein based on the known substrate specificity of elastase. Polypeptides are cleaved by elastase at or near a sequence of small hydrophobic chains. Modifications that render polypeptides more resistant to hydrolysis by elastase are provided. Of the modifications disclosed in Table 5, some are more specific to resistance to protease digestion by elastase. The following is-HIT positions were identified to eliminate elastase sensitive sites and increase protein stability of FIX polypeptides: 3, 4, 10, 12, 24, 28, 31, 35, 38, 39, 46, 48, 53, 59, 60, 61, 66, 68, 76, 79, 86, 87, 90, 93, 102, 103, 107, 108, 110, 112, 114, 118, 123, 127, 128, 133, 135, 136, 137, 138, 140, 141, 144, 146; 148, 149, 153, 156, 158, 159, 161, 163, 164, 168, 169, 171, 172, 174, 179, 181, 182, 183, 184, 187, 190, 196, 197, 200, 202, 204, 207, 208, 209, 210, 211, 216, 217, 218, 219, 220, 223, 225, 226, 227, 229, 230, 231, 232, 233, 234, 238, 241, 244, 250, 251, 253, 254, 261, 262, 263, 270, 271, 280, 283, 285, 286, 288, 290, 291, 296, 298, 303, 304, 305, 307, 308, 309, 311, 313, 317, 319, 320, 322, 328, 331, 334, 335, 339, 340, 343, 344, 351, 352, 356, 357, 360, 363, 365, 366, 367, 370, 371, 373, 375, 376, 377, 380, 381, 382, 383, 384, 386, 390, 393, 396, 397, 399, 401, 402, 405, 408, 412, and 415, corresponding to amino acid positions in a mature FIX polypeptide set forth in SEQ ID NO: 2. Amino acid modifications can be at any one or more positions corresponding to any of the following positions: S3, G4, V10, G12, S24, A28, V31, T35, T38, T39, V46, G48, S53, G59, G60, S61, I66, S68, G76, G79, V86, T87, I90, G93, S102, A103, V107, V108, S110, T112, G114, A118, S123, A127, V128, G133, V135, S136, V137, S138, T140, S141, T144, A146, T148, V149, V153, V156, S158, T159, A161, T163, I164, 1168, T169, S171, T172, S174, T179, V181, V182, G183, G184, A187, G190, V196, V197, G200, V202, A204, G207, G208, S209, I210, V211, I216, V217, T218, A219, A220, V223, T225, G226, V227, I229, T230, V231, V232, A233, G234, I238, T241, T244, V250, I251, I253, I254, A261, A262, I263, I270, A271, V280, S283, V285, T286, I288, I290, A291, T296, I298, G303, S304, G305, V307, S308, G309, G311, V313, G317, S319, A320, V322, V328, V331, A334, T335, S339, T340, T343, I344, A351, G352, G356, G357, S360, G363, S365, G366, G367, V370, T371, V373, G375, T376, S377, T380, G381, I382, I383, S384, G386, A390, G393, I396, I397, T399, V401, S402, V405, I408, T412, and T415, of a mature FIX polypeptide set forth in SEQ ID NO: 2. Other candidate Leads can be obtained by replacement or replacements of amino acids at is-HIT positions such as, but not limited to, amino acid modifications as described for candidate LEADs in Table 6. Candidate leads can be empirically tested to determine those that confer resistance to elastase. Table 6 provides non-limiting examples of amino acid modifications that are more specific to resistance to protease digestion by elastase and, thereby, increase protein stability. Generally, a resulting modified FIX polypeptide retains one or more activities of a mature or unmodified FIX polypeptide. In some embodiments, the modified FIX polypeptide inhibits an activity of an unmodified or wild-type FIX polypeptide and/or acts as an anticoagulant. In Table 6 below, the sequence identifier (SEQ ID NO.) is in parenthesis next to each substitution.

TABLE 6

List of Human FIX Modifications to Increase Resistance to Elastase Proteolysis

S3N (403)
S3Q (404)
S3H (405)
G4N (406)
G4Q (407)
G4H (408)
V10N (409)

V10Q (410)
V10H (411)
G12N (412)
G12Q (413)
G12H (414)
S24N (415)
S24Q (416)

S24H (417)
A28N (418)
A28Q (419)
A28H (420)
V31N (421)
V31Q (422)
V31H (423)

T35N (424)
T35Q (425)
T35H (426)
T38N (427)
T38Q (428)
T38H (429)
T39N (430)

T39Q (431)
T39H (432)
V46N (433)
V46Q (434)
V46H (435)
G48N (436)
G48Q (437)

G48H (438)
S53N (439)
S53Q (440)
S53H (441)
G59N (442)
G59Q (443)
G59H (444)

G60N (445)
G60Q (446)
G60H (447)
S61N (448)
S61Q (449)
S61H (450)
I66N (451)

I66Q (452)
I66H (453)
S68N (454)
S68Q (455)
S68H (456)
G76N (457)
G76Q (458)

G76H (459)
G79N (460)
G79Q (461)
G79H (462)
V86N (463)
V86Q (464)
V86H (465)

T87N (466)
T87Q (467)
T87H (468)
I90N (469)
I90Q (470)
I90H (471)
G93N (472)

G93Q (473)
G93H (474)
S102N (475)
S102Q (476)
S102H (477)
A103N (478)
A103Q (479)

A103H (480)
V107N (481)
V107Q (482)
V107H (483)
V108N (484)
V108Q (485)
V108H (486)

S110N (487)
S110Q (488)
S110H (489)
T112N (490)
T112Q (491)
T112H (492)
G114N (493)

G114Q (494)
G114H (495)
A118N (496)
A118Q (497)
A118H (498)
S123N (499)
S123Q (500)

S123H (501)
A127N (502)
A127Q (503)
A127H (504)
V128N (505)
V128Q (506)
V128H (507)

G133N (508)
G133Q (509)
G133H (510)
V135N (511)
V135Q (512)
V135H (513)
S136N (514)

S136Q (515)
S136H (516)
V137N (517)
V137Q (518)
V137H (519)
S138N (520)
S138Q (521)

S138H (522)
T140N (523)
T140Q (524)
T140H (525)
S141N (526)
S141Q (527)
S141H (528)

T144N (529)
T144Q (530)
T144H (531)
A146N (532)
A146Q (533)
A146H (534)
T148N (535)

T148Q (536)
T148H (537)
V149N (538)
V149Q (539)
V149H (540)
V153N (541)
V153Q (542)

V153H (543)
V156N (544)
V156Q (545)
V156H (546)
S158N (547)
S158Q (548)
S158H (549)

T159N (550)
T159Q (551)
T159H (552)
A161N (553)
A161Q (554)
A161H (555)
T163N (556)

T163Q (557)
T163H (558)
I164N (559)
I164Q (560)
I164H (561)
I168N (562)
I168Q (563)

I168H (564)
T169N (565)
T169Q (566)
T169H (567)
S171N (568)
S171Q (569)
S171H (570)

T172N (571)
T172Q (572)
T172H (573)
S174N (574)
S174Q (575)
S174H (576)
T179N (577)

T179Q (578)
T179H (579)
V181N (580)
V181Q (581)
V181H (582)
V182N (583)
V182Q (584)

V182H (585)
G183N (586)
G183Q (587)
G183H (588)
G184N (589)
G184Q (590)
G184H (591)

A187N (592)
A187Q (593)
A187H (594)
G190N (595)
G190Q (596)
G190H (597)
V196N (598)

V196Q (599)
V196H (600)
V197N (601)
V197Q (602)
V197H (603)
G200N (604)
G200Q (605)

G200H (606)
V202N (607)
V202Q (608)
V202H (609)
A204N (610)
A204Q (611)
A204H (612)

G207N (613)
G207Q (614)
G207H (615)
G208N (616)
G208Q (617)
G208H (618)
S209N (619)

S209Q (620)
S209H (621)
I210N (622)
I210Q (623)
I210H (624)
V211N (625)
V211Q (626)

V211H (627)
I216N (628)
I216Q (629)
I216H (630)
V217N (631)
V217Q (632)
V217H (633)

T218N (634)
T218Q (635)
T218H (636)
A219N (637)
A219Q (638)
A219H (639)
A220N (640)

A220Q (641)
A220H (642)
V223N (643)
V223Q (644)
V223H (645)
T225N (646)
T225Q (647)

T225H (648)
G226N (649)
G226Q (650)
G226H (651)
V227N (652)
V227Q (653)
V227H (654)

I229N (655)
I229Q (656)
I229H (657)
T230N (658)
T230Q (659)
T230H (660)
V231N (661)

V231Q (662)
V231H (663)
V232N (664)
V232Q (665)
V232H (666)
A233N (667)
A233Q (668)

A233H (669)
G234N (670)
G234Q (671)
G234H (672)
I238N (673)
I238Q (674)
I238H (675)

T241N (676)
T241Q (677)
T241H (678)
T244N (679)
T244Q (680)
T244H (681)
V250N (682)

V250Q (683)
V250H (684)
I251N (685)
I251Q (686)
I251H (687)
I253N (688)
I253Q (689)

I253H (690)
I254N (691)
I254Q (692)
I254H (693)
A261N (694)
A261Q (695)
A261H (696)

A262N (697)
A262Q (698)
A262H (699)
I263N (700)
I263Q (701)
I263H (702)
I270N (703)

I270Q (704)
I270H (705)
A271N (706)
A271Q (707)
A271H (708)
V280N (709)
V280Q (710)

V280H (711)
S283N (712)
S283Q (713)
S283H (714)
V285N (715)
V285Q (716)
V285H (717)

T286N (718)
T286Q (719)
T286H (720)
I288N (721)
I288Q (722)
I288H (723)
I290N (724)

I290Q (725)
I290H (726)
A291N (727)
A291Q (728)
A291H (729)
T296N (730)
T296Q (731)

T296H (732)
I298N (733)
I298Q (734)
I298H (735)
G303N (736)
G303Q (737)
G303H (738)

S304N (739)
S304Q (740)
S304H (741)
G305N (742)
G305Q (743)
G305H (744)
V307N (745)

V307Q (746)
V307H (747)
S308N (748)
S308Q (749)
S308H (750)
G309N (751)
G309Q (752)

G309H (753)
G311N (754)
G311Q (755)
G311H (756)
V313N (757)
V313Q (758)
V313H (759)

G317N (760)
G317Q (761)
G317H (762)
S319N (763)
S319Q (764)
S319H (765)
A320N (766)

A320Q (767)
A320H (768)
V322N (769)
V322Q (770)
V322H (771)
V328N (772)
V328Q (773)

V328H (774)
V331N (775)
V331Q (776)
V331H (777)
A334N (778)
A334Q (779)
A334H (780)

T335N (781)
T335Q (782)
T335H (783)
S339N (784)
S339Q (785)
S339H (786)
T340N (787)

T340Q (788)
T340H (789)
T343N (790)
T343Q (791)
T343H (792)
I344N (793)
I344Q (794)

I344H (795)
A351N (796)
A351Q (797)
A351H (798)
G352N (799)
G352Q (800)
G352H (801)

G356N (802)
G356Q (803)
G356H (804)
G357N (805)
G357Q (806)
G357H (807)
S360N (808)

S360Q (809)
S360H (810)
G363N (811)
G363Q (812)
G363H (813)
S365N (814)
S365Q (815)

S365H (816)
G366N (817)
G366Q (818)
G366H (819)
G367N (820)
G367Q (821)
G367H (822)

V370N (823)
V370Q (824)
V370H (825)
T371N (826)
T371Q (827)
T371H (828)
V373N (829)

V373Q (830)
V373H (831)
G375N (832)
G375Q (833)
G375H (834)
T376N (835)
T376Q (836)

T376H (837)
S377N (838)
S377Q (839)
S377H (840)
T380N (841)
T380Q (842)
T380H (843)

G381N (844)
G381Q (845)
G381H (846)
I382N (847)
I382Q (848)
I382H (849)
I383N (850)

I383Q (851)
I383H (852)
S384N (853)
S384Q (854)
S384H (855)
G386N (856)
G386Q (857)

G386H (858)
A390N (859)
A390Q (860)
A390H (861)
G393N (862)
G393Q (863)
G393H (864)

G396N (865)
G396Q (866)
G396H (867)
I397N (868)
I397Q (869)
I397H (870)
T399N (871)

T399Q (872)
T399H (873)
V401N (874)
V401Q (875)
V401H (876)
S402N (877)
S402Q (878)

S402H (879)
V405N (880)
V405Q (881)
V405H (882)
I408N (883)
I408Q (884)
I408H (885)

T412N (886)
T412Q (887)
T412H (888)
T415N (889)
T415Q (890)
T415H (891)

A modified FIX polypeptide provided herein that exhibits increased protease resistance to elastase can contain one or more amino acid modifications corresponding to modifications selected from any of S3Q, S3H, S3N, G4Q, G4H, 04N, V10Q, V10H, V10N, G12Q, G12H, G12N, S24Q, S24H, S24N, A28Q, A28H, A28N, V31Q, V31H, V31N, T35Q, T35H, T35N, T38Q, T38H, T38N, T39Q, T39H, T39N, V46Q, V46H, V46N, G48Q, G48H, G48N, S53Q, S53H, S53N, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, 166Q, 166H, 166N, S68Q, S68H, S68N, G76Q, G76H, G76N, G79Q, G79H, G79N, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, G93Q, G93H, G93N, S102Q, S102H, S102N, A103Q, A103H, A103N, V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, G114Q, G114H, G114N, A118Q, A118H, A118N, S123Q, S123H, S123N, A127Q, A127H, A127N, V128Q, V128H, V128N, G133Q, G133H, G133N, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, T144Q, T144H, T144N, A146Q, A146H, A146N, T148Q, T14811, T148N, V149Q, V149H, V149N, V153Q, V153H, V153N, V156Q, V156H, V156N, S158Q, S158H, S158N, T159Q, T159H, T159N, A161Q, A161H, A161N, T163Q, T163H, T163N, I164Q, I164H, I164N, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, T179Q, T179H, T179N, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q, G183H, G183N, G184Q, G184H, G184N, A187Q, A187H, A187N, G190Q, G190H, G190N, V196Q, V196H, V196N, V197Q, V197H, V19′7N, G200Q, G200H, G200N, V202Q, V202H, V202N, A204Q, A204H, A204N, G207Q, G207H, G207N, G208Q, G208H, G208N, S209Q, S209H, S209N, I210Q, I210H, I210N, V211Q, V21111, V211N, I216Q, I216H, I216N, V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q, A220H, A220N, V223Q, V223H, V223N, T225Q, T225H, T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, I229Q, I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H, V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, I238Q, I238H, I238N, T241Q, T241H, T241N, T244Q, T244H, T244N, V250Q, V250H, V250N, I251Q, I251H, I251N, I253Q, I253H, I253N, I254Q, I254H, I254N, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q, I263H, I263N, I270Q, I270H, I270N, A271Q, A271H, A271N, V280Q, V280H, V280N, S283Q, S283H, S283N, V285Q, V285H, V285N, T286Q, T286H, T286N, I288Q, I288H, I288N, I290Q, I290H, I290N, A291Q, A291H, A291N, T296Q, T296H, T296N, I298Q, I298H, I298N, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H, G305N, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q, G309H, G309N, G311Q, G311H, G311N, V313Q, V313H, V313N, G317Q, G317H, G317N, S319Q, S319H, S319N, A320Q, A320H, A320N, V322Q, V322H, V322N, V328Q, V328H, V328N, V331Q, V331H, V331N, A334Q, A334H, A334N, T335Q, T335H, T335N, S339Q, S339H, S339N, T340Q, T340H, T340N, T343Q, T343H, T343N, I344Q, I344H, I344N, A351Q, A351H, A351N, G352Q, G352H, G352N, G356Q, G356H, G356N, G357Q, G357H, G357N, S360Q, S360H, S360N, G363Q, G363H, G363N, S365Q, S365H, S365N, G366Q, G366H, G366N, G367Q, G367H, G367N, V370Q, V370H, V370N, T371Q, T371H, T371N, V373Q, V373H, V373N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N, T380Q, T380H, T380N, G381Q, G381H, G381N, I382Q, I382H, I382N, I383Q, I383H, I383N, S384Q, S384H, S384N, G386Q, G386H, G386N, A390Q, A390H, A390N, G393Q, G393H, G393N, G396Q, G396H, G396N, I397Q, I397H, I397N, T399Q, T399H, T399N, V401Q, V401H, V401N, S402Q, S402H, S402N, V405Q, V405H, V405N, I408Q, I408H, I408N, T412Q, T412H, T412N, T415Q, T415H, T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2. In some examples, the modifications are in a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 2. Exemplary modified FIX candidate LEAD polypeptides are set forth in any one of SEQ ID NOS: 3-891 or 1025-1034. In some examples, the modifications are in a FIX polypeptide, such as a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 892 or 1035. For example, exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 1036-1926 or 2035-2044.

e. Assessment of FIX Variants with Increased Resistance to Proteolysis

Increased resistance to proteolysis of FIX variants can be assessed by any methods known in the art to assess protein stability, thermal tolerance, protease sensitivity and resistance and/or FIX activity. In one example, protease resistance is measured by incubating a modified FIX polypeptide with one or more proteases and then assessing residual activity compared to an untreated control. A modified FIX can be compared with an unmodified and/or wild-type native FIX treated under similar conditions to determine if the particular variant retains more activity than the unmodified FIX. Activity can be assessed by any methods known in the art, for example by measuring coagulation activities.

Kinetic studies of protease resistance also can be used to assess a modified FIX polypeptide. For example, a modified FIX polypeptide is incubated with one or more proteases and samples are taken over a series of time-points. At each time point, the proteases are inactivated and the samples are then tested for FIX activity. In one embodiment, the modified polypeptide is at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more resistant to proteolysis.

In one exemplary embodiment, FIX variants are assessed for protease resistance with a mixture of proteases and proteolytic conditions including pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. For example, a cytopathic effects (CPE) assay can be used to assess coagulation activity of modified FIX polypeptides compared to unmodified FIX polypeptides. Specifically, coagulation activity of FIX can determined by the capacity of the modified FIX polypeptides to induce formation of a clot in an assay, such as an activated partial thromboplastin time assay. The resistance of the modified FIX polypeptides compared to wild-type FIX against enzymatic cleavage can be analyzed by mixing FIX polypeptides with proteases. After exposure to proteases, coagulation activities can be assessed.

In one example, activity of modified hFIX is assessed in an assay by measuring the capacity of the modified hFIX to modulate coagulation when added to the sample. Prior to the measurement of activity, hFIX molecules can be challenged with proteases (blood, intestinal, etc.) including conditions mimicking administered conditions, such as serum, blood, saliva, or digestive assays (in vitro assays), and/or administered to a subject such as a mouse or human (in vivo assays) during different incubation or post-injection times. The activity measured, corresponds then to the residual activity following exposure to the proteolytic mixtures. Activity can be compared with an unmodified FIX as a measurement of the effect of the modification on protease stability and on the activity. In one example, the unmodified FIX is a wild-type native FIX. In another example, the unmodified FIX is a variant form of FIX that was used as a starting material to introduce further modifications. Modified FIX polypeptides also can be compared with any known FIX polypeptide in any assay known in the art to compare protease sensitivity, thermal tolerance and/or any other activity.

2. Super-LEADs

Modification of FIX polypeptides also can include combining two or more modifications as set forth above. Modified FIX super-LEAD polypeptides are a combination of single amino acid mutations present in two or more of the respective modified FIX LEAD polypeptides. Thus, modified FIX super-LEAD polypeptides have two or more of the single amino acid replacements derived from two or more of the respective modified FIX LEAD polypeptides. As described above and in detail below, modified FIX polypeptides provided herein exhibit increased protein stability manifested as an increased resistance to proteolysis. Typically, modified FIX LEAD polypeptides created are those whose performance has been optimized with respect to the unmodified polypeptide by modification of a single amino acid replacement at one is-HIT position. Modified FIX super-Lead polypeptides are created such that the polypeptide contains two or more FIX LEAD modifications, each at a different is-HIT position. Modifications that increase proteolysis resistance can be added to other modifications provided herein or known in the art to increase proteolysis resistance. In one example, modifications that increase stability can be added to other modifications provided herein or known in the art to increase protein stability. In another example, modifications that increase stability can be added to modifications provided herein or known in the art to increase proteolysis resistance. Modifications that increase protease resistance and/or stability also can be added to modifications to FIX that alter other functionalities including activity, modifications that affect post-translation protein modifications and any other known modifications in the art.

Once the modified LEAD polypeptides have been identified using, for example, 2D-scanning methods, super-LEADs can be generated by combining two or more individual LEADs using methods well known in the art, such as recombination, mutagenesis and DNA shuffling, and by methods such as additive directional mutagenesis, 3D-scanning, and multi-overlapped primer extensions, as provided above.

Exemplary modified FIX super-LEAD polypeptides exhibiting increased protein stability can include FIX polypeptides containing two or more amino acid modifications as compared to an unmodified FIX polypeptide. In some examples, a FIX polypeptide can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified positions. Generally, the resulting FIX polypeptide exhibits increased protein stability and retains at least one activity of an unmodified FIX polypeptide. A modified FIX polypeptide can include any two or more amino acid modifications set forth in Table 5 above. For example, the modified FIX polypeptide can contain two or more amino acid modifications corresponding to any two or more modifications of 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, M19I, M19V, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24H, S24N, F25I, F25V, E26Q, E26H, E26N, E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q, V31H, V31N, F321, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N, 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, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, 166Q, 166H, 166N, S68Q, S68H, S68N, Y69H, Y69I, E70Q, E70H, E70N, W72S, W72H, P74A, P74S, F751, F75V, G76Q, G76H, G76N, F77I, F77V, E78Q, E78H, E78N, G79Q, G79H, G79N, K80N, K80Q, E83Q, E83H, E83N, L84I, L84V, D85N, D85Q, V86Q, V86H, V86N, T87Q, T87H, T87N, 190Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q, G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F98I, F98V, K100N, K100Q, S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q, V107Q, V107H, V10′7N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q, T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I, R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S, A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S, G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H, S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N, S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N, R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H, T148N, V149Q, V149H, V149N, P151A, P151S, 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, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H, T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, F175H, D177N, D177Q, 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, F192IH, 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, E224Q, E224H, E224N, T225Q, T225H, T225N, 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, T241Q, T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, 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, E294Q, E294H, E294N, 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, T371Q, T371H, T371N, E372Q, E372H, E372N, 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, E387H, 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, L414I, L414V, T415Q, T415H, and T415N of a mature FIX polypeptide set forth in SEQ ID NO: 2. In some examples, the modifications are in an unmodified FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 2. Provided herein are modified FIX Super-LEAD polypeptides containing two or more amino acid modifications and exhibiting increased protein stability having a sequence of amino acids set forth in any of SEQ ID NOS: 917-1024. In some examples, the modifications are in a FIX polypeptide, such as a FIX polypeptide having a sequence of amino acids set forth in SEQ ID NO: 892 or 1035. For example, exemplary modified FIX LEAD candidate polypeptides are set forth in any one of SEQ ID NOS: 1927-2034.

In one non-limiting example, a FIX polypeptide can contain a modification at a position corresponding to F1921 of a mature FIX polypeptide set forth in SEQ ID NO: 2, and can contain further amino acid modifications, such as any modification set forth in Table 5. Exemplary super-LEAD FIX polypeptides are set forth in Table 7 with the sequence identifier (SEQ ID NO.) in parenthesis next to each substitution. Resulting super-LEADs can be tested for one or more parameters to assess protein stability (such as, increased resistance to proteases) using any of the assays described herein, such as is described in the Examples.

TABLE 7

Exemplary Modified FIX Super-LEADs to Increase Protein Stability

T163H/F192I (917)
I164Q/F192I (918)
T169H/F192I (919)
S171Q/F192I (920)

D152N/D154N/F192I (921)
S174Q/F192I (922)
S174H/F192I (923)
T179Q/F192I (924)

T179H/F192I (925)
T163N/I164N/F192I (926)
D166Q/F192I (927)
D166N/F192I (928)

D186Q/F192I (929)
D186N/F192I (930)
T148A/D152N/
D154N/F192I (931)

F192I/D203Q (932)
F192I/D203N (933)
D152N/F192I (934)
D152Q/F192I (935)

T148A/D152N/
F192I (936)
D154N/F192I (937)
D154Q/F192I (938)

T163N/I164N (939)
A187N/G190N (940)
V196N/V197N (941)
T140N/T144N (942)

F175I/F178I (943)
F192I/W194I (944)
T148A/D152N (945)
T148A/D152Q (946)

T148A/D154N (947)
T148A/D154Q (948)
D152N/D154N (949)
I168N/T169N/S171N/

T172N/S174N (950)

T179N/V181N/V182N/
D152N/D154N/T148A (952)
T163H/D166Q/F192I (953)
T163H/D166N/F192I (954)

G183N/G184N (951)

T163H/D186Q/F192I (955)
T163H/D186N/F192I (956)
T163H/F192I/D203Q (957)
T163H/F192I/D203N (958)

T148A/D152N/D154N/
T148A/D154N/T163H/
I164Q/D166Q/F192I (961)
I164Q/D166N/F192I (962)

T163H/F192I (959)
F192I (960)

I164Q/D186Q/F192I (963)
I164Q/D186N/F192I (964)
I164Q/F192I/D203Q (965)
I164Q/F192I/D203N (966)

T148A/D152N/D154N/
T148A/D154N/I164Q/
T163N/I164N/D166Q/
T163N/I164N/D166N/

I164Q/F192I (967)
F192I (968)
F192I (969)
F192I (970)

T163N/I164N/D186Q/
T163N/I164N/D186N/
T163N/I164N/F192I/
T163N/I164N/F192I/

F192I (971)
F192I (972)
D203Q (973)
D203N (974)

T148A/D152N/D154N/
T148A/D154N/T163N/
D166Q/T169H/F192I (977)
D166N/T169H/F192I (978)

T163N/I164N/F192I (975)
I164N/F192I (976)

T169H/D186Q/F192I (979)
T169H/D186N/F192I (980)
T169H/F192I/D203Q (981)
T169H/F192I/D203N (982)

T148A/D152N/D154N/
T148A/D154N/T169H/
D166Q/S171Q/F192I (985)
D166N/S171Q/F192I (986)

T169H/F192I (983)
F192I (984)

S171Q/D186Q/F192I (987)
S171Q/D186N/F192I (988)
S171Q/F192I/D203Q (989)
S171Q/F192I/D203N (990)

T148A/D152N/D154N/
T148A/D154N/S171Q/
D166Q/S174Q/F192I (993)
D166N/S174Q/F192I (994)

S171Q/F192I (991)
F192I (992)

S174Q/D186Q/F192I (995)
S174Q/D186N/F192I (996)
S174Q/F192I/D203Q (997)
S174Q/F192I/D203N (998)

T148A/D152N/D154N/
T148A/D154N/S174Q/
D166Q/S174H/F192I (1001)
D166N/S174H/F192I (1002)

S174Q/F192I (999)
F192I (1000)

S174H/D186Q/F192I (1003)
S174H/D186N/F192I (1004)
S174H/F192I/D203Q (1005)
S174H/F192I/D203N (1006)

T148A/D152N/D154N/
T148A/D154N/S174H/
D166Q/T179Q/F192I (1009)
D166N/T179Q/F192I (1010)

S174H/F192I (1007)
F192I (1008)

T179Q/D186Q/F192I (1011)
T179Q/D186N/F192I (1012)
T179Q/F192I/D203Q (1013)
T179Q/F192I/D203N (1014)

T148A/D152N/D154N/
T148A/D154N/T179Q/
D166Q/T179H/F192I (1017)
D166N/T179H/F192I (1018)

T179Q/F192I (1015)
F192I (1016)

T179H/D186Q/F192I (1019)
T179H/D186N/F192I (1020)
T179H/F192I/D203Q (1021)
T179H/F192I/D203N (1022)

T148A/D152N/D154N/
T148A/D154N/T179H/

T179H/F192I (1023)
F192I (1024)

Non-limiting examples of FIX SuperLEAD polypeptides containing two or more amino acid modifications and exhibiting increased resistance to proteolysis can include amino acid replacements at amino acid residues corresponding to T163H/F192I, I164Q/F192I, T169H/F192I, S171Q/F192I, D152N/D154N/F192I, S174Q/F192I, S174H/F192I, T179Q/F192I, T179H/F192I, T163N/I164N/F192I, D166Q/F192I, D166N/F192I, D186Q/F192I, D186N/F192I, T148A/D152N/D154N/F192I, F192I/D203Q, F192I/D203N, D152N/F192I, D152Q/F192I, T148A/D154N/F192I, D154N/F192I, D154Q/F192I, T163N/I164N, A187N/G190N, V196N/V197N, T140N/T144N, F175I/F178I, F192I/W194I, T148A/D152N, T148A/D152Q, T148A/D154N, T148A/D154Q, D152N/D154N, I168N/T169N/S171N/T172N/S174N, T179N/V181N/V182N/G183N/G184N, D152N/D154N/T148A, T163H/D166Q/F192I, T163H/D166N/F192I, T163H/D186Q/F192I, T163H/D186N/F192I, T163H/F192I/D203Q, T163H/F192I/D203N, T148A/D152N/D154N/T163H/F192I, T148A/D154N/T163H/F192I, I164Q/D166Q/F192I, I164Q/D166N/F192I, I164Q/D186Q/F192I, I164Q/D186N/F192I, I164Q/F192I/D203Q, I164Q/F192I/D203N, T148A/D152N/D154N/I164Q/F192I, T148A/D154N/I164Q/F192I, T163N/I164N/D166Q/F192I, T163N/I164N/D166N/F192I, T163N/I164N/D186Q/F192I, T163N/I164N/D186N/F192I, T163N/I164N/F192I/D203Q, T163N/I164N/F192I/D203N, T148A/D152N/D154N/T163N/I164N/F192I, T148A/D154N/T163N/I164N/F192I, D166Q/T169H/F192I, D166N/T169H/F192I, T169H/D186Q/F192I, T169H/D186N/F192I, T169H/F192I/D203Q, T169H/F192I/D203N, T148A/D152N/D154N/T169H/F192I, T148A/D154N/T169H/F192I, D166Q/S171Q/F192I, D166N/S171Q/F192I, S171Q/D186Q/F192I, S171Q/D186N/F192I, S171Q/F192I/D203Q, S171Q/F192I/D203N, T148A/D152N/D154N/S171Q/F192I, T148A/D154N/S171Q/F192I, D166Q/S174Q/F192I, D166N/S174Q/F192I, S174Q/D186Q/F192I, S174Q/D186N/F192I, S174Q/F192I/D203Q, S174Q/F192I/D203N, T148A/D152N/D154N/S174Q/F192I, T148A/D154N/S174Q/F192I, D166Q/S174H/F192I, D166N/S174H/F192I, S174H/D186Q/F192I, S174H/D186N/F192I, S174H/F192I/D203 Q, S174H/F192I/D203N, T148A/D152N/D154N/S174H/F192I, T148A/D154N/S174H/F192I, D166Q/T179Q/F192I, D166N/T179Q/F192I, T179Q/D186Q/F192I, T179Q/D186N/F192I, T179Q/F192I/D203Q, T179Q/F192I/D203N, T148A/D152N/D154N/T179Q/F192I, T148A/D154N/T179Q/F192I, D166Q/T179H/F192I, D166N/T179H/F192I, T179H/D186Q/F192I, T179H/D186N/F192I, T179H/F192I/D203Q, T179H/F192I/D203N, T148A/D152N/D154N/T179H/F192I, and T148A/D154N/T179H/F1921 of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. Exemplary modified FIX SuperLEAD polypeptides containing two or more amino acid modifications and exhibiting increased protease resistance have a sequence of amino acids set forth in any one of SEQ ID NOS: 917-1024 or 1927-2034.

3. Other FIX Modifications

In addition to any one or more amino acid modifications provided herein, a modified FIX polypeptide also can contain one or more other modifications, including those known to those of skill in the art, such as PEGylation, hyperglycosylation, deimmunization and others (see such as U.S. Pat. Nos. 6,277,618, 6,315,995, and 6,531,298 and U.S. Patent Publication Nos. 2004-0102388, 2004-0110675, 2004-0254106, 2005-0100982, and 2006-0040856). Generally, the modification results in increased stability without losing at least one activity, such as blood coagulation activity (i.e. retains at least one activity as defined herein) of an unmodified FIX polypeptide. For example, other further modifications in a FIX polypeptide include one or more additional amino acid modifications and/or one or more chemical modifications. Such modifications include, but are not limited to, those that alter the immunogenicity, glycosylation, activity, or any other known property of a FIX polypeptide. In another example, chemical modifications include post-translational modifications of a protein, such as for example, glycosylation by a carbohydrate moiety; acylation; methylation; phosphorylation; sulfation; prenylation; Vitamin C-dependent modifications such as for example, proline, aspartic acid, lysine hydroxylations and carboxy terminal amidation; Vitamin K-dependent modifications such as for example, carboxylation of glutamic acid residues (i.e. gla residue); and incorporation of selenium to form a selenocysteine. Other protein modifications of a FIX polypeptide include PEGylation. In addition, protein modifications also can include modification to facilitate the detection, purification and assay development of a polypeptide, such as for example, modification of a polypeptide with a Sulfo-NHS-LC-biotin for covalent attachment to a primary amine on a protein, or other similar modification for florescent, non-isotopic or radioactive labels. Exemplary further modifications in a FIX polypeptide are described below. Modified polypeptides that are conjugates and/or labeled also are provided. For example, provided herein are modified polypeptides that are conjugated to a PEG moiety or contain a carbohydrate moiety covalently linked to one or more glycosylation sites on the polypeptide.

In another embodiment, other known properties of a FIX polypeptide can be modified in addition to any one or more amino acid modifications provided herein. Such modifications include, but are not limited to, alteration of the peptidase activity, coagulant activity, esterase activity, protein binding activities (such as FVIII), cofactor binding activities, such as Ca²⁺, phospholipid or cell surface binding activities, and increased resistance to inhibitors, such as heparin and warfarin. Resulting modified FIX polypeptides can be tested for one or more parameters to assess polypeptide properties, such as protein stability (such as, increased resistance to proteases), or polypeptide activities, such as coagulant activity, using any of the assays described herein.

In yet another embodiment, known properties of a FIX polypeptide, as mentioned above, can be modified by generation of chimeric FIX polypeptides, in which all, or portions thereof, of FIX polypeptide domains are replaced by homologous domains of other coagulation factor family members. Such chimeric proteins are known in the art and can be combined with the methods for modification of FIX polypeptides provided herein (International PCT application No. WO 2006/018204 A1, Lin et al. (1990) J. Biol. Chem. 265(1):144-150 and Cheung et al. (1991) J. Biol. Chem. 266(14):8797-880). An exemplary chimeric protein has a sequence of amino acids as set forth in SEQ ID NO: 1035.

a. Immunogenicity

There are many instances where the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. An immune response to a therapeutic protein, such as FIX, proceeds via the MHC class II peptide presentation pathway. Here, exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP type. MHC class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells, amongst others. Engagement of a MHC class II peptide complex by a cognate T-cell receptor on the surface of the T cell, together with the cross binding of certain other co-receptors, such as the CD4 molecule, can induce an activated state within the T cell. Activation leads to the release of cytokines, further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.

The ability of a peptide (T cell epitope) to bind a given MHC class II molecule for presentation on the surface of an APC is dependent on a number of factors, most notably its primary sequence. This will influence its propensity for proteolytic cleavage and also its affinity for binding within the peptide binding cleft of the MHC class II molecule. The MHC class II/peptide complex on the APC surface presents a binding face to a particular T cell receptor (TCR) able to recognize determinants provided by exposed residues of the peptide and the MHC class II molecule.

Formation of inhibitory antibodies to therapeutic FIX polypeptides is known in the art (Herzog et al. (2004) Semin. Thromb. Hemost. 30(2): 215-226). Hence, the combination modified FIX polypeptides provided herein with modifications to decrease overall immunogenicity of the modified FIX polypeptides can improve the therapeutic properties of the FIX polypeptide. The identification of T cell epitopes can be carried out according to methods known in the art (see such as, U.S. Patent Publication No. 20040254106) and can be used to identify the binding propensity of FIX peptides to an MHC class II molecule.

Further modifications to a modified FIX polypeptide provided herein can include modifications of at least one amino acid residue resulting in a substantial reduction in activity of or elimination of one or more T cell epitopes from the protein, i.e. deimmunization of the polypeptide. One or more amino acid modification at particular positions within any of the MHC class II ligands can result in a deimmunized FIX polypeptide with a reduced immunogenic when administered as a therapeutic to a host, such as for example, a human host.

Exemplary amino acid positions for modification of a T cell epitope, and thereby a deimmunized FIX polypeptide with a reduced immunogenic potential, include amino acid modifications at one or more positions corresponding to any of the following positions: Y1, S3, L6, F9, V10, Q11, G12, N13, L14, E15, R16, E17, C18, M19, E20, E21, K22, S24, F25, E26, E27, A28, R29, E30, V31, F32, E33, T35, F41, W42, K43, Q44, Y45, V46, D47, G48, D49, Q50, C51, E52, S53, N54, L57, G60, C62, K63, D65, I66, Y69, C71, W72, F75, F77, L84, V86, I90, K91, N92, G93, R94, C95, E96, Q97, F98, K100, N101, A103, D104, K106, V107, V108, V108, S110, C111, T112, E113, G114, Y115, R116, L117, A118, N120, Q121, S123, V128, F130, V135, V137, S138, T140, S141, K142, L143, R145, A146, T148, V149, F150, D152, V153, D154, Y155, V156, N157, S158, T159, E160, A161, E162, T163, I164, L165, 1168, F175, F178, T179, R180, V181, V182, G183, G184, E185, D186, A187, K188, G190, F192, W194, Q195, V196, V197, L198, N199, G200, K201, V202, D203, A204, F205, G207, G208, S209, I210, V211, N212, E213, K214, W215, I216, V217, A219, V223, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, H236, N237, I238, E239, E240, V250, I251, I253, I254, P255, H256, H257, N258, Y259, N260, A261, A262, I263, N264, K265, Y266, N267, H268, D269, I270, A271, L272, L273, E274, L275, D276, E277, P278, L279, V280, L281, S283, Y284, V285, T286, I288, C289, I290, A291, D292, K293, Y295, T296, N297, I298, F299, L300, K301, F302, G303, S304, G305, Y306, V307, S308, W310, G311, V313, F314, H315, K316, G317, R318, S319, A320, L321, V322, L323, Q324, Y325, L326, R327, V328, L330, V331, D332, R333, A334, T335, C336, L337, R338, S339, K341, F342, T343, I344, Y345, N346, N347, M348, F349, C350, A351, G352, F353, H354, E355, G356, G357, R358, D359, S360, C361, V370, V373, E374, G375, S377, F378, L379, T380, G381, I382, I383, S384, W385, E387, E388, A390, M391, K392, G393, Y395, Y398, K400, V401, S402, R403, Y404, V405, N406, W407, I408, K409, E410, K411, K413, of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035. Exemplary amino acid modifications that can contribute to reduced immunogenicity of a FIX polypeptide include any one or more amino acid modifications corresponding to any one or more modifications set forth in Table 8 corresponding to amino acid positions of a mature FIX polypeptide set forth in SEQ ID NO: 2 or 1035.

TABLE 8

List of human FIX Modifications for Decreased Immunogenicity

Y1A
Y1C
Y1D
Y1E
Y1G
Y1H
Y1K
Y1N
Y1P
Y1Q
Y1R
Y1S

Y1T
S3T
L6A
L6C
L6D
L6E
L6G
L6H
L6K
L6N
L6P
L6Q

L6R
L6S
L6T
L6M
F9A
F9C
F9D
F9E
F9G
F9H
F9K
F9N

F9P
F9Q
F9R
F9S
F9T
F9I
F9M
F9W
V10A
V10C
V10D
V10E

V10G
V10H
V10K
V10N
V10P
V10Q
V10R
V10S
V10T
V10F
V10I
V10M

V10W
V10Y
Q11A
Q11C
Q11G
Q11P
G12D
G12E
G12G
G12H
G12K
G12N

G12P
G12Q
G12R
G12S
G12T
N13A
N13C
N13G
N13H
N13P
N13T
L14A

L14C
L14D
L14E
L14G
L14H
L14K
L14N
L14P
L14Q
L14R
L14S
L14T

L14F
L14I
L14M
L14V
L14W
L14Y
E15D
E15H
E15P
R16A
R16C
R16G

R16P
R16T
E17A
E17C
E17G
E17P
E17T
C18D
C18E
C18G
C18H
C18K

C18N
C18P
C18Q
C18R
C18S
C18T
M19A
M19C
M19D
M19E
M19G
M19H

M19K
M19N
M19P
M19Q
M19R
M19S
M19T
M19F
M19I
M19M
M19V
M19W

M19Y
E20A
E20C
E20G
E20P
E20T
E21A
E21C
E21G
E21P
K22H
K22P

K22T
S24H
S24P
F25A
F25C
F25D
F25E
F25G
F25H
F25K
F25N
F25P

F25Q
F25R
F25S
F25T
F25I
F25M
F25W
F25Y
E26A
E26C
E26G
E26P

E27A
E27C
E27G
E27H
E27P
E27S
E27T
A28C
A28D
A28E
A28G
A28H

A28K
A28N
A28P
A28Q
A28R
A28S
A28T
R29A
R29C
R29G
R29P
E30D

E30H
E30P
V31A
V31C
V31D
V31E
V31G
V31H
V31K
V31N
V31P
V31Q

V31R
V31S
V31T
V31F
V31I
V31W
V31Y
F32A
F32C
F32D
F32E
F32G

F32H
F32K
F32N
F32P
F32Q
F32R
F32S
F32T
E33H
E33N
E33P
E33Q

E33S
E33T
T35A
T35C
T35G
T35P
F41A
F41C
F41D
F41E
F41G
F41H

F41K
F41N
F41P
F41Q
F41R
F41S
F41T
F41M
F41W
F41Y
W42A
W42C

W42D
W42E
W42G
W42H
W42K
W42N
W42P
W42Q
W42R
W42S
W42T
K43A

K43C
K43G
K43P
Q44P
Q44T
Q44
Y45A
Y45C
Y45D
Y45E
Y45G
Y45H

Y45K
Y45N
Y45P
Y45Q
Y45R
Y45S
Y45T
V46A
V46C
V46D
V46E
V46G

V46H
V46K
V46N
V46P
V46Q
V46R
V46S
V46T
V46F
V46I
V46M
V46W

V46Y
D47A
D47C
D47G
D47H
D47P
D47T
G48D
G48E
G48P
G48T
D49H

D49P
D49Q
D49T
Q50A
Q50C
Q50D
Q50G
Q50H
Q50P
Q50T
C51D
C51E

C51G
C51H
C51K
C51N
C51P
C51Q
C51R
C51S
C51T
E52P
E52T
S53A

S53C
S53G
S53H
S53P
S53T
N54H
N54P
N54T
L57A
L57C
L57D
L57E

L57G
L57H
L57K
L57N
L57P
L57Q
L57R
L57S
L57T
L57F
L57I
L57M

L57W
L57Y
G60C
G60D
G60H
G60P
G60T
C62D
C62H
C62P
K63T
D65H

D65T
I66A
I66C
I66D
I66E
I66G
I66H
I66K
I66N
I66P
I66Q
I66R

I66S
I66T
I66M
I66W
I66Y
Y69A
Y69C
Y69D
Y69E
Y69G
Y69H
Y69K

Y69N
Y69P
Y69Q
Y69R
Y69S
Y69T
C71H
C71P
W72A
W72C
W72D
W72E

W72G
W72H
W72K
W72N
W72P
W72Q
W72R
W72S
W72T
W72I
W72Y
F75A

F75C
F75D
F75E
F75G
F75H
F75K
F75N
F75P
F75Q
F75R
F75S
F75T

F77A
F77C
F77D
F77E
F77G
F77H
F77K
F77N
F77P
F77Q
F77R
F77S

F77T
L84A
L84C
L84D
L84E
L84G
L84H
L84K
L84N
L84P
L84Q
L84R

L84S
L84T
L84M
L84W
L84Y
V86A
V86C
V86D
V86E
V86G
V86H
V86K

V86N
V86P
V86Q
V86R
V86S
V86T
I90A
I90C
I90D
I90E
I90G
I90H

I90K
I90N
I90P
I90Q
I90R
I90S
I90T
I90M
I90W
K91A
K91C
K91G

K91P
N92A
N92C
N92G
N92P
N92T
G93D
G93E
G93H
G93K
G93N
G93P

G93Q
G93R
G93S
G93T
R94A
R94C
R94G
R94P
C95D
C95E
C95G
C95H

C95K
C95N
C95P
C95Q
C95R
C95S
C95T
E96P
E96T
Q97A
Q97C
Q97G

Q97P
F98A
F98C
F98D
F98E
F98G
F98H
F98K
F98N
F98P
F98Q
F98R

F98S
F98T
F98M
F98W
F98Y
K100A
K100C
K100G
K100P
N101H
N101T
A103D

A103E
A103H
A103K
A103N
A103P
A103Q
A103R
A103S
A103T
D104T
K106H
K106P

K106T
V107A
V107C
V107D
V107E
V107G
V107H
V107K
V107N
V107P
V107Q
V107R

V107S
V107T
V108A
V108C
V108D
V108E
V108G
V108H
V108K
V108N
V108P
V108Q

V108R
V108S
V108T
V108F
V108M
V108W
V108Y
S110A
S110C
S110G
S110P
C111D

C111E
C111H
C111K
C111N
C111P
C111Q
C111R
C111S
C111T
T112A
T112C
T112G

T112P
E113D
E113H
E113P
G114D
G114E
G114H
G114K
G114N
G114P
G114Q
G114R

G114S
G114T
Y115A
Y115C
Y115D
Y115E
Y115G
Y115H
Y115K
Y115N
Y115P
Y115Q

Y115R
Y115S
Y115T
Y115M
Y115W
R116P
R116T
L117A
L117C
L117D
L117E
L117G

L117H
L117K
L117N
L117P
L117Q
L117R
L117S
L117T
A118D
A118E
A118H
A118K

A118N
A118P
A118Q
A118R
A118S
A118T
N120D
N120H
N120P
Q121T
S123H
S123T

V128A
V128C
V128D
V128E
V128G
V128H
V128K
V128N
V128P
V128Q
V128R
V128S

V128T
F130A
F130C
F130D
F130E
F130G
F130H
F130K
F130N
F130P
F130Q
F130R

F130S
F130T
V135A
V135C
V135D
V135E
V135G
V135H
V135K
V135N
V135P
V135Q

V135R
V135S
V135T
V135W
V135Y
V137A
V137C
V137D
V137E
V137G
V137H
V137K

V137N
V137P
V137Q
V137R
V137S
V137T
V137M
V137W
V137Y
S138H
S138T
T140D

T140H
S141T
K142H
K142P
L143A
L143C
L143D
L143E
L143G
L143H
L143K
L143N

L143P
L143Q
L143R
L143S
L143T
L143F
L143I
L143M
L143V
L143W
L143Y
R145H

R145P
R145T
A146P
A146T
T148H
T148P
V149A
V149C
V149D
V149E
V149G
V149H

V149K
V149N
V149P
V149Q
V149R
V149S
V149T
V149F
V149I
V149M
V149W
V149Y

F150A
F150C
F150D
F150E
F150G
F150H
F150K
F150N
F150P
F150Q
F150R
F150S

F150T
F150M
F150W
F150Y
D152A
D152C
D152G
D152P
D152S
D152T
V153A
V153C

V153D
V153E
V153G
V153H
V153K
V153N
V153P
V153Q
V153R
V153S
V153T
V153F

V153I
V153M
V153W
V153Y
D154A
D154C
D154G
D154P
D154Q
D154S
Y155A
Y155C

Y155D
Y155E
Y155G
Y155H
Y155K
Y155N
Y155P
Y155Q
Y155R
Y155S
Y155T
Y155M

Y155V
Y155W
V156A
V156C
V156D
V156E
V156G
V156H
V156K
V156N
V156P
V156Q

V156R
V156S
V156T
V156I
V156M
V156W
V156Y
N157A
N157C
N157G
N157H
N157P

N157Q
N157T
S158H
S158P
S158T
T159A
T159C
T159G
T159P
E160A
E160C
E160G

E160P
A161C
A161D
A161E
A161H
A161K
A161N
A161P
A161Q
A161R
A161S
A161T

E162P
E162T
T163A
T163C
T163G
T163P
I164A
I164C
I164D
I164E
I164G
I164H

I164K
I164N
I164P
I164Q
I164R
I164S
I164T
L165A
L165C
L165D
L165E
L165G

L165H
L165K
L165N
L165P
L165Q
L165R
L165S
L165T
L165M
L165W
L165Y
I168A

I168C
I168D
I168E
I168G
I168H
I168K
I168N
I168P
I168Q
I168R
I168S
I168T

F175A
F175C
F175D
F175E
F175G
F175H
F175K
F175N
F175P
F175Q
F175R
F175S

F175T
F178A
F178C
F178D
F178E
F178G
F178H
F178K
F178N
F178P
F178Q
F178R

F178S
F178T
F178M
F178W
F178Y
T179A
T179C
T179G
T179P
R180A
R180C
R180D

R180G
R180H
R180P
V181A
V181C
V181D
V181E
V181G
V181H
V181K
V181N
V181P

V181Q
V181R
V181S
V181T
V181F
V181I
V181M
V181W
V181Y
V182A
V182C
V182D

V182E
V182G
V182H
V182K
V182N
V182P
V182Q
V182R
V182S
V182T
V182F
V182I

V182M
V182W
V182Y
G183D
G183E
G183H
G183K
G183N
G183P
G183Q
G183S
G183T

G184D
G184E
G184H
G184K
G184N
G184P
G184Q
G184R
G184S
G184T
E185A
E185C

E185G
E185H
E185P
E185T
D186A
D186C
D186G
D186H
D186P
D186T
A187C
A187D

A187E
A187G
A187H
A187K
A187N
A187P
A187Q
A187R
A187S
A187T
K188A
K188C

K188G
K188H
K188P
K188T
G190D
G190E
G190H
G190K
G190N
G190P
G190Q
G190R

G190S
G190T
F192A
F192C
F192D
F192E
F192G
F192H
F192K
F192N
F192P
F192Q

F192R
F192S
F192T
F192W
F192Y
W194A
W194C
W194D
W194E
W194G
W194H
W194K

W194N
W194P
W194Q
W194R
W194S
W194T
Q195H
Q195P
Q195T
V196A
V196C
V196D

V196E
V196G
V196H
V196K
V196N
V196P
V196Q
V196R
V196S
V196T
V196F
V196I

V196M
V196W
V196Y
V197A
V197C
V197D
V197E
V197G
V197H
V197K
V197N
V197P

V197Q
V197R
V197S
V197T
V197F
V197I
V197M
V197W
V197Y
L198A
L198C
L198D

L198E
L198G
L198H
L198K
L198N
L198P
L198Q
L198R
L198S
L198T
L198I
L198Y

N199A
N199C
N199G
N199H
N199P
N199S
N199T
G200P
G200T
K201A
K201C
K201D

K201E
K201G
K201H
K201N
K201P
K201Q
K201S
K201T
V202A
V202C
V202D
V202E

V202G
V202H
V202K
V202N
V202P
V202Q
V202R
V202S
V202T
V202F
V202I
V202M

V202W
V202Y
D203A
D203C
D203G
D203P
D203T
A204C
A204D
A204E
A204G
A204H

A204K
A204N
A204P
A204Q
A204R
A204S
A204T
F205A
F205C
F205D
F205E
F205G

F205H
F205K
F205N
F205P
F205Q
F205R
F205S
F205T
F205M
F205V
F205W
F205Y

G207H
G207P
G208C
G208D
G208E
G208H
G208K
G208N
G208P
G208Q
G208R
G208S

G208T
S209A
S209C
S209G
S209P
I210A
I210C
I210D
I210E
I210G
I210H
I210K

I210N
I210P
I210Q
I210R
I210S
I210T
I210F
I210W
I210Y
V211A
V211C
V211D

V211E
V211G
V211H
V211K
V211N
V211P
V211Q
V211R
V211S
V211T
V211F
V211I

V211M
V211W
N212A
N212C
N212G
N212P
E213H
E213P
E213S
E213T
K214T
W215A

W215C
W215D
W215E
W215G
W215H
W215K
W215N
W215P
W215Q
W215R
W215S
W215T

I216A
I216C
I216D
I216E
I216G
I216H
I216K
I216N
I216P
1216Q
I216R
I216S

I216T
V217A
V217C
V217D
V217E
V217G
V217H
V217K
V217N
V217P
V217Q
V217R

V217S
V217T
V217I
V217Y
A219H
A219P
A219T
V223A
V223C
V223D
V223E
V223G

V223H
V223K
V223N
V223P
V223Q
V223R
V223S
V223T
V223M
V223W
V223Y
G226P

V227A
V227C
V227D
V227E
V227G
V227H
V227K
V227N
V227P
V227Q
V227R
V227S

V227T
V227F
V227I
V227M
V227W
V227Y
K228A
K228C
K228G
K228H
K228P
I229A

I229C
I229D
I229E
I229G
I229H
I229K
I229N
I229P
I229Q
I229R
I229S
I229T

I229M
I229W
I229Y
T230A
T230C
T230G
T230P
V231A
V231C
V231D
V231E
V231G

V231H
V231K
V231N
V231P
V231Q
V231R
V231S
V231T
V232A
V232C
V232D
V232E

V232G
V232H
V232K
V232N
V232P
V232Q
V232R
V232S
V232T
V232F
V232I
V232M

V232W
V232Y
A233C
A233D
A233E
A233G
A233H
A233K
A233N
A233P
A233Q
A233R

A233S
A233T
A233V
G234D
G234E
G234H
G234K
G234N
G234P
G234Q
G234R
G234S

G234T
E235H
E235N
E235P
E235Q
E235S
E235T
H236A
H236C
H236G
H236P
N237A

N237C
N237G
N237P
N237T
I238A
I238C
I238D
I238E
I238G
I238H
I238K
I238N

I238P
I238Q
I238R
I238S
I238T
E239A
E239C
E239G
E239P
E240H
E240T
V250A

V250C
V250D
V250E
V250G
V250H
V250K
V250N
V250P
V250Q
V250R
V250S
V250T

V250M
V250W
V250Y
I251A
I251C
I251D
I251E
I251G
I251H
I251K
I251N
I251P

I251Q
I251R
I251S
I251T
I253A
I253C
I253D
I253E
I253G
I253H
I253K
I253N

I253P
I253Q
I253R
I253S
I253T
I253M
I253W
I253Y
I254A
I254C
I254D
I254E

I254G
I254H
I254K
I254N
I254P
I254Q
I254R
I254S
I254T
P255H
H256P
H256T

H257A
H257C
H257G
H257P
N258P
N258T
Y259A
Y259C
Y259D
Y259E
Y259G
Y259H

Y259K
Y259N
Y259P
Y259Q
Y259R
Y259S
Y259T
Y259M
Y259W
N260A
N260C
N260G

N260P
A261D
A261E
A261H
A261K
A261N
A261P
A261Q
A261R
A261S
A261T
A262C

A262D
A262E
A262G
A262H
A262K
A262N
A262P
A262Q
A262R
A262S
A262T
I263A

I263C
I263D
I263E
I263G
I263H
I263K
I263N
I263P
I263Q
I263R
I263S
I263T

I263M
I263V
I263W
I263Y
N264A
N264C
N264D
N264G
N264H
N264P
K265A
K265C

K265G
K265H
K265P
Y266A
Y266C
Y266D
Y266E
Y266G
Y266H
Y266K
Y266N
Y266P

Y266Q
Y266R
Y266S
Y266T
Y266M
Y266W
N267A
N267C
N267G
N267H
N267P
N267T

H268P
D269A
D269C
D269E
D269G
D269H
D269N
D269P
D269Q
D269S
D269T
I270A

I270C
I270D
I270E
I270G
I270H
I270K
I270N
I270P
I270Q
I270R
I270S
I270T

I270M
I270W
A271C
A271D
A271E
A271G
A271H
A271K
A271N
A271P
A271Q
A271R

A271S
A271T
L272A
L272C
L272D
L272E
L272G
L272H
L272K
L272N
L272P
L272Q

L272R
L272S
L272T
L272F
L273A
L273C
L273D
L273E
L273G
L273H
L273K
L273N

L273P
L273Q
L273R
L273S
L273T
L273F
L273I
L273M
L273V
L273W
L273Y
E274A

E274C
E274G
E274P
E274T
L275A
L275C
L275D
L275E
L275G
L275H
L275K
L275N

L275P
L275Q
L275R
L275S
L275T
L275W
L275Y
D276P
D276S
D276T
E277A
E277C

E277G
E277P
P278T
L279A
L279C
L279D
L279E
L279G
L279H
L279K
L279N
L279P

L279Q
L279R
L279S
L279T
L279I
L279Y
V280A
V280C
V280D
V280E
V280G
V280H

V280K
V280N
V280P
V280Q
V280R
V280S
V280T
V280F
V280I
V280W
V280Y
L281A

L281C
L281D
L281E
L281G
L281H
L281K
L281N
L281P
L281Q
L281R
L281S
L281T

L281F
L281I
L281V
L281W
L281Y
S283A
S283C
S283G
S283P
Y284A
Y284C
Y284D

Y284E
Y284G
Y284H
Y284K
Y284N
Y284P
Y284Q
Y284R
Y284S
Y284T
Y284M
V285A

V285C
V285D
V285E
V285G
V285H
V285K
V285N
V285P
V285Q
V285R
V285S
V285T

V285M
V285W
V285Y
T286A
T286C
T286G
T286P
I288A
I288C
I288D
I288E
I288G

I288H
I288K
I288N
I288P
I288Q
I288R
I288S
I288T
C289D
C289H
C289P
I290A

I290C
I290D
I290E
I290G
I290H
I290K
I290N
I290P
I290Q
I290R
I290S
I290T

I290Y
A291D
A291E
A291H
A291K
A291N
A291P
A291Q
A291R
A291S
A291T
D292A

D292C
D292G
D292P
D292T
K293H
K293P
K293T
Y295A
Y295C
Y295D
Y295E
Y295G

Y295H
Y295K
Y295N
Y295P
Y295Q
Y295R
Y295S
Y295T
Y295W
T296A
T296C
T296G

T296P
N297A
N297C
N297G
N297P
I298A
I298C
I298D
I298E
I298G
I298H
I298K

I298N
I298P
I298Q
I298R
I298S
I298T
F299A
F299C
F299D
F299E
F299G
F299H

F299K
F299N
F299P
F299Q
F299R
F299S
F299T
L300A
L300C
L300D
L300E
L300G

L300H
L300K
L300N
L300P
L300Q
L300R
L300S
L300T
L300F
L300I
L300M
L300V

L300W
L300Y
K301A
K301C
K301G
K301P
K301T
F302A
F302C
F302D
F302E
F302G

F302H
F302K
F302N
F302P
F302Q
F302R
F302S
F302T
G303H
G303P
G303T
S304A

S304C
S304G
S304P
S304T
G305D
G305E
G305H
G305N
G305P
G305Q
G305S
G305T

Y306A
Y306C
Y306D
Y306E
Y306G
Y306H
Y306K
Y306N
Y306P
Y306Q
Y306R
Y306S

Y306T
V307A
V307C
V307D
V307E
V307G
V307H
V307K
V307N
V307P
V307Q
V307R

V307S
V307T
S308P
S308T
W310A
W310C
W310D
W310E
W310G
W310H
W310K
W310N

W310P
W310Q
W310R
W310S
W310T
G311H
V313A
V313C
V313D
V313E
V313G
V313H

V313K
V313N
V313P
V313Q
V313R
V313S
V313T
F314A
F314C
F314D
F314E
F314G

F314H
F314K
F314N
F314P
F314Q
F314R
F314S
F314T
F314M
F314W
F314Y
H315A

H315C
H315G
H315P
K316A
K316C
K316G
K316P
G317C
G317D
G317E
G317H
G317K

G317N
G317P
G317Q
G317R
G317S
G317T
R318A
R318C
R318G
R318P
S319D
S319H

S319N
S319P
S319Q
A320C
A320D
A320E
A320G
A320H
A320K
A320N
A320P
A320Q

A320R
A320S
A320T
L321A
L321C
L321D
L321E
L321G
L321H
L321K
L321N
L321P

L321Q
L321R
L321S
L321T
V322A
V322C
V322D
V322E
V322G
V322H
V322K
V322N

V322P
V322Q
V322R
V322S
V322T
V322W
V322Y
L323A
L323C
L323D
L323E
L323G

L323H
L323K
L323N
L323P
L323Q
L323R
L323S
L323T
L323F
L323I
L323M
L323V

L323W
L323Y
Q324A
Q324C
Q324G
Q324P
Y325A
Y325C
Y325D
Y325E
Y325G
Y325H

Y325K
Y325N
Y325P
Y325Q
Y325R
Y325S
Y325T
Y325W
L326A
L326C
L326D
L326E

L326G
L326H
L326K
L326N
L326P
L326Q
L326R
L326S
L326T
L326F
L326I
L326M

L326V
L326W
L326Y
R327A
R327C
R327G
R327H
R327P
V328A
V328C
V328D
V328E

V328G
V328H
V328K
V328N
V328P
V328Q
V328R
V328S
V328T
V328F
V328I
V328M

V328W
V328Y
L330A
L330C
L330D
L330E
L330G
L330H
L330K
L330N
L330P
L330Q

L330R
L330S
L330T
L330F
L330I
L330V
L330W
L330Y
V331A
V331C
V331D
V331E

V331G
V331H
V331K
V331N
V331P
V331Q
V331R
V331S
V331T
V331F
V331I
V331M

V331W
V331Y
D332A
D332C
D332G
D332P
R333A
R333C
R333D
R333E
R333G
R333H

R333N
R333P
R333Q
R333R
R333S
R333T
A334C
A334D
A334E
A334G
A334H
A334K

A334N
A334P
A334Q
A334R
A334S
A334T
T335A
T335C
T335G
T335P
C336D
C336E

C336H
C336K
C336N
C336P
C336Q
C336R
C336S
C336T
L337A
L337C
L337D
L337E

L337G
L337H
L337K
L337N
L337P
L337Q
L337R
L337S
L337T
R338A
R338C
R338G

R338P
S339P
S339T
K341A
K341C
K341G
K341P
F342A
F342C
F342D
F342E
F342G

F342H
F342K
F342N
F342P
F342Q
F342R
F342S
F342T
F342M
F342W
T343A
T343C

T343G
T343P
I344A
I344C
I344D
I344E
I344G
I344H
I344K
I344N
I344P
I344Q

I344R
I344S
I344T
Y345A
Y345C
Y345D
Y345E
Y345G
Y345H
Y345K
Y345N
Y345P

Y345Q
Y345R
Y345S
Y345T
Y345M
Y345W
N346A
N346C
N346G
N346P
N347H
N347P

M348A
M348C
M348D
M348E
M348G
M348H
M348K
M348N
M348P
M348Q
M348R
M348S

M348T
F349A
F349C
F349D
F349E
F349G
F349H
F349K
F349N
F349P
F349Q
F349R

F349S
F349T
F349I
F349M
F349W
F349Y
C350D
C350H
C350P
C350T
A351E
A351H

A351N
A351P
A351Q
A351R
A351S
A351T
G352A
G352C
G352P
F353A
F353C
F353D

F353E
F353G
F353H
F353K
F353N
F353P
F353Q
F353R
F353S
F353T
F353I
F353M

F353W
H354A
H354C
H354G
H354P
E355A
E355C
E355D
E355G
E355H
E355K
E355N

E355P
E355Q
E355S
E355T
G356D
G356E
G356H
G356K
G356N
G356P
G356Q
G356R

G356S
G356T
G357D
G357E
G357H
G357K
G357N
G357P
G357Q
G357R
G357S
G357T

R358D
R358E
R358H
R358K
R358N
R358P
R358Q
R358R
R358S
R358T
D359A
D359C

D359G
D359P
D359Q
D359S
D359T
S360A
S360C
S360G
S360P
C361D
C361E
C361H

C361K
C361N
C361P
C361Q
C361R
C361S
C361T
V370A
V370C
V370D
V370E
V370G

V370H
V370K
V370N
V370P
V370Q
V370R
V370S
V370T
V370W
V370Y
V373A
V373C

V373D
V373E
V373G
V373H
V373K
V373N
V373P
V373Q
V373R
V373S
V373T
V373F

V373I
V373M
V373W
E374A
E374C
E374G
E374P
G375H
S377A
S377C
S377G
S377P

F378A
F378C
F378D
F378E
F378G
F378H
F378K
F378N
F378P
F378Q
F378R
F378S

F378T
F378W
L379A
L379C
L379D
L379E
L379G
L379H
L379K
L379N
L379P
L379Q

L379R
L379S
L379T
L379I
L379M
L379W
L379Y
T380A
T380C
T380G
T380P
G381D

G381E
G381H
G381K
G381N
G381P
G381Q
G381R
G381S
G381T
I382A
I382C
I382D

I382E
I382G
I382H
I382K
I382N
I382P
I382Q
I382R
I382S
I382T
I382M
I382W

I382Y
I383A
I383C
I383D
I383E
I383G
I383H
I383K
I383N
I383P
I383Q
I383R

I383S
I383T
S384A
S384C
S384G
S384P
W385A
W385C
W385D
W385E
W385G
W385H

W385K
W385N
W385P
W385Q
W385R
W385S
W385T
W385M
E387A
E387C
E387G
E387H

E387P
E387T
E388H
E388N
E388P
E388Q
E388T
A390C
A390D
A390E
A390G
A390H

A390K
A390N
A390P
A390Q
A390R
A390S
M391A
M391C
M391D
M391E
M391G
M391H

M391K
M391N
M391P
M391Q
M391R
M391S
M391T
M391F
M391I
M391W
M391Y
K392A

K392C
K392G
K392P
G393C
G393D
G393E
G393H
G393K
G393N
G393P
G393Q
G393R

G393S
G393T
Y395A
Y395C
Y395D
Y395E
Y395G
Y395H
Y395K
Y395N
Y395P
Y395Q

Y395R
Y395S
Y395T
Y398A
Y398C
Y398D
Y398E
Y398G
Y398H
Y398K
Y398N
Y398P

Y398Q
Y398R
Y398S
Y398T
K400H
V401A
V401C
V401D
V401E
V401G
V401H
V401K

V401N
V401P
V401Q
V401R
V401S
V401T
V401F
V401I
V401M
V401W
V401Y
S402A

S402C
S402G
S402P
R403A
R403C
R403G
R403P
R403T
Y404A
Y404C
Y404D
Y404E

Y404G
Y404H
Y404K
Y404N
Y404P
Y404Q
Y404R
Y404S
Y404T
V405A
V405C
V405D

V405E
V405G
V405H
V405K
V405N
V405P
V405Q
V405R
V405S
V405T
V405W
V405Y

N406F
N406H
N406I
N406L
N406P
N406W
N406Y
W407D
W407E
W407F
W407H
W407I

W407K
W407N
W407P
W407Q
W407R
W407S
W407T
W407Y
I408D
I408E
I408H
I408K

I408N
I408P
I408Q
I408R
I408S
I408T
K409F
K409H
K409I
K409P
K409T
K409V

K409W
K409Y
E410H
K411A
K411C
K411G
K411I
K411P
K411T
K411V
K411W
K411Y

K413T

b. Glycosylation

Many proteins with therapeutic activity contain one or more glycosylation sites, such as, amino acid sequences that are glycosylated by a eukaryotic cell. There have been various reports of attempts to increase the degree of glycosylation of therapeutic proteins in order to achieve 1) reduced immunogenicity; 2) less frequent administration of the protein; 3) increased protein stability such as increased serum half-life; and 4) reduction in adverse side effects such as inflammation. The glycosylation site(s) provides a site for attachment of a carbohydrate moiety on the subject polypeptide, such that when the subject polypeptide is produced in a eukaryotic cell capable of glycosylation, the subject polypeptide is glycosylated. The further glycosylation of a FIX polypeptide confers one or more advantages including increased serum half-life; reduced immunogenicity; increased functional in vivo half-life; reduced degradation by gastrointestinal tract conditions such as gastrointestinal tract proteases; and increased rate of absorption by gut epithelial cells. An increased rate of absorption by gut epithelial cells and reduced degradation by gastrointestinal tract conditions is important for enteral (such as oral) formulations of a FIX polypeptide.

Glycosylation of proteins results in the formation of glycoproteins due to the covalent attachment of oligosaccharides to a polypeptide. The carbohydrate modifications found in glycoproteins are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. The N-glycosidic linkage is through the amide group of asparagines. The site of carbohydrate attachment to N-linked glycoproteins is found within a consensus sequence of amino acids, N-X-S/T, where X is any amino acid except proline. In N-linked glycosylation, the carbohydrate directly attached to the protein is GlcNAc. Since glycosylation is known to be highly host cell-dependent, the sugar chains associated with N-linked glycosylation of a protein can differ (Kagawa et al., (1988) Journal of Biological Chemistry 263:17508-17515). The β-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. In Ser- and Thr-type O-linked glycoproteins, the carbohydrate directly attached to the protein is GalNAc. A number of O-linked glycosylation sites are known in the art and have been reported in the literature, see such as Ten Hagen et al. (1999) Journal of Biological Chemistry, 274:27867-74; Hanisch et al. (2001) Glycobiology, 11:731-740; and Ten Hagen et al., (2003) Glycobiology, 13:1R-16R.

Modified FIX polypeptides provided herein can further be glycosylated (i.e. hyperglycosylated) as compared to an unmodified FIX polypeptide due to 1) a carbohydrate moiety covalently linked to at least one non-native glycosylation site not found in the unmodified FIX polypeptide or 2) a carbohydrate moiety covalently linked to at least one native glycosylation site found but not glycosylated in the unmodified FIX polypeptide. A hyperglycosylated FIX polypeptide can include O-linked glycosylation, N-linked glycosylation, and/or a combination thereof. In some examples, a hyperglycosylated FIX polypeptide can include 1, 2, 3, 4 or 5 carbohydrate moieties, each linked to different glycosylation sites. The glycosylation site can be a native glycosylation site. In other examples, the hyperglycosylated polypeptide can be glycosylated at a single non-native glycosylation site. In still other examples, the hyperglycosylated polypeptide can be glycosylated at more than one non-native glycosylation site, such as, the hyperglycosylated FIX polypeptide can be glycosylated at 2, 3 or 4 non-native glycosylation sites.

In some instances, a hyperglycosylated FIX polypeptide is glycosylated at a native glycosylation site. For example, FIX, such as for example human FIX having an amino acid sequence set forth in SEQ ID NO: 2, contains N-linked glycosylation sites at N157 and N167 and O-linked glycosylation sites at S53, S61, T159, T169, T172, and T179. The FIX polypeptide can be glycosylated at a single native glycosylation site, or at more than one native glycosylation site, such as, at 2, 3 or 4 native glycosylation sites. A hyperglycosylated FIX polypeptide also can be glycosylated at a native glycosylation site and a non-native glycosylation site. A hyperglycosylated FIX polypeptide also can be glycosylated at multiple native and non-native glycosylation sites.

Modified FIX polypeptides provided herein can have at least one additional carbohydrate moiety not found in the unmodified FIX polypeptide when each is synthesized in a eukaryotic cell that is capable of N- and/or O-linked protein glycosylation. Thus, e.g., compared to an unmodified FIX polypeptide, a hyperglycosylated modified FIX polypeptide can have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional carbohydrate moieties. For example, where an unmodified FIX polypeptide has one covalently linked carbohydrate moiety, a hyperglycosylated FIX polypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, or more covalently linked carbohydrate moieties. In some examples, a hyperglycosylated FIX polypeptide of a modified FIX polypeptide provided herein, lacks a carbohydrate moiety covalently linked to a non-native glycosylation site, and has instead at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional carbohydrate moieties attached to native glycosylation sites. In other examples, a hyperglycosylated FIX polypeptide lacks a carbohydrate moiety covalently linked to a native glycosylation site, and has instead at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more carbohydrate moieties attached to non-native glycosylation sites. In some examples, the compositions of the attached sugars can be modified, such as by increasing or decreasing sialic acid content of the attach sugar moieties.

Whether a subject FIX polypeptide has N-linked and/or O-linked glycosylation is readily determined using standard techniques, see such as, “Techniques in Glycobiology” R. Townsend and A. Hotchkiss, eds. (1997) Marcel Dekker; and “Glycoanalysis Protocols (Methods in Molecular Biology, Vol. 76)” E. Hounsell, ed. (1998) Humana Press. The change in electrophoretic mobility of a protein before and after treatment with chemical or enzymatic deglycosylation (such as, using endoglycosidases and/or exoglycosidases) is routinely used to determine the glycosylation status of a protein. Enzymatic deglycosylation can be carried out using any of a variety of enzymes, including, but not limited to, peptide-N4-(N-acetyl-β-D-glycosaminyl) asparagine amidase (PNGase F); endoglycosidase F1, endoglycosidase F2 and endoglycosidase F3, α(2→3,6,8,9) neuraminidase. For example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein, either pre-treated with PNGaseF or untreated with PNGaseF, is conducted. A marked decrease in band width and change in migration position after treatment with PNGaseF is considered diagnostic of N-linked glycosylation. The carbohydrate content of a glycosylated protein also can be detected using lectin analysis of protein blots (such as, proteins separated by SDS-PAGE and transferred to a support, such as a nylon membrane). Lectins, carbohydrate binding proteins from various plant tissues, have high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans (Cummings (1994) Methods in Enzymol. 230:66-86). Lectins can be detectably labeled (either directly or indirectly), allowing detection of binding of lectins to carbohydrates on glycosylated proteins. For example, when conjugated with biotin or digoxigenin, a lectin bound to a glycosylated protein can be easily identified on membrane blots through a reaction utilizing avidin or anti-digoxigenin antibodies conjugated with an enzyme such as alkaline phosphatase, β-galactosidase, luciferase or horse radish peroxidase, to yield a detectable product. Screening with a panel of lectins with well-defined specificity provides considerable information about a glycoprotein's carbohydrate complement.

Exemplary amino acid positions contemplated herein for modification of a glycosylation site, for attachment of a carbohydrate moiety, include positions corresponding to positions N157 or N167 of a mature FIX polypeptide set forth in SEQ ID NO: 2. Amino acid replacement or replacements can correspond to any of the following positions: N157 or N167 of mature FIX. In a particular embodiment, the amino acid replacement or replacements contributing to hyperglycosylation of modified FIX polypeptides is (are) replacement of amino acids by asparagines (N) or threonine (T). Thus, provided herein are modified FIX polypeptides containing a further modification corresponding to any one or more of N157 or N167 of a mature FIX polypeptide set forth in SEQ ID NO: 2.

c. Additional Modifications

Additional modifications of polypeptides provided herein include chemical derivatization of polypeptides, including but not limited to, acetylation and carboxylation; changes in amino acid sequence that make the protein susceptible to PEGylation or other modification or that alter properties of the FIX polypeptide. Related moieties for modifying FIX polypeptides also are contemplated, including, but not limited to copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidine or polyproline (Abuchowski et al. (1981); Newmark et al. (1982); and Katre et al. (1987)). A modified FIX polypeptide provided herein can be modified with one or more polyethylene glycol moieties (PEGylated). Activated PEG derivatives can be used to interact directly with the FIX polypeptides, and include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups can be used for the modification of sulfhydryl groups, and PEG reagents containing hydrazine or hydrazide groups can be used to modify aldehydes generated by periodate oxidation of carbohydrate groups. In some instances, a modified FIX polypeptide provided herein can contain one or more non-naturally occurring pegylation sites that are engineered to provide PEG-derivatized polypeptides with reduced serum clearance (U.S. Published Patent Application No: 2006-0040856).

Also contemplated are modified FIX polypeptides sequences that have phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine or phosphothreonine.

Other suitable additional modifications of a modified FIX polypeptide provided herein are polypeptides that have been modified using standard chemical techniques and/or recombinant nucleic acid method as to increase their resistance to proteolytic degradation, to optimize solubility properties and/or to render them more suitable as a therapeutic agent. For example, the backbone of the peptide can be cyclized to enhance stability (see such as, Friedler et al. (2000) J. Biol. Chem. 275:23783-23789). Analogs can be used that include residues other than naturally occurring L-amino acids, such as, D-amino acids or non-naturally occurring synthetic amino acids.

Modifications of FIX polypeptides provided herein also can be combined with modifications to improve post-translational processing of the FIX polypeptides. For example, FIX polypeptides can be modified to improve proteolytic cleavage of the signal or propeptides sequences (U.S. Pat. Application No. 2004-0102388), and/or to improve post-translational modifications such as carboxylation, phosphorylation, sulfation, and/or glycosylation, as discussed above. An exemplary modification that improves propeptide cleavage includes, modification of Y1, in particular Y1A (Meulien et al. (1990) Protein. Eng. 3(7): 629-633 and U.S. Pat. Nos. 4,770,999 and 5,521,070).

Modifications of FIX polypeptides provided herein also can be combined with amino acid modifications to further improve stability, binding properties and serum half-life of the FIX polypeptides. A non-limiting example of such modification includes replacement of all or part of the FIX activation peptide (amino acid residues A146 through R180 of a mature FIX polypeptide, SEQ ID NO: 2) with an activation peptide of another vitamin K-dependent polypeptide, such as FVII, FX, or protein C. Another non-limiting example of such modification includes replacement of all or part of the gla domain (amino acid residues Y1 through V46 of a mature FIX polypeptide, SEQ ID NO: 2) with a gla domain of another vitamin K-dependent polypeptide, such as FVII, FX, or protein C. Such modifications and generation of chimeric proteins are described in, for example, published International PCT application No. WO 2006/018204 A1, Lin et al. (1990) J. Biol. Chem. 265(1):144-150 and Cheung et al. (1991) J. Biol. Chem. 266(14):8797-880. In another example, amino acid residues in FIX polypeptides that are involved in binding to collagen, in particular collagen IV in the liver, can be modified to increase circulating levels of the FIX polypeptides in the blood. For example, K5 and V 10 in the γ-carboxyglutamic acid (gla) domain can be modified, such as K5A and/or V10K (Cheung et al. (1992) J. Biol. Chem. 267(29): 20529-20531, Gui et al. (2002) Blood 100(1): 153-158, and Schuettrumpf et al. (2005) Blood 105(6): 2316-2323). In yet another example modified FIX polypeptide can contain modifications to increase resistance to inhibition by heparin. One such modification is described in, for example, U.S. Pat. Application No. 2004-0110675.

Additional modifications that alter properties or activities of FIX polypeptides, such as, peptidase activity, coagulant activity, esterase activity, protein binding activities (such as to FVIII, FX, or FXI), cofactor binding activities, such as Ca2+, phospholipid or cell surface binding activities, and increased resistance to inhibitors, such as heparin and warfarin, are known in the art and can be combined with the modifications provided herein. Exemplary modifications of the peptidase activity include replacement of amino acid residues of the catalytic domain, H221, D269, S365 (U.S. Pat. No. 6,315,995) or replacement of 8338 to enhance clotting activity (U.S. Pat. No. 6,531,298 and Chang et al. (1998) J. Biol. Chem. 273(20): 12089-12094). Peptidase activity also can be altered by modifying substrate binding sites or cofactor sites. For example, modifications of K316 can alter binding of factor X to modified FIX polypeptides (Kolkman and Mertens (2000) Biochem. J. 350: 701-707). Exemplary modifications of FIX polypeptides with altered Ca²⁺ binding include replacement of residues in the EGF-like domains, such as, but not limited to, G60S, or replacement of the EGF-1 domain of FIC with that of FVII (Chang et al. (1995) Thromb and Haemost. Abst 73:1202). Exemplary modifications of FIX polypeptides that alter FVIIIa-dependent FIXa activity include mutations in the α-helical regions 333-339 and 301-333, which interact with FVIIIa. One or more properties of a FIX polypeptide may be altered as a result of a modification or combination of modifications. For example, modification of Ca²⁺ binding may lead to a change in coagulation activity.

F. PRODUCTION OF FIX POLYPEPTIDES

1. Expression Systems

FIX polypeptides (modified and unmodified) can be produced by any methods known in the art for protein production, including the introduction of nucleic acid molecules encoding FIX into a host cell, host animal and expression from nucleic acid molecules encoding FIX in vitro. Expression hosts include E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.

Expression in eukaryotic hosts can include expression in yeasts such as Saccharomyces cerevisiae and Pichia Pastoria, insect cells such as Drosophila cells and lepidopteran cells, plants and plant cells such as tobacco, corn, rice, algae and lemna. Eukaryotic cells for expression also include mammalian cells lines such as Chinese hamster ovary (CHO) cells. Eukaryotic expression hosts also include production in transgenic animals, for example, including production in serum, milk and eggs. Transgenic animals for the production of wild-type FIX polypeptides are known in the art (U.S. Patent Publication Nos. 2002-0166130 and 2004-0133930) and can be adapted for production of modified FIX polypeptides provided herein.

Many expression vectors are available for the expression of FIX. The choice of expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vectors in the cells.

Methods of production of FIX polypeptides can include coexpression of one or more additional heterologous polypeptides that can aid in the generation of the FIX polypeptides. For example, such polypeptides can contribute to the post-translation processing of the FIX polypeptides. Exemplary polypeptides include, but are not limited to, peptidases that help cleave FIX precursor sequences, such as the propeptide sequence, and enzymes that participate in the modification of the FIX polypeptide, such as by glycosylation, hydroxylation, carboxylation or phosphorylation, for example. An exemplary peptidase that can be coexpressed with FIX is PACE/furin (or PACE-SOL), which aids in the cleavage of the FIX propeptide sequence (Harrison et al. (1998) Semin Hematol. 35(2 Suppl 2): 4-10). An exemplary protein that aids in the carboxylation of the FIX polypeptide is the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR), which produces reduced vitamin K for utilization as a cofactor by the vitamin K-dependent γ-carboxylase (Wajih et al., J. Biol. Chem. 280(36)31603-31607). A subunit of this enzyme, VKORC1, can be coexpressed with the modified FIX polypeptide to increase the γ-carboxylation The one or more additional polypeptides can be expressed from the same expression vector as the FIX polypeptide or from a different vector.

a. Prokaryotic Expression

Prokaryotes, especially E. coli, provide a system for producing large amounts of FIX (see, for example, Platis et al (2003) Protein Exp. Purif. 31(2): 222-30; and Khalizzadeh et al. (2004) J. Ind. Microbiol. Biotechnol. 31(2): 63-69). Transformation of E. coli is a simple and rapid technique well known to those of skill in the art. Expression vectors for E. coli can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λP_Lpromoter.

FIX can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreitol and β-mercaptoethanol and denaturants (such as, such as guanidine-HCl and urea) can be used to resolubilize the proteins. An alternative approach is the expression of FIX in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases leading to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility. Typically, temperatures between 25° C. and 37° C. are used. Mutations also can be used to increase solubility of expressed proteins. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells.

b. Yeast

Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris are useful expression hosts for FIX (see for example, Skoko et al. (2003) Biotechnol. Appl. Biochem. 38(Pt3):257-65). Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7, and GAL5 and metallothionein promoters such as CUP1. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble and co-expression with chaperonins, such as Bip and protein disulfide isomerase, can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from Saccharomyces cerevisiae and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site (such as, the Kex-2 protease) can be engineered to remove the fused sequences from the polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs.

c. Insects and Insect Cells

Insects and insect cells, particularly using a baculovirus expression system, are useful for expressing polypeptide such as FIX (see, for example, Muneta et al. (2003) J. Vet. Med. Sci. 65(2):219-23). Insect cells and insect larvae, including expression in the haemolymph, express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typically, expression vectors use a promoter such as the polyhedrin promoter of baculovirus for high level expression. Commonly used baculovirus systems include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), and the bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation patterns similar to mammalian cell systems.

An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes albopictus) can be used for expression. The Drosophila metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin.

d. Mammalian Cells

Mammalian expression systems can be used to express FIX polypeptides. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter, and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha 1-antitrypsin, beta-globin, myelin basic protein, myosin light chain-2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase and thymidine kinase. Fusion with cell surface signaling molecules such as TCR-ζ and Fc_εRI-γ can direct expression of the proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include, but are not limited to, CHO, Balb/3T3, HeLa, MT2, mouse NS0 (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. One such example is the serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42). Expression of recombinant wild-type FIX polypeptides exhibiting similar structure and post-translational modifications as plasma-derived FIX are known in the art (see, such as, Bon et al. (1998) Semin Hematol. 35 (2 Suppl 2): 11-17). Methods of optimizing Factor IX expression are known. For example, supplementation of vitamin K in culture medium or co-expression of vitamin K-dependent γ-carboxylases (Wajih et al., J. Biol. Chem. 280(36)31603-31607) can aid in post-translational modification of FIX polypeptides. Multiple purification steps also can aid in enriched subpopulations of FIX polypeptides with desired post-translational modifications including, γ-carboxylation, β-hydroxylation, and glycosylation.

e. Plants

Transgenic plant cells and plants can be used for the expression of FIX. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements, and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce proteins (see, for example, Mayfield et al. (2003) PNAS 100:438-442). Because plants have different glycosylation patterns than mammalian cells, this can influence the choice to produce FIX in these hosts.

2. Purification

Methods for purification of FIX polypeptides from host cells depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary the proteins can be extracted and further purified using standard methods in the art.

FIX can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography and ionic exchange chromatography. Multiple chromatographic steps can be employed to improve selection of FIX polypeptides with particular posttranslational modifications. For example, multiple chromatographic steps can be used to separate γ-carboxylated FIX polypeptides (see such as, Cott et al. (2004) Papers in Biotechnology 1-21). Such techniques also can be used to enrich for FIX polypeptides with or without modifications at particular sites, including modifications such as, but not limited to, γ-carboxylation of particular glutamic acid residues in the gla domain, phosphorylation (e.g., at S158), sulfation (such as, at Y155), and glycosylation (see such as, Kaufman et al. (1986) J Biol Chem 261: 9622-8; Harrison et al. (1998) Semin Hematol 35 (Suppl. 2): 4-10; Bond et al. (1998) Semin Hematol 35 (Suppl. 2): 11-7).

Affinity purification techniques also can be used to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind FIX can be used in affinity purification. Expression constructs also can be engineered to add an affinity tag such as a myc epitope, GST fusion or His₆and affinity purified with myc antibody, glutathione resin, and Ni-resin, respectively, to a protein. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques.

3. Fusion Proteins

Fusion proteins containing a targeting agent and a modified FIX polypeptide also are provided. Pharmaceutical compositions containing such fusion proteins formulated for administration by a suitable route are provided. Fusion proteins are formed by linking in any order the modified FIX polypeptide and an agent, such as an antibody or fragment thereof, growth factor, receptor, ligand, and other such agent for directing the mutant protein to a targeted cell or tissue. Linkage can be effected directly or indirectly via a linker. The fusion proteins can be produced recombinantly or chemically by chemical linkage, such as via heterobifunctional agents or thiol linkages or other such linkages. The fusion proteins can contain additional components, such as E. coli maltose binding protein (MBP) that aid in uptake of the protein by cells (see, International PCT application No. WO 01/32711). In another embodiment the modified FIX is fused to polypeptides that aid in stability, such as albumin (Sheffield et al. (2004) Br. J. Haemotol. 126(4): 565-573).

4. Polypeptide Modification

Modified FIX polypeptides can be prepared as naked polypeptide chains or as a complex. For some applications, it can be desirable to prepare modified FIX in a “naked” form without post-translational or other chemical modifications. Naked polypeptide chains can be prepared in suitable hosts that do not post-translationally modify FIX. Such polypeptides also can be prepared in in vitro systems and using chemical polypeptide synthesis. For other applications, particular modifications can be desired including pegylation, albumination, glycosylation, carboxylation, hydroxylation, phosphorylation, or other known modifications. Modifications can be made in vitro or, for example, by producing the modified FIX in a suitable host that produces such modifications.

5. Nucleotide Sequences

Nucleic acid molecules encoding modified FIX polypeptides or the fusion protein operationally linked to a promoter, such as an inducible promoter for expression in mammalian cells also are provided. Such promoters include, but are not limited to, CMV and SV40 promoters; adenovirus promoters, such as the E2 gene promoter, which is responsive to the HPV E7 oncoprotein; a PV promoter, such as the PBV p89 promoter that is responsive to the PV E2 protein; and other promoters that are activated by the HIV or PV or oncogenes.

Modified FIX polypeptides provided herein also can be delivered to cells in gene transfer vectors. The transfer vectors can encode additional therapeutic agent(s) for treatment of diseases or disorders, such as treatments for hemophilia, inherited disorders and others for which FIX is administered. Transfer vectors encoding modified FIX polypeptides can be used systemically by administering the nucleic acid to a subject. For example, the transfer vector can be a viral vector, such as an adenoviral vector. Vectors encoding FIX also can be incorporated into stem cells and such stem cells administered to a subject, for example, by transplanting or engrafting the stem cells at sites for therapy. For example, mesenchymal stem cells (MSCs) can be engineered to express a modified FIX and such MSCs engrafted at a tumor site for therapy.

G. ASSESSING MODIFIED FIX POLYPEPTIDE PROPERTIES AND ACTIVITIES

FIX activities and properties can be assessed in vitro and/or in vivo. Assays for such assessment are known to those of skill in the art and are known to correlate tested activities and results to therapeutic and in vivo activities. In one example, FIX variants can be assessed in comparison to unmodified and/or wild-type FIX. In other examples, a modified FIX polypeptide can be assessed for biological activity following in vitro or in vivo exposure to protein stability-altering conditions (i.e. exposure to proteases, or denaturing agents such as temperature or pH). In vitro assays include any laboratory assay known to one of skill in the art, such as for example, cell-based assays including coagulation assays, protein assays, and molecular biology assays. In vivo assays include FIX assays in animal models as well as administration to humans. In some cases, activity of FIX in vivo can be determined by assessing blood, serum, or other bodily fluid for assay determinants. FIX variants also can be tested in vivo to assess an activity or property, such as stability (such as, half-life) and therapeutic effect.

1. In Vitro Assays

Exemplary in vitro assays include assays to assess polypeptide stability and activity. Stability assays include assays that assess protease resistance or thermal stability or other physical property indicative of stability of the polypeptide in vivo or in vitro. Stability also can be assessed by protein structure and conformational assays known in the art. Assays for activity include, but are not limited to, measurement of FIX interaction with other coagulation cofactors, such as factor VIII, enzymatic assays to determine proteolytic activity of FIX polypeptides, and cell-based assays to determine the effect of FIX polypeptides variants on coagulation.

Concentrations of modified FIX polypeptides can be assessed by methods well-known in the art, including but not limited to enzyme-linked immunosorbant assays (ELISA), SDS-PAGE; Bradford, Lowry, BCA methods; UV absorbance, and other quantifiable protein labeling methods, such as, but not limited to, immunological, radioactive and fluorescent methods and related methods.

Assessment of cleavage products of proteolysis reactions, including cleavage of FIX polypeptides or products produced by FIX protease activity, can be performed using methods including but not limited to, chromogenic substrate cleavage, HPLC, SDS-PAGE analysis, immunohistochemistry, immunoprecipitation, NH₂-terminal sequencing, and protein labeling.

FIX polypeptides that have been activated via proteolytic cleavage after R145 and R180 can be prepared in vitro. The FIX polypeptides can be first prepared by any of the methods of production described herein, including, but not limited to, production in mammalian cells followed by purification. Cleavage of the FIX polypeptides into the active protease form of FIX can be accomplished by incubation with activated factor XI (FXIa). The activated polypeptides can be used in any of the assays to measure FIX activities described herein.

Modified FIX polypeptides can be tested for proteolytic activity. Activated forms of the modified FIX polypeptides (FIXa) can be used in the assay. Assays using a synthetic substrate, such as a CH₃SO₂-LGR-pNA peptide, can be employed to measure enzymatic cleavage activity of the FIXa polypeptides. Hydrolysis of CH₃SO₂-LGR-pNA in the presence of FIXa can be measured by assessing the production of p-nitroanaline (pNA) from the cleavage reaction sample. The amount of pNA in the sample is proportional to the absorbance of the sample at 405 nm and thus indicates the extent of proteolytic activity in the FIXa sample. Additional exemplary fluorogenic substrates that can be used to assess FIXa cleavage activity include, but are not limited to, Mes-D-CHD-Gly-Arg-AMC (Pefafluor FIXa10148) and H-D-Leu-PHG-Arg-AMC (Pefafluor FIXa3688), wherein cleavage is assessed by release of AMC. Molecules that enhance FIXa catalytic activity, such as ethylene glycol, can be employed in such assays (Sturzebecher et al. (1997) FEBS Lett. (412) 295-300).

Proteolytic activity of FIXa also can be assessed by measuring the conversion of factor X (FX) into activated factor X (FXa). Modified FIXa polypeptides can be incubated in the presence of FX polypeptides in the presence of phospholipids vesicles (phosphatidylserine and/or phosphatidylcholine) and Ca²⁺, and cleavage of FX to produce FXa can be assayed using a chromogenic substrate, such as S2222 or S2765, which is specifically cleaved by FXa (Chromogenics AB, Molndal, Sweden).

Inhibition assays can be used to measure resistance of modified FIX polypeptides to FIX inhibitors. Exemplary inhibitors include, but are not limited to, antithrombin, p-aminobenzamidine, serine protease inhibitors, and FIX-specific antibodies. Inhibitors such as antithrombin which bind to the active site of FIXa can be used in inhibition assays, such as competition assays, wherein the ability of the inhibitor to compete with another substrate is measured.

FIX polypeptides can be tested for binding to other coagulation factors and inhibitors. For example, FIX direct and indirect interactions with proteins of the tenase complex, including factors VIIIa, IXa and X, and inhibitors, such as antithrombin III and heparin can be assessed using any binding assay known in the art, including, but not limited to, immunoprecipitation, column purification, non-reducing SDS-PAGE, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), isothermal titration calorimetry (ITC), circular dichroism (CD), protein fragment complementation assays (PCA), Nuclear Magnetic Resonance (NMR) spectroscopy, light scattering, sedimentation equilibrium, small-zone gel filtration chromatography, gel retardation, Far-western blotting, fluorescence polarization, hydroxyl-radical protein footprinting, phage display, and various two-hybrid systems.

FIX polypeptides can be tested for coagulation activity by using assays well known in the art. For example, some of the assays include, but are not limited to, a two stage clotting assay, (Leibman, H. A. et al. (1985) Proc. Natl. Acad. Sci., USA, 82, 3879-3883); an assay based on the single stage activated partial thromboplastin time (“aPTT”), Smith, K. J. et al. (1988) Blood, 72, 1269-1277; and assays which are modifications of the aPTT test, for example, Jenny, R. et al. (1986) Preparative Biochemistry, 16, 227-245, whole blood partial thromboplastin time (PTT), activated clotting time (ACT), recalcified activated clotting time, or the Lee-White Clotting time. For the aPTT assay, coagulation activity of a FIX polypeptide can be assessed by using equal volumes of activated partial thromboplastin reagent, FIX-deficient plasma (obtained from either a patient with hemophilia B using sterile phlebotomy techniques well known in the art or by using FIX immunodepleted plasma), and normal pooled plasma as standard, or the sample. In this assay, one unit of activity is defined as that amount present in one milliliter of normal pooled plasma. Further, an assay for biological activity based on the ability of FIX to reduce the clotting time of plasma from FIX-deficient patients to normal can be performed as described in, for example, Proctor and Rapaport (1961) Amer. J. Clin. Path. 36: 212.

FIX polypeptides also can be assessed for presence of post-translational modifications. Such assays are known in the art and include assays to measure glycosylation, hydroxylation, sulfation, carboxylation, and phosphorylation. In an exemplary assay for glycosylation, carbohydrate analysis can be performed, for example, with SDS page analysis of FIX polypeptides exposed to hydrazinolysis or endoglycosidase treatment. Hydrazinolysis releases N- and O-linked glycans from glycoproteins by incubation with anhydrous hydrazine, while endoglycosidase release involves PNGase F, which releases most N-glycans from glycoproteins. Hydrazinolysis or endoglycosidase treatment of FIX polypeptides generates a reducing terminus that can be tagged with a fluorophore or chromophore label. Labeled FIX polypeptides can be analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The fluorescent tag for glycans also can be used for monosaccharide analysis, profiling or fingerprinting of complex glycosylation patterns by HPLC. Exemplary HPLC methods include hydrophilic interaction chromatography, electronic interaction, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Exemplary glycan probes include, but are not limited to, 3-(acetylamino)-6-aminoacridine (AA-Ac) and 2-aminobenzoic acid (2-AA). Carbohydrate moieties also can be detected through use of specific antibodies that recognize the glycosylated FIX polypeptide.

An exemplary assay to measure carboxylation comprises reverse phase HPLC analysis of FIX polypeptides that have been subjected to alkaline hydrolysis (Przysiecki et al. (1987) PNAS 84: 7856-7860). Exemplary assays to measure phosphorylation include use of phosphospecific antibodies to phospho-serine and/or -tyrosine amino acid residues or to a serine-phosphorylated FIX polypeptide. ³²P metabolic labeling of cells that produce the FIX polypeptide also can be used to assess phosphorylation, wherein the labeled FIX polypeptide can be purified and analyzed for incorporation of radioactive phosphate. An exemplary assay for tyrosine sulfation includes ³⁵S labeling of cells that produce the FIX polypeptide. In such method, cells are incubated with either ³⁵S—S₂SO₄or ³⁵S-methionine and incorporation of the ³⁵S is determined by normalization to the ³⁵S-methionine sample.

Structural properties of modified FIX polypeptides also can be assessed. For example, X-ray crystallography, nuclear magnetic resonance (NMR), and cryoelectron microscopy (cryo-EM) of modified FIX polypeptides can be performed to assess three-dimensional structure of the FIX polypeptides and/or other properties of FIX polypeptides, such as Ca²⁺or cofactor binding.

Additionally, the presence and extent of FIX degradation can be measured by standard techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting of electrophoresed FIX-containing samples. FIX polypeptides that have been exposed to proteases also can be subjected to N-terminal sequencing to determine location or changes in cleavage sites if the modified FIX polypeptides.

2. Non-Human Animal Models

Non-human animal models can be used to assess activity and stability of modified FIX polypeptides. For example, non-human animals can be used as models for a disease or condition. Non-human animals can be injected with disease and/or phenotype-inducing substances prior to administration of FIX variants to monitor the effects on disease progression. Genetic models also are useful. Animals, such as mice, can be generated which mimic a disease or condition by the overexpression, underexpression or knock-out of one or more genes. Such animals can be generated by transgenic animal production techniques well-known in the art or using naturally-occurring or induced mutant strains. Examples of useful non-human animal models of diseases associated with FIX include, but are not limited to, models of bleeding disorders, in particular hemophilia, or thrombotic disease. These non-human animal models can be used to monitor activity of FIX variants compared to a wild type FIX polypeptide.

Animal models also can be used to monitor stability, half-life, and clearance of modified FIX polypeptides. Such assays are useful for comparing modified FIX polypeptides and for calculating doses and dose regimens for further non-human animal and human trials. For example, a modified FIX polypeptide can be injected into the tail vein of mice. Blood samples are then taken at time-points after injection (such as minutes, hours and days afterwards) and then the level of the modified FIX polypeptides in bodily samples including, but not limited to, serum or plasma can be monitored at specific time-points for example by ELISA or radioimmunoassay. Blood samples also can be tested for coagulation activity in methods, such as the aPTT assay.

Modified FIX polypeptides can be tested for immune tolerance using animal models. Animal models for immune tolerance of FIX are known in the art and include rhesus macaque, mouse, rat, and dog models to test long term expression of FIX via injection of polypeptides or gene transfer vectors (Lozier et al. (1999) Blood 93(6) 1875-1881 and Keith et al., (1995) Thromb. Haemost. 73(1): 101-105).

Modified FIX polypeptides can be tested for therapeutic effectiveness using animal models for hemophilia. In one non-limiting example, an animal model such as a mouse can be used. Mouse models of hemophilia are available in the art and include FIX deficient mice and mice expressing mutant FIX polypeptides, and can be employed to test modified FIX polypeptides (Wang et al. (1997) PNAS 94:11563-11566, Lin et al. (1997) Blood 90:3962-3966, Kundu et al. (1998) Blood 92: 168-174, Sabatino et al. (2004) Blood 104(9): 2767-2774 and Jin et al. (2004) Blood 104:1733-1739)

Other models of FIX deficiencies include hemophilic dogs that express defective FIX or that have been hepatectomized (Evans et al. (1989) PNAS 86:10095, Mauser et al. (1996) Blood 88:3451, and Kay et al. (1994) PNAS 91:2353-2357).

Modified FIX polypeptides can be tested for therapeutic effectiveness using animal models for thrombotic disease (Dodds (1987) Ann N Y Acad. Sci. 516: 631-635). Modified FIX polypeptides that have antithrombotic activity can be tested in such models. In one non-limiting example, an animal model such as a rat or mouse model of ischemic stroke can be used to test the effectiveness of modified FIX polypeptides. Such animal models are known in the art (Beech, et al. (2001) Brain Res 895: 18-24, Buchan et al. Stroke 23: 273-9 (1992), Carmichael et al. (2005) NeuroRx 2: 396-409, Chen et al. (1986) Stroke 17: 738-43, Gerriets et al. (2003) J Neurosci Methods 122: 201-11, Koizumi et al. (1986) Jpn. J. Stroke 8: 1-8, Longa et al. (1989) Stroke 20: 84-91, Mayzel-Oreg et al. (2004) Magn Reson Med 51: 1232-8, Tamura, et al. (1981) J Cereb Blood Flow Metab 1: 53-60, Watson et al. (1985) Ann Neurol 17: 497-504, Zhang et al. (1997) J Cereb Blood Flow Metab 17: 123-35).

3. Clinical Assays

Many assays are available to assess activity of FIX for clinical use. Such assays can include assessment of coagulation, protein stability, and half-life in vivo and phenotypic assays. Phenotypic assays and assays to assess the therapeutic effect of FIX treatment include assessment of blood levels of FIX (such as measurement of serum FIX prior to administration and time-points following administrations including, after the first administration, immediately after last administration, and time-points in between, correcting for the body mass index (BMI)), phenotypic response to FIX treatment including amelioration of symptoms over time compared to subjects treated with an unmodified and/or wild type FIX or placebo. Examples of clinical assays to assess FIX activity can be found such as in Franchini et al. (2005) Thromb Haemost. 93(6): 1027-1035, Shapiro et al. (2005) Blood 105(2): 518-525, and White et al. (1997) Thromb Haemost. 78(1): 261-265. Patients can be monitored regularly over a period of time for routine or repeated administrations, following administration in response to acute events, such as hemorrhage, trauma, or surgical procedures.

H. FORMULATION/PACKAGING/ADMINISTRATION

Pharmaceutical compositions containing an optimized polypeptide produced using methods described herein, including FIX variant (modified) polypeptides, modified FIX fusion proteins or encoding nucleic acid molecules, can be formulated in any conventional manner by mixing a selected amount of the polypeptide with one or more physiologically acceptable carriers or excipients. Selection of the carrier or excipient is within the skill of the administering profession and can depend upon a number of parameters. These include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, local, topical or any other mode) and disorder treated. The pharmaceutical compositions provided herein can be formulated for single dosage (direct) administration or for dilution or other modification. The concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment. Typically, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

1. Administration of Modified FIX Polypeptides

The polypeptides can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients. The polypeptides can be targeted for delivery, such as by conjugation to a targeting agent, such as an antibody. Liposomal suspensions, including tissue-targeted liposomes, also can be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Pat. No. 4,522,811. Liposomal delivery also can include slow release formulations, including pharmaceutical matrices such as collagen gels and liposomes modified with fibronectin (see, for example, Weiner et al. (1985) J Pharm Sci. 74(9): 922-5).

The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration can be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein. The active compounds can be administered by any appropriate route, for example, oral, nasal, pulmonary, parenteral, intravenous, intradermal, subcutaneous, or topical, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. In a particular embodiment, the FIX polypeptide is administered orally. FIX polypeptides can be formulated with additional coagulation factors.

The modified FIX and physiologically acceptable salts and solvates can be formulated for administration by inhalation (either through the mouth or the nose), oral, transdermal, pulmonary, parenteral, or rectal administration or injection. For administration by inhalation, the modified FIX can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer with the use of a suitable propellant, such as, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of such as, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of a therapeutic compound and a suitable powder base such as lactose or starch.

FIX polypeptides can be formulated as liquid or powder. In the case of a liquid, the modified polypeptides can be injected from a syringe or an auto-injector. In the case of a powder, the modified polypeptides can be reconstituted with a pharmaceutically acceptable excipient, such as pharmaceutically-acceptable saline, prior to administration. Administration can be by a medical professional or self-administration.

For pulmonary administration to the lungs, the modified FIX can be delivered in the form of an aerosol spray presentation from a nebulizer, turbonebulizer, or microprocessor-controlled metered dose oral inhaler with the use of a suitable propellant. Generally, the particle size is small, such as in the range of 0.5 to 5 microns. In the case of a pharmaceutical composition formulated for pulmonary administration, detergent surfactants are not typically used. Pulmonary drug delivery is a promising non-invasive method of systemic administration. The lungs represent an attractive route for drug delivery, mainly due to the high surface area for absorption, thin alveolar epithelium, extensive vascularization, lack of hepatic first-pass metabolism, and relatively low metabolic activity.

The modified FIX polypeptides exhibit increased resistance to proteolysis and half-life in the gastrointestinal tract. Thus, preparations for oral administration can be suitably formulated without the use of protease inhibitors, such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor, aprotinin and camostat.

The modified FIX polypeptides can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The modified FIX can be formulated, for example, for parenteral administration by injection (such as, by bolus injection or continuous infusion). Formulations for injection can be presented in unit dosage form (such as, in ampoules or in multi-dose containers) with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder-lyophilized form for constitution with a suitable vehicle, such as, sterile pyrogen-free water, before use.

Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in a conventional manner.

The pharmaceutical compositions can be formulated for local or topical application, such as for topical application to the skin (transdermal) and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, can be formulated as 0.01%-10% isotonic solutions and pH about 5-7 with appropriate salts. The compounds can be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma).

The concentration of active compound in the drug composition depends on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. As described further herein, dosages can be determined empirically using dosages known in the art for administration of unmodified FIX, and comparisons of properties and activities (such as, stability and activities) of the modified FIX compared to the unmodified and/or native FIX.

The compositions, if desired, can be presented in a package, in a kit or dispenser device, that can contain one or more unit dosage forms containing the active ingredient. The package, for example, contains metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The compositions containing the active agents can be packaged as articles of manufacture containing packaging material, an agent provided herein, and a label that indicates the disorder for which the agent is provided.

a. Oral Administration

Among the modified FIX polypeptides provided herein are FIX polypeptides modified to increase protein stability to conditions amendable to oral delivery. Oral delivery can include administration to the mouth and/or gastrointestinal tract. Such modifications can include increased protein-half life under one or more conditions such as exposure to saliva, exposure to proteases in the gastrointestinal tract, exposure to increased temperature, and exposure to particular pH conditions, such as the low pH of the stomach and/or pH conditions in the intestine. For example, modifications can include resistance to one or more proteases including pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, elastase, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. Modifications also can include increasing overall stability to potentially denaturing or conformation-altering conditions such as thermal tolerance, and tolerance to mixing and aeration (such as, chewing).

FIX polypeptides modified for suitability to oral delivery can be prepared using any of the methods described herein. For example, 2D- and 3D-scanning mutagenesis methods for protein rational evolution (see, co-pending U.S. Publication No. US 2005-0202438 A1 and U.S. Publication No. US-2004-0132977-A1 and published International applications WO 2004/022593 and WO 2004/022747) can be used to prepare modified FIX. Modification of FIX polypeptides for suitability for oral delivery can include removal of proteolytic digestion sites and/or increasing the overall stability of the protein structure. Such FIX variants exhibit increased protein half-life compared to an unmodified and/or wild-type native FIX in one or more conditions for oral delivery. For example, a modified FIX can have increased protein half-life and/or bioavailability in the mouth, throat (such as, through the mucosal lining), the gastrointestinal tract or systemically.

In one embodiment, the half-life of the modified FIX polypeptides provided herein is increased by an amount at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more, when compared to the half-life of a native FIX polypeptide exposed to one or more conditions (i.e. proteases, pH, temperature) for oral delivery. In other embodiments, the half-life in vitro or in vivo (protein stability) of the modified FIX polypeptides provided herein is increased by an amount of at least 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, or more, compared to the half-life of native FIX exposed to one or more conditions for oral delivery (i.e. proteases, pH, temperature).

In one example, half-life of the modified FIX polypeptide is assessed by increased half-life in the presence of one or more proteases. Proteases include, but are not limited to, proteases in blood, serum, the gastrointestinal tract, and the stomach. For example, proteases include, but are not limited to, pepsin, trypsin, chymotrypsin, elastase, aminopeptidase, gelatinase B, gelatinase A, α-chymotrypsin, carboxypeptidase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, trypsin, luminal pepsin, microvillar endopeptidase, dipeptidyl peptidase, enteropeptidase, hydrolase, NS3, elastase, factor Xa, Granzyme B, thrombin, plasmin, urokinase, tPA and PSA. The modified polypeptide can be mixed with one or more proteases and then assessed for activity and/or protein structure after a suitable reaction time. In one embodiment, the modified polypeptide is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% more resistant to proteolysis.

Assessment of half-life also can include exposure to increased temperature, such as the body temperature of a subject; exposure to gastric juices and/or simulated gastric juices; exposure to particular pH conditions and/or a combination of two or more conditions. Following exposure to one or more conditions, activity and/or assessment of protein structure can be used to assess the half-life of the modified FIX in comparison to an appropriate control (i.e., an unmodified and/or wild-type FIX polypeptide).

For oral administration, the pharmaceutical compositions can take the form of, for example, tablets, pills, liquid suspensions, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (such as, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as, magnesium stearate, talc or silica); disintegrants (such as, potato starch or sodium starch glycolate); or wetting agents (such as, sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically-acceptable saline, pharmaceutically acceptable additives such as suspending agents (such as, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (such as, lecithin or acacia); non-aqueous vehicles (such as, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (such as, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations also can contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

The modified FIX polypeptides can be formulated for oral administration, Oral formulations include tablets, capsules, liquids or other suitable vehicle for oral administration. In some examples, the capsules or tablets are formulated with an enteric coating to be gastro-resistant. Preparation of pharmaceutical compositions containing a modified FIX for oral delivery can include formulating modified FIX polypeptides with oral formulations known in the art and/or those described herein. The compositions as formulated do not require addition of protease inhibitors and/or other ingredients that are necessary for stabilization of unmodified (for protease resistance) and wild-type FIX polypeptides upon exposure to proteases, such as selecting pH and other conditions to minimize protease cleavage. For example, such compositions exhibit stability in the absence of compounds such as actinonin or epiactinonin and derivatives thereof; Bowman-Birk inhibitor and conjugates thereof; aprotinin and camostat. In other examples, the preparations for oral administration can include protease inhibitors.

Additionally, because modified FIX polypeptides provided herein exhibit increased protein stability, there is more flexibility in the administration of pharmaceutical compositions than their unmodified counterparts. Typically, orally ingested polypeptides are administered in the morning before eating (i.e., before digestive enzymes are activated). The modified polypeptides provided herein exhibit protease resistance to digestive enzymes and can offer the ability to administer pharmaceutical compositions containing a modified FIX polypeptide at other periods during the day and under conditions when digestive enzymes are present and active.

For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (such as, pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as, magnesium stearate, talc or silica); disintegrants (such as, potato starch or sodium starch glycolate); or wetting agents (such as, sodium lauryl sulphate). The active ingredient present in the capsule can be in, for example, liquid or lyophilized form. The tablets or capsules can be coated by methods well known in the art. Tablets and capsules can be coated, for example, with an enteric coating. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (such as, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (such as, lecithin or acacia); non aqueous vehicles (such as, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (such as, methyl or propyl-p hydroxybenzoates or sorbic acid). The preparations also can contain buffer salts, flavoring, coloring and/or sweetening agents as appropriate.

The compositions for oral administration can be formulated, for example, as gastro-resistant capsules or tablets. Such gastro-resistant capsules are modified release capsules that are intended to resist the gastric fluid and to release their active ingredient or ingredients in the intestinal fluid. They are prepared by providing hard or soft capsules with a gastro-resistant shell (enteric capsules) or by filling capsules with granules or with particles covered with a gastro-resistant coating.

The enteric coating is typically, although not necessarily, a polymeric material. Enteric coating materials can contain bioerodible, gradually hydrolyzable and/or gradually water-soluble polymers. The “coating weight,” or relative amount of coating material per capsule, generally dictates the time interval between ingestion and drug release. Any coating should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile can be used as an enteric coating to achieve delivery of the active ingredient to the lower gastrointestinal tract. The selection of the specific enteric coating material will depend on the following properties: resistance to dissolution and disintegration in the stomach; impermeability to gastric fluids and drug/carrier/enzyme while in the stomach; ability to dissolve or disintegrate rapidly at the target intestine site; physical and chemical stability during storage; non-toxicity; ease of application as a coating (substrate friendly); and economical practicality.

Suitable enteric coating materials include, but are not limited to: cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, such as formed from acrylic acid, met acrylic acid, methyl acrylate, ammonium methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (such as, those copolymers sold under the trade name EUDRAGIT); vinyl polymers and copolymers, such as polyvinyl pyrrolidone (PVP), polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac (purified lac). Combinations of different coating materials also can be used to coat a single capsule. Exemplary of such gastro-resistant capsules are hard gelatin capsules (sold by Torpac or Capsugel) size 9, coated with cellulose acetate phthalate (CAP) at 12% in acetone.

The enteric coating provides for controlled release of the active agent, such that drug release can be accomplished at some generally predictable location in the lower intestinal tract below the point at which drug release would occur without the enteric coating. The enteric coating also prevents exposure of the hydrophilic therapeutic agent and carrier to the epithelial and mucosal tissue of the buccal cavity, pharynx, esophagus, and stomach, and to the enzymes associated with these tissues. The enteric coating therefore helps to protect the active agent and a patient's internal tissue from any adverse event prior to drug release at the desired site of delivery. Furthermore, the coated capsules can permit optimization of drug absorption, active agent protection, and safety. Multiple enteric coatings targeted to release the active agent at various regions in the lower gastrointestinal tract would enable even more effective and sustained improved delivery throughout the lower gastrointestinal tract.

The coating optionally can contain a plasticizer to prevent the formation of pores and cracks that would permit the penetration of the gastric fluids. Suitable plasticizers include, but are not limited to, triethyl citrate (CITROFLEX 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (CITROFLEC A2), CARBOWAX 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. In particular, a coating comprised of an anionic carboxylic acrylic polymer will typically contain less than about 50% by weight, such as less than about 30%, 10% to about 25% by weight, based on the total weight of the coating, of a plasticizer, particularly dibutyl phthalate, polyethylene glycol, triethyl citrate and triacetin. The coating also can contain other coating excipients, such as detackifiers, antifoaming agents, lubricants (such as, magnesium stearate), and stabilizers (such as, hydroxypropylcellulose, acids and bases) to solubilize or disperse the coating material, and to improve coating performance and the coated product.

The coating can be applied to the capsule or tablet using conventional coating methods and equipment. For example, an enteric coating can be applied to a capsule using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. Detailed information concerning materials, equipment and processes for preparing coated dosage forms are described in Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6^thEdition (Media, Pa.: Williams & Wilkins, 1995). The coating thickness, as noted above, must be sufficient to ensure that the oral dosage form remains intact until the desired site of topical delivery in the lower intestinal tract is reached.

Preparations for oral administration can be formulated to give controlled or sustained release or for release after passage through the stomach or in the small intestine of the active compound. For oral administration the compositions can take the form of tablets, capsules, liquids, lozenges and other forms suitable for oral administration Formulations suitable for oral administration include lozenges and other formulations that deliver the pharmaceutical composition to the mucosa of the mouth, throat and/or gastrointestinal tract. Lozenges can be formulated with suitable ingredients including excipients for example, anhydrous crystalline maltose and magnesium stearate. As noted, modified polypeptides herein exhibit resistance to blood or intestinal proteases and can exhibit increased half-life in the gastrointestinal tract. Thus, preparations of oral administration can be suitably formulated without additional protease inhibitors or other protective compounds, such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor, aprotinin and camostat. Preparations for oral administration also can include a modified FIX resistant to proteolysis formulated with one or more additional ingredients that also confer proteases resistance, or confer stability in other conditions, such as particular pH conditions.

2. Administration of Nucleic Acids Encoding Modified FIX Polypeptides (Gene Therapy)

Also provided are compositions of nucleic acid molecules encoding the modified FIX polypeptides and expression vectors encoding them that are suitable for gene therapy. Rather than deliver the protein, nucleic acid can be administered in vivo, such as systemically or by other route, or ex vivo, such as by removal of cells, including lymphocytes, introduction of the nucleic therein, and reintroduction into the host or a compatible recipient.

Modified FIX polypeptides can be delivered to cells and tissues by expression of nucleic acid molecules. Modified FIX polypeptides can be administered as nucleic acid molecules encoding modified FIX polypeptides, including ex vivo techniques and direct in vivo expression. Nucleic acids can be delivered to cells and tissues by any method known to those of skill in the art. The isolated nucleic acid sequences can be incorporated into vectors for further manipulation.

Methods for administering modified FIX polypeptides by expression of encoding nucleic acid molecules include administration of recombinant vectors. The vector can be designed to remain episomal, such as by inclusion of an origin of replication or can be designed to integrate into a chromosome in the cell. Modified FIX polypeptides also can be used in ex vivo gene expression therapy using non-viral vectors. For example, cells can be engineered to express a modified FIX polypeptide, such as by integrating a modified FIX polypeptide encoding-nucleic acid into a genomic location, either operatively linked to regulatory sequences or such that it is placed operatively linked to regulatory sequences in a genomic location. Such cells then can be administered locally or systemically to a subject, such as a patient in need of treatment.

Viral vectors, include, for example adenoviruses, adeno-associated viruses (AAV), poxviruses, herpes viruses, retroviruses and others designed for gene therapy can be employed. The vectors can remain episomal or can integrate into chromosomes of the treated subject. A modified FIX polypeptide can be expressed by a virus, which is administered to a subject in need of treatment. Viral vectors suitable for gene therapy include adenovirus, adeno-associated virus (AAV), retroviruses, lentiviruses, vaccinia viruses and others noted above. For example, adenovirus expression technology is well-known in the art and adenovirus production and administration methods also are well known. Adenovirus serotypes are available, for example, from the American Type Culture Collection (ATCC, Rockville, Md.). Adenovirus can be used ex vivo, for example, cells are isolated from a patient in need of treatment, and transduced with a modified FIX polypeptide-expressing adenovirus vector. After a suitable culturing period, the transduced cells are administered to a subject, locally and/or systemically. Alternatively, modified FIX polypeptide-expressing adenovirus particles are isolated and formulated in a pharmaceutically-acceptable carrier for delivery of a therapeutically effective amount to prevent, treat or ameliorate a disease or condition of a subject. Typically, adenovirus particles are delivered at a dose ranging from 1 particle to 1014 particles per kilogram subject weight, generally between 106 or 108 particles to 1012 particles per kilogram subject weight. In some situations it is desirable to provide a nucleic acid source with an agent that targets cells, such as an antibody specific for a cell surface membrane protein or a target cell, or a ligand for a receptor on a target cell. FIX also can be targeted for delivery into specific cell types. For example, adenoviral vectors encoding FIX polypeptides can be used for stable expression in nondividing cells, such as liver cells, and skeletal muscle cells (Arruda et al. (2001) Blood 97(1): 130-138 and Yao et al. (1992) PNAS 89: 3357-3361). In another example, viral or nonviral vectors encoding FIX polypeptides can be transduced into isolated cells for subsequent delivery. Additional cell types for expression and delivery of FIX are known in the art and include but are not limited to, fibroblasts and endothelial cells (Palmer et al. (1989) Blood 73: 438-445; Yao et al. (1991) PNAS 88: 8101-8105).

The nucleic acid molecules can be introduced into artificial chromosomes and other non-viral vectors. Artificial chromosomes, such as ACES (see, Lindenbaum et al. Nucleic Acids Res. 2004 Dec. 7; 32(21):e172) can be engineered to encode and express the isoform. Briefly, mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure using an ACE System. Using this approach, specific loading of one or two gene targets has been achieved in LMTK(−) and CHO cells.

Another method for introducing nucleic acids encoding the modified FIX polypeptides is a two-step gene replacement technique in yeast, starting with a complete adenovirus genome (Ad2; Ketner et al. (1994) Proc. Natl. Acad. Sci. USA 91: 6186-6190) cloned in a Yeast Artificial Chromosome (YAC) and a plasmid containing adenovirus sequences to target a specific region in the YAC clone, an expression cassette for the gene of interest and a positive and negative selectable marker. YACs are of particular interest because they permit incorporation of larger genes. This approach can be used for construction of adenovirus-based vectors bearing nucleic acids encoding any of the described modified FIX polypeptides for gene transfer to mammalian cells or whole animals.

The nucleic acids can be encapsulated in a vehicle, such as a liposome, or introduced into cells, such as a bacterial cell, particularly an attenuated bacterium or introduced into a viral vector. For example, when liposomes are employed, proteins that bind to a cell surface membrane protein associated with endocytosis can be used for targeting and/or to facilitate uptake, such as capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.

For ex vivo and in vivo methods, nucleic acid molecules encoding the modified FIX polypeptide is introduced into cells that are from a suitable donor or the subject to be treated. Cells into which a nucleic acid can be introduced for purposes of therapy include, for example, any desired, available cell type appropriate for the disease or condition to be treated, including but not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, such as, such as stem cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources thereof.

For ex vivo treatment, cells from a donor compatible with the subject to be treated or the subject to be treated cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the subject. Treatment includes direct administration, such as, for example, encapsulated within porous membranes, which are implanted into the patient (see, such as, U.S. Pat. Nos. 4,892,538 and 5,283,187 each of which is herein incorporated by reference in its entirety). Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes and cationic lipids (such as, DOTMA, DOPE and DC-Chol) electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation methods. Methods of DNA delivery can be used to express modified FIX polypeptides in vivo. Such methods include liposome delivery of nucleic acids and naked DNA delivery, including local and systemic delivery such as using electroporation, ultrasound and calcium-phosphate delivery. Other techniques include microinjection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer and spheroplast fusion.

In vivo expression of a modified FIX polypeptide can be linked to expression of additional molecules. For example, expression of a modified FIX polypeptide can be linked with expression of a cytotoxic product such as in an engineered virus or expressed in a cytotoxic virus. Such viruses can be targeted to a particular cell type that is a target for a therapeutic effect. The expressed modified FIX polypeptide can be used to enhance the cytotoxicity of the virus.

In vivo expression of a modified FIX polypeptide can include operatively linking a modified FIX polypeptide encoding nucleic acid molecule to specific regulatory sequences such as a cell-specific or tissue-specific promoter. Modified FIX polypeptides also can be expressed from vectors that specifically infect and/or replicate in target cell types and/or tissues. Inducible promoters can be used to selectively regulate modified FIX polypeptide expression. An exemplary regulatable expression system is the doxycycline-inducible gene expression system, which has been used to regulate recombinant FIX expression (Srour et al. Thromb Haemost. 90(3): 398-405 (2003)).

Nucleic acid molecules, as naked nucleic acids or in vectors, artificial chromosomes, liposomes and other vehicles can be administered to the subject by systemic administration, topical, local and other routes of administration. When systemic and in vivo, the nucleic acid molecule or vehicle containing the nucleic acid molecule can be targeted to a cell.

Administration also can be direct, such as by administration of a vector or cells that typically targets a cell or tissue. For example, tumor cells and proliferating cells can be targeted cells for in vivo expression of modified FIX polypeptides. Cells used for in vivo expression of a modified FIX polypeptide also include cells autologous to the patient. Such cells can be removed from a patient, nucleic acids for expression of an modified FIX polypeptide introduced, and then administered to a patient such as by injection or engraftment.

Polynucleotides and expression vectors provided herein can be made by any suitable method. Further provided are nucleic acid vectors comprising nucleic acid molecules as described above, including a nucleic acid molecule comprising a sequence of nucleotides that encodes the polypeptide as set forth in any of SEQ ID NOS: 3-891, 917-1034, 1036-2044 or a fragment thereof. Further provided are nucleic acid vectors comprising nucleic acid molecules as described above and cells containing these vectors.

I. THERAPEUTIC USES

The modified FIX polypeptides and nucleic acid molecules provided herein can be used for treatment of any condition for which unmodified FIX is employed. Modified FIX polypeptides have therapeutic activity alone or in combination with other agents. The modified polypeptides provided herein are designed to retain therapeutic activity but exhibit modified properties, particularly increased stability. Such modified properties, for example, can improve the therapeutic effectiveness of the polypeptides and/or can provide for additional routes of administration, such as oral administration. The modified FIX polypeptides and encoding nucleic acid molecules provided herein can be used for treatment of any condition for which unmodified FIX is employed. This section provides exemplary uses of and administration methods. These described therapies are exemplary and do not limit the applications of modified FIX polypeptides.

The modified FIX polypeptides provided herein can be used in various therapeutic as well as diagnostic methods in which FIX is employed. Such methods include, but are not limited to, methods of treatment of physiological and medical conditions described and listed below. Modified FIX polypeptides provided herein can exhibit improvement of in vivo activities and therapeutic effects compared to wild-type FIX, including lower dosage to achieve the same effect, a more sustained therapeutic effect and other improvements in administration and treatment.

The modified FIX polypeptides described herein exhibit increased protein stability and improved half-life. Thus, modified FIX polypeptides can be used to deliver longer-lasting, more stable therapies. Examples of therapeutic improvements using modified FIX polypeptides include, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects and increased therapeutic effects.

In particular, modified FIX polypeptides, are intended for use in therapeutic methods in which FIX has been used for treatment. Such methods include, but are not limited to, methods of treatment of diseases and disorders, such as, but not limited to, blood coagulation disorders, hematologic diseases, hemorrhagic disorders, hemophilias, in particular hemophilia B, and acquired blood disorders, such as caused by liver disease. Modified FIX polypeptides also can be used in the treatment of additional bleeding diseases and disorders, such as, but not limited to, thrombocytopenia (such as, idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (such as, storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation. In some embodiments, the bleedings to be treated by FIX polypeptides occur in organs such as the brain, inner ear region, eyes, liver, lung, tumor tissue, gastrointestinal tract. In other embodiments, the bleeding is diffuse, such as in haemorrhagic gastritis and profuse uterine bleeding. Patients with bleeding disorders are often at risk for hemorrhage and excessive bleeding during surgery or trauma. Such patients often have acute haemarthroses (bleedings in joints), chronic haemophilic arthropathy, haematomas, (such as, muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (bleeding from the renal tract), cerebral hemorrhage, surgery (such as, hepatectomy), dental extraction, and gastrointestinal bleedings (such as, UGI bleeds), that can be treated with modified FIX polypeptides. In one embodiment, the modified FIX polypeptides can be used to treat bleeding episodes due to trauma, or surgery, or lowered count or activity of platelets, in a subject. Exemplary methods for patients undergoing surgery include treatments to prevent hemorrhage and treatments before, during, or after surgeries such as, but not limited to, heart surgery, angioplasty, lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular surgery, dental surgery, or organ transplant surgery, including transplantation of heart, lung, pancreas, or liver.

FIX polypeptides lacking functional peptidase activity have been used in therapeutic methods to inhibit blood coagulation (U.S. Pat. No. 6,315,995). Modified FIX polypeptides provided herein that inhibit or antagonize blood coagulation can be used in anticoagulant methods of treatment for ischemic disorders, such as a peripheral vascular disorder, a pulmonary embolus, a venous thrombosis, deep vein thrombosis (DVT), superficial thrombophlebitis (SVT), arterial thrombosis, a myocardial infarction, a transient ischemic attack, unstable angina, a reversible ischemic neurological deficit, an adjunct thrombolytic activity, excessive clotting conditions, reperfusion injury, sickle cell anemia or stroke disorder. In patients with an increased risk of excessive clotting, such as DVT or SVT, during surgery, protease inactive modified FIX polypeptides provided herein can be administered to prevent excessive clotting in surgeries, such as, but not limited to heart surgery, angioplasty, lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular surgery, or organ transplant surgery, including transplantation of heart, lung, pancreas, or liver. In some cases treatment is performed with FIX alone. In some cases, FIX is administered in conjunction with additional coagulation or anticoagulation factors as required by the condition or disease to be treated.

Treatment of diseases and conditions with modified FIX polypeptides can be effected by any suitable route of administration using suitable formulations as described herein including, but not limited to, injection, pulmonary, oral and transdermal administration. If necessary, a particular dosage and duration and treatment protocol can be empirically determined or extrapolated. For example, exemplary doses of recombinant and native FIX polypeptides can be used as a starting point to determine appropriate dosages. For example, a recombinant FIX polypeptide, BeneFIX®, has been administered to patients with hemophilia B, at a dosage of 50 I.U./kg over a 10 minute infusion, resulting in a mean circulating activity of 0.8±0.2 I.U./dL per I.U./kg infused with a mean half-life of 19.4±5.4 hours. Modified FIX polypeptides that are more stable and have an increased half-life in vivo, can be effective at reduced dosage amounts and or frequencies. For example, because of the improvement in properties such as serum stability, dosages can be lower than comparable amounts of unmodified FIX. Dosages for unmodified FIX polypeptides can be used as guidance for determining dosages for modified FIX polypeptides. Factors such as the level of activity and half-life of the modified FIX in comparison to the unmodified FIX can be used in making such determinations. Particular dosages and regimens can be empirically determined.

Dosage levels and regimens can be determined based upon known dosages and regimens, and, if necessary can be extrapolated based upon the changes in properties of the modified polypeptides and/or can be determined empirically based on a variety of factors. Such factors include body weight of the individual, general health, age, the activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician. The active ingredient, the polypeptide, typically is combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form or multi-dosage form can vary depending upon the host treated and the particular mode of administration.

The effect of the FIX polypeptides on the clotting time of blood can be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.

Upon improvement of a patient's condition, a maintenance dose of a compound or compositions can be administered, if necessary; and the dosage, the dosage form, or frequency of administration, or a combination thereof can be modified. In some cases, a subject can require intermittent treatment on a long-term basis upon any recurrence of disease symptoms or based upon scheduled dosages. In other cases, additional administrations can be required in response to acute events such as hemorrhage, trauma, or surgical procedures.

The following are some exemplary conditions for which FIX has been used as a treatment agent alone or in combination with other agents.

1. Hemophilia

Hemophilia is a bleeding disorder that is caused by a deficiency in one or more blood coagulation factors. It is characterized by a decreased ability to form blood clots at sites of tissue damage. Congenital X-linked hemophilias include hemophilia A and hemophilia B, or Christmas disease, which are caused by deficiencies in FVIII and FIX, respectively. Hemophilia A occurs at a rate of 1 out of 10,0000 males, while hemophilia B occurs in 1 out of 50,000 males.

Patients with hemophilia suffer from recurring joint and muscle bleeds, which can be spontaneous or in response to trauma. The bleeding can cause severe acute pain, restrict movement, and lead to secondary complications including synovial hypertrophy. Furthermore, the recurring bleeding in the joints can cause chronic synovitis, which can cause joint damage, destroying synovium, cartilage, and bone.

Hemophilia B can be effectively managed with administration of FIX therapeutics. Patients can often achieve normal life span with proper treatment to control bleeding episodes. Administration of FIX can aid in controlling bleeding during surgery, trauma, during dental extraction, or to alleviate bleeding associated with hemarthroses, hematuria, mucocutaneous bleeding, such as epistaxis or gastrointestinal tract bleeding, cystic lesions in subperiosteal bone or soft tissue, or hematomas, which cause neurological complications such as intracranial bleeding, spinal canal bleeding. Death in patients with hemophilia is often the result of bleeding in the central nervous system. Other serious complications in hemophilic patients include development of inhibitors to coagulation factor therapeutics and disease, such as progressive hepatitis with hepatic failure or AIDS caused by use of contaminated blood products.

The most frequent alterations in the FIX gene are point mutations, in particular missense mutations. Most of the identified FIX mutations occur in amino acid residues in the coding region of the FIX gene, often affecting evolutionarily conserved amino acids. The severity of the hemophilia depends upon the nature of the mutation. Mutations in the coding region can affect a number of different properties or activities of the FIX polypeptide including alteration of protease activity, cofactor binding, signal peptide or propeptide cleavage, post-translational modifications, and inhibition of cleavage of FIX into its activated form. Other types of point mutations include nonsense mutations that produce an unstable truncated FIX polypeptide, and frameshift mutations (small deletions and insertions) that result in a terminally aberrant FIX molecule. In addition, FIX point mutations can be found in the promoter region, which can disrupt the recognition sequences for several specific gene regulatory proteins, resulting in reduced transcription of coagulation factor IX. Decreased FIX as a result of transcriptional abnormalities is called Hemophilia B Leyden. An exemplary mutation in the promoter region includes disruption of the HNF-4 binding site, which affect regulation of FIX transcription by the androgen receptor. The severity of this type of hemophilia is governed by the levels of androgen in the blood, which increase during puberty and partially alleviate the FIX transcriptional deficiency (Kurachi et al. (1995)). Other missense nucleotide changes affect the processing of factor IX primary RNA transcript. For example, some mutations occur at evolutionarily conserved donor-splice (GT), and acceptor-splice (AG) consensus sequences, which can create cryptic splice junctions and disrupt assembly of spliceosomes. Some severe cases of hemophilia (approximately 10%) present with large deletions in the FIX gene (Gianelli et al. (1998). Treatment of FIX deficiency most often involves administration of FIX in the form of purified plasma concentrates, purified FIX preparations, or recombinant forms of FIX. Occasionally patients with large deletions produce anti-FIX antibodies, or inhibitors, in response to replacement therapy (Gianelli et al. (1983)). Among purified FIX therapeutics available for the treatment of hemophilia are: MonoNine® (Aventis Behring), Alpha-nine-SD® (Alpha therapeutic), and BeneFix® (Genetics Institute). MonoNine® and Alpha-nine-SD® are FIX preparations derived from human plasma, while BeneFix® is a recombinant FIX generated from the native FIX polypeptide sequence. FIX complex therapeutics include Bebulin® VII, a purified concentrate of FIX with FX and low amounts of FVII; Konyne® 80 (Bayer), a purified concentrate of FIX, with FII, FX, and low levels of FVII; PROPLEX® T (Baxter International), a heat treated product prepared from pooled normal human plasma containing FIX with FII, FVII, and FX; and Profilnine SD® (Alpha Therapeutic Corporation), a solvent detergent treated, concentrate of FIX with FII, FX and low levels of FVII. All such products can be modified as described herein and/or replaced with modified FIX polypeptides provided herein.

The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in therapies for hemophilia, including treatment of bleeding conditions associated with hemophilia. The modified FIX polypeptides provided herein can be used, for example, to control or prevent spontaneous bleeding episodes or to control or prevent bleeding in response to trauma or surgical procedures. In one embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of hemophilia B. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for hemophilia. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example FIX-deficient mice, or any other known disease model for hemophilia, can be treated with modified FIX polypeptides. Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX.

The modified FIX polypeptide can be used to deliver longer lasting, more stable hemophilia B therapies. Thus, the modified FIX polypeptides provided herein can be administered at lower dosages and/or less frequently then unmodified or native FIX polypeptides, such as MonoNine® or Alpha-nine-SD®, or recombinant forms of FIX, such as BeneFix®, while retaining one or more therapeutic activities and/or having one or more fewer/decreased side effects.

2. Thrombotic Diseases and Conditions

Thrombotic diseases are characterized by hypercoagulation, or the deregulation of hemostasis in favor of development of blot clots. Exemplary thrombotic diseases and conditions include arterial thrombosis, venous thrombosis, venous thromboembolism, pulmonary embolism, deep vein thrombosis, stroke, ischemic stroke, myocardial infarction, unstable angina, atrial fibrillation, renal damage, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, artificial organs, shunts or prostheses, and other acquired thrombotic diseases, as discussed below. Typical therapies for thrombotic diseases involve anticoagulant therapies, including inhibition of the coagulation cascade.

The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in anticoagulant therapies for thrombotic diseases and conditions, including treatment of conditions involving intravascular coagulation. The modified FIX polypeptides provided herein that can inhibit blood coagulation can be used, for example, to control or prevent formation of thromboses. In a particular embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of an arterial thrombotic disorder. In another embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of a venous thrombotic disorder, such as deep vein thrombosis. In a particular embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used for treatment of an ischemic disorder, such as stroke. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for thrombotic diseases and conditions. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example mouse models of ischemic stroke, or any other known disease model for a thrombotic disease or condition, can be treated with modified FIX polypeptides (Dodds (1987) Ann NY Acad Sci 516: 631-635). Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX.

a. Arterial Thrombosis

Arterial thrombi form as a result of a rupture in the arterial vessel wall. Most often the rupture occurs in patients with vascular disease, such as atherosclerosis. The arterial thrombi usually form in regions of disturbed blood flow and at sites of rupture due to an atherosclerotic plaque, which exposes the thrombogenic subendothelium to platelets and coagulation proteins, which in turn activate the coagulation cascade. Plaque rupture may also produce further narrowing of the blood vessel due to hemorrhage into the plaque. Nonocclusive thrombi can become incorporated into the vessel wall and can accelerate the growth of atherosclerotic plaques. Formation arterial thrombi can result in ischemia either by obstructing flow or by embolism into the distal microcirculation. Anticoagulants and drugs that suppress platelet function and the coagulation cascade can be effective in the prevention and treatment of arterial thrombosis. Such classes of drugs are effective in the treatment of arterial thrombosis. Arterial thrombosis can lead to conditions of unstable angina and acute myocardial infarction. Modified FIX polypeptides provided herein that inhibit coagulation can be used in the treatment and/or prevention of arterial thrombosis and conditions, such as unstable angina and acute myocardial infarction.

b. Venous Thrombosis and Thromboembolism

Venous thrombosis is a condition in the which a blood clot forms in a vein due to imbalances in the signals for clot formation versus clot dissolution, especially in instances of low blood flow through the venous system. Results of thrombus formation can include damage to the vein and valves of the vein, though the vessel wall typically remains intact. The clots can often embolize, or break off, and travel through the blood stream where they can lodge into organ areas such as the lungs (pulmonary embolism), brain (ischemic stroke, transient ischemic attack), heart (heart attack/myocardial infarction, unstable angina), skin (purpura fulminans), and adrenal gland. In some instances the blockage of blood flow can lead to death. Patients with a tendency to have recurrent venous thromboembolism are characterized as having thrombophilia. Risk factors for developing thromboembolic disease include trauma, immobilization, malignant disease, heart failure, obesity, high levels of estrogens, leg paralysis, myocardial infarction, varicose veins, cancers, dehydration, smoking, oral contraceptives, and pregnancy. Genetic studies of families with thrombophilia have shown inheritable high levels coagulation factors, including FVIII, FIX, and FXI (Lavigne et al. (2003) J. Thromb. Haemost. 1:2134-2130).

Deep vein thrombosis (DVT) refers the formation of venous blot clot in the deep leg veins. The three main factors that contribute to DVT are injury to the vein lining, increased tendency for the blood to clot and slowing of blood flow. Collectively, these factor are called Virchow's triad. Veins can become injured during trauma or surgery, or as a result of disease condition, such as Buerger's disease or DIC, or another clot. Other contributing factors to development of DVT are similar to that of more general thromboembolic diseases as discussed above. The clot that forms in DVT causes only minor inflammation, thus, allowing it to break loose into the blood stream more easily. Often the thrombus can break off as a result of minor contraction of the leg muscles. Once the thrombus becomes an embolus it can become lodged into vessels of the lungs where is can cause a pulmonary infarction. Patients with high levels of active FIX in their bloodstream are at an increased risk of developing deep vein thrombosis (Weltermann et al. (2003) J. Thromb. Haemost. 1(1): 16-18).

Thromboembolic disease can be hereditary, wherein the disease is caused by hereditary abnormalities in clotting factors, thus leading to the imbalance in hemostasis. Several congenital deficiencies include antithrombin III, protein C, protein S, or plasminogen. Other factors include resistance to activated protein C (also termed APC resistance or Factor V leiden effect, in which a mutation in factor V makes it resistant to degradation by protein C), mutation in prothrombin, dysfibrinogemia (mutations confer resistance to fibrinolysis), and hyperhomocysteinemia. Development of thrombombolic disease in younger patients is most often due to the congenital defects described above and is called Juvenile Thrombophilia.

Treatments for venous thromboembolic disease and DVT typically involve anticoagulant therapy, in which oral doses of heparin and warfarin are administered. Heparin is usually infused into patients to control acute events, followed by longer term oral anticoagulant therapy with warfarin to control future episodes. Other therapies include direct thrombin inhibitors, inhibitors of platelet function, such as aspirin and dextran, and therapies to counteract venous stasis, including compression stockings and pneumatic compression devices. Modified FIX polypeptides provided herein that inhibit blood coagulation can be used in anticoagulant therapies for thromboembolic disease and/or DVT. In some embodiments, modified FIX polypeptides provided herein that inhibit blood coagulation can be used in prevention therapies for thromboembolic disease and/or DVT in patients exhibiting risk factors for thromboembolic disease and/or DVT. Modified FIX polypeptides provided herein that inhibit coagulation can be used in anticoagulation therapies for thromboembolic disease and/or DVT due to high levels of FIX in the blood stream.

i. Ischemic Stroke

Ischemic stroke occurs when the blood flow to the brain is interrupted, wherein the sudden loss of circulation to an area of the brain results in a corresponding loss of neurologic function. In contrast to a hemorrhagic stroke, which is characterized by intracerebral bleeding, an ischemic stroke is usually caused by thrombosis or embolism. Ischemic strokes account for approximately 80% of all strokes. In addition to the causes and risk factors for development a thromboembolism as discussed above, processes that cause dissection of the cerebral arteries (such as, trauma, thoracic aortic dissection, arteritis) can cause thrombotic stroke. Other causes include hypoperfusion distal to a stenotic or occluded artery or hypoperfusion of a vulnerable watershed region between 2 cerebral arterial territories. Treatments for ischemic stroke involve anticoagulant therapy for the prevention and treatment of the condition. Modified FIX polypeptides provided herein that inhibit coagulation can be used in the treatment and/or prevention or reduction of risk of ischemic stroke.

3. Acquired Coagulation Disorders

Acquired coagulation disorders are the result of conditions or diseases, such as vitamin K deficiency, liver disease, disseminated intravascular coagulation (DIC), or development of circulation anticoagulants. The defects in blood coagulation are the result of secondary deficiencies in clotting factors caused by the condition or disease. For example, production of coagulation factors from the liver is often impaired when the liver is in a diseased state. Along with decreased synthesis of coagulation factors, fibrinolysis becomes increased and thrombocytopenia (deficiency in platelets) is increased. Decreased production of coagulation factors by the liver also can result from fulminant hepatitis or acute fatty liver of pregnancy. Such conditions promote intravascular clotting which consumes available coagulation factors. Modified FIX polypeptides provided herein can be used in the treatment of acquired coagulation disorders in order to alleviate deficiencies in blood clotting factors.

a. Disseminated Intravascular Coagulation (DIC)

Disseminated intravascular coagulation (DIC) is a disorder characterized by a widespread and ongoing activation of coagulation. In DIC, there is a loss of balance between thrombin activation of coagulation and plasmin degradation of blot clots. Vascular or microvascular fibrin deposition as a result can compromise the blood supply to various organs, which can contribute to organ failure. Prolonged activation of the coagulation system impairs the synthesis of new coagulation factors and increases the degradation of existing coagulation factors, thus leading to decreased levels of procoagulant proteins. As a result, patients with DIC are at an increased risk of serious bleeding, especially during surgery and trauma situations. In acute, massive DIC, thrombocytopenia and depletion of coagulation factors, which promote severe bleeding, are accompanied by secondary fibrinolysis, wherein fibrin degradation products are generated that interfere with normal platelet function and impairs proper fibrin formation. The fibrinolysis is associated with high levels of plasmin which also contributes to the hemorrhagic state by degradation of coagulation factors, such as FIX, FVII, and FV. Treatments for acute DIC, therefore, aid in correcting the coagulation factor deficiencies in order to restore normal coagulation. In sub-acute or chronic DIC, patients present with a more hypercoagulatory phenotype, with thromboses from excess thrombin formation, and the symptoms and signs of venous thrombosis can be present. In contrast to acute DIC, sub-acute or chronic DIC is treated by methods of alleviating the hyperthrombosis, including heparin, anti-thrombin III and activated protein C treatment.

The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in therapies for acute DIC. In one embodiment, the modified FIX polypeptides herein, and nucleic acids encoding modified FIX polypeptides can be used in combination with other coagulation factors to treat deficiencies in clotting factors caused by acute DIC. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for DIC. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example FIX-deficient mice, or any other known disease model for DIC, can be treated with modified FIX polypeptides. Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX.

b. Bacterial Infection and Periodontitis

Systemic infection with microorganisms, such as bacteria, are commonly associated with DIC. The upregulation of coagulation pathways can be mediated in part by cell membrane components of the microorganism (lipopolysaccharide or endotoxin) or bacterial exotoxins (such as staphylococcal alpha toxin) that cause inflammatory responses leading to elevated levels of cytokines. The cytokines, in turn, can influence induction of coagulation.

Bacterial pathogens, such as Porphyrus gingivalis, are well-known as causative agents for adult periodontitis. The Porphyrus gingivalis bacterium produces arginine-specific cysteine proteinases that function as virulence factors (Grenier et al. (1987) J. Clin. Microbiol. 25:738-740, Smalley et al. (1989) Oral Microbiol. Immunol. 4:178-181, Marsh, et al. (1989) FEMS Microbiol. 59:181-185, and Potempa et al. (1998) J. Biol. Chem. 273:21648-21657. Porphyrus gingivalis generated two proteinases that are referred to as 50 kDa and 95 kDa gingipains R (RgpB and HRgpA, respectively). The protease can proteolytically cleave and hence activate coagulation factors, such as FIX. During bacterial infection release of the gingipains R into the blood stream can thus lead to uncontrolled activation of the coagulation cascade leading to overproduction of thrombin and increase the possibility of inducing disseminated intravascular coagulation (DIC). The large increases in thrombin concentrations can furthermore contribute alveolar bone resorption by osteoclasts at sites of periodontitis.

The modified FIX polypeptides provided herein that inhibit blood coagulation, and nucleic acids encoding modified FIX polypeptides can be used in treatment of periodontitis. The modified FIX polypeptides herein provide increased protein stability and improved half-life. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies. Examples of therapeutic improvements using modified FIX polypeptides include, for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness for airway responsiveness in periodontitis models. Such models are available in animals, such as nonhuman primates, dogs, mice, rats, hamsters, and guinea pigs (Weinberg and Bral (1999) J. of Periodontology 26(6), 335-340). Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX.

c. Liver Disease

The liver is the site of production of most coagulation factors of the coagulation cascade. It also is responsible for clearing degraded and inactivated components of coagulation. As such, diseases of the liver can have a profound effect on hemostasis. In some cases, liver disease can be associated with hypersplenism and suppression of platelet production in the bone marrow. Liver disease can be characterized as either an acute or chronic condition. Acute hepatic necrosis is caused by factors, such as infection, shock, or toxins, wherein the necrosis of the liver leads to the inability to produce sufficient quantities of coagulation factors. Chronic liver disease is caused by factors, such as, but not limited to, alcohol, viral infection and autoimmune disease. Hemostatic defects in chronic liver disease include decreased coagulation factors due to decreased production, decreased platelet count due to sequestration in the spleen, and increased fibrin degradation products (FDP) due to decreased clearance, which can lead to conditions, such as DIC, as described above.

The modified FIX polypeptides provided herein and the nucleic acids encoding the modified FIX polypeptides provided herein can be used in therapies for liver disease, including treatment of deficiencies in coagulation factors. The modified FIX polypeptides provided herein can be used, for example, to control or prevent spontaneous bleeding episodes or to control or prevent bleeding in response to trauma or surgical procedures. The modified FIX polypeptides herein provide increased protein stability and increased protein half-life. Of particular interest are FIX polypeptides that are resistant to proteases. Thus, modified FIX polypeptides can be used to deliver longer lasting, more stable therapies for liver disease. Examples of therapeutic improvements using modified FIX polypeptides include for example, but are not limited to, lower dosages, fewer and/or less frequent administrations, decreased side effects, and increased therapeutic effects. Modified FIX polypeptides can be tested for therapeutic effectiveness, for example, by using animal models. For example animal models of autoimmune liver disease and alcohol induced liver disease are available and can be treated with modified FIX polypeptides provided herein (such as Nanji et al. (2003) Alcohol Res Health. 27(4):325-30; Heneghan and McFarlane (2005) Hepatology 42(1): 149-55). Progression of disease symptoms and phenotypes is monitored to assess the effects of the modified FIX polypeptides. Modified FIX polypeptides also can be administered to animal models as well as subjects such as in clinical trials to assess in vivo effectiveness in comparison to placebo controls and/or controls using unmodified FIX.

d. Warfarin and Heparin-Induced Bleeding

Patients undergoing anticoagulant therapies for the treatment of conditions, such as thromboembolism, can exhibit bleeding episodes upon acute administration of anticoagulants or develop hemorrhagic disorders as a result long term usage of such therapies. Treatments for bleeding episodes typically include administration of procoagulants, such as vitamin K, plasma, concentrates of FIX polypeptides, and protamines to neutralize heparin. The modified FIX polypeptides provided herein can be used in treatments to control bleeding episodes in patients with acquired bleeding disorders due to anticoagulant treatments.

e. Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a type of stroke characterized by spontaneous bleeding into the brain. In contrast to ischemic stroke, which can be treated effectively with anticoagulant therapies, the progressive bleeding induced in ICH is more difficult to treat. Mortality from hemorrhagic stroke at 30 days is seven times higher than ischemic strokes. The most common cause of ICH is degeneration and rupture of small arteries or arterioles due to sustained hypertension. In the elderly, cerebral amyloid angiopathy also can induce ICH Therapies for ICH involves the use of coagulation factor therapies, in particular recombinant factor VII. The modified FIX polypeptides provided herein, either alone or in combination with other coagulation factors, can be used in treatments to control bleeding episodes in patients with ICH.

J. COMBINATION THERAPIES

Any of the modified FIX polypeptides, and nucleic acid molecules encoding modified FIX polypeptides described herein can be administered in combination with, prior to, intermittently with, or subsequent to, other therapeutic agents or procedures including, but not limited to, other biologics, small molecule compounds and surgery. For any disease or condition, including all those exemplified above, for FIX is indicated or has been used and for which other agents and treatments are available, FIX can be used in combination therewith. Hence, the modified FIX polypeptides provided herein similarly can be used. Depending on the disease or condition to be treated, exemplary combinations include, but are not limited to combination with other plasma purified or recombinant coagulation factors, procoagulants, anticoagulants, anti-coagulation antibodies, glycosaminoglycans, heparins, heparinoids, heparin derivatives, heparin-like drugs, coumarins, such as warfarin and coumarin derivatives. Additional procoagulants that can be used in combination therapies with modified FIX polypeptides include, but are not limited to, vitamin K, vitamin K derivatives, and protein C inhibitors. Additional anticoagulants that can be used in combination therapies with modified FIX polypeptides include, but are not limited to, β2 adrenoreceptor antagonists, neuropeptide V2 antagonists, prostacyclin analogs, thromboxane synthase inhibitors, calcium agonists, elastase inhibitors, non-steroidal anti-inflammatory molecules, thrombin inhibitors, lipoxygenase inhibitors, FVIIa inhibitors, FXa inhibitors, phosphodiesterase III inhibitors, fibrinogen, vitamin K antagonists, and glucoprotein IIb/IIIa antagonists.

K. ARTICLES OF MANUFACTURE AND KITS

Pharmaceutical compounds of modified FIX polypeptides for nucleic acids encoding modified FIX polypeptides, or a derivative or a biologically active portion thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating a FIX-mediated disease or disorder, and a label that indicates that modified FIX polypeptide or nucleic acid molecule is to be used for treating a FIX-mediated disease or disorder.

The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,033,252 and 5,052,558, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any FIX-mediated disease or disorder.

Modified FIX polypeptides and nucleic acid molecules also can be provided as kits. Kits can include a pharmaceutical composition described herein and an item for administration. For example a modified FIX can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The kit can, optionally, include instructions for application including dosages, dosing regimens and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of FIX or a FIX regulated system of a subject.

L. EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the embodiments provided herein.

Example 1
Cloning and Generation FIX Mutants

A. Cloning of cDNA Encoding FIX and Insertion into a Mammalian Expression Vector

The nucleotide sequence comprising the coding sequence of FIX was cloned into the plasmid pCR-Blunt II-TOPO (Invitrogen, SEQ ID NO: 915) by polymerase chain reaction (PCR), using standard techniques known in the art, from plasmid pCMV5.8 (ATCC Number: 79829), using the following primers:

FIXHIND 5′-GCATTAGGATAAGCTTGGATGCAGCGCGTGAACATG-3′ (SEQ ID NO: 913) and

FIXXBA 5′-GCATCGGCGCAGATCTCTGTTAATTTTCAATTCC-3′ (SEQ ID NO: 914). After confirming the sequence of the FIX fragment contained in the pTOPO-PCR bluntII plasmid by sequencing, the FIX fragment was subcloned by restriction digest using EcoRI into plasmid pNaut to produce plasmid pNaut-FIX-EcoRI (SEQ ID NO: 916). The encoded mature form of the FIX polypeptide has a sequence of amino acids as set forth in SEQ ID NO: 1035. This sequence differs from SEQ ID NO: 2 at five positions: K39T, L40E, 143K, S44Q and S46V. These changes do not affect the clotting activity of the polypeptide.

B. Generation of FIX Mutants

A collection of pre-designed, targeted mutants was generated such that each individual mutant was created and processed individually, and physically separated from each other and in addressable arrays. 2D-scanning technology, described herein and also described in published U.S. Application Nos. US-2004-0132977-A1 and US 2005-0202438 A1) was used to design and obtain FIX mutants with improved resistance to proteolysis. Is-HITs were identified based upon (1) the protein property to be evolved (such as, resistance to proteolysis or stability); (2) the amino acid sequence; and (3) the properties of individual amino acids.

1. LEADS Created for Higher Resistance to Proteolysis of FIX

Variants were designed using 2D-scanning to identify positions conferring resistance to proteolysis. Positions selected (is-HITs) on human FIX (SEQ ID NO: 2) were (numbering corresponds to amino acid positions in the mature FIX polypeptide set forth in SEQ ID NO: 2) 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, E36, R37, T38, T39, E40, F41, W42, K43, Y45, V46, D47, G48, D49, E52, S53, P55, L57, N58, G59, G60, S61, K63, D64, D65, 166, S68, Y69, E70, W72, P74, F75, G76, F77, E78, G79, K80, E83, L84, D85, V86, T87, I90, K91, N92, G93, R94, E96, F98, K100, S102, A103, D104, K106, V107, V108, S110, T112, E113, G114, Y115, R116, L117, A118, E119, K122, S123, E125, P126, A127, V128, P129, P131, G133, R134, V135, S136, V137, S138, T140, S141, K142, L143, T144, R145, A146, E147, T148, V149, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, S171, T172, S174, F175, D177, 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, E224, T225, G226, V227, K228, I229, T230, V231, V232, A233, G234, E235, I238, E239, E240, T241, E242, T244, E245, K247, 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, 1290, A291, D292, K293, E294, 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, I327, 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, T371, E372, 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, L414, and T415. The native amino acid at each of the is-HIT positions listed above was replaced by residues as listed in Table 9.

TABLE 9

Amino acid at
Replacing

is-HIT
ammo acids

R
H, Q

E
H, N, Q

K
N, Q

D
N, Q

M
I, V

P
A, S

Y
I, H

F
I, V

W
H, S

L
I, V

N
Q, S

A
H, N, Q

G
H, N, Q

I
H, N, Q

S
H, N, Q

T
H, N, Q

V
H, N, Q

The FIX variants generated for testing increased resistance to proteolysis are listed in Table 10 (SEQ ID NOS: 3-891, 1025-1034, 1036-1926, and 2035-2044).

TABLE 10

List of human FIX variants for testing increased resistance to proteolysis

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
M19I
M19V
E20Q
E20H

E20N
E21Q
E21H
E21N
K22N
K22Q
S24Q
S24H
S24N
F25I

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
E36Q
E36H
E36N
R37H

R37Q
T38Q
T38H
T38N
T39Q
T39H
T39N
E40Q
E40H
E40N

F41I
F41V
W42S
W42H
K43N
K43Q
Y45H
Y45I
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
S61Q
S61H
S61N
K63N

K63Q
D64N
D64Q
D65N
D65Q
I66Q
I66H
I66N
S68Q
S68H

S68N
Y69H
Y69I
E70Q
E70H
E70N
W72S
W72H
P74A
P74S

F75I
F75V
G76Q
G76H
G76N
F77I
F77V
E78Q
E78H
E78N

G79Q
G79H
G79N
K80N
K80Q
E83Q
E83H
E83N
L84I
L84V

D85N
D85Q
V86Q
V86H
V86N
T87Q
T87H
T87N
I90Q
I90H

I90N
K91N
K91Q
N92Q
N92S
G93Q
G93H
G93N
R94H
R94Q

E96Q
E96H
E96N
F98I
F98V
K100N
K100Q
S102Q
S102H
S102N

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
E119Q
E119H
E119N
K122N
K122Q
S123Q

S123H
S123N
E125Q
E125H
E125N
P126A
P126S
A127Q
A127H
A127N

V128Q
V128H
V128N
P129A
P129S
P131A
P131S
G133Q
G133H
G133N

R134H
R134Q
V135Q
V135H
V135N
S136Q
S136H
S136N
V137Q
V137H

V137N
S138Q
S138H
S138N
T140Q
T140H
T140N
S141Q
S141H
S141N

K142N
K142Q
L143I
L143V
T144Q
T144H
T144N
R145H
R145Q
A146Q

A146H
A146N
E147Q
E147H
E147N
T148Q
T148H
T148N
V149Q
V149H

V149N
P151A
P151S
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

D166N
D166Q
I168Q
I168H
I168N
T169Q
T169H
T169N
S171Q
S171H

S171N
T172Q
T172H
T172N
S174Q
S174H
S174N
F175I
F175V
F175H

D177N
D177Q
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
F192IH

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
E224Q
E224H
E224N
T225Q
T225H
T225N
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
T241Q
T241H
T241N
E242Q

E242H
E242N
T244Q
T244H
T244N
E245Q
E245H
E245N
K247N
K247Q

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

E294Q
E294H
E294N
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
T371Q
T371H
T371N
E372Q

E372H
E372N
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
E387H
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
L414I
L414V
T415Q
T415H
T415N

Example 2
Production of Native and Modified Human FIX Polypeptides (Proteins) in Mammalian Cells and Yield Determination

Human fetal kidney 293 HEK EBNA fibroblast cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2 mM glutamine and 10% SVF. Cells were plated at 5×10⁵cells per well in 6-well plate for 2 days, washed two times with PBS, and then switched to serum free medium. Cells were transfected with 2 μg of DNA (FIX variants in pNaut-FIX plasmid) using transfection reagent according to the instructions of the manufacturer (Perfectin, GTS). Cell medium was replaced after 4 hours with serum free media containing 50 μg/ml of vitamin K and 1×-ITS (insulin, transferrin, selenium) mixture. Cell medium was collected 72 hours after transfection and stored at −80° C.

ASSERACHROM® IX: Ag ELISA (Diagnostica Stago, France) was used to determine concentration of FIX in supernatants from cell production according to the instructions of the manufacturer. Samples were diluted 20-fold using kit dilution reagent. Absorbance at 492 nm was measured using a spectrophotometer (Spectramax, Molecular Devices). Results were obtained by interpolation of the absorbance values from the calibration curve obtained from patients' plasma dilutions and plotted in log-log graph. FIX level at 100% corresponded approximately to 4 mg/l. Yields of FIX native and mutant proteins produced in 293 HEK cells are shown in Table 11. The data are the results from two productions.

TABLE 11

Yield of FIX native and mutant proteins

Nemo

Code #
Mutation
FIX (mg/L)

1
Native FIX
9.2

2
T163N
14.1

3
T163Q
11.2

4
T163H
10.1

5
I164N
8.9

6
I164Q
13.3

7
I164H
14.9

8
T163N/I164N
10.6

9
I168N
11.9

10
I168Q
11.9

11
I168H
12.6

12
T169N
7.5

13
T169Q
9.1

14
T169H
9.7

15
S171N
11.3

16
S171Q
11.7

17
S171H
10.2

18
T172N
17.9

19
T172Q
12.6

20
T172H
16.3

21
S174N
14.3

22
S174Q
11.5

23
S174H
12.1

24
I168N/T169N/S171N/T172N/S174N
12.9

25
T179N
10.0

26
T179Q
13.3

27
T179H
8.3

28
V181N
9.8

29
V181Q
20.3

30
V181H
13.2

31
V182N
7.5

32
V182Q
7.6

33
V182H
12.1

34
G183N
14.1

35
G183Q
15.4

36
G183H
11.6

37
G184N
7.6

38
G184Q
10.7

39
G184H
8.9

40
T179N/V181N/V182N/G183N/G184N
4.9

41
A187N
1.3

42
A187Q
3.0

43
A187H
1.6

44
G190N
1.1

45
G190Q
0.4

46
G190H
5.4

47
A187N/G190N
1.1

48
V196N
0.5

49
V196Q
0.1

50
V196H
0.1

51
V197N
0.6

52
V197Q
1.4

53
V197H
3.1

54
V196N/V197N
0.1

55
R180H
9.9

56
R180Q
8.1

57
K188N
10.5

58
K188Q
13.4

59
K201N
8.3

60
K201Q
6.7

61
L165I
7.0

62
L165V
11.3

63
F175I
7.0

64
F175V
9.3

65
F178I
10.3

66
F178V
8.7

67
F192I
2.9

68
F192V
1.7

69
W194H
0.1

70
W194I
0.1

71
L198I
6.5

72
L198V
9.8

73
D166Q
7.6

74
D166N
6.8

75
D177Q
8.4

76
D177N
7.7

77
D186Q
9.7

78
D186N
14.6

79
D203Q
4.5

80
D203N
1.0

81
T140N
12.9

82
T140Q
11.4

83
T140H
7.8

84
T144N
9.3

85
T144Q
1.8

86
T144H
9.3

87
T140N/T144N
6.3

88
L165Q
13.9

89
L165H
8.3

90
F175L/F178I
8.1

91
F175H
10.7

92
F178H
4.5

93
F192I/W194I
<0.1

94
F192H
0.8

95
L198Q
0.1

96
L198H
1.5

97
D152N/D154N
7.2

98
D152N
11.8

99
D152Q
7.0

100
D154N
4.4

101
D154Q
6.7

Example 3
Determination of Specific Activity of Human FIX by Coagulation Assay

The collection of human FIX mutants was tested for coagulation activity. Coagulation activity (expressed as clotting time T₅₀) was determined as the concentration needed for formation of a fibrin clot. Clotting activity of native and mutant FIX molecules was measured by the one stage activated partial thromboplastin time (aPTT) clotting assay (Cephascreen, Diagnostica Stago, France) using FIX immunodepleted human plasma (Deficient IX, Diagnostica Stago, France).

Briefly, 20 μl of production supernatant containing 60 ng of native FIX or mutant FIX was diluted with 60 μl of immunodepleted plasma. 80 μl of Cephascreen reagent containing cephalin (platelet substitute) prepared from rabbit cerebral tissues and a polyphenolic activator in a buffered medium was added to diluted FIX samples and incubated at 37° C. for 15 minutes. 80 μl of pre-warmed CaCl₂(0.025M, Diagnostica Stago, France) was added to mixture of FIX/Cephascreen reagent samples, and the time required for clot formation was immediately monitored using spectrophotometer (Spectramax, Molecular devices) at 350 nm. Serial dilution of normal human pooled plasma representing 100 to 120% of FIX activity (System control N, Diagnostica Stago, France) was used as the standard curve in order to validate linearity of clotting time detection. Clotting activity of FIX native and mutant proteins by clotting assay is shown in Tables 12a and 12b. The T₅₀values are displayed in seconds and represent the time required to achieved 50% clotting. The data are the result from two assays done in duplicate. Tables 12a and 12b below depict two separate experiments measuring the coagulation activity of exemplary non-limiting modified FIX polypeptides and native FIX. Table 12c depicts the protease specificity groups as indicated for each mutant.

TABLE 12a

Clotting activity of FIX native and mutant proteins

Bioactivity

Nemo

Clotting Assay

Code #
Mutation
T50 (sec)

1
Native FIX
116

2
T163N
111

3
T163Q
117

4
T163H
108

5
I164N
105

6
I164Q
113

7
I164H
118

8
T163N/I164N
108

9
I168N
112

10
I168Q
119

11
I168H
112

12
T169N
104

13
T169Q
104

14
T169H
111

15
S171N
116

16
S171Q
112

17
S171H
110

18
T172N
133

19
T172Q
111

20
T172H
111

21
S174N
113

22
S174Q
115

23
S174H
113

24
I168N/T169N/S171N/T172N/S174N
112

25
T179N
115

26
T179Q
112

27
T179H
110

28
V181N
178

29
V181Q
141

30
V181H
179

31
V182N
174

32
V182Q
156

33
V182H
176

34
G183N
146

35
G183Q
155

36
G183H
170

37
G184N
177

38
G184Q
178

39
G184H
179

40
T179N/V181N/V182N/G183N/G184N
nd

41
A187N
180

42
A187Q
169

43
A187H
201

44
G190N
148

45
G190Q
162

46
G190H
120

47
A187N/G190N
194

48
V196N
183

49
V196Q
176

50
V196H
183

51
V197N
161

52
V197Q
157

53
V197H
123

54
V196N/V197N
190

55
R180H
191

56
R180Q
186

57
K188N
124

58
K188Q
130

59
K201N
119

60
K201Q
118

61
L165I
109

62
L165V
120

63
F175I
116

64
F175V
118

65
F178I
140

66
F178V
122

67
F192I
134

68
F192V
145

69
W194H
183

70
W194I
168

71
L198I
124

72
L198V
168

73
D166Q
119

74
D166N
118

75
D177Q
118

76
D177N
182

77
D186Q
127

78
D186N
117

79
D203Q
109

80
D203N
113

81
T140N
118

82
T140Q
143

83
T140H
119

84
T144N
151

85
T144Q
nd

86
T144H
117

87
T140N/T144N
133

88
L165Q
117

89
L165H
118

90
F175I/F178I
155

91
F175H
153

92
F178H
127

93
F192I/W194I
175

94
F192H
154

95
L198Q
183

96
L198H
178

97
D152N/D154N
125

98
D152N
148

99
D152Q
126

100
D154N
119

101
D154Q
150

nd = not determined

TABLE 12b

Clotting activity of FIX native and mutant proteins

Bioactivity

Nemo

Clotting Assay

Code #
Mutation
T50 (sec)

1
Native FIX
114

2
T163N
113

3
T163Q
118

4
T163H
110

5
I164N
107

6
I164Q
114

7
I164H
115

8
T163N/I164N
110

9
I168N
111

10
I168Q
119

11
I168H
111

12
T169N
110

13
T169Q
108

14
T169H
111

15
S171N
116

16
S171Q
111

17
S171H
110

18
T172N
143

19
T172Q
112

20
T172H
113

21
S174N
113

22
S174Q
115

23
S174H
112

24
I168N/T169N/S171N/T172N/S174N
113

25
T179N
116

26
T179Q
114

27
T179H
115

28
V181N
200

29
V181Q
120

30
V181H
196

31
V182N
180

32
V182Q
165

33
V182H
180

34
G183N
150

35
G183Q
162

36
G183H
182

37
G184N
195

38
G184Q
187

39
G184H
192

40
T179N/V181N/V182N/G183N/G184N
169

41
A187N
200

42
A187Q
163

43
A187H
202

44
G190N
148

45
G190Q
158

46
G190H
123

47
A187N/G190N
201

48
V196N
191

49
V196Q
183

50
V196H
191

51
V197N
155

52
V197Q
148

53
V197H
130

54
V196N/V197N
203

55
R180H
202

56
R180Q
200

57
K188N
124

58
K188Q
130

59
K201N
121

60
K201Q
118

61
L165I
109

62
L165V
121

63
F175I
119

64
F175V
120

65
F178I
166

66
F178V
122

67
F192I
113

68
F192V
134

69
W194H
192

70
W194I
188

71
L1981
128

72
L198V
175

73
D166Q
118

74
D166N
116

75
D177Q
118

76
D177N
195

77
D186Q
125

78
D186N
116

79
D203Q
112

80
D203N
110

81
T140N
116

82
T140Q
124

83
T140H
117

84
T144N
132

85
T144Q
123

86
T144H
114

87
T140N/T144N
128

88
L165Q
117

89
L165H
119

90
F175I/F178I
147

91
F175H
131

92
F178H
122

93
F192I/W194I
193

94
F192H
154

95
L198Q
198

96
L198H
199

97
D152N/D154N
121

98
D152N
131

99
D152Q
121

100
D154N
112

101
D154Q
134

102
D152N
123

103
D152Q
121

104
D154N
120

105
D154Q
118

TABLE 12c

Protease specificity groups for FIX mutant proteins

Nemo

Specificity

Code #
Mutation
Group

1
Native FIX
—

2
T163N
E

3
T163Q
E

4
T163H
E

5
I164N
E

6
I164Q
E

7
I164H
E

8
T163N/I164N
E

9
I168N
E

10
I168Q
E

11
I168H
E

12
T169N
E

13
T169Q
E

14
T169H
E

15
S171N
E

16
S171Q
E

17
S171H
E

18
T172N
E

19
T172Q
E

20
T172H
E

21
S174N
E

22
S174Q
E

23
S174H
E

24
I168N/T169N/S171N/T172N/S174N
E

25
T179N
E

26
T179Q
E

27
T179H
E

28
V181N
E

29
V181Q
E

30
V181H
E

31
V182N
E

32
V182Q
E

33
V182H
E

34
G183N
E

35
G183Q
E

36
G183H
E

37
G184N
E

38
G184Q
E

39
G184H
E

40
T179N/V181N/V182N/G183N/G184N
E

41
A187N
E

42
A187Q
E

43
A187H
E

44
G190N
E

45
G190Q
E

46
G190H
E

47
A187N/G190N
E

48
V196N
E

49
V196Q
E

50
V196H
E

E

51
V197N
E

52
V197Q
E

53
V197H
E

54
V196N/V197N
E

55
R180H
T

56
R180Q
T

57
K188N
T

58
K188Q
T

59
K201N
T

60
K201Q
T

61
L165I
CT

62
L165V
CT

63
F175I
CT

64
F175V
CT

65
F178I
CT

66
F178V
CT

67
F192I
CT

68
F192V
CT

69
W194H
CT

70
W194I
CT

71
L198I
CT

72
L198V
CT

73
D166Q
EAsp

74
D166N
EAsp

75
D177Q
EAsp

76
D177N
EAsp

77
D186Q
EAsp

78
D186N
EAsp

79
D203Q
EAsp

80
D203N
EAsp

81
T140N
CT

82
T140Q
CT

83
T140H
CT

84
T144N
CT

85
T144Q
CT

86
T144H
CT

87
T140N/T144N
CT

88
L165Q
CT

89
L165H
CT

90
F175I/F178I
CT

91
F175H
CT

92
F178H
CT

93
F192I/W194I
CT

94
F192H
CT

95
L198Q
CT

96
L198H
CT

97
D152N/D154N
EAsp

98
D152N
EAsp

99
D152Q
EAsp

100
D154N
EAsp

101
D154Q
EAsp

102
D152N
EAsp

103
D152Q
EAsp

104
D154N
EAsp

105
D154Q
EAsp

E = elastase; T = trypsin; CT = chymotrypsin; EAsp = Endoproteinase AspN

Example 4
Resistance to Proteolysis

The collection of human FIX mutants was tested for their resistance to proteolysis as measured by retention of coagulation activity following treatment with proteases. Equal amounts of FIX native or mutant proteins (60 ng per assays) were treated with selected proteases (1.5% w/w trypsin, chymotrypsin, endoproteinase AspN, and endoproteinase GluC; and 6% w/w elastase). Aliquots of the samples were taken at different time points between 0.083 and 8 hrs and the proteolysis reaction was stopped by adding antiprotease cocktail (Roche Cat No. 11836170001). Samples were conserved at −20° C. until determination of clotting time could be performed. Clotting activity of the treated samples was assayed as described in Example 3 to determine residual clotting activity at each time point. Resistance to proteases for exemplary non-limiting modified FIX polypeptides is displayed in Table 13 as either no change in (N) or increased (+) resistance to proteases as compared to the residual coagulation activity of native FIX under the same protease treatment conditions.

The data are not meant to be representative of all proteases, but are exemplary data showing the resistance to proteolysis to an exemplary protease cocktail containing the proteases as described above. Thus, the data are not comprehensive and are not meant to be indicative that other FIX polypeptides do not exhibit protease resistance.

TABLE 13

Resistance to proteolysis of FIX native and mutant proteins

Nemo

Resistance

Code #
Mutation
to proteases

1
Native FIX
—

2
T163N
N

3
T163Q
N

4
T163H
+

5
I164N
N

6
I164Q
+

7
I164H
+

8
T163N/I164N
+

9
I168N
N

10
I168Q
+

11
I168H
+

12
T169N
N

13
T169Q
N

14
T169H
+

15
S171N
+

16
S171Q
+

17
S171H
N

18
T172N
N

19
T172Q
+

20
T172H
N

21
S174N
N

22
S174Q
+

23
S174H
+

24
I168N/T169N/S171N/T172N/S174N
N

25
T179N
+

26
T179Q
+

27
T179H
+

28
V181N
N

29
V181Q
+

30
V181H
N

31
V182N
+

32
V182Q
N

33
V182H
N

34
G183N
N

35
G183Q
N

36
G183H
N

37
G184N
+

38
G184Q
N

39
G184H
N

40
T179N/V181N/V182N/G183N/G184N
N

41
A187N
N

42
A187Q
+

43
A187H
N

44
G190N
N

45
G190Q
N

46
G190H
N

47
A187N/G190N
N

48
V196N
N

49
V196Q
N

50
V196H
N

51
V197N
N

52
V197Q
N

53
V197H
+

54
V196N/V197N
N

55
R180H
N

56
R180Q
N

57
K188N
N

58
K188Q
N

59
K201N
N

60
K201Q
N

61
L1651
N

62
L165V
N

63
F175I
N

64
F175V
N

65
F178I
N

66
F178V
N

67
F192I
+

68
F192V
N

69
W194H
N

70
W194I
N

71
L198I
+

72
L198V
+

73
D166Q
+

74
D166N
+

75
D177Q
+

76
D177N
+

77
D186Q
+

78
D186N
+

79
D203Q
+

80
D203N
+

81
T140N
N

82
T140Q
N

83
T140H
N

84
T144N
N

85
T144Q
N

86
T144H
N

87
T140N/T144N
+

88
L165Q
N

89
L165H
N

90
F175I/F178I
+

91
F175H
+

92
F178H
N

93
F192I/W194I
N

94
F192H
N

95
L198Q
N

96
L198H
N

97
D152N/D154N
+

98
D152N
N

99
D152Q
+

100
D154N
+

101
D154Q
N

N = no change; + = Increased resistance to proteolysis

In one exemplary assay, native FIX had a large increase in its T₅₀clotting time from about 140 seconds to about 200 seconds after only 0.5 hours of incubation with proteases, whereas exemplary modified FIX polypeptides with increased resistance to proteases, such as D203Q, had an increase in T₅₀clotting time from about 150 seconds to about 160 seconds after 0.5 hours of incubation with proteases. After 2 hours of incubation with proteases the T₅₀clotting time for D203Q only increased to about 173 seconds, and after 3 hours, 190 seconds. Similarly, exemplary modified FIX polypeptides D154N, and D152N/D154N exhibited T₅₀clotting times of 160 seconds and 170 seconds, respectively, after 0.5 hours of incubation with proteases, and 170 seconds and 178 seconds, after 2 hours of incubation with proteases. The results demonstrate that exemplary modified FIX polypeptides are more resistant to protease treatment than native FIX. Thus, the modified FIX polypeptides indicated to have an “increase” protease resistance as compared to native FIX have a greater half-life and are more stable than wild-type FIX polypeptides following incubation with one or more proteases.

Example 5
SuperLEAD Clotting Activity and Resistance to Proteolysis

Based on the data obtained above for protease resistance of FIX LEAD polypeptides, selected FIX SuperLEAD polypeptides were generated and tested for their clotting activity and resistance to proteolysis as measured by retention of coagulation activity following treatment with proteases. A schematic representation of the combinations used to generate triple SuperLEADs is shown in Table 14.

TABLE 14

Generation of SuperLEAD Combinations

2^ndmutation

NEMO code
(Endoproteinase AspN)

67
73
74
77
78
79
80
97
100
←

4
141
142
143
144
145
146
147
148

6
149
150
151
152
153
154
155
156

8
157
158
159
160
161
162
163
164

14
165
166
167
168
169
170
171
172

16
173
174
175
176
177
178
179
180

22
181
182
183
184
185
186
187
188

23
189
190
191
192
193
194
195
196

26
197
198
199
200
201
202
203
204

27
205
206
207
208
209
210
211
212

↑

3^rd

mutation

(Elastase)

Clotting activity of exemplary non-limiting FIX SuperLEAD polypeptides was assayed as described above in Example 3, and the data is shown in Table 14b. The T₅₀values are displayed in seconds and represent the time required to achieved 50% clotting.

TABLE 14b

Clotting activity of FIX SuperLEAD mutant proteins

Bioactivity

Nemo

Clotting Assay

Code #
Mutation
T₅₀(sec)

107
T163H/F192I
99

108
I164Q/F192I
101

109
T163N/I164N/F192I
106

110
T169H/F192I
94

111
S171Q/F192I
105

112
S174Q/F192I
99

113
S174H/F192I
101

114
T179Q/F192I
94

115
T179H/F192I
101

116
D166Q/F192I
93

117
D166N/F192I
104

118
D186Q/F192I
113

119
D186N/F192I
111

120
F192I/D203Q
90

121
F192I/D203N
181

122
T148A/D152N/D154N/F192I
90

123
T148A/D154N/F192I
105

141
T163H/D166Q/F192I
97

142
T163H/D166N/F192I
89

143
T163H/D186Q/F192I
123

144
T163H/D186N/F192I
103

145
T163H/F192I/D203Q
90

146
T163H/F192I/D203N
89

147
T148A/D152N/D154N/T163H/F192I
101

148
T148A/D154N/T163H/F192I
92

149
I164Q/D166Q/F192I
ND

150
I164Q/D166N/F192I
ND

151
I164Q/D186Q/F192I
109

152
I164Q/D186N/F192I
101

153
I164Q/F192I/D203Q
96

154
I164Q/F192I/D203N
87

155
T148A/D152N/D154N/I164Q/F192I
97

156
T148A/D154N/I164Q/F192I
95

157
T163N/I164N/D166Q/F192I
103

158
T163N/I164N/D166N/F192I
100

159
T163N/I164N/D186Q/F192I
128

160
T163N/I164N/D186N/F192I
102

161
T163N/I164N/F192I/D203Q
101

162
T163N/I164N/F192I/D203N
95

163
T148A/D152N/D154N/T163N/I164N/F192I
100

164
T148A/D154N/T163N/I164N/F192I
97

165
D166Q/T169H/F192I
99

166
D166N/T169H/F192I
96

167
T169H/D186Q/F192I
114

168
T169H/D186N/F192I
100

169
T169H/F192I/D203Q
86

170
T169H/F192I/D203N
97

171
T148A/D152N/D154N/T169H/F192I
95

172
T148A/D154N/T69H/F192I
87

173
D166Q/S171Q/F192I
104

174
D166N/S171Q/F192I
97

175
S171Q/D186Q/F192I
126

176
S171Q/D186N/F192I
116

177
S171Q/F192I/D203Q
90

178
S171Q/F192I/D203N
90

179
T148A/D152N/D154N/S171Q/F192I
ND

180
T148A/D154N/S171Q/F192I
104

181
D166Q/S174Q/F192I
93

182
D166N/S174Q/F192I
95

183
S174Q/D186Q/F192I
129

184
S174Q/D186N/F192I
109

185
S174Q/F192I/D203Q
95

186
S174Q/F192I/D203N
96

187
T148A/D152N/D154N/S171Q/F192I
97

188
T148A/D154N/S171Q/F192I
91

189
D166Q/S174H/F192I
93

190
D166N/S174H/F192I
102

191
S174H/D186Q/F192I
110

192
S174H/D186N/F192I
102

193
S174H/F192I/D203Q
98

194
S174H/F192I/D203N
99

195
T148A/D152N/D154N/S174H/F192I
98

196
T148A/D154N/S174H/F192I
102

197
D166Q/T179Q/F192I
100

198
D166N/T179Q/F192I
89

199
T179Q/D186Q/F192I
120

200
T179Q/D186N/F192I
107

201
T179Q/F192I/D203Q
92

202
T179Q/F192I/D203N
94

203
T148A/D152N/D154N/T179Q/F192I
106

204
T148A/D154N/T179Q/F192I
98

205
D166Q/T179H/F192I
103

206
D166N/T179H/F192I
97

207
T179H/D186Q/F192I
130

208
T179H/D186N/F192I
113

209
T179H/F192I/D203Q
94

210
T179H/F192I/D203N
96

211
T148A/D152N/D154N/T179H/F192I
103

212
T148A/D154N/T179H/F192I
102

Resistance to proteolysis of FIX SuperLEADs was assayed as described above in Example 4. For testing of FIX SuperLEADs, the concentration of selected proteases (trypsin, chymotrypsin, endoproteinase AspN, endoproteinase GluC, and elastase) was increased to 6% w/w. Resistance to proteases for exemplary non-limiting modified FIX SuperLEAD polypeptides is displayed in Table 15a. The data is expressed as relative resistance to proteases among the samples tested: (+), (++), or (+++), with (+++) indicating the highest resistance to proteases.

TABLE 15a

Resistance to proteolysis of FIX SuperLEAD mutant proteins

Nemo

Resistance

Code #
Mutation
to proteases

107
T163H/F192I
(+)

108
I164Q/F192I
(+)

109
T163N/I164N/F192I
(+)

110
T169H/F192I
(+)

111
S171Q/F192I
(+)

112
S174Q/F192I
(+)

113
S174H/F192I
(+)

114
T179Q/F192I
(+)

115
T179H/F192I
(+)

116
D166Q/F192I
(+)

117
D166N/F192I
(+)

118
D186Q/F192I
(++)

119
D186N/F192I
(+)

120
F192I/D203Q
(+)

121
F192I/D203N
(+)

122
T148A/D152N/D154N/
(++)

F192I

123
T148A/D154N/F192I
(+)

141
T163H/D166Q/F192I
(++)

142
T163H/D166N/F192I
(+)

143
T163H/D186Q/F192I
(+)

144
T163H/D186N/F192I
(+)

145
T163H/F192I/D203Q
(++)

146
T163H/F192I/D203N
(+)

147
T148A/D152N/D154N/
(+)

T163H/F192I

148
T148A/D154N/T163H/
(++)

F192I

149
I164Q/D166Q/F192I
(+)

150
I164Q/D166N/F192I
(+)

151
I164Q/D186Q/F192I
(+)

152
I164Q/D186N/F192I
(+)

153
I164Q/F192I/D203Q
(++)

154
I164Q/F192I/D203N
(++)

155
T148A/D152N/D154N/
(+)

I164Q/F192I

156
T148A/D154N/I164Q/
(+)

F192I

157
T163N/I164N/D166Q/
(+)

F192I

158
T163N/I164N/D166N/
(+)

F192I

159
T163N/I164N/D186Q/
(+)

F192I

160
T163N/I164N/D186N/
(+)

F192I

161
T163N/I164N/F192I/
(+)

D203Q

162
T163N/I164N/F192I/
(+)

D203N

163
T148A/D152N/D154N/
(+)

T163N/I164N/F192I

164
T148A/D154N/T163N/
(++)

I164N/F192I

165
D166Q/T169H/F192I
(+)

166
D166N/T169H/F192I
(+)

167
T169H/D186Q/F192I
(+)

168
T169H/D186N/F192I
(+)

169
T169H/F192I/D203Q
(+)

170
T169H/F192I/D203N
(++)

171
T148A/D152N/D154N/
(+)

T169H/F192I

172
T148A/D154N/T169H/F192I
(+)

173
D166Q/S171Q/F192I
(+)

174
D166N/S171Q/F192I
(+)

175
S171Q/D186Q/F192I
(+)

176
S171Q/D186N/F192I
(+)

177
S171Q/F192I/D203Q
(+)

178
S171Q/F192I/D203N
(+)

179
T148A/D152N/D154N/
(+)

S171Q/F192I

180
T148A/D154N/S171Q/F192I
(+)

181
D166Q/S174Q/F192I
(+)

182
D166N/S174Q/F192I
(+)

183
S174Q/D186Q/F192I
(+)

184
S174Q/D186N/F192I
(+)

185
S174Q/F192I/D203Q
(+++)

186
S174Q/F192I/D203N
(+)

187
T148A/D152N/D154N/
(+)

S174Q/F192I

188
T148A/D154N/S174Q/F192I
(+)

189
D166Q/S174H/F192I
(+)

190
D166N/S174H/F192I
(+)

191
S174H/D186Q/F192I
(+)

192
S174H/D186N/F192I
(+++)

193
S174H/F192I/D203Q
(+)

194
S174H/F192I/D203N
(+)

195
T148A/D152N/D154N/
(+)

S174H/F192I

196
T148A/D154N/S174H/F192I
(+)

197
D166Q/T179Q/F192I
(+)

198
D166N/T179Q/F192I
(+)

199
T179Q/D186Q/F192I
(+)

200
T179Q/D186N/F192I
(+)

201
T179Q/F192I/D203Q
(+++)

202
T179Q/F192I/D203N
(+)

203
T148A/D152N/D154N/
(+)

T179Q/F192I

204
T148A/D154N/T179Q/F192I
(+)

205
D166Q/T179H/F192I
(+)

206
D166N/T179H/F192I
(+)

207
T179H/D186Q/F192I
(+)

208
T179H/D186N/F192I
(+)

209
T179H/F192I/D203Q
(+)

210
T179H/F192I/D203N
(+)

211
T148A/D152N/D154N/
(+)

T179H/F192I

212
T148A/D154N/T179H/F192I
(+)

All variants tested exhibited increased protease resistance relative to the native FIX polypeptide. Several FIX SuperLEAD polypeptides exhibited particularly high resistance to proteases including D186Q/F 192I, Ti 48A/D 152N/D 154N/F 192I, T163H/D166Q/F192I, T163H/F192I/D203Q, T148A/D154N/T163H/F192I, I164Q/F192I/D203 Q, I164Q/F192I/D203N, T148A/D154N/T163N/I164N/F192I, T169H/F 192I/D203N, S174Q/F192I/D203Q, S174H/D186N/F192I, and T179Q/F192I/D203 Q.

In a separate experiment, protease resistance of selected SuperLEADs was assayed by the same methods as described above. In this experiment, the data for resistance to proteases was compared in terms of t_1/2clotting time fold increase at 50% of T₀compared to native Factor IX. Data for this analysis is shown in Table 15b.

TABLE 15b

Resistance to proteolysis of FIX SuperLEAD mutant proteins

protease resistance

Nemo

protease specificity
clotting time
(50% t_1/2clotting fold-

code
mutations
of leads
(seconds)
increase versus native)

161
T163N/I164N/F192I/D203Q
EndAsp + CT + E
101
4.2

194
S174H/F192I/D203N
EndAsp + CT + E
99
4.2

195
T148A/D152N/D154N/S174H/F192I
EndAsp + CT + E
98
4

145
T163H/F192I/D203Q
EndAsp + CT + E
90
3.3

107
F192I-T163H
EndAsp + CT
99
3.2

120
F192I-D203Q
EndAsp + CT
90
3.2

147
T148A/D152N/D154N/T163H/F192I
EndAsp + CT + E
101
3

170
T169H/F192I/D203N
EndAsp + CT + E
97
2.8

177
S171Q/F192I/D203Q
EndAsp + CT + E
90
2.8

203
T148A/D152N/D154N/T179Q/F192I
EndAsp + CT + E
106
2.8

153
I164Q/F192I/D203Q
EndAsp + CT + E
96
2.7

196
T148A/D154N/S174H/F192I
EndAsp + CT + E
102
2.7

157
T163N/I164N/D166Q/F192I
EndAsp + CT + E
103
2.5

180
T148A/D154N/S171Q/F192I
EndAsp + CT + E
104
2.5

193
S174H/F192I/D203Q
EndAsp + CT + E
98
2.5

204
T148A/D154N/T179Q/F192I
EndAsp + CT + E
98
2.5

146
T163H/F192I/D203N
EndAsp + CT + E
89
2.3

169
T169H/F192I/D203Q
EndAsp + CT + E
86
2.3

179
T148A/D152N/D154N/S171Q/F192I
EndAsp + CT + E
97
2.3

192
S174H/D186N/F192I
EndAsp + CT + E
102
2.3

172
T148A/D154N/T169H/F192I
EndAsp + CT + E
87
2.2

173
D166Q/S171Q/F192I
EndAsp + CT + E
104
2.2

202
T179Q/F192I/D203N
EndAsp + CT + E
94
2.2

185
S174Q/F192I/D203Q
EndAsp + CT + E
95
2

187
T148A/D152N/D154N/S174Q/F192I
EndAsp + CT + E
97
1.8

190
D166N/S174H/F192I
EndAsp + CT + E
102
1.8

119
F192I-D186N
EndAsp + CT
111
1.7

205
D166Q/T179H/F192I
EndAsp + CT + E
103
1.7

148
T148A/D154N/T163H/F192I
EndAsp + CT + E
92
1.5

175
S171Q/D186Q/F192I
EndAsp + CT + E
126
1.5

155
T148A/D152N/D154N/I164Q/F192I
EndAsp + CT + E
97
1.3

native FIX

114
1

All variants tested exhibited increased protease resistance relative to the native FIX polypeptide. Exemplary FIX SuperLEAD polypeptides that exhibited particularly high resistance to proteases in this analysis included T163N/I164N/F192I/D203Q, S174H/F192I/D203N, T148A/D152N/D154N/S174H/F192I, T163H/F192I/D203Q, T163H/F192I, F192I/D203Q, T148A/D152N/D154N/T163H/F192I, T169H/F192I/D203N, S171Q/F192I/D203Q, and T148A/D152N/D154N/T179Q/F192I.

The data above for the protease resistance are exemplary showing the resistance to proteolysis to an exemplary protease cocktail containing the proteases as described above. Thus, the data are not comprehensive and are not meant to be indicative that other FIX polypeptides do not exhibit protease resistance.

Example 6
SuperLEAD Clotting Activity and Resistance to Proteolysis Using an Alternative FIX Polypeptide Background

As mentioned in Example 1, the starting FIX polypeptide on which the FIX variants were generated is shown in SEQ ID NO: 1035. This sequence differs from another wild-type FIX polypeptide shown in SEQ ID NO: 2 at five positions: K39T, L40E, I43K, S44Q and S46V. These changes, however, do not affect the clotting activity of the FIX polypeptide. In order to compare the activity of the FIX variants between the two polypeptide backgrounds, site-directed mutagenesis was performed to convert the SuperLEAD variants to the alternative FIX background (BFIX-T148). The mutants that were generated are shown in Table 16.

TABLE 16

FIX SuperLEAD Variants Produced on BFIX-T148 Background

NFIX code
BFIX-T148 code
mutations

161
249
T163N/I164N/F192I/D203Q

194
250
S174H/F192I/D203N

195
251
T148A/D152N/D154N/S174H/F192I

145
252
T163H/F192I/D203Q

107
253
T163H/F192I

120
254
F192I/D203Q

147
255
T148A/D152N/D154N/T163H/F192I

170
256
T169H/F192I/D203N

177
257
S171Q/F192I/D203Q

203
258
T148A/D152N/D154N/T179Q/F192I

153
259
I164Q/F192I/D203Q

196
260
T148A/D154N/S174H/F192I

157
261
T163N/I164N/D166Q/F192I

180
262
T148A/D154N/S171Q/F192I

193
263
S174H/F192I/D203Q

204
264
T148A/D154N/T179Q/F192I

146
265
T163H/F192I/D203N

169
266
T169H/F192I/D203Q

179
267
T148A/D152N/D154N/S171Q/F192I

192
268
S174H/D186N/F192I

172
269
T148A/D154N/T169H/F192I

173
270
D166Q/S171Q/F192I

202
271
T179Q/F192I/D203N

185
272
S174Q/F192I/D203Q

187
273
T148A/D152N/D154N/S174Q/F192I

190
274
D166N/S174H/F192I

119
275
D186N/F192I

205
276
D166Q/T179H/F192I

148
277
T148A/D154N/T163H/F192I

175
278
S171Q/D186Q/F192I

155
279
T148A/D152N/D154N/I164Q/F192I

Human fetal kidney 293 HEK EBNA fibroblast cell line was grown in Dulbecco's modified eagle medium supplemented with 2 mM of glutamine and 10% of fetal bovine serum (FBS). Cells were plated at 5×10⁵cells per well in 6-well plates for 2 days, washed twice with PBS and then placed in serum-free medium. Cells were transfected with 2 μg of DNA using transfection reagent (PEI, NaCl). The cell medium was replaced after 4 hours by serum-free medium containing 50 μg/mL of K vitamin and 1×-ITS (Insulin, Transferrin, Selenium) mixture. The cell medium was collected 72 h after transfection and stored at −80° C.

Asserachrom® IX:Ag Elisa kit (Diagnostica Stago, France) was used to determine the concentration of the 31 selected mutant cell productions according to the instructions of the manufacturer. Samples were diluted 20-fold using the dilution reagent. Absorbance at 492 nm was measured using a spectrophotometer (Spectramax, Molecular Devices). Results were obtained by interpolation of the absorbance values from the calibration curve obtained from patients' plasma dilutions and plotted in a log-log graph. Factor IX level at 100% corresponds approximately to 4 mg/L. Data are presented in Table 17.

TABLE 17

Yields for BFIX T148 Super LEAD variants.

embedded image

Mean of production corresponds to 3 separated productions (except for mutants marked with * = only one production). Lower produced mutants are highlighted in grey.

In order to determine the bioactivity of each mutant, the clotting activity was measured by the one-stage activated partial thromboplastin time (aPTT) clotting assay (Cephascreen, Diagnostica Stago, France) using Factor IX immunodepleted human plasma (Deficient IX, Diagnostica Stago, France). Supernatants containing 60 ng of native or mutant Factor IX molecules from transient transfection production were diluted with 60 μL of immunodepleted plasma. 80 μL of Cephascreen reagent containing cephalin (platelet substitute) prepared from rabbit cerebral tissues and a polyphenolic activator in a buffered medium was added to dilute Factor IX samples and incubated at 37° C. for 15 minutes. Thus, 80 μL of pre-warmed CaCl₂(0.025M, Diagnostica Stago, France) was added to Factor IX samples/Cephascreen reagent mix. The time required for clot formation was immediately monitored using a spectrophotometer (Spectramax, Molecular devices) at 350 nm. A serial dilution of normal human pooled plasma representing 100 to 120% of Factor IX activity (System control N, Diagnostica Stago, France) was used as the standard curve in order to validate the linearity of the clotting time detection. Data are presented in Table 18.

TABLE 18

Clotting activity of BFIX T148 SuperLEAD variants

mutant number
Production 1
Production 2
Production 3
MEAN OF T50

in BFIX-T148
T50 (clotting)
conversion
T50 (clotting)
conversion
T50 (clotting)
conversion
(clotting)

Rank
matrix
(seconds)
(μg/l)
(seconds)
(μg/l)
(seconds)
(μg/l)
(seconds)

1
249
122
38.2
100
46.7
110
22.9
111

2
250
121
34.0
114
24.0
99
40.2
111

3
251
131
21.2
132
10.7
99
40.2
121

4
252
105
33.5
122
18.7
106
43.9
111

5
253
113
22.8
128
13.8
136
12.4
125

6
254
114
21.7
128
13.4
130
15.6
124

7
255
137
18.9
103
39.1
110
23.9
117

8
256
140
17.1
107
32.4
108
26.2
118

9
257
nd
nd
nd
nd
114
30.4
114*

10
258
121
32.2
116
24.1
98
42.5
112

11
259
nd
nd
109
29.6
130
15.7
119

12
260
128
23.4
120
17.9
101
36.7
117

13
261
144
14.9
115
21.8
110
23.1
123

14
262
nd
nd
nd
nd
133
13.9
133*

15
263
118
23.3
100
51.3
115
18.7
111

16
264
118
37.7
108
34.9
109
39.2
111

17
265
nd
nd
106
34.1
120
23.0
113

18
266
138
19.6
115
21.5
100
39.6
118

19
267
115
21.3
123
16.7
128
16.5
122

20
268
nd
nd
nd
nd
126
18.5
126*

21
269
115
24.9
110
28.8
105
30.5
110

22
270
120
19.8
118
19.8
116
17.5
118

23
271
133
11.4
106
34.2
115
18.1
118

24
272
125
15.5
116
21.8
120
14.9
120

25
273
nd
nd
125
14.6
128
16.8
126

26
274
118
21.7
102
45.3
109
24.4
109

27
275
nd
nd
135
10.5
147
8.1
141

28
276
nd
nd
123
17.1
135
12.9
129

29
277
127
14.8
116
21.6
102
34.5
115

30
278
136
17.8
129
12.6
135
12.6
133

31
279
128
28.4
102
39.9
113
20.7
114

BFIX
107
48
103
44

105

BFIX-T148
116
32
104
42
120
15.0
113

NFIX
128
19
117
21

123

Mean of production corresponds to 3 separated clotting time assays (except for mutants marked with * = only one production). Conversion in μg/L was based from standard curves obtained from normal human sera.

All selected mutants in BFIX-T148 background were active by clotting time assays and were similar to BFIX-T148 native sequence (t_1/2between 111 sec and 133 sec). Only mutant 275 exhibited a clotting time higher than 140 sec.

Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

	Number	Date	Country
Parent	11818985	Jun 2007	US
Child	13694290		US

Modified coagulation factor IX polypeptides and use thereof for treatment

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

RELATED APPLICATIONS

Provisional Applications (1)

Continuations (1)