MX2010011345A - Modified factor ix polypeptides and uses thereof. - Google Patents

Abstract

Disclosed are modified Factor IX polypeptides such as Factor IX polypeptides with one or more introduced glycosylation sites. The modified Factor IX polypeptides may exhibit increased in vitro or in vivo stability such as a longer plasma half-life. Also, methods are disclosed of making modified Factor IX polypeptides, and methods of using modified Factor IX polypeptides, for example, to treat patients afflicted with hemophilia B.

Description

MODIFIED OLIPEPTIDES OF THE FACTOR IX AND USES OF THE MIS This application claims benefit of the US provisional application. CO 124,567; filed on April 16, 2008, and provisional application of series 61 / 045,961; filed on April 17, 2008, which is included in the present descriptive report by reference in its entirety.

FIELD OF THE INVENTION The invention relates to modified factor IX polypeptides, factor IX strains with one or more glycosylation sites introduced. The effects of factor IX may exhibit an increase in in vitro stability or a longer plasma half-life. The invention also provides for the manufacture of modified factor IX polypeptides and factor IX procedures to, for example, treat patients affected by heme.

BACKGROUND OF THE INVENTION Hemophilia B affects one in 34,500 males and is caused by tumors (Giangrande, Expert Opin, Pharmacother, 6: 1517-1524, 2005). or (derived from plasma or recombinant) has an approximate half-life, FIX levels decrease to less than 1% of levels nor 3 to 4 days after the bolus injection that needs to repeat the injection or days to achieve effective prophylaxis Giangrande , Expert Opin. Ph 24, 2005). Such frequent intravenous injection is a problem and an obstacle to effective prophylaxis (Petrini, Haemoph 2007), especially in children. A FIX protein with a half-life plus a less frequent administration and, therefore, would be a benevolent one.

BRIEF DESCRIPTION OF THE INVENTION The invention provides FIX polypeptides (also referred to as FIX os, FIX muteins or FIX variants), which comprise two that have been modified by the introduction of one or more ion. In some embodiments, the one or more N-linked glycosylation glycosylation sites. In some embodiments, the n, the hydrocarbon chain may have a fero chain structure. In some embodiments, the hydrocarbon chain can be a hydrocarbon chain of a human being. In some forms of r a hydrocarbon chain to one or more of the glycosylation sites mention the serum half-life of the polypeptide in, for example, at least the polypeptide lacking the introduced glycosylation sites. In some embodiments, the attachment of a hydrocarbon chain to one or more of the ion introduced does not reduce the amount of polypeptide secreted in, by e with respect to the amount of the secreted polypeptide lacking ion introduced. In some embodiments, the binding of a urine to one or more of the introduced glycosylation sites does not inhibit the in or at least one of Factor VIII (FVIII), Factor XI (FXI) or Factor X more than 50% respect to the interaction of the polypeptide lacking in urine at the glycosylation sites introduced with FVIII, FXI or FX. In some embodiments, the modified polypeptide may have a specific activity of 100 units per mg of polypeptide.

One or more glycosylation sites can be introduced through S3T; S3N and K5T; G4N and L6T; K5N and E7T; L6N and E8T; E7N and F9T; F9N and IN and N13T; G12N and L14T; N13 and E15T; L14N and Rl 6T; 17T; M19N and E21T; E20N and K22T; S24N and E26T; F25N and E27T; E26N and A28N and E30T; R29N and V31T; E30N and F32T; V31 N and E33T; F32N and N 8N and E40T; T39N and F41T; E40N and W42T; F41 N and K43T; W42N and 0 4N and V46T; Y45N and D47T; V46N and G48T; E52N and N54T; S53N and P55T; G 65T; I66N and S68T; S68N and E70T; G76N and E78T; E78N and K80T; E83N and D 0N and N92T; KIOON and S102T; S102N and D104T; A103N and N105T; D10 V108T; Rl 16N and AI 18T; The 19N and Q121T; Q121 N and S123T; A127N and P12 136N and S138T; V137N and Q139T; Q139N and S141T; T140N and K142T; S14 V149T; T148N and F150T; V149N and P151T; P151N and V153T; D152N and D15 154N and V156T; Y155N and N157T; V156N and S158T; S158N and E160T; T15 E162T; E162N and I164T; T163N and L165T; I164N and D166T; L165N and N16 68N and Q170T; T169N and S171T; S171N and Q173T; T172N and S174T; Q17 N176T; G184N and D186T; E185N and A187T; D186N and K188T; A187N and P18 K122T, and 1251 T; D85N,? 122 ?, and? 242 ?; ? 125 ?,? 126 ?, and A127T; P12 148N, F150T, and P151A; F150N, P151A, and D152T; P151 N, V153T, and A1 T172N; V153N, Y155T, and E294N; ?? 72 ?, G226N, and K228T; F353N, H35 354I, and E355T; V370N, T371V, and E372T; V370N, T371 I, and E372T; ? 391 85N, P151 N, V153T, and K228N; D85N, P151 N, V153T, and E242N; K1 K228N; K122T, P151 N, V153T, and E242N; K122T, P151 N, V153T, and I2 226N, and K228T; P151 N, V153T, T172N, and R338A; P151 N, V153T, D17 153T, G226N, and K228T; T172N, G226N, K228T, and R338A; D85N, K1 E242N; D85N, P151, V153T, G226N, and K228T; K122T, P151 N, V153 138N, P151 N, V153T, G226N, and K228T; T148N, F150T, G226N, K228 153T, T172N, G226N, and K228T; P151 N, V153T, D177E, F178T, and R3 226N, K228T, and R338A; and P151 N, V153T, T172N, G226N, K228T, combination thereof. ? one or more glycosylation sites can be introduced into the cat domain of activation. Exemplary embodiments include polypeptide in one or more substitutions, such as, among others: R37N; D85N; K1 A161 N; Q170N; T172N; D177N; F178T; K201 N; K228N; E239N; E2 Y345T; and E372N and E374T; Fl 50N, Pl 5 IA, and D152T. In some n, the modified polypeptides may further comprise n, such as, for example, R338A and V86A. In some forms of modified areas can also comprise both substitutions, R338 s one or more glycosylation sites can be introduced by O-linked convoliosylation at an N-linked glycosylation site. Example include substitutions such as, among others, T169N; T172N; T148 A161T. In some embodiments, the substitutions may F150T; and T159N and A161T. In some embodiments, they may also comprise at least one substitution, such as, V86A. In some embodiments, the polypeptides modify to understand both substitutions, R338A and V86A. s one or more glycosylation sites can be introduced by amino acid inserts 1 and 12 between amino acid residues 160-164. ions can be introduced between amino acid residues 160-161, between acids 161-162, between amino acid residues 162-163 or between the two 163-164. In some embodiments, SEQ ID NO: 2 may be embodiments, the polypeptide may further comprise its two P151N, V153T, and T172N. In some embodiments, the polypeptide also substitutes amino acids T148N and F150T. In some n, the polypeptide may further comprise amino acid substitutions. In some embodiments, the modified polypeptides may at least one substitution, such as R338A and V86A. In some n, the modified polypeptides may further comprise both s V86A.

? Examples of embodiments are provided FIX polypeptides ignite one or more substitutions, such as: D85N; K122T; S138N; T1 239N; E242N; I251T; A262T; E294N; G59N and S61T; G76N and E78T; S10 N105T; D104N and K106T; E119N and Q121T; Q121 N and S123T; S136N and S; T140N and K142T; T148N and F150T; V149N and P151T; P151 N and V153T; D15 E160T; E162N and I164T; T163N and L165T; T172N and S174T; Q173N and F17 225N and V227T; G226N and K228T; I253N and P255T; A262N and N264T; V28 E374T; F150N, P151A, and D152T; and insertion of SEQ ID No. 2 between Al K228T, and an insertion of SEQ ID No. 2 between A161 and E162. In some in administering to a subject in need thereof a therapeutic amount pharmaceutical rations described in the present specification, The application also provides DNA sequences encoding os, as well as eukaryotic host cells transfected with the The application also provides methods for producing polypeptides comprising (i) modifying the amino acid sequence of the introduction of one or more glycosylation sites; (ii) expressing the p e allowing glycosylation at the one or more glycosylation sites; and (iii o.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts a multiple sequence alignment of the sequential activation peptide of eight species.

Figure 2 depicts Western blot analysis of media ansfected with FIX glycosylation site muteins. I am procuring an anti-FIX-HRP antibody. in the form of a percentage of the FIX-R338A mutein. The constructs ra 3.

Figure 5 depicts Western blot analysis of medi ransfected with the glycosylation site mutein G226N, K228T FIX was detected using an anti-FIX-HRP antibody.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides FIX polypeptides that include one or ion linked by O or N. The increase in glycosylation of the sarse proteins to achieve 1) reduction of immunogenicity; 2) administer the protein; 3) increase in the stability of the protein, such as in / ida in serum; and 4) reduction of adverse side effects, ion.

Upon request it provides variants of human FIX with one or more sites of them. Modified FIX polypeptides can also be presented in plasma that would provide, for example, an extended prot 0 time, these modification sites can be combined with alterations that are specific to FIX, including, among others, the R338A substitution and / or substitution. col., J. Biol. Chem. 273: 12089-12094, 1998 and Chang, et al., J. 3-25399, 2002). The combination with one or both of the R3 substitutions at any reduction in activity resulting from the addition of sites of do that the specific activity of the modified polypeptides may be that of wild-type FIX.

Once expressed, the wild-type FIX is a mono-valent glycoprotein 55,000 daltons. Structurally we can consider that: the domain rich in gamma carboxyglutamate or Gla; the activation regions; and the catalytic domain containing the active site (Thor 72, 1986). FIX is synthesized in the liver as a monocate two polypeptide and undergoes extensive posttranslational modification during the Golgi and endoplasmic reticulum (Nemerson, et al., CRC Crit. Rev. Bi; Stenflo, et al., Annu. Rev. Biochem. 46: 157-172, 1977). Both the sec propeptide are removed, resulting in a protein m? activated factor VII (FVII) initiates the normal hemostatic process forming tissue actor (TF), exposed as a result of a damage in the parei, the complex activates the FIX; the active form is called FlXa. The FIX is eliminated by proteolytic cleavage at two sites by media A) or the tissue factor (TF) / Factor VI complex to generate active mind, Factor IXa (FlXa). The factors FlXa and Villa (FIBA) convert Xa (FXa), which, in turn, converts prothrombin into thrombin. Then the fibrinogen in fibrin, which results in the formation of a Since wild-type FIX has numerous post-translational modifications it has been suggested that it plays a role in the pharmacokinetic profile of producing an ectopic glycosylation site in a position that does not affect ions. Once produced, the FIX should retain the nar activity with FVIII, FXI and FX in order to be an effective treatment for the glycosylation introduced will not alter these interactions and function. In part, modifications of the FIX that are likely to have two modified FIX The application provides FIX polypeptides comprising one or an introduced ion, ie, modified FIX polypeptides. "Factor IX", c descriptive memory, refers to a plasma FIX glycoprotein belonging to the intrinsic coagulation pathway and is essential for a. It should be understood that this definition includes forms both before the FIX glycoprotein of human plasma. Unless otherwise, FIX, as used herein, means protein of functional human FIX in its normal role in the coagulation, fragment, analog, variant and derivative thereof. The terms "," analogous "," mutein "and" variant ", when referring to the polypeptides, mean fragments, derivatives, analogs, muteins and variants thereof substantially substantiating the same function or biological activity.

Non-limiting compounds of FIX polypeptides include FIX, FlXa and FIX that have FIX activity. Biological deletion fragments, substitution variants or addition variants of any amino acid with another amino acid having properties for example, changes from alanine to serine; arginine to lysine; a or histidine; aspartate to glutamate; cysteine to serine; glutamine a or aspartate; glycine to proline; histidine to asparagine or glutamine; isoleuc leucine to valine or isoleucine; lysine to arginine; methionine to leucine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. In some embodiments, the FIX polypeptides of SEQ ID NO: 1 comprise 1-30, of conservative amino acid 1s, in addition to the introduction of one ion. to abbreviation of a single letter for a concrete amino acid, its corrido and the abbreviation of three letters are the following: A, alanine (Ala); C, asyrtactic C (Asp); E, glutamic acid (Glu); F, phenylalanine (Phe); G, gly (His); I, isoleucine (He); K, lysine (Lys); L, leucine (Leu); M, methionine (Asn); P, proline (Pro); Q, glutamine (Gln); R, arginine (Arg); S, it (Thr); V, valine (Val); W, tryptophan (Trp); And, tyrosine (Tyr); and norleucine (Nl amino acid except for Pro; X3 is Ser or Thr; and X4 is optionally He In some embodiments, X1 is optionally Asp; X2 e is GIn In some embodiments, X1 is Asp; X2 is He X3 is the location of the N-linked glycosylation sites in a FIX polypeptide the amino acid sequence so that one or more of the as described above is introduced.

O-linked lysylation refers to the binding of one of loas ctosamine, galactose or xylose in a hydroxyamino acid, with higher threonine, although it is also possible to bind 5-hydroxyproline or 5-hyd to O-linked glycosylation sites in A FIX polypeptide can be the amino acid sequence so that one or more of r is introduced.

The glycosylation sites can be introduced by, for example, the amino acid residues, by substituting one or more amino acid residues with another amino acid (s) or by adding one or more amino acid residues and an amino acid residue can be two amino acid residues) of the human FIX polypeptide, position 338 of SEQ ID No. 1 cn 384 of SEQ ID No. 26.

? FIX residue number system used herein for the mature human FIX protein in which residue 1 represents the mature FIX polypeptide after elimination of the sequence or. Native or wild FIX is the mature human FIX molecule of length shown in SEQ ID No. 1. ? some embodiments, the glycosylation sites are subjected to i locations that do not nullify the function of the protein or its expression e? of designing FIX polypeptides comprising one or more sites of os may apply several criteria. In some embodiments, it is exposed on the surface. The surface exposure on the basis of the solvent accessible surface area as described, (J. Thromb. Haemost, 3: 2044-56, 2005). In some forms of embodiment introduced, it does not introduce a mutation that is known to be present. 95 Y? H 400 K? N 407 W 96 G? R 402 S? Y 408 I? 97 I? L 403 R? W 412 T? 98 Y? S 404 Y? H 413 K L? 99 T? N 405 V? F 414 G, I It is desirable to compare the properties of the modified polypeptides or more glycosylation sites introduced into a control polypeptide. Stopping include, for example, solubility, activity, plasma half-life and binding properties. In some embodiments, the polypeptides may be glycosylated. It is within the scope of an expert to the most suitable control polypeptide to be compared. In some n, the control polypeptide may be identical to the modified polypeptide at or more introduced glycosylation sites. Examples of polypeptides or wild-type FIC and FIX polypeptides comprising one or more ace, such as R338A and / or V86A.

The term "half-life", as used herein and to isolate a polypeptide drug to a patient, is defined as the time plasma concentration of a drug in a patient is reduced to that in the art. the determination of half-life and stability in vivo. The teachings in Kenneth, et al., Chemical Stability of Pharmaceuticals: A H ists and in Peters and coi, Pharmacokinetc analysis: A Practical Appro refers to "Pharmacokinetics", M Gibaldi & D Perron, publish a Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta and the area under the curve (AUC). activity of the modified FIX polypeptides can be described as biolume, such as in units, or as a percentage of the activity of some embodiments, the FIX modified polypeptides have a specific pity that has not been reduced by more than about 10, 20, 80% with respect to a control protein. For example, a polypeptide can have a specific activity that has not been reduced more than about a polypeptide of FIX control, if the polypeptide modified FIX deficiency. Such lines are available from one such as, without limitation, the Division of Laboratories and Researchers of Public Health, Albany, N. Y. and the Department of Pathology, Univer Chapel Hill, N.C. Both sources provide, for example, canine mofilia B animals. Alternatively, mice with d atino, et al., Blood 104: 2767-2774, 2005) are also available. In order to analyze the polypeptide assay activity in the sick animal, a small bleeding time is performed with that of a healthy control.

? Wild human FIX has a specific activity of approximate per mg. A unit of FIX has been defined as the amount of FIX for normal (cumulative) human plasma (corresponding to a level of IFs embodiments, the modified FIX polypeptides can be specific to at least about 100 units per mg p. embodiments, the modified FIX polypeptides can be specific to at least about 120, 140, 160, 180, 200, 220 ades per mg of FIX polypeptide In some embodiments, a of FIX can be measured using the APTT or third control time For example, a modified FIX polypeptide can be secreted that has not been reduced by more than about 80% with respect to its control, if the modified polypeptide is secreted in an amount of 20% compared to The amount of the polypeptide can be measured by, for example, the determination of extracellular levels using any procedure known in traditional methods for quantification. n Protein include mass etria electrophoresis and antibody binding. Examples of procedures for roteics in a biological sample include techniques based on anti-transfer (western blot), immunohistochemical assay or enzyme-linked absorption (ELISA) or radioimmunoassay (RIA).

In some embodiments, the modified FIX polypeptides and one of FVIII, FXI or FX at a non-reduced level more than about 80% relative to the interaction of a control protein with at least one example, a modified FIX polypeptide. can interact with the m I, or FX at a non-reduced level more than about 80% re or FIX control, if the modified polypeptide interacts with at least natural glycosylation in FIX they are located in the EGF1 domain (two if 53 and Ser61 ) and in the activation peptide (four sites bound to O in Thrl79 T and 2 sites bound to N in Asnl57 and Asn 167). Thus, six of the natural illation are located in the activation peptide. The sites of os may have less than a negative impact on activity or introduce them into regions of the FIX that have sites of activation glycosylation. In addition, the activation peptide is cleaved when so much, it is not required for the catalytic activity of FlXa. However, n is present in the zymogen, which makes the activation peptide to introduce the glycosylation sites to improve the half-life in ci. upon request it also provides, in part, polypeptides comprising glycosylation. In certain embodiments polypeptide is provided in one or more G4T-selected substitutions; E33N; E36T; E36N; 83T; D85N; V86A; K91T; A103T; V107T; K122N; K122T; S138N; A1 > 151 N; T159N; A161T; T169N; Q170N; T172N; D177N; D177E; F178T; K201 N; K201T; K21 Q44N and V46T; Y45N and D47T; V46N and G48T; E52N and N54T; S53N and P 3N and D65T; I66N and S68T; S68N and E70T; G76N and E78T; E78N and K80T; E 86T; I90N and N92T; KIOON and S102T; S102N and D104T; A103N and N10 106N and V108T; Rl 16N and Al 18T; The 19N and Q121T; Q121 N and S123T; A12 V137T; S136N and S138T; V137N and Q139T; Q139N and S141 T; T140N and K14 147? and V149T; T148N and F150T; V149N and P151T; P151 N and V153T; D15 Y155T; D154N and V156T; Y155N and N157T; V156N and S158T; S158N and E16 160N and E162T; E162N and I164T; T163N and L165T; I164N and D166T; L16 I168T; I168N and Q170T; T169N and S171T; S171 N and Q173T; T172N and S17 174N and N176T; G184N and D186T; E185N and A187T; D186N and K188T; A18 Q191T; G200N and V202T; K201N and D203T; V202N and A204T; E224N and G G226N and K228T; V227N and I229T; H236N and I238T; I238N and E240T; E24 H243T; H243N and E245T; K247N and N249T; V250N and R252T; 125 IN and I2 261 N and I263T; A262N and N264T; D276N and P278T; V280N and N282T; F30 Y306T; R312N and F314T; V313N and H315T; F314N and K316T; H315N and G31 317N and S319T; S319N and L321T; A320N and V322T; R327N and P329T; P32 A334T; L337N and S339T; S339N and K341T; T340N and F342T; T343N and Y34 E372T; M391 N, K392V, and G393T; D85N, P151 N, V153T, and K228N; D E242N; K122T, P151 N, V153T, and K228N; K122T, P151 N, V153T, and E2 153T, and 1251 T; T148N, F150T, G226N, and K228T; P151 N, V153T, T172 153T, D177E, and F178T; P151 N, V153T, G226N, and K228T; T172N, G22 D85N, K122T, P151 N, V153T, and E242N; D85N, P151 N, V153T, G226 I5 IN, V153T, G226N, and K228T; S138N, P151 N, V153T, G226N, and K2 226N, K228T, and R338A; P151 N, V153T, T172N, G226N, and K228T; P1 178T, and R338A; P151 N, V153T, G226N, K228T, and R338A; and P151 N, V1 228T, and R338A; or any combination thereof.

? In some embodiments, FIX polypeptides are provided which substitutes selected from E33N; E36T; E36N; R37N; F75N; F77T; E83T; D85N; V86A; K91T; A1 K122T; S138N; A146N; T148N; F150T; P151 N; T159N; A161T; A16 T172N; D177N; D177E; F178T; K201 N; K201T; K214T; V223N; G2 K228T; E239N; E242N; 1251 T; A262T; E294N; R338A; R338N; K3 354I; E355T; V370N; T371V; T371 I; E372T; E374N; M391 N; K392V; G3 414I; Q139T; Q139N and S141T; T140N and K142T; S141 N and L143T; E147N and V14 149N and P151T; P151 N and V153T; D152N and D154T; V153N and Y155T; D15 N157T; V156N and S158T; S158N and E160T; T159N and A161T; E160N and E16 163N and L165T; I164N and D166T; L165N and N167T; D166N and I168T; ? 168 ; S171 N and Q173T; T172N and S174T; Q173N and F175T; S174N and? 176 185N and A187T; D186N and K188T; A187 and P189T; P189N and Q191T; G20 D203T; V202N and A204T; E224N and G226T; T225N and V227T; G226N and K22 236N and I238T; I238N and E240T; E240N and E242T; T241 N and H243T; H24 N249T; V250N and R252T; 1251 N and I253T; I253N and P255T; A261 and I26 276N and P278T; V280N and N282T; F302N and S304T; S304N and Y306T; R31 315 ?; F314N and K316T; H315N and G317T; K316N and R318T; G317N and S31 320N and V322T; R327N and P329T; P329N and V331T; D332N and A334T; L33 K341T; T340N and F342T; T343N and Y345T; G352N and H354T; F353N and E35 355N and G357T; G356N and R358T; G357N and D359T; E372N and E374T; W38 E388T; A390N and K392T; , K122T, and I251T; D85N, K122T, and E242N; E125N, P126A, and A127T; P12 ( 153T, T172N, G226N, and 228T; P151 N, V153T, D177E, F178T, and R3 226N, K228T, and R338A; and P151 N, V153T, T172N, G226N, K228T, combination thereof.

The application provides, in part, FIX polypeptides comprising glycosylation introduced by conversion of a glycosylation site into an N-linked glycosylation site. It has been reported that the N-linked glycosylation sites are more lly than the glycosylation sites tested. that a higher content of sialic acid confers a protein increment. In general, it is believed that the increase in the swimming content by the additional N-linked glycosylation may be responsible for the blood influx (White, et al., Thromb. Haemost 78: 261-265, 1997). In order to carry out said modified FIX polypeptides are the following embodiments, FIX polypeptides are provided which comprise a In some embodiments, polypeptides are provided in a T172N substitution. In some embodiments, of FIX comprising a T148N substitution and a substitution embodiments, FIX polypeptides are provided which all have additional amino acids (between 7 and 10 residues) in which they are not found in other species (human beings, rhesus, perr Figure 1). These additional sequences are located between E160 and E This suggests that the insertion of at least 10 amino acid residues of the FIX at this site. This region was the target for the additional intr and was found to be a good location, since it was significantly. In some embodiments 8, 16, 14 or 12 amino acid residues may be inserted between the uman amino acid residues. In some embodiments up to 10 embodiments can be inserted up to 9 amino acids can be inserted.

Depending on the criteria used to perform the FIX sequence alignment of eight species, the apparent site in which the s are found in rats, mice and guinea pigs can vary such that site 60 and A161, between A161 and E162, enter E162 and T163 or between T163 e 1 In some embodiments, the inserted amino acid sequence may be SEQ ID No. 2. In some embodiments, the s two inserted between A161 and E162 may be SEQ ID NO: 2 In some 1164. In some of the embodiments, 30, 25, 20, 18, 16, 14, or 12 amino acid residues may be inadvertently available to amino acids 160 to 164 of the human FIX. In some of the forms d insert up to about 10 amino acids in total between the two 160 to 164 of the human FIX. In some of the embodiments, approximately 9 amino acids total between the residues of human amin FIX. In some embodiments, one glycosylation can be inserted. on request further provides FIX polypeptides comprising introduced glycosylation moieties disclosed herein for FIX nan polypeptides comprising at least one site of or in the catalytic domain and at least one glycosylation site introduced. FIX polypeptides comprising at least ion introduced into the catalytic domain are provided. Polypeptides are provided in at least two glycosylation sites introduced into the peptide of embodiments, the FIX polypeptides may comprise the following: R37N; D85N; K122T; S138N; A146N; A161 N; Q17 343N and Y345T; E372N and E374T; F 150N, P151A, and D152T; and inserted it between A161 and E162. In some embodiments, the other polypeptides comprise at least one substitution, such as, for example, R3 as embodiments, the modified polypeptides may also be substituted, R338A and V86A.

The application further provides modified FIX polypeptides that co-site glycosylation introduced at the C-terminus of the FIX polypeptide (amino acid 415 of the FIX polypeptide). Den polypeptide is provided for at least two, at least three, at least four or more glyco C sites of the FIX polypeptide. In some embodiments, the polypeptide will learn the addition of the amino acid sequence of SEQ ID NO: 4 and FIX lipeptide. In some embodiments, the polypeptide der the addition of the amino acid sequence of SEQ ID NO: 5 in the FIX e. In some embodiments, the FIX polypeptide may from the amino acid sequence of SEQ ID NO: 6 at the C-terminus in some embodiments, the FIX polypeptide may comprise amino acid sequence of SEQ ID NO: 7 at the C-terminus of the polypeptide. In some embodiments, the FIX polypeptides may contain R338A and a V86A substitution. some embodiments, the ener a specific activity of at least about 200, 300, 4, 900, 1000, 1100, 1200, 1400, 1600, 1800 or 2000 units per mg of specific p can be determined as described above, such using the APTT assay. These polypeptides are useful as mind agents in patients affected by hemophilia B. These polypeptides can substitutions or modifications, such as memory glycosylation sites.

The application aspect provides modified polypeptides of the fa in the following amino acid sequence: EEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQ YVDGDQCE KDDINSYECWCPFGFEGKNCELXd5X86TCNIKNGRCEQFCKNSAX104N YRLAENX12iKSCEPAVPFPCGRVSVX138QTSKLTRAEX14eVX15oXi5iXi52X 9EZ1X16iZ2EZ3TZ4lLD IX169QSX172QXi74FNX177Xi78TRWGGEDAKPGQF KVDAFCGGSIVNEKWIVTMHCVETX226VX228ITWAGEHNIEETEHTEQK HHNYNAAIN YNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGY ¡) X148 isNyX150 is S; X151 is selected from A, P and T; X151 and X153 are selected from: ) X151 is PyX153 is V; i) X151 isNyX153esT; Y ii) X151 is yX153esS; Zi, Z2, Z3) and Z4 are independently selected from zero to twelve amino acid residues and i) SEQ ID No. 2; X152 is selected from D, N and T; X159 and X161 are selected from: (i) Xi5gesTyX16i is A; (ii) Xi59esNyX161esT; and (iii) Xisges N yX161 is S; Xi69 is selected from T and N; X72 is selected from T and N; X74 is selected from S and T; 353, X354 and X355 are selected from: ) X353 is F, X354 is H, X355 is E; i) X353 is N, X354 is V, X355 is T; ii) X353 is N, X354 is I, X355 is T; Y ) X353 is N, X354 is H, V, or I, X355 is S; X370 X371 and X372 are selected from: ) X370 is V, X371 is T, X372 is E; i) X370 is N, X371 is V, X372 is T; ii) X370 is N, X371 is I, X372 is T; Y ) X370 is N, X351 is T, V, or I, X372 is S; X391, X392 and X393 are selected from: ) X391 is M, X392 is K, X393 is G; i) X391 is N, X392 is K, X393 is T; ii) X391 is N, X392 is V, X393 is T; Y v) X391 is N, X392 is V or K, X393 is S; X413 and X414 are selected from: (i) X413 is K and X414 is L; (ii) X413 is N and X4 and X4i4 is I; Y ion of modified FiX polypeptides The amino acid residues can be inserted, removed or replaced with a non-native glycosylation site. For example, the glycosylation sites altering the amino acid sequence of FIX. The alteration of the s two can be achieved by various techniques, such as, pion of the corresponding nucleic acid sequence by site. Techniques for site-specific mutagenesis are either c and are described in, for example, Zoller et al., (DNA 3: 479-488, 1984) or H: 61-68, 1989, pp. 630-748. 61-68). Therefore, using the nuclei sequences of the FIX, the alteration (s) of choice can be introduced. The methods for preparing a polymerase chain reaction with specific primers are well known in the art (Protocols, 1990, Academic Press, San Diego, California, USA). ? nucleic acid construct encoding the FIX polypeptide also synthetically by established standard procedures, preferably with phosphoramidite described by e, et al., (Gene Amplif.Anal.3: 1-26, 1983). According to the process DNA sequences encoding the FIX polypeptides can be combined using recombinant DNA methods. The choice will depend on the host cell into which the vector is to be introduced. The autonomous replication ctor or an integration vector. A vector d exists in the form of an extrachromosomal entity and its replication is chromosome in lication, for example a plasmid. An integer vector is integrated into the genome of the host cell and replicated together (s) in which it has been integrated. ? vector can be an expression vector in which the DNA-containing sequence is operably linked to additional segments required, translation or processing of DNA, such as promoters, ter polyadenylation. In general, the expression vector can be derived from DNA and contain elements of both. The expression "operably linked" are arranged so as to work in concert for the intended ends, for example the transcription is initiated in a promoter and proc uence of DNA encoding the polypeptide. expression vectors for use in the expression of Bio! polypeptides, 2: 1304-1319, 1982).

Similarly, the DNA sequences encoding the FIX polypeptide may be operably connected to a suitable terminator, r of the human growth hormone (Palmiter, et al., Science 2 TPII Terminators (Alber et al., J Mol. Appl. Gen. 1: 419-434, 19 t, et al., EMBO J. 4: 2093-2099, 1985.) Expression vectors are a polyadenylation signal localized after the insertion site. include the early or late polyadenylation signal of SV40, the Elb region of adenovirus 5, the terminator of the human gene (DeNoto, et al., Nucí Acid Res. 9: 3719-3730, 1981), or of the human FIX gene The expression vectors can also be enhancers, such as the SV40 enhancer.

In order to target the FIX polypeptides of the present invention to the secondary path, the native FIX secretory signal can be used. How to alternate a secretory signal sequence (also known as secu to prepro or pre-sequence) in the recombinant vector. The sequence refers to the DNA sequences encoding the FIX analogues in?, For example, Kaufman, et al., (J. Mol. Biol. 159: 601-621, 1982); Southe l. Genet 1: 327-341, 1982); Loyter, et al., (Proc. Nati. Acad. Sci. USA igler, et al., (Cell 14: 725-731, 1978); Corsaro, et al., (Somatic Cell Ge 1), Graham, et al. ., (Virology 52: 456-467, 1973), and Neumann, et al., (EM 2) describe transfection procedures of mammalian cells and DNAs introduced into cells. mammal by, for example, lipofection, by DEAE-dextran, microinjection, protoplast fusion, precípitació retrovirus release, electroporation, sonoporation, fection radiation, natural transformation and biolistic transformation (see, Umbert, et al., Adv. Drug Deliv. Rev. 57: 733-753, 2005). To identify which exogenous DNA is expressed, a selection phenotype (a selectable marker) is normally introduced into the cells together with the selection arcator gene include, for example, conferring genes such as neomycin, pyromycin, hygromycin and methotrexate. Able may be an amplifiable selectable marker that allows the designer and the exogenous DNA when the sequences are linked. , essential amino acids, essential sugars, vitamins, salts, and growth factors and, in the case of DIX-dependent proteins, vitamin K can also be provided. Then, it is applied to select the growth of cells that are expressing a stable mode. For the cells that have been transfected, I will select amplifiable, the concentration of the drug may increase a greater number of copies of the cloned sequences, than the levels of expression. Thereafter, transferable cell clones are screened for expression of the polypeptide from mammalian cell lines for use in the present invention COS-1 (ATCC CRL 1650), from hamster pup kidney (B ol. , J. Biomed, Sci, 9: 631-638, 2002) and HEK-293 (ATCC CRL 1573; Grah? 36: 59-72, 1977). In addition, cell phones can be used in the present invention, including rat Hep I cells (rat hepatoma, ATCC ep II (rat hepatoma; ATCC CRL 1548), TCMK-I (ATCC CCL 139), He), NCTC 1469 (ATCC CCL 9.1), CHO-KI (ATCC CCL 61) and CHO-DUKX roc. Nati Acad. Sci. USA 77: 4216-4220, 1980). high performance liquid matography. Other methods are known in the art and can be applied to the purification of modific polypeptides. eg, Scopes, R., Protein Pu fication, Springer-Verlag, N. Y., 1982).

Generally, "purified" will refer to a protein composition or peptide to fractionation to eliminate other various components and its expressed biological activity. When the "purified ally" is used, this designation will refer to a composition or peptide forms the main component of the composition, so that 50%, approximately 60%, approximately 80%, approximately 90%, approximately 99% or more of the proteins in the composition, Various methods for quantifying the polypeptide are known to those skilled in the art. These include, for example, determining the activity and active ion or evaluating the amount of polypeptides in a medi E fraction. An example of a procedure for evaluating the purity of a specific fraction of the fraction, comparing the activity with the specific activity therefore, calculate the degree of purity, evaluated in this memorandum. CHO cells do not endogenously express the enzyme beta-giallyltransferase, which is used to add sialic acid in the 2,6-binding to the alpha-1, 3- To add sialic acid in this bond to a CHO protein. , CHO cells can be transfected with a functional alpha-2,6-sialyltransferase g to allow incorporation of ace 2.6 to the galactose as desired (see, for example, Lee, and 4: 13848-13855, 1989). similarly, transfection of a host cell which does not produce endogenously with the functional gene for the N-acetylglucosaminyltransferase d enzyme that catalyzes the formation of a bisectional GicNAc structure can be added to the FIX N-acetylglucosamine (GcINAc). protein systems expressing proteins in plant cells that produce mammalian glycosylation (see, for example, bryophyte cells, / 057002).

For the N-linked glycosylation, the final structures of the N-gluc of the organism in which the polypeptide is produced. In general, s in bacteria are completely glycosylated. The polypeptides that glucan resembles, or are identical, to that present in the form.

Various methods have been proposed in the art for adapting ion to a polypeptide (see, e.g., WO 99/22764; doc; WO 99/54342; US Publication No. 2008 / 00 and US No. 5,047,335). Essentially, many of the enzymes required in vitro for polypeptides have been cloned and sequenced. In some they have been used in vitro to add specific sugars to a ta molecule in a polypeptide. In other cases, the cells have been subjected to engineering a combination of desired enzymes and polypeptides so that the desired sugar to an expressed polypeptide is produced within the O-linked glycosylation, the glucans-0 are principally bound and threonine and are formed by the stepwise addition of nucleotide sugars (Tanner, et al., Biochim, Biophys, Acta 906: 81-99, 1987) or conj. J. 13: 19-26, 1996). The function of the polypeptide can be seen afe of the glycans bound to O present. For a review of the is linked to O, see, for example, Schachter and Brockhausen, The Bio including, among others, the peptide-N4- (N-acetyl-beta-D-glycosaminil) (PNGase F), endoglycosidase F1, endoglycosidase F2, endoglicosi. For example, electrophoresis analysis can be performed on sodium p-lphate (SDS-PAGE) of the protein, or pre-treated with PNGaseF asaF. A marked decrease in the width of the band and the changes and igration after treatment with PNGasaF can be considered as an N-linked diary. The hydrocarbon content of a glycosylated protein tectar using lectin analysis of the protein strands (p. by SDS-PAGE and transferred to a support, such as a nas, carbohydrate binding proteins of various vegetale affinity tissues and a narrow specificity for a wide range of epitopes found in glycoprotein glycans (Cummings, Metho 6, 1994). The lectins can be labeled (either directly or indirectly), the ion of the binding of lectins to carbohydrates on the glycoproteins when conjugated with biotin or with digoxigenin, a lectin bound to a can be easily identified in membrane transfers to trionic acid. mammal. In some embodiments, the human glycosylation standard hydrat chain. As used herein, "glycosylation" refers to the representation of ol structures within a given population of FIX polypeptides. Non-standard examples include the relative proportion of oligosaccharide chains that sialic acid residue; (ii) lack of any residual neutral acid); (iii) have at least one terminal galactose residue; (V) thien or terminal N-acetylgalactosamine; (v) have at least one antenna "without at least one galactose residue or terminal N-acetylgalactosamine, or a mucosa bound at alpha 1-> 3 to a residue of N-acetylglucosamine in anti-glycosylation pattern can be determined using any p in the art, including, without limitations: High capillary electrophoresis liquid chromatography (CE), nuclear magnetic resonance (NMR), spec EM) using ionization techniques such as rapid bombardment bulge or matrix assisted laser desorption (ALDI) chromatography treatment with exoglycosidases together with anionic exchange for size exclusion (SEC) or EM (see, eg, Weber, r these conditions, effective dosing of the polypeptides of the present easily determine for the treatment of each Desired indication, active tooth to be administered in the treatment of one of the widely used according to considerations such as the dosage polypeptide used, the The administration period, the sex period of the patient treated and the nature and extent of the condition treated request provides, in part, compositions comprising polypeptide or more glycosylation sites introduced as described in the descriptive. The compositions may be suitable for administering pyrogen. The compositions may also comprise a tetically acceptable. The phrase "pharmaceutically or pharmacologically to molecular entities and compositions that do not produce a reaction or detrimental when administered to an animal or a human being." Descriptive memory, "pharmaceutically acceptable carrier" or of the solvents, dispersion media, coatings, anti-aging agents. It is well known that active agents can be prepared for administration in the form of pharmacologically acceptable salts in water, suitable mixed, such as hydroxypropylcellulose. liquid liquors and their mixtures can be prepared, and in oils Under conditions of use and use, these preparations contain a preservative for microorganisms.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions and sterile powders for external preparation or sterile injectable dispersions. The form must be sterile and easily injected. It must be stable in the conditions and storage and should be preserved against the action contismos such as bacteria and fungi. The carrier can be a dispersion containing, for example, water, ethanol, polyol (eg, licol and liquid polyethylene glycol and the like) sucrose, L-histidine, suitable polysorba, and vegetable oils. The proper fluidity can be achieved by the use of a coating such as lecithin, by the required particle size in the case of dispersion and by the use of t followed by filtered sterilization.

In general, the dispersions can be prepared by incorporating sterilized active ingredients in a sterile vehicle containing n basic and the other ingredients required from those enumerated in the sterile powders for the preparation of injectable solutions. Preparation preparations include, for example, Vacuum drying and lyophilization and active ingredient plus any additional desired ingredient to the same previously filtered to sterilize.

In the formulation, the solutions can be administered in a dosage-wise manner and such an amount as is therapeutically effective is used herein to make a polypeptide that is necessary to provide a desired level of interest. The precise quantity will depend, for example, on the specific FIX polypeptide, the components and the therapeutic composition, the population of patients to which it is administered, the considerations of each individual patient and whether or not You can easily determine it on the basis of the healthy information they have a FIX unit for my plasma, that is, 100% hemophilia B are defined by plasma cntrations of FIX between derates of 1-5% and severe cases, which they represent approximately cases of hemophilia B, they have less than 1% FIX.Prior treatment of small hemorrhages usually requires raising FIX levels. poorly, the treatment of moderate hemorrhages requires re-elevates 30-50%, while the treatment of large trauma pu levels from 50 to 100%. The total number of units required for e e of a patient can be determined as follows: 1, 0 unit (kg) x desired percentage increase (% of normal). The administration is carried out with an initial bolus, followed by a continuous infusion for therapeutic purposes of the pharmacological product. In some forms, administer between 15 to 150 units / kg of the FIX polypeptide. The former will easily optimize dosages and regimens of administration by good medical practice and the clinical condition of the individual patient. Frequency of dosing will depend on the pharmacokinetic parameters of the administration routes. One skilled in the art can determine the ation and assays disclosed in the present specification, to macokinetics observed in animal clinical trials or to dosing regimens include, without limitation, administration five times a day, three times a day, two times a day, a day, three times a week, a week, twice a month, a month on them.

They can determine appropriate dosages through the use of them to determine blood coagulation levels together with S dose response. The physician in charge of the care can be the final user, considering factors that modify the action of the specific activity of the drug, the severity of the damage and the capacity, age, condition, body weight, sex and diet of the patient. patient, fection, time of administration and other clinical factors, The composition may also include an antimicrobial agent to avoid microbial. Non-limiting examples of antimicrobial agents according to the invention include benzalkonium chloride, benzethonium chloride, alcohol and cetylpyridinium, chlorobutanol, phenol, phenylethyl alcohol, phenylmerc 80 nitrate (polyoxyethylene sorbitan monooleate) and pluronics such as F68 and is in BASF, Mount Olive, NJ.); sorbitan esters; lipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, steroidal fatty acids such as cholesterol; and chelating agents such as EDTA, suitable.

? The composition may be present as acids or bases as excipients which may be used include hydrochloric acid, acetic acid, acid, malic acid, lactic acid, formic acid, trichloroacetic acid, acid, phosphoric acid, sulfuric acid, fumaric acid and combinations thereof. Suitable bases include, without limitation, sodium hydroxide, ammonium ace, potassium hydroxide, ammonium acetate, potassium acetate, fo otas, sodium citrate, sodium formate, sodium sulfate, potassium sulfate and combinations thereof.

To the amount of any individual excipient in the composition can be the activity of the excipient and the specific needs of the entity, the optimal amount of any individual excipient can be routinely tested, i.e. by the preparation of component 8); and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3 Editio eutical Association, Washington, D.C., 2000. of uses The compositions described herein may be associated with functional defects of the FIX or as a shortening of the in vivo half-life of the FIX, alteration of the FIX, genetic defects of FIX and reduction of the concentration in pituitary FIX genes comprise, for example, deletions, additions and / or in the nucleotide sequence encoding FIX. In a form of hemorrhagic r hemophilia B may be, the symptoms of such disorders for example, severe epistaxis, oral mucosal hemorrhage, hemarthrosis, persistent, gastrointestinal hemorrhage, retroperitoneal hemorrhage, tropharyngeal, intracranial hemorrhage and hemorrhage associated with traumatis s compositions of the present invention can be used for as. In some embodiments, the modified FI polypeptides bind a subject susceptible or otherwise at risk of a condition to enhance the coagulation capacity of the subject itself. Said to which the scope of the invention is not limited by the scope of the examples in the art will recognize that the invention can be practiced with various disclosed materials, compositions and methods, and said within the scope of the invention.

The following examples are presented to illustrate the described and descriptive invention but should not be construed as limiting the scope of mode.

EXAMPLES In order that this invention be better understood, S are set forth. These examples are for illustrative purposes only and are not to be construed within the scope of the invention in any way. All descriptive memory publications are incorporated by reference in their entirety. 1: Analysis in silico e carried out an in silito analysis of the FIX sequence for access, the known secondary structure, the location of mutations with B, the proximity to predicted sites of interaction with FVIII and FX and the effect given after the stability of the protein FIX, in order to identify TABLE 2 mutein re Sequence 226N, K228T sequence Gly226-Val227-Lys228 Asn226-Val22 N, H354V, E355T Phe353-His354-Glu355 Asn353-Val35 N, H354I, E355T Asn353-Ile354 ?, T371 V, E372T Val370-Thr371-Glu372 Asn370-Val37 0N, T371I, E372T Asn370-Ile371 391N, G393T Met391-Lys392-Gly393 Asn391- Lys392 N, K392V, G393T Asn391- Val39 K413N Lys413-Leu414-Thr415 Asn413-Leu41 K413N, L414I Asn413-Ile41 4 2: Activation peptide sequence alignment The conserved and non-conserved residues in the activation peptide are a multiple sequence sequence alignment as shown in the consensus sequence of the N-linked glycosylation sites is Asn-XS s any amino acid except proline or perhaps aspartic acid, which) . To design new N-linked glycosylation sites in the peptide, the amino acids within the activation peptide were used, as well as the consensus sequences for other sites linked to N and l 3: Insertion of amino acids to generate glycosylation sites Multiple sequence (Figure 1) also revealed that rató have additional amino acids between residues A161 and E162 respec hands, rhesus monkeys, pigs, dogs and rabbits. These amino acids range from 7 to 10 among the three species and contain an overrepresentation of Asp and, to some extent, lie. This observation demonstrates that additional rats, mice and guinea pigs are tolerated on this site, on the activity of FIX. Depending on the criteria used for multiple sequence between the FIX of eight species, the apparent site of the additional amino acids in rats, mice and guinea pigs may be that the site may be between E160 and A161, between A161 and E162, between E1 3 and 1164 of the human FIX. and inserted nine additional amino acids with the sequence N-S-T-N ° 2) into the activation peptide between A161 and E162. The sequences are N-linked consensus sequences of the FIX activation peptide, followed by a glutamine (Q) and preceded by an acid TABLE 4a Substitution Description Activity of the D85N Create 1 new site linked to 99 N in EGF1 K122T Create 1 new site linked to 137 N in EGF2 S138N Create 1 new site linked to 75 N in EGF2-linker region AP KI28N Create 1 new site linked to 67 N in catalytic domain E242N Create 1 new site linked to 183 N in catalytic domain KIOIT Create 1 new site linked to 21 N in catalytic domain E4I0N Create 1 new consensus 1040 joined to N in domain catalytic K413N Create 1 new consensus 388 joined to N in domain catalytic E83T Create 1 new consensus NA joined to N in domain EGF1 KI14T Create 1 new consensus NA structo Substitution Description N-linked activity in domain catalytic 415 -. 415 -. 415 -. 415 -. 415 - T-N-S-T-T (SEQ ID Add 1 new sequence 57 No. 4) consensus attached to N in the C extreme 415 - T-N-S-T-T-Q-N-l-T - Add 2 new 42 T (SEQ ID N ° 5) consensus sequences attached to N at the end C 415 - T-N-S-T-Q-N-l-T-G- Add 3 new NA N-D-T-E-K-T (SEQ ID N ° consensus sequences 6) attached to N at the end C 415 - T-N-S-T-Q-N-l-T-G- Add 4 new 45 N-D-T-E-N-G-T-K-T (SEC consensus sequences ID N ° 7) attached to N at the end C ad specific was calculated by dividing the activity measured in the supernatant by the antigenic concentration of the FIX in the same supernatant of a percentage of the control FIX molecule that is not new. TABLE 4b ruct Substitution of waste Specific activity Activity e (% of WT) (% of c P151N, V153T, G226N, 270 40 K228T T172N, G226N, K228T 300 47 Insertion of SEQ ID NO: 350 55 2 between A161 and E162, G226N, K228T S138, P151N.V153T, 200 26 G226N, K228T P151N, V153T, T172N, 190 38 G226N, K228T D85N, P151N, V153T, 200 26 G226N, K228T 3 D85N, P151N, V153T, 200 20 K228N 3 K122T, P151N, V153T, 400 40 K228N 4 D85N, P151N, V153T, 210 21 E242N 4 122T.P151N, V153T, 800 8 TABLE 4c Expression Activity Constructo Substitution (with respect to control) * (with respect to control) * Cl YIN, S3T 4 1 C2 G4T 5 1 C3 S3N, K5T 4 1 C4 G4N, L6T 4 1 C5 K5N, E7T 4 1 C6 L6N, E8T 4 1 C7 E7N, F9T 4 1 C8 E8N, V10T 2 1 C9 F9N, Q11T 3 1 C10 V10N, G12T 3 1 Cll Q11N.N13T 3 1 C12 G12N, LHT 4 1 C13 N13, E15T 4 C14 L14N, R16T 4 1 C15 E15N, E17T 5 1 C16 M19N, E21T 5 1 C17 E20N, K22T 4 1 C18 S24N, E26T 4 1 T39N, F41T 4 1 E40N, W42T 4 1 F41N, K43T 3 1 W42N, Q44T 4 1 K43N, Y45T 3 1 Q44N, V46T 4 2 Y45N, D47T 3 1 V46N, G48T 2 1 D47N, D49T 1 1 G48N, Q50T 2 1 E52N, N54T 1 2 S53N, P55T 1 2 L57N, G59T 1 1 G60T 1 1 G59N, S61T 4 4 K63N, D65T 3 2 D64N, I66T 2 1 D65N, N67T 3 1 I66N, S68T 2 1 Y69T 1 1 S68N, E70T 4 1 P74N, G76T 1 1 C68 A103T C69 S102N, D104T C70 A103N, N105T C71 D104N, K106T C72 V107T C73 K106N, V108T C74 R116N, A118T C75 L117N, E119T C76 A118N.N120T C77 E119N.Q121T C78 K122T C79 Q121N, S123T E125N, P126A, C80 A127T P126N, V128T, C81 P129A C82 A127KP129T V128N, P129A, C83 F130T, P131A C84 P129N, P131T C85 R134N, S136T C86 V135N, V137T C98 P151N, V153T 4 4 C99 D152N, D154T 3 4 100 V153N, Y155T 4 4 101 D154N, V156T 3 4 C102 Y155N.N157T 3 4 C103 V156N, S158T 3 4 C104 N157 3 4 C105 S158N, E160T 3 4 C106 T159N, A161T 3 4 C107 E160N, E162T 3 3 C108 A161N 5 4 C109 E162N, I164T 6 4 C110 T163N, L165T 5 4 Clll I164N, D166T 5 4 C112 L165N.N167T 5 4 C113 D166N, I168T 5 4 C114 N167 5 4 C115 I168N, Q170T 6 4 C116 T169N, S171T 5 3 C117 Q170N 5 3 C118 S171N.Q173T 5 3 C119 T172N, S174T 5 3 K201T 2 2 G200N, V202T 2 1 K201N, D203T 3 3 V202N, A204T 3 2 I210N, N212T 1 1 V21 1N, E213T 1 1 K214T 1 1 E213N, W215T 1 1 V223N 1 1 E224N, G226T 4 2 T225N, V227T 3 3 G226N, 228T 2 2 V227N, I229T 1 3 K228N 3 3 H236N, I238T 1 1 E239T 2 3 1238N5 E240T 1 1 E239N 3 3 E240N, E242T 1 1 T241N, H243T 2 3 E242N 1 2 H243N, E245T 2 2 165 P255N, H257T 1 2 i66 H256N, N258T 1 1 167 H257N, Y259T 1 1 i68 N260T 1 1 169 Y259N, A261T 1 1 i70 A262T 4 3 ni A261N, I263T 2 3 i72 A262N, N264T 3 3 173 I263N, K265T 1 1 I74 Y266T 1 1 I75 K265N, N267T 1 1 176 Y266N, H268T 1 1 177 E274N, D276T 1 1 L275N, E277T, 178 P278A 1 1 179 D276N, P278T 1 2 E277N, P278A, 180 L279T 1 1 181 P278N, V280T 1 3 182 L279N, L281T 1 1 183 V280N, N282T 3 184 L281N, S283T 1 1 C198 R312N, F314T 5 1 C199 V313N, H315T 5 1 C200 F314N, K316T 4 1 C201 H315N, G317T 5 1 C202 K316N, R318T 4 1 C203 G317N, S319T 4 1 C204 R318N, A320T 3 1 C205 S319N, L321T 5 1 C206 A320N, V322T 4 1 L326N, V328T, C207 P329A 1 1 C208 R327N, P329T 4 1 V328N, P329A, C209 L330T 1 1 C210 P329N, V331T 3 1 C211 L330N, D332T 1 1 C212 V331N, R333T 1 1 C213 D332N, A334T 3 1 C214 R333N 1 1 C215 L337N, S339T 2 1 C216 R338N 4 1 C217 S339N, 341T 4 1 C231 T371N, V373T 1 1 C232 E372N, E374T 2 C233 V373N, G375T 1 1 C234 E374N 5 C235 W385N, E387T 3 1 C236 G386N, E388T 3 1 C237 A390N, K392T 3 1 C238 391N, G393T 3 1 C239 K392N, 394T 4 1 C240 G393N, Y395T 1 1 C241 R403N, V405T 2 1 C242 Y404N, N406T 1 1 C243 V405N, W407T 1 1 C244 I408T 1 1 C245 W407, K409T 1 1 C246 I408N, E410T 1 1 C247 K409N, 411 T 1 1 C248 E410N 2 C249 411 ?, 413? eleven C250 T412N, L414T 1 1 C251? 413? > ? 415 2 1 TABLE 5 5: Expression of FIX variants in HKB11 cells In order to determine whether the GIX genes with protein sequences alter and secrete in mammalian cells and to determine the effects on the coagulation activity of FIX, expression plasmids of FIX were transfected into HKB11 cells. HKB11 is one generated by the fusion of HEK293 cells and a B cell lymphoma. In addition to the substitutions at the glycosylation site it was combined with a, R338A, as shown for from HG1 to HG8 in Figure 3. The substitution the specific activity of FIX in 3-4 times, measured by the assay, additional combinations of two muteins 6, HG7, HG8, and HG3 were created and analyzed, plus HG8 had a coagulation activity that R338A and at least 300% of the Wild FIX. 6: Glycosylation of factor IX variants in HKB11 cells n initial analysis of the muteins HG1 to HG8 (Figure 3) and HG9 (Figure, HG3, HG5, HG6, HG8, and HG9 had higher glycosylation, such as or by an increase in apparent molecule weight in SD gels additional FIX polypeptides containing several combinations present in HG2, HG3, HG5, HG6, and HG8, For example, the following ions: HG2 / HG3, HG8 / HG2, HG8 / HG3, HG8 / HG2 / HG3, Ot ions include, for example, HG3 / HG5, HG5 / HG8, HG3 / HG5 / HG8, HG8 / HG6, HG3 / HG5 / HG6, HG3 / HG6 / HG8, HG5 / HG6 / HG8, and HG3 / HG can generate combinations with HG9. glycosylation increases the molecular weight of a protein and this can be reduced mobility in SDS-PAGE gels. As demonstrated in substitutions performed on HG2, HG3, HG5, HG6, HG8, and HG9, you have reduced mobility on an SDS-PAGE gel. Among these oc, HG8, which has two consensus sequences linked to additional Ns 338A. Since the fact that it would be expected that the substitution in H nidus to O and, therefore, increase the mobility on the gel, the finding in mobility suggests that the N-linked site introduced can be combinations of substitutions present in HG2 / HG3, HG8 / HG2, 2 / HG3 resulted in more significant reductions in the mutation with the individual muteins, indicating that the various ion sites present in these combinations were functional when linked to the FIX molecule. The mutein HG8 / HG2 / HG3 containing a total of 4 exhibited the greatest decrease in mobility. 7: Combination of substitutions R338A and V86A ? FIX amino acid V86 was changed to alanine by direct mutagenesis of wild FIX (WT-FIX) or FIX-R338A. The vectors of these constructs were transfected into HKB11 cells and the subsequent media and analyzed to determine the levels of FIX protein coagulation activity of FIX by an aPTT assay. Both on levels similar to WT-FIX and FIX-R338A. The data of an experiment in Table 6a. Table 6b summarizes the average of three experiments.

TABLE 6b The results show that the substitution V86A alone has as r to about 1.8 times the specific activity, while q results in an increase of 4.5 times the specific activity. La and V86A resulted in an 8.1-fold increase in ion activity with wild-type FIX. These results show that the positive effects R338A and V86A are additive and result in a mutein 8-fold increase in specific activity compared to WT-FIX 86A would have improved the therapeutic benefit for heme patients an 8-fold lower dose of protein to achieve the same recombinant ter effect currently available. In addition, the increase of a of R338A-V86A is beneficial when creating licosylated forms of AGCTTGATTAGTTAGTGAGA GGCCCTG) (SEQ ID No. 9) contained 22 nia of FIX that places 45 nucleotides 3 'of the end of the region co-produced by a Hindlll site. Amplification of the first strand of normal DNA (Stratagene, San Diego CA) using these primers and high fidelity polymers (Invitrogen, Carlsbad, CA) resulted in a single predicted for the human FIX cDNA (1464 bp). After co-digestion of the PCR it was gel purified and then cloned into the HindIII site. The clones into which the FIX cDNA was inserted in direct orientation of CV in the vector were identified by digestion of double-stranded DNA was performed for the insertion of several clones and the derivation with the published sequence of the FIX showed that the A year with threonine at amino acid 148 of the mature protein. This pAGE16-Factor IX (pAGE16-FIX). 9: Generation of modified FIX polypeptides to change several amino acids within the human FIX sequence of erasers using the Quickchange ™ ne design program. These primers were used to generate mutations in the plasmids TABLE 7 Sense oligonucleotide sequence N / K228T CTGCTGCCCACTGTGTTGAAACTAACGTTACCATTACA GTTGTCGC AGGTGAAC (SEQ ID NO: 10) N / H354V / E355T CACCATCTATAACAACATGTTCTGTGCTGGCAAGGTGA CCGGAGGTAGAGATTCATGTCAAGGAGATAGTG (SEQ ID NO: 1 1) N / T371V / E372T GATTCATGTCAAGGAGATAGTGGGGGACCCCATAACG TGACCGTGGAAGGGACCAGTTTCTTAACTGGAATTA (SEQ ID NO: 12) 1N / K392V / G393T GGAATTATTAGCTGGGGTGAAGAGTGTGCAAACGTGA CCAAATATGGAATATATACCAAGGTATCCCGG (SEQ ID NO: 13) N TCAACTGGATTAAGGAAAAAACAAACCTCACTTAATG AAAGATGGATTTC (SEQ ID NO: 14) N CTGATGTGGACTATGTAAATTCTACTGAAAATGAAACC ATTTTGGATAACATCAC (SEQ ID NO: 15) E / F178T AGTCAAAGCACCCAATCATTTAATGAGACCACTCGGGT TGTTGGTGG (SEQ ID NO: 16) N / V153T ACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTAATGA TACGGACTATGTAAATTCTACTG (SEQ ID NO: 17) N GAAGCTGAAACCATTTTGGATAACATCAATCAAAGCA The underlined ones are those that changed with respect to the FIX salvajce consensus for N-linked glycosylation.

In addition to the substitutions described above, an innocuous S was inserted containing 2 consensus sequences for the N-glycosylation between residues A161 and E162. To generate this variant of the restriction FI unique for SnaBI and Xbal in Y155 and 1164 without altering the s by site-directed mutagenesis with primers t8216c "g821. The resulting plasmid was digested with SnaBI and Xbal to remove the fragment to residues V156 to 1164 and then ligated to a fragment by hybridization of the 2PrimerF and 2PrimerR oligonucleotides (a of the resulting plasmid was determined by sequencing of AD to a 27 bp insertion encoding nine amino acids with ITQ (SEQ ID NO: 2) containing two consensus sequences for glycos).

TABLE 8 10: Cell culture and transient transfection HKB11 cells (a hybrid of HEK293 and a lymphoma cell line cultured in suspension culture on an orbital shaker (100-125 r to C02 (5%) at 37 ° C in serum-free medium (RF No. 277) supplemented with Soluble K3 (Sigma-Aldrich, St. Louis, MO) and maintained at a density of 106 cells / ml. s cells for transfection were collected by centrifugation for 5 minutes, then suspended in Free n expression medium, Carlsbad, CA) at 1.1 x 106 i. The cells were seeded in 6-well plates (4.6 ml / well) and irradiated (125 rpm) in a C02 incubator at 37 ° C. For each well, 5 or were mixed with 0.2 ml of Opti-MEM® I medium (Invitrogen). For 293fectin ™ body (Invitrogen), they were gently mixed with 0m2 ml of and incubated at room temperature for 5 minutes. The reagent was added to the solution with diluted DNA, mixed gently, in room environment for 20-30 minutes, and then 5 x 106 (4.6 ml) of HKB11 cells were added to each well. Afterwards, the cells were incubated. 11: Western blot for FIX.

The supernatant of the cell culture (50 μ?) was mixed with 20 μ? of AGE pigment, heated at 95 ° C for 5 minutes, loaded on NuP A E gels and then transferred to nitrocellulose membranes. After milk OIVO for 30 minutes, the membranes were incubated with goat ul labeled HRP against human FIX (US Biological, usetts, catalog number F0017-07B) for 60 minutes at wash temperature with phosphate buffered saline with 0.1% HRP buffer was detected using SuperSignal® Peak (Pierce, Rockford, IL) and X-rays. 12: FIX ELISA s FIX antigen levels in the culture supernatants were used using a FIX ELISA kit (Hyphen Biomed / Aniara), but before the cell culture was diluted in sample diluent buffer (supply a signal within the ranges of the standard curve. Human plasma ((Hyphen Biomed / Aniara, catalog number RK032 at 196 U / mg) diluted in sample diluent was used to create a commercially available FIX cuvette (Haemochrom Diagnostica Gm) according to the manufacturer's instructions. The ™ plates (Nunc ™, Rochester, NY) were coated with tt germ agglutinin Louis, MO.) The wells were blocked, washed and then washed, and after further washing the detection was carried out using antibody p. side to HRP (Haemochrom Diagnostica GmbH, Essen, Germany). 13: FIX coagulation assay FIX coagulation activity was determined using a human-deficient FIX assay performed on an 1800C automated analyzer (Beckman Coulter, Fullerton, CA). Briefly, by inserting dilutions of supernatant samples in coagulation diluent aron 100 μ? with 100 μ? of plasma deficient in FIX (Aniara, Mason, OH) for automatic aPTT (brain phospholipid and micronized silica (bio NC), after the addition of 100 μl of 25 mM CaCl2 solution and the formation of the clot. a standard curve for each rs in series of the same purified human FIX (Hyphen Biomed / Aniara) the ELISA assay.The standard curve was, routinely, a line is in the fiber knob domain, it does not bind to FIX. it has a liver cells and produce liver toxicity in vivo signif, which deduces that FIX plays a major role in targeting liver cells (Shayakhmetov, et al., 2005). Liver cells can be blocked with protein-HSPG inhibitions (Shayakhmetov, et al., 2005).

In addition, the HSPG-mediated uptake of FIX contributes to a FIX ivo and, consequently, it can be expected that the interference with the G will increase the half-life of the FIX. Therefore, the in vitro uptake of FIX in hepatocytes is measured and it is expected that the variants with lower capture x half-life in vivo.

To measure the half-life of FIX in vitro, mammalian cells are used in the presence or absence of FIX or FIX variants. Wild viral uptake and is measured by expression of the encoded reporter gene and example green fluorescent protein (GFP) or luciferase expression. The adenovirus ion in the presence of FIX variants is measured in the form of the reporter gene, for example reduction of the fluorescence of the GFP eve a correlation between the lower cellular uptake and the in vitr assay and the in vivo assay. the publications and patents mentioned in the specification n in the present specification by reference. Various modi? Of the described methods of the invention described will be evident in the art without deviating from the scope and spirit of the invention. The invention has been described in connection with enderse embodiments that the invention, as claimed, should not be limited to specific embodiments. In fact, it is intended that various methods described in the foregoing for carrying out the invention which are in the field of biochemistry or related fields are within the following disclosures. Those skilled in the art will recognize, or may or more than routine experimentation, many equivalents to the specific n of the invention described herein are intended to be within the following claims.

Claims (3)

CLAIMS . A factor IX polypeptide characterized in that it comprises a factor that has been modified by introducing one or more substitutions or in two. . The polypeptide according to claim 1, characterizing the amino acids has been modified by introducing one or more glycine sites . The polypeptide according to claim 2, characterized by glycosylation Ss are selected from the sites of N-linked glycosylation and O-linked ion. . The polypeptide according to claims 2 and 3, characterized in that it comprises a carbohydrate chain attached to the one or mion introduced. . The polypeptide according to any one of the claims E30N and F32T; V31 N and E33T; F32N and N34T; T35N and R37T; T38N and E 0N and W42T; F41N and K43T; W42N and Q44T; K43N and Y45T; Q44N and V 6N and G48T; E52N and N54T; S53N and P55T; G59N and S61T; K63N and D65T; 1 70T; G76N and E78T; E78N and K80T; E83N and D85T; L84N and V86T; I90N and N S102N and D104T; A103N and N105T; D104N and K106T; K106N and V108T; Rl 1 Q121 T; Q121 N and S123T; A127N and P129T; V135N and V137T; S136N and S13 139N and S141T; T140N and K142T; S141 N and L143T; E147N and V149T; T14 P151T; P151 N and V153T; D152 and D154T; V153N and Y155T; D154N and V15 156N and S158T; S158N and E160T; T159N and A161T; E160N and E162T; E16 L165T; I164N and D166T; L165N and N167T; D166N and I168T; I168N and Q17 171N and Q173T; T172N and S174T; Q173N and F175T; S174N and N176T; G18 A187T; D186N and K188T; A187N and P189T; P189N and Q191T; G200N and V20 202N and A204T; E224N and G226T; T225N and V227T; G226N and K228T; V2 I238T; I238N and E240T; E240N and E242T; T241 N and H243T; H243N and E24 250N and R252T; 125 IN and I253T; I253N and P255T; A261 N and I263T; A26 P278T; V280N and N282T; F302N and S304T; S304N and Y306T; R312N and F31 314N and K316T; H315N and G317T; K316N and R318T; G317N and S319T; S31 1251 T; T148N, F150T, G226N, and K228T; P151N, V153T, T172N, and R3 177E, and F178T; P151, V153T, G226N, and K228T; T172N, G226N, K228 122T, P151 N, V153T, and E242N; D85N, P151 N, V153T, G226N, and K2 153T, G226N, and K228T; S138N, P151 N, V153T, G226N, and K228T; T1 228T, and R338A; P151N, V153T, T172N, G226N, and K228T; P151N, V1 R338A; P151 N, V153T, G226N, K228T, and R338A; and P151 N, V153T, T1 R338A; and any combination thereof. . The polypeptide according to any one of the claims because the one or more amino acid substitutions is selected 122T; S138N; A146N; A161 N; Q170N; T172N; D177N; F178T; K20 242N; 1251 T; A262T; E294N; E374N; E410N; G59N and S61T; K63N and D 02N and D104T; A103N and N105T; D104N and K106T; The 19N and Q121T; Q12 S138T; Q139N and S141T; T140N and K142T; T148N and F150T; V149N and P15 152N and D154T; V153N and Y155T; D154N and V156T; V156N and S158T; S15 E162T; E162N and I164T; T163N and L165T; I164N and D166T; D166N and I1 171 N and Q173T; T172N and S174T; Q173N and F175T; S174N and N176T; K20 . The polypeptide according to any one of those claimed because the one or more amino acid substitutions is selected as 138N; T172N; K201 N; K228N; E239N; E242N; 1251 T; A262T; E294N; G 78T; S102N and D104T; A103N and N105T; D104N and K106T; The 19N and Q12 136N and S138T; Q139N and S141T; T140N and K142T; T148N and F150T; V14 V153T; D152N and D154T; S158N and E160T; E162N and I164T; T163N and L16 173N and F175T; K201 N and D203T; T225N and V227T; G226N and K228T; I25 N264T; V280N and N282T; E372N and E374T; and D85N, K122T, and E242N; D8 150N, P151A, and D152T; T172N, G226N, and K228T; D85N, P151N, V153 151 N, V153T, and E242N; K122T, P151 N, V153T, and K228N; K122T, P151 K122T, P151N, V153T, and 1251 T; P151, V153T, G226N, and K228T; D 153T, and E242N; D85N, P151 N, V153T, G226N, and K228T; S138N, P1 K228T; P151 N, V153T, T172N, G226N, and K228T. . A factor IX polypeptide characterized in that it comprises the s EEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQ YVDGDQCE Xs6 is selected from A, E, P, S, and V; X104 is selected from D, N, and T X12i is selected from N, Q, and T; X38 is selected from N, S, and T; Xi4s and Xi5o are selected from: ) Xi48esTyX150 is F; i)? 14ß is N and X150 is T; Y i) Xi48 isNyX150 is S; X151 is selected from A, P and T; Xi5i and X153 are selected from: ) X15i esPyX153esV; i) X15i is N and X153 is T; Y Z-i, Z2, Z3, and Z4 are independently selected from zero to twelve amino acid residues and i) SEQ ID No. 2; Xi52 is selected from D, N and T; ü) X177 is E or D and X178 is S; X226 and X228 are selected from: i) X226 is N and X228 is T; Y ii) X226 is N and X228 is S; X338 is selected from R and A; X353, X354 and X355 are selected from: ) X353 is F, X354 is H, X355 is E; i) X353 is N, X354 is V, X355 is T; ii) X353 is N, X354 is I, X355 is T; Y ) X353 is N, X354 is H, V, or I, X355 is S; X370 »X371 and X372 are selected from: ) X370 is V, X371 is T, X372 is E; i) X370 is N, X371 is V, X372 is T; ii) X370 is N, Xa71 is I, X372 is T; Y ) X370 is N, X371 is T, V, or I, X372 is S; and the polypeptide comprises at least one site of glycosylation int ion with the FIX polypeptide having SEQ ID No. 1. . The polypeptide according to claim 6 or 7, characterized by substitutions of selected amino acids of R338A and V86 . The polypeptide according to any one of those claimed because the amino acid sequence has been modified by introducing the amino acid between amino acid residues 160-164 of factor IX or results in the introduction of one or more glycosylation sites.

1. The polypeptide according to claim 10, characterized in amino acids are inserted between amino acid residues 160-1 of amino acids 161-162, between amino acid residues 162-163 of amino acids 163-164.

2. The polypeptide according to claim 11, characterized . The polypeptide according to any one of those claimed because the amino acid sequence has been modified by introducing the amino acid at the C-terminus of the Factor IX polypeptide having co-tion of one or more glycosylation sites. . The polypeptide according to claim 14, characterized in that it comprises the amino acid sequence selected from SEC I EC ID No. 6 and SEQ ID NO: 7 is introduced at the C-terminus of the polypeptide. . A factor IX polypeptide characterized in that it comprises a V86A substitution. . The polypeptide according to any one of those claimed in that the binding of a carbohydrate chain in one or more introduced ilaments increases the serum half-life of the polypeptide in . A method of treating hemophilia B, characterized in that a subject in need thereof has a therapeutically effective amount of the ttca of claim 19. 1. A DNA sequence characterized in that it encodes the polypeptide of claims 1-18. . A eukaryotic host cell characterized in that it is transferable from DNA according to claim 20 so that a factor IX polypeptide is allowed to express.

3. A method for producing a factor IX C polypeptide comprises (i) modifying the amino acid sequence of the ion polypeptide of one or more glycosylation sites; (ii) expressing the glycosylation polypeptide at the one or more glycosylation sites; and (iii) purifying the poly