WO2017050820A1

WO2017050820A1 - Fviii fusion proteins

Info

Publication number: WO2017050820A1
Application number: PCT/EP2016/072414
Authority: WO
Inventors: Gert Bolt; Marianne Hjortnaes Kjalke; Ditte Maria Karpf; Jakob Ewald Rasmussen; Lars Thim
Original assignee: Novo Nordisk A/S
Priority date: 2015-09-22
Filing date: 2016-09-21
Publication date: 2017-03-30

Abstract

The present invention relates to FVIII fusion molecules and pharmaceutical compositions suitable for treatment of blood clotting diseases.

Description

FVIII FUSION PROTEINS

TECHNICAL FIELD

The present invention relates to treatment and/or prophylaxis of haemophilia using FVIII fusion proteins. BACKGROUND

Haemophilia A is an inherited bleeding disorder wherein formation of the blood clot in the patients occurs normally but the clot is unstable due to a lack of secondary thrombin formation. The disease is treated by intravenous injection of coagulation factor VIII (FVIII) isolated from blood or produced recombinantly. Current haemophilia treatment

recommendations are moving from traditional on-demand treatment towards prophylaxis.

The circulatory in vivo half life of endogenous FVIII is 12-14 hours and prophylactic treatment with FVIII is thus to be performed several times a week in order to obtain a virtually symptom-free life for the patients.

Recombinant FVIII variants with a prolonged in vivo circulatory half life (e.g.

heterologous FVIII:Fc fusions) are becoming available for the haemophilia patients. Another frequently used strategy for prolonging FVIII circulatory half life is conjugation of FVIII, employing chemical or enzymatic methods. There is, however, still a need in the art for improved safe and convenient Haemophilia A therapies offering compounds with significantly prolonged circulatory half life that are preferably relatively simple to produce on an industrial scale.

SUMMARY

The present invention relates to FVIII fusion proteins comprising a FVIII molecule and a fusion partner, wherein said fusion partner is inserted at the FVIII light chain N- terminus at position D1658, wherein said fusion partner thus replaces amino acids E1649- S1657 according to SEQ ID NO 1. Amino acids E1649-S1657 are thus absent from the FVIII fusion proteins according to the invention.

The present invention furthermore relates to pharmaceutical compositions suitable for treatment of blood clotting diseases.

DESCRIPTION

The inventors of the present invention have made the surprising observation that certain FVIII fusion proteins are associated with unexpected advantages. Definitions

The term "treatment", as used herein, refers to the medical therapy of any human or other vertebrate subject in need thereof. Said subject is expected to have undergone physical examination by a medical practitioner, or a veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to treating a disease in said human or other vertebrate. The timing and purpose of said treatment may vary from one individual to another, according to the subject's health. Thus, said treatment may be prophylactic, palliative, symptomatic and/or curative.

Mode of administration: Compounds and pharmaceutical compositions according to the invention may be administered parenterally, such as e.g. intravenously or extravascularly (such as e.g. intradermally, intramuscularly, subcutaneously, etc). Compounds and pharmaceutical compositions according to the invention may be administered

prophylactically and/or therapeutically and/or on demand.

Factor VIII: Factor VIII (FVIII) is a large, complex glycoprotein that is primarily produced by endothelial cells including liver sinusoidal endothelial cells (LSECs) and possibly also hepatocytes. Human FVIII codes for 2351 amino acids, including a signal peptide, and contains several distinct domains as defined by homology. There are three A- domains, a unique B-domain, and two C-domains. The domain order can be listed as NH2- A1 -A2-B-A3-C1-C2-COOH. Small acidic regions C-terminal of the A1 (the a1 region) and A2 (the a2 region) and N-terminal of the A3 domain (the a3 region) play important roles in FVIII interaction with other coagulation proteins, including thrombin and von Willebrand factor (VWF). FVIII domains and regions are listed in table a below.

During cellular processing furin cleaves prior to the a3 region. The resulting A1 -a1- A2-a2-B chain is termed the heavy chain (HC) while the a3-A3-C1 -C2 is termed the light chain (LC). The chains are connected by bivalent metal ion-bindings. Table a: FVIII domains and regions. The numbering of domains, regions and single amino acid residues in the Factor VIII molecule follow the numbering of full length Factor VIII (also if the B-domain is truncated or if a fusion partner is added to the molecule).

*) The numbering of domains, regions and single amino acid residues is in accordance with uniprot: P00451 . Other FVIII alleles with FVIII activity exist as well in human populations and are also part of the present invention.

^**) The nucleotide sequence encoding full length Factor VIII encodes a B domain of 908 amino acid residues. During protein synthesis, the B-domain in full-length FVIII is processed, resulting in a mixture of heavy chain with different length of B-domains attached (Jankowski MA et al. Haemophilia 2007; 13: 30-37). rFVIII with truncated B domains may comprise B domains being significantly shorter than 908 amino acids - one example of a truncated B domain is the 21 amino acid B domain linker according to SEQ ID NO 2.

^***) Some naturally occurring FVIII variants comprise an a3 region spanning amino acids 1655-1689 and 1658-1689 (Lind P et al. Eur J Biochem 1995; 232: 19-27). Such FVIII proteins, as well as other naturally occurring FVIII variants, are also part of the present invention.

Endogenous FVIII molecules circulate in vivo as a pool of molecules with B domains of various sizes, the shortest having C-terminal at position 740, i.e. at the C-terminal of A2- a2, and thus contains no B domain. FVIII molecules with B-domains of different length all maintain procoagulant activity. Upon activation with thrombin, FVIII is cleaved C-terminal of A1 -a1 at position 372, C-terminal of A2-a2 at position 740, and between a3 and A3 at position 1689, the latter cleavage releasing the a3 region with concomitant loss of affinity for VWF. The activated FVIII molecule is termed FVIIIa. The activation allows interaction of FVIIIa with phospholipid surfaces like activated platelets and activated factor IX (FIXa), i.e. the tenase complex is formed, allowing efficient activation of factor X (FX) resulting in thrombin generation and ultimately formation of a fibrin-stabilized haemostatic clot.

"Wildtype(wt)/native FVIII" is the human FVIII molecule derived from the full length sequence as shown in SEQ ID NO: 1 (amino acid 1 -2332). "FVIII" includes natural allelic variants of FVIII with FVIII activity. It follows that FVIII fusion proteins according to the present invention are recombinantly produced proteins (rFVIII), using well known methods of production and purification. The degree and location of glycosylation, tyrosine sulfation and other post-translation modifications of FVIII occurring in the cell may therefore vary, depending on the chosen host cell and its growth conditions.

Following activation, FVIII fusion proteins herein are capable of functioning in the coagulation cascade in a manner that - on a molar basis - is functionally similar, or equivalent, to wt/endogenous FVIII, inducing the formation of FXa via interaction with FIXa on an activated platelet and supporting the formation of a blood clot. FVIII(a) activity can be assessed in vitro using techniques well known in the art. Clot analyses, FX activation assays (often termed chromogenic assays), thrombin generation assays and whole blood thrombo- elastography are examples of such in vitro techniques. FVIII molecules according to the present invention have FVIII activity that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 100% or even more than 100% of that of native human FVIII. "Native human FVIII" is herein understood to be either plasma derived purified FVIII or recombinant FVIII corresponding closely to SEQ ID NO 1 , or a B domain truncated version thereof.

Endogenous full length FVIII is synthesized as a single-chain precursor molecule. Prior to secretion, the precursor is cleaved into the heavy chain and the light chain.

Recombinant FVIII can be produced by means of two different strategies. Either the heavy chain with or without the B-domain and the light chain are synthesized individually as two different polypeptide chains (two-chain strategy) or the B domain-truncated FVIII is synthesized as a single precursor polypeptide chain (single-chain strategy) that is cleaved into the heavy and light chains in the same way as the full-length FVIII precursor. In a B domain-truncated FVIII (precursor) fusion polypeptide according to the invention, produced by the single-chain strategy, the heavy and light chain moieties are often separated by a linker. To minimize the risk of introducing immunogenic epitopes in the B domain-truncated FVIII, the sequence of the linker is preferably derived from the FVIII B-domain. The C- terminal part of the linker contains a furin recognition site resulting in intracellular processing into a heavy and a light chain. In the B domain of full length FVIII, amino acid 1644-1648 constitutes this recognition site. The thrombin cleavage site leading to removal of the linker during activation of B domain-deleted FVIII is located in the a2 region C-terminal to the A2 domain. Thus, the size and amino acid sequence of the linker is unlikely to influence its removal from the remaining FVIII molecule by thrombin activation. Truncation of the B domain is often considered to be an advantage for recombinant production of FVIII.

Nevertheless, parts of the B domain can be included in the B domain linker herein without reducing the productivity.

SEQ ID NO: 1 : wt human FVIII (Ser750 residue shown in bold and underline) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEAN RPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSL NACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEID YDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQS GSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFY SSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKD VHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSVVYFTENMERNCRAPCNIQME DPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRVVYLLSMGSNENIHSIHFSGHVFTVRKKEE YKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHI RDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFS SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAW RPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGK VKVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

The terms "B domain truncated" and "B domain deleted" (BDD) FVIII are used interchangeably herein. The B domain in FVIII spans amino acids 741-1648 of SEQ ID NO: 1. The B domain undergoes endo-proteolysis at several different sites, generating large heterogeneity in circulating plasma FVIII molecules as explained above and in Jankowski et al, Haemophilia 2007; 13: 30-37 and D'Amici et al, Electrophoresis 2010; 31 : 2730-2739 . While the B-domain plays a role in intracellular expression of FVIII, the exact extracellular function of the heavily glycosylated B domain, if any, is unknown. What is known is that the B domain is dispensable for FVIII activity in the coagulation cascade. Recombinant FVIII is thus frequently produced in the form of B domain-truncated variants. In one embodiment, the FVIII fusion protein is produced by an expression vector encoding a FVIII molecule comprising a 21 amino acid residue linker (B domain linker) sequence with the following sequence: SEQ ID NO 2: SFSQNSRHPSQNPPVLKRHQR. An O-glycan is attached to the underlined S in SEQ ID NO 2 - this residue corresponds to position S750 in SEQ ID N01 . In another embodiment, the FVIII fusion protein herein comprises a linker sequence with the following sequence: SEQ ID NO: 3: SFSQNSRHPSQNPPVLKRHQ. In another embodiment, the FVIII fusion protein herein comprises a linker sequence with the following sequence: SEQ ID NO: 4: FSQNSRHPSQNPPVLKRHQR. In another embodiment, the FVIII fusion protein herein are B domain truncated FVIII variants comprising an O-glycan attached to the Ser 750 residue shown in SEQ ID NO 1 - the B domain linker sequence may in that case optionally be selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.

FVIII fusion proteins herein may be FVIII molecules comprising the full B domain, or it may be comprise a FVIII molecule comprising a truncated B domain - the FVIII molecule herein furthermore has FVIII activity. FVIII molecules according to the invention comprise a B domain of 908, 4-908, 10-908, 15-908, 4-900, 5-900, 10-900, 15-900, 20-900, 25-900, 50- 900, 100-900, 200-900, 300-900, 400-900, 500-900, 600-900, 700-900, 10-700, 15-700, 20- 700, 25-700, 50-700, 100-700, 200-700, 300-700, 400-700, 500-700, 600-700, 10-500, 15- 500, 20-500, 25-500, 50-500, 100-500, 200-500, 300-500, 400-500, 10-400, 15-400, 20-400, 25-400, 50-400, 100-400, 200-400, 300-400, 10-300, 15-300, 20-300, 25-300, 50-300, 100- 300, 200-300, 10-250, 15-250, 20-250, 25-250, 50-250, 100-250, 200-250, 10-200, 15-200, 20-200, 25-200, 50-200, 100-200, 10-100, 15-100, 20-100, 25-100, 50-100, 10-50, 15-50, 20-50, 10-25, 10-20, 10-15, 15-25, 15-20, 20-21 , 20-25, or 20-30 amino acids.

The inventors of the present invention have made the surprising observation that the FVIII fusion proteins herein results in a FVIII fusion protein with a better yield and/or more simple production process, and/or improved therapeutic properties. The FVIII part of this fusion protein may be a B domain truncated FVIII molecule comprising a B domain linker with an amino acid sequence selected from SEQ ID No 2, 3, or 4. The FVIII part of the protein may alternatively be a full length FVIII protein comprising the entire B domain. A relatively simple and safe way of obtaining therapeutic FVIII molecules with advantageous properties is thus provided with the present invention.

Fusion proteins according to the present invention are proteins created through the in-frame joining of two or more DNA sequences which originally encoded FVIII and the FVIII fusion partner. Translation of the fusion protein DNA sequence will result in a single protein sequence which may have functional properties derived from each of the original proteins or peptides. DNA sequences encoding fusion proteins may be created by standard molecular biology methods such as overlapping PCR or DNA ligation - the FVIII fusion partner according to the present invention is preferably inserted at the FVIII light chain N-terminus at position D1658 - and thus replacing positions E1649-S1657 (according to the sequence in SEQ ID NO 1 - amino acid positions according to SEQ ID NO 1 ). The resulting fusion protein DNA sequence may be inserted into an appropriate expression vector that supports fusion protein expression in a standard host organism.

Fusion proteins may furthermore contain a linker or spacer peptide sequence that separates the protein or peptide parts which define the fusion protein. The linker or spacer peptide sequence may facilitate the correct folding of the individual protein or peptide parts and may make it more likely for the individual protein or peptide parts to retain their individual functional properties. Linker or spacer peptide sequences may be inserted into fusion protein DNA sequences during the in frame assembly of the individual DNA fragments that make up the complete fusion protein DNA sequence i.e. during overlapping PCR or DNA ligation. Examples of linker sequences according to the invention comprising repetitive GS residues include e.g.: (GS)_n, (GGGS)_n, (GGGGS)_n, (GGGS)_n(GGG), etc. The linker can thus be very short, e.g. 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids or somewhat longer, e.g. 5-10, 5-15, 5-20, 5- 30, 5-50, 10-15, 10-20, 10-30, 10-40, 10-50, 15-20, 15-30, 15-40, 15-50 amino acids.

FVIII fusion protein: The fusion proteins according to the present invention are FVIII proteins fused to a fusion partner. The fusion partner (or optionally a linker sequence bridging FVIII and the fusion partner) is fused to the FVIII light chain terminus at position D1658 - thus replacing/deleting residues E1649-S1657 from the FVIII molecule (relative to the amino acid sequence according to SEQ ID NO 1 ). The fusion partner may alternatively be fused to position Q1659, E1660, E1661 , 11662, or even position D 1663 relative to the amino acid sequence according to SEQ ID NO 1 . However, in connection with the present invention, it may not be desirable to fuse a fusion partner to a position in FVIII beyond E1661 , 11662, or D1663 in the a3 domain (and thus deleting more than 9 amino acids from the FVIII light chain N-terminal). At least amino acids E1649-S1657 according to SEQ ID NO 1 are thus absent from the FVIII fusion proteins herein.

If the FVIII DNA sequence encodes amino acid residue E1649-R1689 (according to the sequence set forth in SEQ ID NO 1 ), a significant fraction - up to about 90% depending on production method - of FVIII protein has an N-terminus at position D1658 - likely due to endo-proteolytical processing of the S1657-D1658 sequence in the production cell. It is possible that processing at the N-terminal FVIII light chain is done by a metallo-protease - in which case it may also take place at one of the D1658 neighbouring amino acid residues. In connection with the present invention, this protease site is thus presumed to be removed by deletion. It follows that an alternative solution to the light chain endo-proteolytical problem may be to introduce mutations in the FVIII light chain N-terminus in order to remove the protease site - a preferred solution is, however, to remove the protease site by deletion.

In connection with production of recombinant FVIII proteins - in particular FVIII fusion proteins fused to the FVIII light chain N-terminus - endo-proteolytical cleavage is undesirable. Firstly, it results in a heterogenous population of molecules. Secondly, in connection with fusion to the FVIII light chain N-terminus, part of the resulting protein does not contain the fusion partner thus either resulting in a heterogenous product with a relatively low in vivo circulatory half life or requiring additional purification step(s) to remove the degraded material from the final product. In any event, endo-proteolytical processing at D1658 (or a neighbouring residue) results in a lower yield of FVIII fusion protein - and/or a heterogenous rFVIII product.

The inventors have herein shown that it is possible to solve these problems by fusing the fusion partner (or the linker attached to the fusion partner) e.g. at position D1658 (according to the sequence in SEQ ID NO 1 ). Deletion of amino acids E1649-S1657 is not associated with any negative impact - the resulting molecules are safe in use, have FVIII activity, are produced with good yields, etc. Furthermore, position Y1664 and the acidic amino acid residues surrounding Y1664 are maintained. This is desirable because sulphation of Y1664 is reported to be required for activation of FVIII by thrombin (Michnick DA et al., J Biol Chem 1994; 269: 20095-20102), and the acidic environment of Tyr residues are required for sulphation. Therefore the structural requirements for thrombin activation of the resulting "FVIII-A(1649-1657)-LCN-linked-fusion protein" are preserved.

FVIII B domain fusion partner: A "FVIII B domain fusion partner" according to the present invention is derived from the FVIII B domain. Preferably, the only modification compared to the wt FVIII B domain is that the FVIII B domain fusion partner may be truncated, i.e. a region has been removed/deleted relative to the wt FVIII B domain. In other embodiments, two, three or more truncations were made in the FVIII B domain fusion partner

- relative to the wt FVIII B domain. In some embodiments, minor amino acid modifications (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 point mutations in the form of amino acid alterations, -additions and/or -deletions) may be present in the FVIII B domain fusion partner

- relative to the corresponding positions in the wt FVIII B domain. The FVIII fusion proteins fused to a FVIII B domain fusion partner according to the present invention are thus structurally similar to the native FVIII molecule in that the fusion partner is derived from the FVIII B domain rather than being derived from a heterologous fusion molecule. FVIII fusion proteins fused to a FVIII B domain fusion partner can thus be said to be a homologous type of FVIII fusion protein with a desirable safety profile.

The FVIII B domain fusion partner/B domain fusion partner according to the present invention can be regarded as an "additional FVIII B domain" having a size of 100-908 amino acids. Alternatively, the size of the FVIII B domain fusion partner is 100-900, 100-800, 100- 700, 100-600, 100-500, 100-400 amino acids, preferably 150-650, more preferably 150-600, more preferably 150-550, more preferably 150-500, more preferably 150-450, more preferably 150-400, more preferably 150-350, more preferably 200-700, more preferably 200-600, more preferably 200-500, more preferably 200-400, more preferably 200-300, and most preferably about 200 to 250 amino acids.

In some embodiments, the FVIII fusion proteins according to the invention may even comprise more than one FVIII B domain fusion partners, such as e.g. two, three, or even four FVIII B domain fusion partners fused head-to-tail to the FVIII light chain N-terminus as described herein and even fused to or within different domains of the FVIII molecule. FVIII fusion molecules according to the present invention may thus comprise a FVIII molecule fused to two, three, or four identical or different FVIII B domain fusion partners as defined herein.

The FVIII in vivo circulatory half life can be increased by fusing a FVIII derived B domain fusion partner to a FVIII molecule. This effect may be caused by an ability of this additional FVIII B domain(-s) to interfere with various FVIII clearance receptors. An explanation of the apparent ability of FVIII B domain fusion partners to interfere with FVIII clearance may be that the FVIII B domain tends to fold in a less structured and compact way compared to other proteins - thus mimicking the effect of large bulky groups frequently attached to therapeutic proteins to prolong the half life thereof, such as e.g. polymeric groups (e.g. poly ethylen glycol, poly sialic acid, dextran, starch, heparosan, etc.), XTEN™ polypeptides, etc. It thus follows that FVIII B domain fusion partners according to the present invention may have a relatively low tendency to form secondary and/or tertiary protein structures such as e.g. alpha helix, beta sheets and folded domains. Secondary structures can be measured by e.g. circular dichroism (CD). Another feature of the FVIII B domain is a tendency to be heavily glycosylated with N-glycans but likely also several O-glycans - this structure may provide the B domain with a large hydrodynamic volume in a similar manner as e.g. PEG polymers, polysaccharide polymers, etc.

Some FVIII B domain fusion partners according to the present invention may furthermore have the advantage of being relatively homogenous in structure - in the production cell line as well as in in vivo circulation. An example thereof is the 226 amino acid B domain fusion partner corresponding to SEQ ID NO 1 1 - this FVIII B domain fusion partner tends to avoid endo-proteolytical processing. The tendency to undergo this processing can be analysed and quantified using established techniques such as e.g. HPLC, SDS-PAGE, etc.

Examples of B domain fusion partners according to the present invention include: SEQ ID no. 5 (FVIII amino acids 741 -769):

SFSQNSRHPSTRQKQFNATTIPENDIEKT

SEQ ID no. 6 (FVIII amino acids 741 -794):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLL

SEQ ID no. 7 (FVIII amino acids 741 -857):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL

SEQ ID no. 8 (FVIII amino acids 741 -903):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSS

SEQ ID no. 9 (FVIII amino acids 741 -914):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYD

SEQ ID no. 10 (FVIII amino acids 741 -954):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMN

SEQ ID no. 11 (FVIII amino acids 741 -966) "226" amino acid B domain:

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSS

SEQ ID no. 12 (FVIII amino acids 741 -968):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTE

SEQ ID no. 13 (FVIII amino acids 741 -1003):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKT

SEQ ID no. 14 (FVIII amino acids 741 -1009):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSAT

SEQ ID no. 15 (FVIII amino acids 741 -1018):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGP SEQ ID no. 16 (FVIII amino acids 741 -1020):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSL

SEQ ID no. 17 (FVIII amino acids 741 -1070):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LI H D RM LM D KN ATALRLN H MSN KTTS SEQ ID no. 18 (FVIII amino acids 741 -1079):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LI H D RM LM D KN ATALRLN H MSN KTTSSKN M EM VQQ

SEQ ID no. 19 (FVIII amino acids 741 -1206):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQ

SEQ ID no. 20 (FVIII amino acids 741 -1230):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LL SEQ ID no. 21 (FVIII amino acids 741 -1261):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRT

SEQ ID no. 22 (FVIII amino acids 741 -1301):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNT

SEQ ID no. 23 (FVIII amino acids 741 -1309):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQ SEQ ID no. 24 (FVIII amino acids 741 -1386):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRS

SEQ ID no. 25 (FVIII amino acids 741 -1395):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSS

SEQ ID no. 26 (FVIII amino acids 741 -1444):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLS

SEQ ID no. 27 (FVIII amino acids 741 -1440):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKK SEQ ID no. 28 (FVIII amino acids 741 -1461):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGS SEQ ID no. 29 (FVIII amino acids 741 -1514):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGS

SEQ ID no. 30 (FVIII amino acids 741 -1524):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGS SEQ ID no. 31 (FVIII amino acids 741 -1554):

FSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQS PTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLR LNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTL FGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGP ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLI HDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESA RWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKWVGKGEFTKDVGLKEM VFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLFLL STRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKY ACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLT QIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLP AASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENT VLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANR PGKVPFLRVATESS

SEQ ID no. 32 (FVIII amino acids 741 -1595):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEAN RPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKK SEQ ID no. 33 (FVIII amino acids 741 -1643):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEAN RPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSL NACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVL

SEQ ID no. 34 (FVIII amino acids 741 -1648):

SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQ SPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTT LFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHG PALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVWGKGEFTKDVGLKE MVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLF LLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTL TQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHL PAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEAN RPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSL NACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQR Fc fusion protein: The term "Fc fusion protein" or "FVIII-Fc fusion protein" is herein meant to encompass FVIII fused to an Fc domain that can be derived from any antibody isotype. An IgG Fc domain will often be preferred due to the relatively long circulatory half-life of IgG antibodies. The Fc domain may furthermore be modified in order to modulate certain effector functions such as e.g. complement binding and/or binding to certain Fc receptors. Fusion of FVIII with an Fc domain, which has the capacity to bind to FcRn receptors, will generally result in a prolonged in vivo circulatory half-life. Mutations in positions 234, 235 and 237 in an IgG Fc domain will generally result in reduced binding to the FcyRI receptor and possibly also the FcyRlla and the FcyRIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes a long circulatory in vivo half-life by an endocytic recycling pathway. Preferably, a modified IgG Fc domain of a fusion protein according to the invention comprises one or more of the following mutations that will result in decreased affinity to certain Fc receptors (L234A, L235E, and G237A) and in reduced C1 q-mediated complement fixation (A330S and P331 S), respectively. Alternatively, the Fc domain may be an lgG4 Fc domain, preferably comprising the S241 P/S228P mutation.

Fc receptor fusion protein: In another embodiment, the fusion partner can be an Fc receptor that assumingly can extend the FVIII half life by interaction with immunoglobulins. Fc receptors are cell surface receptors that recognize and bind the Fc portion of antibodies. Based on structure, cell distribution and affinity to IgG, the Fc receptors are divided into three classes: FcyRI (CD64), FcvRII (CD32), and FcvRII I (CD16). Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. In one embodiment, the Fc receptor can be FcyRI (CD64 -Cluster of Differentiation 64) or a portion thereof, e.g. the extracellular portion. In another embodiment, the Fc receptor can be FcyRII or FcyRIII, or a portion thereof, e.g. the extracellular portion.

Various other fusion partners can be used in connection with the present invention in the FVIII fusion molecule. In one embodiment, the FVIII fusion protein according to the invention comprises a

FVIII molecule fused to albumin. FVIII-albumin fusion proteins delay in vivo clearance of FVIII by interaction/binding with the FcRn receptor.

In another embodiment, FVIII is fused to a fusion partner to delay in vivo clearance of FVIII by interaction with platelets. A non-limiting example thereof includes single-chain (SC) antibodies as well as Fab fragments of antibodies binding to proteins on the platelet surface such as e.g. GPIIIa SC antibodies.

In another embodiment, FVIII can be fused to a fusion partner to delay in vivo clearance of FVIII by shielding. A non-limiting example thereof includes polypeptides with stretches of non-hydrophobic amino acids ("unstructured polypeptides") such as Sequence A (seq A - a non-limiting example is set forth in SEQ ID NO 39).

In another embodiment, FVIII can be fused to a fusion partner to delay in vivo clearance of FVIII by unknown mechanisms. Non-limiting examples thereof include: growth hormone binding protein (GHBP), parts of vWF, vWF binding protein, and parts of chorion gonadotropin. In one embodiment, FVIII can be fused to a fragment derived from VWF: VWF(amino acids 764-828), VWF(amino acids 764-865), VWF(amino acids 764-1035), VWF(amino acids 764-1041 ), VWF(amino acids 764-1045), VWF(amino acids 764-1 128), VWF(amino acids 764-1 198), VWF(amino acids 764-1242), VWF(amino acids 764-1250), VWF(amino acids 764-1261 ), VWF(amino acids 764-1268), VWF(amino acids 764-1464). The amino acid sequence of full length VWF is shown in SEQ ID NO 40.

Examples of various fusion partners according to the present invention include:

SEQ ID NO 35 - Human serum albumin (the Cys residue in position 34 (shown with bold) may alternatively be substituted with another amino acid such as e.g. Ser or Ala):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACL LPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPAD LPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKCCAA ADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVE VSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRR PCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO 36: extracellular region of human FcyRI (CD64):

QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSY RITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKL VYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPV

LNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDS

GLYWCEAATEDGNVLKRSPELELQVLGLQLPTP SEQ ID NO 37: Human lgG1 Fc domain:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO 38: The C-terminal 28 amino acids of the beta-chain of human chorion gonadotropin (hCG C-terminus):

SSSSKAPPPSLPSPSRLPGPSDTPILPQ SEQ ID NO 39: Sequence A / XTEN:

GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPG

SEQ ID NO 40: wt Human von Willebrand factor (VWF):

AEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSIIGDFQ NGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVA RIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQ WCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECA GGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHIN EMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWI CSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAV CTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQM DWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDC QDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGR ECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEE CNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHC TMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLEC MSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCRDRKWN CTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPS VKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEWESGRYIILLLGKALSWWDRHLSIS WLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDS SPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTI AAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPL ACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQI CHCDWNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSS RLSEAEFEVLKAFVVDMMERLRISQKWVRVAWEYHDGSHAYIGLKDRKRPSELRRIASQV KYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKVI VIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPHMA QVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSI HVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGD REQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPR EAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKAN IGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSE MHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDS NWKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPD GQTLLKTHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFK LTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRL VSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDE NGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAEC HKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYN HCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLE AWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDP VSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCC DEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCD VCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRG DSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTS ACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPL GYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKR VTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQ GKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKC SK

Examples of fusion proteins according to the invention include:

SEQ ID NO 41 : F8-500-A(1649-1657)-LCN-linked-Fc (hlgG1) (B domain deleted/truncated FVIII fused to the Fc domain of human lgG1 in the light chain terminal end at position D1658):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYVVYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDY GMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDN IMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFD CKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE NMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLA PMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIF NPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATW SPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY

SEQ ID NO 42: F8-500-A(1649-1657)-LCN-linked-hCG C-terminus (B domain deleted/truncated FVIII fused to the C-terminal 28 amino acids of the beta-chain of human chorion gonadotropin in the light chain N-terminal end at position D1658):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRSSSSKAPPPSLPSPSRLPGPSDTPILPQDQEEIDY DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSG SVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMED PTFKENYRFHAINGYIMDTLPGLVMAQDQRI RWYLLSMGSNENIHSIHFSGHVFTVRKKEEY KMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIR DFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSS LYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRS TLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWR PQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV KVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

SEQ ID NO 43: F8-500-A(1649-1657)-LCN-linked-albumin (B domain

deleted/truncated FVIII fused to human albumin in the light chain N-terminal end at position D1658):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC PFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFF AKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVA RLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECC EKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPD YSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLH EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQT ALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLDQE EIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRA QSGSVPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPY SFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGAR QKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPT HYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGR SNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFF QNGKVKVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

SEQ ID NO 44: F8-500-A(1649-1657)-LCN-linked-FcyRI (B domain

deleted/truncated FVIII fused to extracellular region of FcyRI in the light chain N- terminal end at position D1658):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLP GSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVS SRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGM GKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSK TLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPDQEEIDYD DTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGS VPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSS LISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVH SGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDP TFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIR DFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSS LYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRS TLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWR PQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV KVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

SEQ ID NO 45: F8-500-A(1649-1657)-LCN-linked-FVIII(741 -966) (B domain deleted/truncated FVIII fused to a 741 -966 FVIII B domain fusion partner in the light chain N-terminal end at position D1658):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFA HRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTH FRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTD NTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSW GKNVSSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS SSPHVLRNRAQSGSVPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMV TFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENM ERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIH FSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSN KCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK ARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLY Pharmaceutical compositions:

The present invention provides compositions and formulations comprising one or more FVIII proteins, preferably fusion proteins of the invention, formulated together with one or more pharmaceutically acceptable carrier(-s).

Accordingly, one object of the invention is to provide a pharmaceutical formulation comprising a protein according to the invention present in a concentration from 0.25 mg/ml to 250 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such a formulation is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 % w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-dried

formulation, to which the physician or the patient adds solvents and/or diluents prior to use. In a further aspect, the pharmaceutical formulation comprises an aqueous solution.

Pharmaceutical compositions according to the present invention are preferably suitable for intravenous (IV) and/or extravascular administration (e.g. subcutaneous (sc) or intradermal administration) in prophylactic/therapeutic treatment of haemophilia.

"Haemophilia": Haemophilia/hemophilia/blood clotting diseases is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation ("bleeding disorders"), which is used to stop bleeding when a blood vessel is broken. Haemophilia A (clotting factor VIII deficiency) is the most common form of the disorder, present in about 1 in 5,000-10,000 male births. List of embodiments:

It is understood that all aspects and embodiments of the invention can be combined and that they are not to be understood in any limiting way.

Embodiment 1 : A FVIII fusion protein comprising, or consisting of, a FVIII molecule

(preferably with a B domain - a full length B domain or a truncated B domain), and a fusion partner, wherein said fusion partner is inserted at the FVIII light chain N-terminus at position D1658, wherein said fusion partner thus replaces amino acids E1649-S1657 according to SEQ ID NO 1. The FVIII fusion protein according to the invention may furthermore comprise a linker between the FVIII protein and the fusion partner. Amino acids E1649-S1657 are thus absent from said FVIII fusion protein.

Embodiment 2: A FVIII protein, wherein amino acids E1649-S1657 are deleted from the FVIII light chain N-terminus.

Embodiment 3: A FVIII protein, wherein amino acids E1649-D1658 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position Q1659).

Embodiment 4: A FVIII protein, wherein amino acids E1649-Q1659 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position E1660).

Embodiment 5: A FVIII protein, wherein amino acids E1649-E1660 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position E1661 ).

Embodiment 6: A FVIII protein, wherein amino acids E1649-E1661 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position 11662).

Embodiment 7: A FVIII protein, wherein amino acids E1649-11662 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position D1663).

Embodiment 8: A FVIII protein, wherein amino acids E1649-D1663 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position Y1664).

Embodiment 9: A FVIII fusion protein according to the invention, wherein said fusion protein comprises a fusion partner selected from the group consisting of: albumin (preferably human serum albumin), an Fc domain, an Fc receptor (preferably FcyRI or the extracellular domain/region of FcyRI), and a FVIII B domain fusion partner (preferably amino acids 741 -966). The fusion proteins according to the invention may have (or comprise or consist of) the sequence as set forth in e.g. SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, or SEQ ID NO 44.

Embodiment 10: A FVIII protein/fusion protein according to the invention, wherein the B domain of said FVIII molecule comprises 4-908 or 15-25 amino acids.

Embodiment 11 : A FVIII protein/fusion protein according to the invention, wherein the FVIII B domain fusion partner is a truncated FVIII B domain comprising at least 200 amino acids and up to 500 amino acids.

Embodiment 12: A FVIII protein/fusion protein according to the invention, wherein said FVIII molecule comprises, or has, a B domain amino acid sequence/linker of 15-25 amino acids. Preferably, the sequence of said FVIII B domain is selected from the amino acid sequence in the group consisting of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.

Embodiment 13: A FVIII fusion protein according to the invention, wherein said FVIII molecule comprisesan amino acid sequence selected from the list consisting of: SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 1 1 , SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21 , SEQ ID No 22, SEQ ID No 23, SEQ ID No 24, SEQ ID No 25, SEQ ID No 26, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31 , SEQ ID No 32, SEQ ID No 33, SEQ ID No 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, and SEQ ID NO 45.

Embodiment 14: A FVIII fusion protein according to the invention, wherein said FVIII B domain fusion partner has a low degree of secondary and tertiary structures.

Secondary and tertiary structure can be measured by e.g. Circular Dichroism (CD). Based on e.g. a CD wavelength spectrum from 190-260 nm, wherein the fingerprint of the FVIII B domain fusion partner resembles that of a disordered polypeptide having a signal minimum of 204 nm. Preferably, the FVIII B domain fusion partner is homogenous in structure in in vivo circulation upon administration to the patient in need thereof. An example of a protein according to the invention having a homogenous structure is a FVIII protein fused to the FVIII B domain fusion partner according to SEQ ID NO 1 1 .

Embodiment 15: A pharmaceutical composition comprising a FVIII protein/fusion protein according to the invention.

Embodiment 16: A method for making a FVIII protein/fusion protein according to the invention, wherein said method comprises the step of incubating a host cell under appropriate conditions, wherein said host cell comprises an expression vector that encodes a FVIII protein/fusion protein according to the invention.

Embodiment 17: A nucleotide molecule encoding a FVIII protein/fusion protein according to the invention.

Embodiment 18: An expression vector comprising a nucleotide molecule according to the invention.

Embodiment 19: A host cell comprising an expression vector according to the invention.

Embodiment 20: A FVIII protein/fusion protein according to the invention, or a pharmaceutical formulation according to the invention, for use as a medicament for treatment of haemophilia.

Embodiment 21 : A FVIII protein/fusion protein according to the invention, or a pharmaceutical composition according to the invention, for use in treatment of haemophilia by subcutaneous administration.

Embodiment 22: A FVIII protein/fusion protein according to the invention, or a pharmaceutical formulation according to the invention, for use in treatment of haemophilia by intravenous administration.

Embodiment 23: A method of treatment of haemophilia, wherein said method comprises administering the FVIII protein/fusion protein according to the invention, or a pharmaceutical formulation according to the invention, to a patient in need thereof.

EXAMPLES

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art.

Example 1 : Expression of FVIII proteins and fusion proteins:

A FVIII B domain deleted construct was expressed in CHO cells, wherein the DNA sequence encoding the full length B-domain (aa 741 -1648) has been replaced with a DNA sequence encoding an 21 amino acid residue truncated B-domain with the SEQ ID NO 2. The resulting B domain deleted/truncated FVIII molecule is named turoctocog alfa. An expression vector encoding turoctocog alfa was thus inserted into a CHO host cell for recombinant production thereof. The resulting recombinant turoctocog alfa protein was subsequently purified as previously described (Thim et al, Haemophilia 2010; 16: 349-359) and subjected to N- terminal amino acid sequences analysis. This analyse was carried out by automated Edman degradations using an Applied Biosystem Model 494 Protein Sequencer essentially as described by the manufacturer: the protein is degraded by the removal of one amino acid residue at a time starting from the N-terminal of the protein. The removed amino acid residue is converted to phenylthiohydantoin (PTH) amino acid derivative and subsequently identified and quantified by HPLC analysis. Thus, if the protein consists of one chain only, one amino acid residue will be removed in each cycle. If the protein consist of two chains two amino acid residues will be removed in each cycle. By measuring the amount of PTH-amino acid (quantified in pmol) in each cycle the ratio between different chains of the protein can be calculated. Table 1 shows the results of an N-terminal amino acid sequencing of turoctocog alfa. :

Table 1 : Automated Edman degradation of purified turoctocog alfa. Yields of the individual PTH-amino acids are given in each cycle. Numbers in brackets indicate that the same amino acid occurs from more than one sequence. HC, heavy chain. LC, light chain.

Cycle no FVIII HC AA1- FVIII LC AA1- FVIII LC AA10-

PTH-aa pmol PTH-aa pmol PTH-aa pmol

1 Ala 37 Glu 16 Asp 9

2 Thr 34 He 20 Gin 9

3 Arg 26 Thr 20 Glu 10

4 Arg (40) Arg (40) Glu 12

5 Tyr 40 Thr 19 He 11

6 Tyr 42 Thr 19 Asp 11

7 Leu (64) Leu (64) Tyr 18

8 Gly 31 Gin 21 Asp 13

9 Ala 38 Ser 15 Asp 15

10 Val 44 Asp 21 Thr 14

11 Glu 35 Gin 20 lie 16

12 Leu 52 Glu 22 Ser 13

13 Ser 24 Glu 22 Val 24

14 Trp 16 He 22 Glu 20

15 Asp (31) Asp (31) Met 13

16 Tyr (37) Tyr (37) Lys 21

17 Met 26 Asp 23 Lys 23

18 Gin 28 Asp 24 Glu 20

19 Ser 21 Thr 19 Asp 23

20 Asp 28 He 22 Phe 25 As can be seen in table 1 , the heavy chain of turoctocog alfa was found to be a uniform product having the expected (and encoded) N-terminal sequence. The light chain, however, was found to consist of two species: (i) one species having the N-terminal sequence of full length light chain and (ii) one species having a sequence starting at amino acid residue number 10 of the light chain.

In the present example, the short form of the light chain was found in approx. 33% of the molecules. The degree to which the shorter form is produced appears to be non- predictable and to depend on e.g. the type of FVIII construct, the nature of the mammalian cell (CHO, BHK, HEK, etc.), cell culturing conditions (e.g. Fill and Draw process versus Alternating Tangential Flow (ATP) process), etc.

In order to prepare a Factor VIII fusion protein where the fusion partner is linked to the N-terminal of the light chain, it is therefore advantageous to place the fusion partner linked to residue no. 10 of the light chain instead of residue number 1. This gives higher yield and/or a more homogeneous FVIII fusion protein product.

Example 2: Presence of FVIII light chain (LC), with and without a fusion partner attached:

Recombinant FVIII fusion proteins were produced using constructs encoding FVIII with the light chain having N-terminus at E1649. FVIII samples were loaded on 7% Tris- Acetate gels (NuPAGE, Novex, Life technologies) with 7 lU/lane for coomassie-stained gels, 0.5 lU/lane for silver staining or 0.1 lU/lane for Western blots. Gel electrophoresis was performed according to the manufacturer's instructions, and either Coomassie stained using SimplyBlue Safestain (Invitrogen, Life Technologies), silver stained using SilverXpress (Invitrogen, Life Technologies) or blotted to nitrocellulose membrane using iBIot (Invitrogen, Life Technologies). The blots were blocked 1 h with 3% skim milk in TBS/Tween (50 mM

Tris, 150 mM NaCI, pH 7.5, containing 0.1 % Tween 20) and incubated overnight at 4°C with 10 ug/mL anti- FVIII-LC antibody FVIII-4F36 (Novo Nordisk) in TBS/Tween. Next, the blot was washed 3 times with TBS/Tween, incubated with peroxidalse-labelled rabbit anti-mouse IgG (Dako), washed and developed using Super Signal West Pico chemiluminescent substrat (Pierce) as described by the manufacturers. The gels were scanned using HP Scanjet G2710 and bands on gels and the blot quantified using CLIQS 1 D software

(Totallab).

FVIII fusion protein with the fusion partner attached to the N-terminal of the LC had a band with the same mobility as the LC of FVIII without any fusion partner attached

(turoctocog alfa, Thim et al, Haemophilia 2010; 16: 349-359). The density of the FVIII light chain band on the coomassie-stained gel corresponded to 5.7 % of the total band density for that protein.

However, when the same fusion partner was attached to the truncated LC lacking the first nine amino acids (LC delta(1649-1657)), no bands could be seen at the position of LC without fusion partner (by visual inspection of the coomassie- and silver-stained gels or on the western blot). When the coomassie-stained gel was scanned and bands quantified, the program reported a minor band at the position of the LC in FVIII without fusion partner with a density of 1.0 - 1.5 % of the total band density.

These data thus demonstrate that the yield of FVIII with a LC-N-terminally attached fusion partner can be increased by attaching the fusion partner to a LC lacking the first 9 amino acids.

Example 3: Specific FVIII activity measured in cell culture supernatants:

The FVIII activity (FVIILC) of the rFVIII compounds in cell culture supernatant were measured in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: a FVIII standard e.g. rFVIII (turoctocog alfa, Novoeight^®) calibrated against the 7th international FVIII standard from NIBSC (2009 # 07/350),were diluted to 5 - 4 - 3 - 2 - 1 - 0.5 - 0.25 mll/mL in Coatest assay buffer (50 mM Tris, 150 mM NaCI, 1 % BSA, pH 7.3, with preservative). Cell culture supernatants were diluted minimum 10-fold in the same buffer. At least two dilutions were analysed. Fifty μΙ of samples, standards, and buffer negative control were added to 96-well microtiter plates in duplicates. The factor IXa/factor X reagent, the phospholipid reagent and CaCI₂ from the Coatest SP kit were mixed 5:1 :3 (vol:vol:vol) and 75 μΙ of this added to the wells. After 15 min incubation at room temperature 50 μΙ of the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions incubated 5 min at room temperature before 25 μΙ 1 M citric acid, pH 3, was added. The absorbance at 405 nm was measured on a microtiter plate reader with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and calibrators and the activity of the samples calculated based on a calibration curve prepared by plotting the absorbance values vs. FVIII concentration of the FVIII calibrator dilutions.

The FVIII antigen concentration was determined in an ELISA (Factor VIII antigen (FVIILC), Affinity Biologicals) using a polyclonal anti-FVIII antibody both for catching and detection. Microtiter plates (96 well plates, NUNC Maxisorp) were coated overnight at 4°C with 100 μΙ capture antibody from the kit (F8C-EIA-C). After 5 times washing in PBS + 0.05 % Tween 20 and 15 min incubation with the buffer at room temperature the wells were blocked 30 min at room temperature with 3% casein. After washing were 100 μΙ cell culture supernatant diluted minimum 10-fold, FVIII calibrator (turoctocog alfa, Novoeight^®) diluted to 100 - 33 - 1 1 - 3.70 -1.23 - 0.41 - 0.14 ng/mL (corresponding to 600 - 200 - 67 - 22 - 7.4 - 2.5 - 0.8 pM) in 0.1 M Hepes, 0.1 M NaCI, 10 g/L BSA and 0.1 % Tween 20, pH 7.0, and buffer negative control added in duplicates to the wells and incubated 1-1.5 h at room temperature. Plates were washed, and 100 μΙ_ HRP-labelled antibody from the kit (F8C-EIA- D) added to wells and incubated 1 h. Plates were washed, 100 μΙ_ TMB One™ Substrate (Kem-En-Tek) added, and colour development allowed to take place for approx 5 min before the reaction was stopped by adding 100 μΙ_ 4 M Phosphoric acid. The absorbance at 450 nm with 620 nm as reference was measured on a microtiter plate reader. The value for the negative control was subtracted from all samples and calibrators, and FVIII antigen in the samples calculated relative to the FVIII calibrator.

The specific activity of the FVIII fusion protein was calculated by dividing the activity of the samples with the FVIII antigen concentration. From table 2 below it can be seen that the activity of the LC-N linked FVIII fusion protein with the fusion partner attached to amino acid 1658 (the deltal 649-1657 light chain) had maintained FVIII activity. The molar specific activity was 67% of FVIII without fusion partner. The specific activity of FVIII in cell culture supernatant is lower than when measured on purified protein (see below) likely due to the presence of excess light chain in the cell culture supernatant.

Table 2. Specific activity of LC-(delta 1649-1657)-N-linked FVIII fusion protein and FVIII without fusion partner in cell culture supernatants.

Example 4: Specific FVIII activity measured for purified proteins:

The activity of purified FVIII fusion proteins were measured as described in example

3 above except that the colour development was measured continuously after adding the S- 2765/1-2581 mix. The initial rate of colour development was used for further calculations.

The protein concentration can be determined in an ELISA as described above or by HPLC by applying approximately 10 μg FVIII fusion protein on a Daiso 300 A, 5 mm, 2.1 x 250 mm column (FeF Chemicals A/S) equilibrated with 1 % trifluoro acetic acid (TFA) at a flow of 0.25 mL/min and a temperature of 40°C using an Agilent 1 100 instrument. The protein was eluted with a 30 min gradient of 35-84% of 80% acetonitrile in 0.09 % TFA. The concentration of the sample was determined by integrating the area under the peaks determined at 214 nm and compare with the area of the peaks in a parallel analysis of a rFVIII protein (turoctocog alfa, Novoeight^®), where the concentration was determined by amino acid analyses. The molar concentration is calculated by dividing the concentration in mg/mL with the molecular weight of the protein. Table 3 includes the protein concentration of the LC-(delta1649-1657)-N-linked FVIII fusion protein by ELISA. The protein concentration by ELISA was in agreement with the protein concentration determined by HPLC (2.6 mg/mL corresponding to 15705 nM).

The specific molar activity of the purified FVIII fusion protein was determined by dividing the activity with the molar protein concentration. The data shown in table 3 are based on concentration determination by ELISA. The data demonstrate that the specific molar activity was maintained for the FVIII fusion protein with fusion partner attached to the LC lacking the first nine amino acids.

Table 3. Specific activity of purified LC-(delta 1649-1657)-N-linked FVIII fusion protein.

Example 5: FVIII fused with a fusion partner at the N-terminus of FVIII light chain:

Four plasmids encoding FVIII fusion proteins were constructed. In the fusion proteins encoded by these plasmids, the fusion partner is fused to the N-terminus of the FVIII light chain without omitting any amino acids - these proteins are thus not part of the present invention:

• Human serum albumin (SEQ ID NO 35) inserted between amino acid 1648 and 1649 of the "F8-500 FVIII molecule with the B domain linker sequence according to SEQ ID NO 2 (SEQ ID NO 46). • The extracellular region of human FcyRI (CD64 - SEQ ID NO 36) inserted between amino acid 1648 and 1649 of the "F8-500" FVIII molecule (SEQ ID NO 47). · Human FVIII amino acids 741 -966 (SEQ ID NO 1 1 ) inserted between amino acid 1648 and 1649 of the "F8-500" FVIII molecule (SEQ ID NO 48).

• Human lgG1 Fc domain (SEQ ID NO 37) inserted between amino acid 1648 and 1649 of the "F8-500" FVIII molecule (SEQ ID NO 49).

SEQ ID NO 46: F8-500-LCN-linked-albumin (B domain deleted/truncated FVIII fused to human albumin in the light chain N-terminus end at position D1649):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSWYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC PFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFF AKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVA RLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECC EKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPD YSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF QNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLH EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQT ALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLEITR TTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSS PHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAW AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMER NCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFS GHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNK CQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIA RYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDG HQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLY

SEQ ID NO 47: F8-500-LCN-linked-FcgRI (B domain deleted/truncated FVIII fused to extracellular region of FcgRI in the light chain N-terminal end at position D1649):

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLP GSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVS SRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGM GKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSK TLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPEITRTTLQ SDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPL GMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHL QGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQW TLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

SEQ ID NO 48: F8-500-LCN-linked-FVIII(741-966) (B domain

deleted/truncated FVIII fused to a 741-966 FVIII B domain fusion partner in the light chain N-terminal end at position D1649) :

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFA HRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTH FRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTD NTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSW GKNVSSEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVER LWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRA EVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPT KDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMG SNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGM STLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI KVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVHQIAL RMEVLGCEAQDLY

SEQ ID NO 49: F8-500-LCN-linked-Fc (hlgGl) (B domain

deleted/truncated FVIII fused to the Fc domain of human IgG l in the light chain N-terminal end at position D1649) :

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGR KYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRP LYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFSQNSRHPSQNPPVLKRHQRDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIA AVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGP YIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLL SMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPF SWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSY FTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY

Example 6: FVIII fused with a fusion partner at the N-terminus of FVIII light chain without FVIII amino acid 1649-1657:

Four plasmids encoding FVIII fusion proteins were constructed by modification of the plasmids described in Example 5: the 27 nucleotides encoding amino acid 1649 to 1657 were deleted. Thus, the resulting modified plasmids encode the same fusion proteins as those in Example 5 except that amino acid 1649 to 1657 have been omitted. These proteins are thus part of the present invention:

• Human serum albumin inserted between amino acid 1648 and 1658 of the "F8-500" FVIII molecule (SEQ ID NO 43).

• The extracellular region of human FcyRI (CD64) inserted between amino acid 1648 and 1658 of the "F8-500" FVIII molecule (SEQ ID NO 44).

• Human FVIII amino acids 741 -966 inserted between amino acid 1648 and 1658 of the F8-500 FVI 11 molecule (SEQ ID NO 45).

• Human lgG1 Fc domain inserted between amino acid 1648 and 1658 of the "F8-500" FVIII molecule (SEQ ID NO 41 ). Example 7: Expression and analysis of FVIII fusion proteins:

CHOEBNALT85 cell cultures were transiently transfected with plasmids encoding the fusion proteins of Examples 5+6. Portions of 1 X 107 CHOEBNALT85 cells in 700 ul 1 :1 CD CHO and SFM II medium

(Thermo Fisher Scientific) were electroporated with 10 ug plasmid in a Gene Pulser Cuvette (BioRad) using a GenePulser Xcell (Biorad). Subsequently, the cells were transferred to 125 ml Erlenmeyer flasks with 30 ml of the above growth medium and incubated in a shaker incubator at 36.5 °C and 8 % C02. Five days after transfection, the culture volumes were increased 20 % by addition of CHO CD Efficient Feed B (Thermo Fisher Scientific), and the incubation temperature was reduced to 30 °C. After 4 days of incubation at 30 °C, cell culture supernatants were collected and frozen in aliquots with 20 uM imidazol pH 7.0 (Sigma) and 0.2 % Tween 80 (Merck). The supernatants were utilized for Western blotting as described below.

A vial with supernatant from each of the transfected cultures were thawn and 7.5 ul of each supernatant were transferred to individual tubes along with 2.5 ul 4 X NuPage LDS sample buffer (Thermo Fisher Scientific) and 1.1 ul 10 X NuPAGE sample Reducing Agent (Thermo Fisher Scientific). The tubes were incubated for 10 minutes at 70° C on a block heater and then centrifuged 20.000 X g for 2 minutes. The content of each tube was loaded in the wells of a NuPAGE 4-12 % Bis-Tris gel with 15 wells (Thermo Fisher Scientific). The samples were electrophorized in the gel for 2 hours at 200 volt in MOPS running buffer (Thermo Fisher Scientific) with NuPAGE Antioxidant (Thermo Fisher Scientific) added to the running buffer in the inner electrophoresis chamber.

After electrophoresis, the gel was blotted for 7 minutes in an iBIot module (Thermo Fisher Scientific) to a nitrocellulose membrane using the iBIot Gel Transfer Stacks

Nitrocellulose Mini kit (Thermo Fisher Scientific). After blotting, the membrane was incubated for 1 hour at room temperature with gentle agitation in TBS with 2 % Tween 20. Subsequently, the membrane was washed 4 times for 3 minutes in TBS with 0.1 % Tween 20 as described above. Then, the membrane was incubated with gentle agatation overnight in a cold store with polyclonal sheep anti-FVIII antibody (Cedarlane Laboratories Limited) diluted 1 :500 in TBS with 0.1 % Tween 20.

Subsequently, the membrane were washed 4 times for 3 minutes at room temperature with gentle agitation in TBS with 0.1 % Tween 20. Then, the membranes were incubated for 45 minutes as described above but protected against light with donkey anti- sheep Alexa fluor 680 conjugate (Molecular Probes) diluted 1 :10000. Subsequently, the membranes were washed as describesd above 4 times for 3 minutes in TBS with 0.1 % Tween 20. Finally, the membranes were scanned in an Odyssey reader at 680 nM.

On the Western blot, the FVIII heavy chain of all FVIII proteins migrated as protein with a molecular weight between the 80 kDa and 100 kDa molecular weight markers. The wild-type FVIII light chain of F8-500 was visualized as 4 bands with higher electrophoretic mobility than the FVIII HC. The wild-type FVIII light chain consists of 4 individual bands to due to N-terminus heterogeneity (amino acid E1649 or D1658) and due to incomplete utilization of a glycosylation site at N1810. For each of the fusion proteins containing FVIII amino acid 1649 to1657 (the fusion proteins in Example 5), two FVIII light chain bands were detected. One light chain band migrated between the 100 kDa and 220 kDa molecular weight markers and represented FVIII light chain with fusion partner. The other light chain band co- migrated with wild-type FVIII light chain and represented FVIII light chain without fusion partner. According to densitometric scanning of the bands, the FVIII light chain without fusion partner constituted 1 1.4 - 23.2 % of the total amount of FVIII light chain. In contrast, bands of FVIII light chain without fusion partner were not seen for the fusion proteins without FVIII amino acid 1649-1657 (the fusion proteins in Example 6). This demonstrates that removal of amino acid 1649-1657 prevents or dramatically reduces the cleavage events removing fusion partners from the N-terminus of the FVIII light chain. The production yield and quality and fusion proteins is therefore significantly improved compared to regular N-terminal fusion.

Claims

1. A FVIII fusion protein comprising a FVIII molecule and a fusion partner, wherein said fusion partner is inserted at the FVIII light chain N-terminus at position D1658, wherein said fusion partner thus replaces amino acids E1649-S1657 according to SEQ ID NO 1.

2. A FVIII fusion protein according to claim 1 , wherein said fusion protein comprises a fusion partner selected from the group consisting of: albumin, an Fc receptor, an Fc domain, and a B domain fusion partner.

3. A FVIII fusion protein according to claim 2, wherein the fusion partner is albumin.

4. A FVIII fusion protein according to claim 3, wherein the fusion partner is an Fc receptor.

5. A FVIII fusion protein according to any one of claims 1 and 2, wherein the fusion partner is a FVIII B domain fusion partner, wherein said B domain fusion partner comprises a truncated FVIII B domain.

6. A FVIII fusion protein according to any one of claims 1 -2, wherein said FVIII B domain fusion partner is derived from the FVIII B domain and comprises 100-908 amino acids.

7. A FVIII fusion protein according to any one of claims 1 or 2, wherein the protein comprisesan amino acid sequence selected from the group consisting of: SEQ ID NO 5,

SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 32, SEQ ID NO 33, and SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45.

8. A FVIII fusion protein according to any one of the preceding claims, wherein the B domain of said FVIII molecule comprises 15-25 amino acids.

9. A FVIII fusion protein according to any one of the preceding claims, wherein the B domain of said FVIII molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.

10. A pharmaceutical composition comprising a FVIII fusion protein according to any one of claims 1 -19.

11. A FVIII fusion protein according to any one of claims 1-9, or a pharmaceutical formulation according to claim 10, for use as a medicament for treatment of haemophilia.

12. A FVIII fusion protein according to any one of claims 1-9, or a pharmaceutical composition according to claim 10, for use in treatment of haemophilia by subcutaneous administration.

13. A FVIII fusion protein according to any one of claims 1-9, or a pharmaceutical formulation according to claim 10, for use in treatment of haemophilia by intravenous administration.