Hyperglycosylated human coagulation factor ix

Abstract

The invention relates to hyperglycosylated human coagulation factor IX polypeptides, to processes for preparing said polypeptides, to pharmaceutical compositions comprising said polypeptides and to the use of the compounds for the treatment of diseases alleviated by human coagulation factor IX, in particular, but not exclusively hemophilia.

1 . A human factor IX polypeptide analogue having one or more mutations wherein said mutations result in the introduction of one or more glycosylation sites in the polypeptide, wherein said glycosylation comprises N-glycosylation. 
     
     
         2 . The polypeptide of  claim 1  wherein the one or more mutations result in no more than a 0, 1, 2, 5, 10, 20, 50 or 100 fold reduction of proteolytic activity and/or clot activity of the factor IX polypeptide analogue when compared with the wild-type polypeptide. 
     
     
         3 . The polypeptide of  claim 1  wherein the one or more mutations comprise incorporation of one or more N—X—S/T motifs. 
     
     
         4 . The polypeptide of  claim 3  wherein the one or more N—X—S/T motifs are established at positions where the N residue has a relative side-chain surface accessibility of more than 25% or more than 50%. 
     
     
         5 . The polypeptide of  claim 4  wherein said one or more mutations are selected from the group consisting of: Y1N, S3N+K5S/T, G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T, G12N+L14S/T, R16N+C18S/T, K22N, R29N+V31S/T, T35N+R37S/T, R37N, T39N+F41S/T, F41N+K43S/T, W42N+Q44S/T, Q44N+V46S/T, V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T, D49N+C51S/T, Q50N+E52S/T, E52N+N54T, S53N+P55S/T, C56S/T, L57N+G59S/T, G60S/T, S61N+K63S/T, K63N+D65S/T, D65N+N67S/T, I66N+S68S/T, Y69S/T, Y69N+C71S/T, S68N+E70S/T, E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, F77N+G79S/T, G79N+N81S/T, K80N+C82S/T, E83S/T, E83N+D85S/T, L84N+V86S/T, D85N, V86N+C88S/T, T87N+N89S/T, I90N+N92S/T, K91S/T, I90N+N92S/T, K91N+G93S/T, R94S/T, R94N+E96S/T, K100N, A103S/T, S102N+D104S/T, A103N+N105S/T, D104N+K106S/T, V107S/T, K106N+V108S/T, V108N+V110S/T, S110N, E113N+Y115S/T, G114N+R116S/T, R116N+A118S/T, E119N+Q121S/T, K122S/T, Q121N+S123S/T, K122N+C124S/T S123N+E125S/T, E125N+A125S/T, P126N+V128S/T, V128N+F130S/T, P129N+P131S/T, F130N+C132S/T, R134N, V135N+V137S/T, S136N, S138N, Q139N, T140N+L142S/T, S141N+L143S/T, K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T, F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T, D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T, E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T, L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N, S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T, F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T, G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T, P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T, E213N+W215S/T, E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N, E239N, E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T, I251S/T, I251N+I253S/T, R252N+I254S/T, I253N+P255S/T, P255N+H257S/T, H257N+Y259S/T, N260S/T, A262S/T, A261N+I263S/T, A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, A266N+H268S/T, D276N+P278S/T, P278N+V280S/T, E277N+L279S/T, V280N+N282S/T, Y284S/T, S283N+V285S/T, Y284N, D292N+K294S/T, K293N+Y295S/T, E294N, F299S/T, I298N+L300S/T, K301N+G303S/T, F302N, G303N+G305S/T, S304N+Y306S/T, Y306N+S308S/T, R312N+F314S/T, F314N+K316S/T, H315N+G317S/T, K316N+R138S/T, G317N, R318N+A320S/T, S319N+L321S/T, L321N+L323S/T, V322N+Q324S/T, Y325N+R327S/T, R327N+P329S/T, P329N+V331S/T, L330N+D332S/T, D332N+A334S/T, R333N, A334N+C336S/T, T335N+L337S/T, L337N, R338N, K341N, F342N+I344S/T, T343N+Y345S/T, Y345N+N347S/T, M348S/T, H354N+G356S/T, E355N+G357S/T, G357N+D359S/T, R358N, Q362N+D364S/T, E372N+E374S/T, E374N, G375N, E388N+A390S/T, M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T, K394N+G396S/T, R403N+V405S/T, I408S/T, K409N+K411S/T, E410N, K411N+K413S/T, K413N, and combinations thereof. 
     
     
         6 . The polypeptide of  claim 4 , wherein said one or more mutations are selected from the group consisting of: Y1N, S3N+K5S/T, G4N+L6S/T, F9N+Q11S/T, V10N+G12S/T, K22N, R37N, Q44N+V46S/T, V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T, Q50N+E52S/T, E52N+N54T, S53N+P55S/T, C56S/T, L57N+G59S/T, S61N+K63S/T, I66N+S68S/T, Y69S/T, S68N+E70S/T, E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, K80N+C82S/T, L84N+V86S/T, T87N+N89S/T, K91S/T, I90N+N92S/T, K91N+G93S/T, R94N+E96S/T, K100N, A103S/T, S102N+D104S/T, A103N+N105S/T, D104N+K106S/T, V107S/T, K106N+V108S/T, V108N+V110S/T, E113N+Y115S/T, R116N+A118S/T, E119N+Q121S/T, K122S/T, Q121N+S123S/T, S123N+E125S/T, E125N+A125S/T, P129N+P131S/T, S138N, T140N+L142S/T, S141N+L143S/T, K142N, A146N+A148S/T, E147N+V149S/T, A148N+F150S/T, V149N+P515S/T, F150N+D152S/T, P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T, D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T, E160N+E162S/T, A161N, E162N+I164S/T, T163N+L165S/T, I164N+D166S/T, L165N+N167S/T, D166N+I168S/T, I168N+Q170S/T, T169N, Q170N, S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T, F178S/T, D177N, F178N+R180S/T, T179N+V181S/T, R180N+V182S/T, G183+E185S/T, E185N+A187S/T, D186N+K188S/T, K188N+G190S/T, P189N+Q181S/T, K201N+D203S/T, V202N+A204S/T, D203N+F205S/T, E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, K228N, E239N, E240N+E242S/T, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T, I251S/T, R252N+I254S/T, P255N+H257S/T, H257N+Y249S/T, A262S/T, A261N+I263S/T, A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, E277N+L279S/T, V280N+N282S/T, D292N+K294S/T, K293N+Y295S/T, E294N, K301N+G303S/T, G303N+G305S/T, F314N+K316S/T, K316N+R138S/T, R318N+A320S/T, L321N+L323S/T, R327N+P329S/T, D332N+A334S/T, R333N, R338N, K341N, F342N+I344S/T, T343N+Y345S/T, H354N+G356S/T, E355N+G357S/T, G357N+D359S/T, E372N+E374S/T, E374N, G375N, M391N+G393S/T, K392N+K394S/T, G393N+Y395S/T, I408S/T, E410N, K413N, and combinations thereof. 
     
     
         7 . The polypeptide of  claim 1 , wherein said one or more mutations are selected from the group consisting of: Y1N, K22N, R37N, C56S/T, G60S/T, Y69S/T, E83S/T, D85N, K91S/T, R94S/T, K100N, A103S/T, V107S/T, S110N, K122S/T, R134N, S136N, S138N, Q139N, K142N, V156N, A161N, T169N, Q170N, T172N, F178S/T, D177N, K228N, E239N, I251S/T, N260S/T, A262S/T, Y284S/T, Y284N, E294N, F299S/T, F302N, G317N, R333N, L337N, R338N, K341N, M348S/T, G358N, E374N, G375N, I408S/T, E410N, K413N, and combinations thereof. 
     
     
         8 . The polypeptide of  claim 1 , wherein said one or more mutations are selected from the group consisting of: T172N, K228N, I251T, A262T, and combinations thereof. 
     
     
         9 . A hyperglycosylated human factor IX polypeptide analogue, wherein said polypeptide is glycosylated at one or more positions other than N157 or N167 relative to wild-type human factor IX polypeptide. 
     
     
         10 . The polypeptide of  claim 9  wherein said polypeptide is glycosylated at one or more positions selected from the group consisting of: Y1, S3, G4, F9, V10, Q11, G12, R16, K22, R29, T35, R37, T39, F41, W42, Q44, V46, D47, G48, D49, Q50, E52, S53, N54, L57, N58, G59, S61, K63, D65, I66, N67, S68, Y69, E70, W72, P74, F77, G79, K80, N81, E83, L84, D85, V86, T87, N89, I90, K91, N92, R94, K100, N101, S102, A103, D104, N105, K106, V108, S110, E113, G114, R116, E119, N120, Q121, K122, S123, E125, P126, V128, P129, F130, R134, V135, S136, S138, Q139, T140, S141, K142, A146, E147, A148, V149, F150, P151, D152, V153, D154, Y155, V156, S158, T159, E160, A161, E162, T163, I164, L165, D166, I168, T169, Q170, S171, T172, Q173, S174, F175, N176, D177, F178, T179, R180, G183, E185, D186, K188, P189, K201, V202, D203, E213, E224, T225, G226, K228, E239, E240, T241, H243, K247, N249, I251, R252, I253, P255, H257, N258, N260, A261, A262, I263, N264, K265, A266, D276, E277, P278, V280, N282, S283, Y284, D292, K293, E294, N297, I298, K301, F302, G303, S304, Y306, R312, F314, H315, K316, G317, R318, S319, L321, V322, Y325, R327, P329, L330, D332, R333, A334, T335, L337, R338, K341, F342, T343, Y345, N346, H354, E355, G357, R358, Q362, E372, E374, G375, E388, M391, K392, G393, K394, R403, N406, K409, E410, K411, K413, and combinations thereof. 
     
     
         11 . The polypeptide of  claim 9  wherein said polypeptide is glycosylated at one or more positions selected from the group consisting of: T172, K228, N249, N260, and combinations thereof. 
     
     
         12 . The polypeptide of  claim 9  wherein the hyperglycosylated polypeptide comprises at least one glycan. 
     
     
         13 . A pharmaceutical composition comprising the polypeptide of  claim 1 . 
     
     
         14 . A process for preparing the polypeptide analogue of  claim 1  comprising the steps of:
 (a) performing site directed mutagenesis of a polynucleotide encoding a human factor IX polypeptide to incorporate one or more N—X—S/T motifs into the polynucleotide, thereby forming a nucleic acid construct; 
 (b) transfecting the nucleic acid construct into a producer cell; and 
 (c) purifying of the polypeptide analogue from the transfected producer cell. 
 
     
     
         15 . A nucleic acid construct encoding the human factor IX polypeptide analogue of  claim 1 . 
     
     
         16 . A pharmaceutical composition comprising the polypeptide of  claim 9 . 
     
     
         17 . A process for preparing the polypeptide analogue of  claim 1  comprising the steps of:
 (a) performing site directed mutagenesis of a polynucleotide encoding a human factor IX polypeptide to incorporate one or more N—X—S/T motifs into the polynucleotide, thereby forming a nucleic acid construct; 
 (b) transfecting the nucleic acid construct into a producer cell; and 
 (c) purifying the polypeptide analogue from the transfected producer cell. 
 
     
     
         18 . A nucleic acid construct encoding the human factor IX polypeptide analogue of  claim 1 .