AU2013202563A1 - Method of increasing the in vivo recovery of therapeutic polypeptides - Google Patents

Abstract

The present invention relates to the field of modified therapeutic polypeptides with increased in vivo recovery compared to their non-modified parent polypeptide, i.e., the invention relates to fusions of therapeutic polypeptides with recovery enhancing polypeptides connected directly or optionally connected by a linker peptide.

Description

5 Method of increasing the in vivo recovery of therapeutic polypeptides Field of the invention: 10 The present invention relates to the field of modified therapeutic polypeptides with increased in vivo recovery compared to their non-modified parent polypeptide. I.e., the invention relates to fusions of therapeutic polypeptides with recovery enhancing polypeptides connected directly or optionally connected by a linker peptide. 15 The gist of the invention is demonstrated in particular by vitamin K-dependent polypeptides like e.g. human Factor VII, human Factor Vila, human Factor IX, and human protein C as the therapeutic polypeptide and albumin as the recovery enhancing polypeptide. Therefore, in particular, the invention also relates to cDNA 20 sequences coding for any of the vitamin K-dependent polypeptides and derivatives genetically fused to a cDNA coding for human serum albumin which may be linked by oligonucleotides which code for intervening peptidic linkers, such encoded derivatives exhibiting improved in vivo recovery, recombinant expression vectors containing such cDNA sequences, host cells transformed with such recombinant 25 expression vectors, recombinant polypeptides and derivatives which do have biological activities comparable to the unmodified wild type polypeptide but having improved in vivo recovery and processes for the manufacture of such recombinant polypeptides and their derivatives. The invention also covers a transfer vector for use in human gene therapy, which comprises such modified DNA sequences useful 30 to increase product levels in vivo.

frame in a way that expression in a host cell in which said genetic construct is introduced, generates a protein in which the therapeutic polypeptide is linked by peptide linkage to the recovery enhancing polypeptide. Optionally the therapeutic polypeptide and the recovery enhancing polypeptide can also be connected by a 5 short peptidic linker. Vitamin K-dependent polypeptides Vitamin K-dependent polypeptides which are posttranslational modified by gamma carboxylation and comprise e.g. the blood coagulation factors 1I (prothrombin), Vil, 10 IX, and X, the anticoagulant proteins C and S, and the thrombin-targeting protein Z, the bone protein osteocalcin, the calcification inhibiting matrix protein, the cell growth regulating growth arrest specific gene 6 protein (Gas6), and the four transmembrane Gla proteins (TMGPs) the function of which is at present unknown. Among those polypeptides some are used to treat certain types of hemophilia and 15 bleeding disorders. Hemophilia A is an inherited bleeding disorder. It results from a chromosome X-inked deficiency of blood coagulation Factor Vill and the clinical manifestation is an increased bleeding tendency. The disease is treated by injection of FVIll concentrates from plasma or recombinant sources. Hemophilia B is caused by non-functional or missing Factor IX and is treated with Factor IX concentrates 20 from plasma or a recombinant form of Factor IX, In both hemophilia A and in hemophilia B the most serious medical problem in treating the disease is the generation of alloantibodies against the replacement factors. Up to about 30% of all hemophilia A patients develop antibodies to Factor Vill. Antibodies to Factor IX are less frequent. 25 The current model of coagulation states that the physiological trigger of coagulation is the formation of a complex between tissue Factor (TF) and Factor Vila (FVIla) on the surface of TF expressing cells, which are normally located outside the vasculature. This leads to the activation of Factor IX and Factor X ultimately 30 generating some thrombin. In a positive feedback loop thrombin -directly or indirectly- activates Factor Vill and Factor IX, the so-called "intrinsic" arm of the IXa, Factor Vila, Factor Seven Activating Protease (FSAP) and thrombin. Mollerup et al. (Biotechnol. Bioeng. (1995) 48: 501-505) reported that some cleavage also occurs in the heavy chain at Arg290 and or Arg315. 5 Factor Vil is present in plasma in a concentration of about 500 ng/ml. About 1% or 5 ng/ml of Factor VII are present as Factor Vila. Plasma half-life of Factor Vil was found to be about 4 hours and that of Factor Vila about 2 hours. The half-life of Factor Vila of 2 hours constitutes a severe drawback for the therapeutic use of Factor Vila, as it leads to the need of multiple i.v. injections or continuous infusion 10 to achieve hemostasis. This results in very high treatment cost and inconvenience for the patient. Both, improvement in plasma half-life and in vivo recovery, would bring benefit to the patient. Up to now no pharmaceutical preparation of a Factor Vila with improved in vivo recovery is commercially available nor have any data been published showing FVII/FVIlIa variants with improved in vivo recovery. As 15 Factor VIINIla has the potential to be used as a universal hemostatic agent, a high medical need still exists to develop forms of Factor Vila which have an improved in vivo recovery. Factor IX 20 Human FIX is a single-chain glycoprotein with a molecular weight of 57 kDa, which is secreted by liver cells into the blood stream as an inactive zymogen of 415 amino acids. It contains 12 yearboxy-glutamic acid residues localized in the N-terminal Gla-domain of the polypeptide. The Gla residues require vitamin K for their biosynthesis. Located C-terminal to the Gla domain are two epidermal growth factor 25 domains and an activation peptide followed by a trypsin-type serine protease domain. Further posttranslational modifications of FIX encompass hydroxylation (Asp 64), N- (Asn157 and Asn167) as well as 0-type glycosylation (Ser53, Ser61, Thr159, Thr169, and Thr172), sulfation (Tyr155), and phosphorylation (Ser158). 30 FIX is converted to its active form Factor IXa by proteolysis of the activation peptide at Arg145-Ala146 and Arg18O-Val181 leading to the formation of two polypeptide therapeutic polypeptides also Factor IX and FVII/FVIla are mentioned as examples of the invention. Ballance et al. is silent about in the vivo recovery of such fusion proteins. 5 In vivo recovery of vitamin K-dependent polypeptides In vivo recovery of recombinant FIX (BeneFIX, Genetics Institute) of 0.84 - 0.86 IU/dL per IU/kg has been reported to be significantly lower in haemophilia B patients than that of plasma derived FIX like Mononine of 1.17 - 1.71 IU/dL per IU/kg (White G. et al., Semin Hematol 35 (Suppl. 2): 33-38 (1998); Ewenstein B.M. 10 et al., Transfusion 42(2): 190-197 (2002)). As a consequence, at least 20% higher amounts of recombinant FIX have to be applied in comparison to plasma derived FIX to achieve comparably efficient treatment of hemophilia B. Sheffield (Sheffield WP et al. (2004) Br. J. Haematol. 126:565-573) expressed a 15 human Factor IX albumin fusion polypeptide and showed in pharmacokinetic experiments that in FIX knockout mice, the in vivo recovery of the human FIX albumin fusion protein was significantly lower (less than half) than the unfused human FIX molecule. 20 In vivo recovery of recombinant FVIla (NovoSeven, Novo Nordisk) has been reported to be about 19 to 22% in FVI deficient patients (Berrettini M et al. 2001. Haematologica 86:640-645) and about 46-48% in hemophilia patients (Lindley CM et al, 1994. Clin. Pharmacol. Ther. 55:638-648). Likewise the in vivo recovery of rFVlla was described at about 34% in hemophilia A dogs and about 44% in 25 hemophilia B dogs, respectively (Brinkhous KM et al., 1989. Proc. Nat. Acad. Sci. 86:1382-1386). 80 Another aspect of the invention are vitamin K-dependent polypeptides fused to the N- or C-terminus of albumin or any other recovery enhancing polypeptide. The fusion proteins display a significant increase of the in vivo recovery of the respective recombinantly produced, wild-type vitamin K dependent polypeptides. 5 A further aspect of the invention are fusion proteins in which Factor VIINIla polypeptides are fused to the N-terminus of albumin which display a significant increase of the in vivo recovery as compared to unfused, recombinantly produced Factor VIlNIla. 10 Another aspect of the invention are fusion proteins in which Factor IX polypeptides are fused to the N-terminus of albumin which display a significant increase of the in vivo recovery as compared to unfused Factor IX. 15 One aspect of the invention are therefore vitamin K dependent polypeptides fused to the N- or C-terminus of albumin increasing the in vivo recovery compared to the corresponding recombinant non-fused polypeptide by at least 10%, preferably more than 25%, even more preferably more than 40%. 20 The invention encompasses therapeutic polypeptides, in particular vitamin K dependent polypeptides linked to the N- or C-terminus of a recovery enhancing polypeptide like albumin, compositions, pharmaceutical compositions, formulations and kits. The invention also encompasses the use of said recovery enhancing polypeptide linked therapeutic polypeptides in certain medical indications in which 25 the unfused therapeutic polypeptides also would be applicable. The invention also encompasses nucleic acid molecules encoding the recovery enhancing polypeptides linked therapeutic polypeptides of the invention, as well as vectors containing these nucleic acids, host cells transformed with these nucleic acids and vectors, and methods of making the recovery enhancing polypeptides linked 30 therapeutic polypeptides of the invention using these nucleic acids, vectors, and/or host cells.

dependent polypeptide is joined in-frame to the 5'end of a polynucleotide encoding all or a portion of albumin optionally linked by a polynucleotide which encodes a linker sequence, introducing a linker peptide between the vitamin K dependent polypeptide moiety and the albumin moiety). 5 In one embodiment, the invention provides a vitamin K dependent polypeptide albumin fusion polypeptide comprising, or alternatively consisting of biologically active or activatable and/or therapeutically active or activatable vitamin K dependent polypeptide fused to the N-terminus of a serum albumin polypeptide. 10 in other embodiments, the invention provides an albumin fusion polypeptide comprising, or alternatively consisting of, a biologically active or activatable and/or therapeutically active or activatable fragment of a vitamin K dependent polypeptide and a peptidic linker fused to the N-terminus of a serum albumin. 15 In other embodiments, the invention provides a vitamin K dependent polypeptide albumin fusion polypeptide comprising, or alternatively consisting of, a biologically active or activatable and/or therapeutically active or activatable variant of a vitamin K dependent polypeptide fused to the N-terminus of a serum albumin 20 polypeptide and optionally a peptidic linker. In further embodiments, the invention provides a vitamin K dependent polypeptide albumin fusion polypeptide comprising, or alternatively consisting of, a biologically active or activatable and/or therapeutically active or activatable fragment or variant 25 of a vitamin K dependent polypeptide fused to the N-terminus of a fragment or variant of serum albumin and optionally a peptidic linker. In some embodiments, the invention provides an albumin fusion polypeptide comprising, or alternatively consisting of, the mature portion of a vitamin K 30 dependent polypeptide fused to the N-terminus of the mature portion of serum albumin and optionally a peptidic linker.

25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of specific domains of HA. The albumin portion of the albumin-linked polypeptides of the invention may be a 5 variant of normal HA. The vitamin K dependent polypeptide portion of the albumin linked polypeptides of the invention may also be variants of the vitamin K dependent polypeptides as described herein. The term "variants" includes insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the active site, or active domain 10 which confers the therapeutic activities of the vitamin K dependent polypeptides. In particular, the albumin-linked polypeptides of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin. The albumin may be derived from any vertebrate, especially any mammal, for 15 example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin-linked polypeptide may be from a different animal than the vitamin K dependent polypeptide portion. Generally speaking, an albumin fragment or variant will be at least 20, preferably at 20 least 40, most preferably more than 70 amino acids long. The albumin variant may preferentially consist of or alternatively comprise at least one whole domain of albumin or fragments of said domains, for example domains I (amino acids 1-194 of SEQ ID NO:20), 2 (amino acids 195-387 of SEQ ID NO: 20), 3 (amino acids 388-585 of SEQ ID NO: 20), 1 + 2 (1-387 of SEQ ID NO: 20), 2 + 3 (195-585 of 25 SEQ ID NO: 20) or I + 3 (amino acids 1-194 of SEQ ID NO: 20 + amino acids 388-585 of SEQ ID NO: 20). Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to G1u119, Glu292 to ValI315 and Glu492 to Ala5 11. 30 variants differ in one or more amino acid residues from the wild type sequence. Examples of such differences may include truncation of the N- and/or C-terminus by one or more amino acid residues (e.g. I to 10 amino acid residues), or addition of one or more extra residues at the N- and/or C-terminus, as well as conservative 5 amino acid substitutions, i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, and (6) aromatic amino acids. Examples of such conservative substitutions are shown in table 1. 10 Table I (1) Alanine Glycine (2) Aspartic acid Glutamic acid (3a) Asparagine Glutamine (3b) Serine Threonine (4) Arginine Histidine Lysine (5) Isoleucine Leucine Methionine Valine (6) Phenylalanine Tyrosine Tryptophane The vitamin K dependent polypeptide albumin fusions of the invention have at least 10%, preferably at least 25% and more preferably at least 40% increased in vivo 15 recovery compared to unfused vitamin K dependent polypeptides. The in vivo recovery of the Factor VII albumin linked polypeptides of the invention is usually at least about 10%, preferably at least about 25%, more preferably at least about 40% higher than the in vivo recovery of the wild type form of human Factor 20 VII.

polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. The polynucleotide may be single- or double-stranded DNA, single or double stranded RNA. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs that comprise one or more modified bases and/or unusual bases, such as 5 inosine. It will be appreciated that a variety of modifications may be made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for 10 example, simple and complex cells. The skilled person will understand that, due to the degeneracy of the genetic code, a given polypeptide can be encoded by different polynucleotides. These "variants" are encompassed by this invention. 15 Preferably, the polynucleotide of the invention is an isolated polynucleotide. The term "isolated" polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides may be purified 20 from a host cell. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides. Yet another aspect of the invention is a plasmid or vector comprising a 25 polynucleotide according to the invention. Preferably, the plasmid or vector is an expression vector. In a particular embodiment, the vector is a transfer vector for use in human gene therapy. Still another aspect of the invention is a host cell comprising a polynucleotide of the 30 invention or a plasmid or vector of the invention.

domain, disulfide bond formation, asparagine-linked glycosylation, O-linked glycosylation, and other post-translational modifications as well as secretion into the cultivation medium. Examples of other post-translational modifications are tyrosine O-sulfation, hydroxyiation, phosphorylation, proteolytic processing of the nascent 5 polypeptide chain and cleavage of the propeptide region. Examples of cell lines that can be use are monkey COS-cells, mouse L-cells, mouse C127-cells, hamster BHK-21 cells, human embryonic kidney 293 cells, and hamster CHO-cells. The recombinant expression vector encoding the corresponding cDNAs can be 10 introduced into an animal cell line in several different ways. For instance, recombinant expression vectors can be created from vectors based on different animal viruses. Examples of these are vectors based on baculovirus, vaccinia virus, adenovirus, and preferably bovine papilloma virus. 15 The transcription units encoding the corresponding DNAs can also be introduced into animal cells together with another recombinant gene which may function as a dominant selectable marker in these cells in order to facilitate the isolation of specific cell clones which have integrated the recombinant DNA into their genome. Examples of this type of dominant selectable marker genes are Tn5 amino 20 glycoside phosphotransferase, conferring resistance to geneticin (G418), hygromycin phosphotransferase, conferring resistance to hygromycin, and puromycin acetyl transferase, conferring resistance to puromycin. The recombinant expression vector encoding such a selectable marker can reside either on the same vector as the one encoding the cDNA of the desired polypeptide, or it can be 25 encoded on a separate vector which is simultaneously introduced and integrated to the genome of the host cell, frequently resulting in a tight physical linkage between the different transcription units. Other types of selectable marker genes which can be used together with the cDNA 30 of the desired protein are based on various transcription units encoding dihydrofolate reductase (dhfr). After introduction of this type of gene into cells An example of such purification is the adsorption of the recombinant protein to a monoclonal antibody or a binding peptide, which is immobilised on a solid support. After desorption, the protein can be further purified by a variety of chromatographic techniques based on the above properties. 5 It is preferred to purify the therapeutic polypeptide e.g. the vitamin K dependent polypeptide albumin fusion of the present invention to greater than 80% purity, more preferably greater than 95% purity, and particularly preferred is a pharmaceutically pure state that is greater than 99.9% pure with respect to 10 contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, an isolated or purified therapeutic polypeptide e.g. a vitamin K dependent polypeptide albumin fusion of the invention is substantially free of other polypeptides. 15 The therapeutic polypeptide, respectively vitamin K dependent polypeptide albumin fusion described in this invention can be formulated into pharmaceutical preparations for therapeutic use. The purified proteins may be dissolved in conventional physiologically compatible aqueous buffer solutions to which there may be added, optionally, pharmaceutical excipients to provide pharmaceutical 20 preparations. Such pharmaceutical carriers and excipients as well as suitable pharmaceutical formulations are well known in the art (see for example "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & 25 Francis (2000) or "Handbook of Pharmaceutical Excipients" 3 rd edition, Kibbe et al Pharmaceutical Press (2000)). In particular, the pharmaceutical composition comprising the polypeptide variant of the invention may be formulated in lyophilized or stable soluble form. The therapeutic polypeptide may be lyophilized by a variety of procedures known in the art. Lyophilized formulations are reconstituted prior to 30 use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

The modified DNA's of this invention may also be integrated into a transfer vector for use in the human gene therapy. Another aspect of the invention is the use of a therapeutic polypeptide of the 5 invention e.g. an albumin-linked vitamin K dependent polypeptide as described herein, of a polynucleotide of the invention, of a plasmid or vector of the invention, or of a host cell of the invention for the manufacture of a medicament for the treatment or prevention of bleeding disorders. Bleeding disorders include but are not limited to hemophilia A. In another embodiment of the invention, the treatment 10 comprises human gene therapy. The invention also concerns a method of treating an individual in one or more of the following indications: "Haemophilia A or B", "bleeding episodes in patients with inherited or acquired coagulation deficiencies", "vascular occlusion episodes like 15 e.g. thrombosis in patients with inherited or acquired factor deficiencies", "sepsis", "bleeding episodes and surgery in patients with inherited or acquired hemophilia with inhibitors to coagulation Factors (FVIII or FIX)", "reversal of hemostasis deficits developed as consequence of drug treatments such as anti-platelet drugs or anti coagulation drugs", "improvement of secondary hemostasis", "hemostasis deficits 20 developed during infections or during illnesses such as Vitamin K deficiency or severe liver disease", "liver resection", "hemostasis deficits developed as consequences of snake bites", "gastro intestinal bleeds". Also preferred indications are "trauma", "consequences of massive transfusion (dilutional coagulopathy)", "coagulation factor deficiencies other than FVIll and FIX", "VWD", "Fl deficiency", 25 "FV deficiency", "Fy1i deficiency", "FX deficiency", "FXIII deficiency", "HUS", "inherited or acquired platelet diseases and disorders like thrombocytopenia, ITP, TTP, HELLP syndrome, Bernard-Soulier syndrome, Glanzmann Thrombasthenia, HIT", "Chediak-Higahi Syndrom", "Hermansky-Pudlak-Syndrome", "Rendu-Osler Syndrome", "Henoch-Schontein purpura", "Wound Healing", and "Sepsis". The 30 method comprises administering to said individual an efficient amount of the vitamin K-dependent albumin linked polypeptide as described herein. In another EXAMPLES: Example 1: Generation of cDNAs encoding FVII and FVII - albumin fusion proteins 5 Factor VII coding sequence was amplified by PCR from a human liver cDNA library (ProQuest, Invitrogen) using primers We1303 and We1304 (SEQ ID NO I and 2). After a second round of PCR using primers We1286 and We1287 (SEQ ID NO 3 and 4) the resulting fragment was cloned into pCR4TOPO (Invitrogen). From there the FVI1 cDNA was transferred as an EcoRI Fragment into the EcoRI site of 10 plRESpuro3 (BD Biosciences) wherein an internal Xhol site had been deleted previously. The resulting plasmid was designated pFVII-659. Subsequently an Xhol restriction site was introduced into pFVII-659 at the site of the natural FVIl stop codon (figure 1) by site directed mutagenesis according to 15 standard protocols (QuickChange XL Site Directed Mutagenesis Kit, Stratagene) using oligonucleotides We1643 and We 1644 (SEQ ID NO 5 and 6). The resulting plasmid was designated pFVII-700. Oligonucleotides We1731 and We1732 (SEQ ID NO 7 and 8) were annealed in 20 equimolar concentrations (10 pmol) under standard PCR conditions, filled up and amplified using a PCR protocol of a 2 min. initial denaturation at 940C followed by 7 cycles of 15 sec. of denaturation at 94*C, 15 sec. of annealing at 55*C and 15 sec. of elongation at 72*C, and finalized by an extension step of 5 min at 720C. The resulting fragment was digested with restriction endonucleases Xhol and Notl and 25 ligated into pFVII-700 digested with the same enzymes. The resulting plasmid was designated pFVII-733, containing coding sequence for FVII and a C-terminal extension of a thrombin cleavable glycine/serine linker. Based on pFVII-733 other linkers without thrombin cleavage site and additional N 30 glycosylation sites were inserted. For that primer pairs We2148 and We2149 (SEQ ID NO 9 and 10), We2148 and We2151 (SEQ ID NO 9 and 11), We2152 and 31 and 32) deleting the stop codon and introducing an Xhol site instead. The resulting FIX fragment was digested with restriction endonucleases EcoRI and Xhol and ligated into an EcoRi / BamHl digested pIRESpuro3 together with one Xhol / BamH1 digested linker fragment as described below. 5 Two different glycine / serine linker fragments were generated: Oligonucleotides We2148 and We2150 (SEQ ID NO 9 and 33) were annealed in equimolar concentrations (10 pmol) under standard PCR conditions, filled up and amplified using a PCR protocol of a 2 min. initial denaturation at 94*C followed by 7 cycles of 10 15 sec. of denaturation at 94"C, 15 sec. of annealing at 550C and 15 sec. of elongation at 72*C, and finalized by an extension step of 5 min at 720C. The same procedure was performed using oligonucleotides We2156 and We2157 (SEQ ID NO 15 and 16). 15 The resulting linker fragments were digested with restriction endonucleases Xhol and BamH1 and used separately in the above described ligation reaction. The resulting two plasmids therefore contained the coding sequence for FIX and a C terminal extension of a glycine/serine linker. In the next cloning step these plasmids were digested with BamH1 and a BamHl fragment containing the cDNA of mature 20 human albumin was inserted. This fragment had been generated by PCR on an albumin cDNA sequence using primers We1862 and We1902 (SEQ ID NO 17 and 18) under standard conditions. The final plasmids were designated pFIX-980 and pFIX-986, respectively. Their linker sequences and the C-terminal FIX and N terminal albumin sequences are outlined in figure 3. 25 For efficient processing of the propeptide in cells expressing FIX in high amounts coexpression of furin is required (Wasley LC et al. 1993. PACE/Furin can process the vitamin K-dependent pro-factor IX precursor within the scretory pathway. J. Biol. Chem. 268:8458-8465). Furin was amplified from a liver cDNA library (Ambion) 30 using primers We1791 and We1792 (SEQ ID NO 34 and 35). A second round of PCR using primers We1808 and We1809 (SEQ ID NO 36 and 37) yielded a furin S29 FVIl antigen and activity were determined as described in example 5. Example 5: Determination of FVII activity and antigen. FVIl activity was determined using a commercially available chromogenic test kit 5 (Chromogenix Coaset F\11) based on the method described by Seligsohn et al. Blood (1978) 52:978-988. FVIla activity was determined using a commercially available test kit (STACLOT*)Vila-rTF, Diagnostica Stago) based on the method described by 10 Morissey et al. (1993) Blood 81:734-744. FVIli antigen was determined by an ELISA whose performance is known to those skilled in the art. Briefly, microplates were incubated with 120 pL per well of the capture antibody (sheep anti human FVIl IgG, Cedarlane CL20030AP, diluted 15 1:1000 in Buffer A [Sigma C3041]) overnight at ambient temperature. After washing plates three times with buffer B (Sigma P3563), each well was incubated with 200 pL buffer C (Sigma P3688) for one hour at ambient temperature. After another three wash steps with buffer B, serial dilutions of the test sample in buffer B as well as serial dilutions of standard human plasma (Dade Behring; 50 - 0.5 mU/mL) in 20 buffer B (volumes per well: 100 pL) were incubated for two hours at ambient temperature. After three wash steps with buffer B, 100 pL of a 1:5000 dilution in buffer B of the detection antibody (sheep anti human FVII IgG, Cedarlane CL20030K, peroxidase labelled) were added to each well and incubated for another two hours at ambient temperature. After three wash steps with buffer B, 100 pL of 25 substrate solution (TMB, Dade Behring, OUVF) were added per well and incubated for 30 minutes at ambient temperature in the dark. Addition of 100 pL undiluted stop solution (Dade Behring, OSFA) prepared the samples for reading in a suitable microplate reader at 450 nm wavelength. Concentrations of test samples were then calculated using the standard curve with standard human plasma as reference. 30 incubated for 30 minutes at ambient temperature in the dark. Addition of 100 pL undiluted stop solution (Dade Behring, OSFA) prepared the samples for reading in a suitable microplate reader at 450 nm wavelength. Concentrations of test samples were then calculated using the standard curve with standard human plasma as 5 reference. Example 8: Comparison of FVil and FVil - albumin fusion proteins in respect to in vivo recovery Recombinant FVIl wild-type and FVIl albumin fusion polypeptides described above 10 were administered intravenously to narcotised CD / Lewis rats (6 rats per substance) with a dose of 100 pg/kg body weight. Blood samples were drawn at appropriate intervals starting at 5 minutes after application of the test substances from the arteria carotis. FVI1 antigen content was subsequently quantified by an Elisa assay specific for human Factor Vil (see above). The mean values of the 15 respective rat groups were used to calculate in vivo recovery. The in vivo recovery was determined 5 min after application of the products (table 2). The FV11 resp. FVIla antigen levels measured per mL of plasma 5 min after intravenous application via the tail vein were related to the amount of product 20 applied per kg. Alternatively, a percentage was calculated by relating the determined antigen level (IU/mL) 5 min post infusion to the theoretical product level expected at 100% recovery (product applied per kg divided by a theoretical plasma volume of 40 mL per kg). 25 The in vivo recoveries of the FV11 fusion proteins determined accordingly in rats were found to be significantly increased in comparison to the non-fused recombinant wild type FVI. It was between 2.3 and 7.9 fold increased over wild type FV11 depending on the construct used. 30 33 Calculated in vivo recoveries 5 min post-infusion are summarized in table 3. The FIX antigen levels measured per mL of plasma 5 min after intravenous application via the tail vein were related to the amount of product applied per kg. Alternatively, a percentage was calculated by relating the determined antigen level (IU/mL) 5 min 5 post infusion to the theoretical product level expected at 100% recovery (product applied per kg divided by an assumed plasma volume of 40 mL per kg). In rats as well as in rabbits the in vivo recoveries of the FIX fusion proteins surprisingly were found to be significantly increased in comparison to the non-fused 10 recombinant F IX prepared in house or the commercially available FIX product BeneFIX. The increase over BeneFIX was 49.7, 69.4 or 87.5%, depending on the animal species or construct used, Compared to the corresponding wild type FIX, the recovery increases of the FIX fusion proteins were even higher. 15

Claims (7)

1. Method of increasing the in vivo recovery of a therapeutic polypeptide in humans or animals wherein, a) the therapeutic polypeptide is fused directly or via a linker peptide to a 10 recovery enhancing protein and b) the in vivo recovery is increased to at least 110% of the in vivo recovery of the non-fused therapeutic polypeptide.

2. Method according to claim 1, wherein the recovery enhancing 15 polypeptide is albumin, a variant or a fragment thereof.

3. Method according to claim I or 2, wherein the therapeutic polypeptide comprises a vitamin K-dependent protein. 20

4, Method according to claims I to 3, wherein the therapeutic polypeptide comprises Factor IX, Factor Vil or Factor Vila or a fragment or variant thereof.

5. Method according to claim 3 and 4, wherein the therapeutic polypeptide moiety is fused to the N-terminus of the albumin moiety. 25

6. Method according to claim 5, wherein the therapeutic polypeptide is Factor Vil or Factor Vila.

7. Method according to claim 6, characterized in that a peptidic linker 30 separates the Factor VIlI or Factor Vila moiety from the albumin moiety.