Factor VIII Fusion Protein

Abstract

The present invention relates to modified coagulation factors. In particular, the present invention relates to conjugated Factor VIII molecules fused to a polypeptide such as e.g. an antibody binding protein or a Fc domain.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 13/574,686, filed Oct. 5, 2012, which is a 35 U.S.C. .sctn.371 National Stage application of International Application PCT/EP2011/051959 (WO 2011/101284), filed Feb. 10, 2011, which claims priority to European Patent Application 10153715.7, filed Feb. 16, 2010; this application claims priority under 35 U.S.C. .sctn.119 to U.S. Provisional Application 61/306,177; filed Feb. 19, 2010; the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to modified coagulation factors. In particular, the present invention relates to Factor VIII molecules fused to a non-homologous polypeptide such as e.g. an antibody binding polypeptide, such as e.g. an Fc receptor or an Fc domain. The invention furthermore relates to use of such molecules as well as methods for producing such molecules.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 19, 2012 and modified Sep. 30, 2015, is named 7920US01_SeqList_ST25.txt and is 90,020 bytes in size.

BACKGROUND OF THE INVENTION

[0004] Haemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII) activity. The clinical manifestation is not on primary haemostasis--formation of the blood clot occurs normally--but the clot is unstable due to a lack of secondary thrombin formation. The disease is treated by intravenously injection of coagulation factor FVIII which is either isolated from blood or produced recombinantly.

[0005] Current treatment recommendations are moving from traditional on-demand treatment towards prophylaxis. The circulatory half life of endogenous FVIII bound to von Willebrandt Factor is 12-14 hours and prophylactic treatment is thus to be performed several times a week in order to obtain a virtually symptom-free life for the patients. IV administration is for many, especially children and young persons, associated with significant inconvenience and/or pain.

[0006] Various methods have been employed in the development of a Factor VIII variant with significantly prolonged circulatory half life. A number of these methods relate to conjugation of Factor VIII with hydrohphilic polymers such as e.g. PEG (poly ethylene glycol).

[0007] There is thus a need in the art for novel Factor VIII products with factor VIII activity that comprise one or more of the following features: preferably homogenous in structure, preferably safe, preferably biologically degradable, and preferably have a significantly prolonged circulatory half life in order to reduce the number of factor VIII administration per week. There is likewise a need in the art for relatively simple methods for providing such molecules.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a recombinant Factor VIII molecule, wherein said Factor VIII molecule is a fusion protein. The invention furthermore relates to methods for making such molecules as well as use of such molecules. Such molecules preferably have a modified circulatory half life.

DESCRIPTION OF THE INVENTION

Definitions

[0009] Fusion protein: Fusion proteins/chimeric proteins, are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Factor VIII molecules according to the present invention may be fused to another polypeptide. Preferably, the Factor VIII fusion protein will have a longer circulatory half life compared to the non-fused Factor VIII molecule.

[0010] A wide range of fusion partners can be joined to the FVIII part. These fusion partners can alter the properties of the fusion protein relative to wild-type FVIII by various mechanisms.

[0011] A number of fusion partners are presumed to delay in vivo clearance of FVIII by interaction with the neonatal Fc receptor (FcRn). Non-limiting examples of fusion partners that assumingly protracts FVIII by interaction with FcRn are immunoglobulin Fc domains, human serum albumin (hSA), and transferrin or parts of these proteins. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with FcRn are shown in Table 2. "Fc fusion derivatives" or "Fc fusion proteins" is herein meant to encompass FVIII variants according to the invention fused to an Fc domain that can be derived from any antibody isotype, although 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 a FVIII with an Fc domain, having the capacity to bind to FcRn receptors, will generally result in a prolonged circulatory half life of the fusion protein compared to the half life of the wt FVIII. Mutations in positions 234, 235 and 237 in an IgG Fc domain will generally result in reduced binding to the Fc.gamma.RI receptor and possibly also the Fc.gamma.RIIa and the Fc.gamma.RIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes a long circulatory 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 C1q-mediated complement fixation (A330S and P331S), respectively.

[0012] However, the present invention also includes FVIII variants fused to Fc domains having altered binding properties to e.g. the neonatal Fc receptors. If e.g. Fc domain variants have increased affinity to e.g. the neonatal Fc receptor, it is plausible that the in vivo circulatory half life of the fusion protein will be further improved. Examples of amino acid replacements in human IgG believed to modulate the affinity of the Fc domain to the neonatal Fc receptor are T250Q, M252Y, S254T, T256E, P257I, T307A, Q311I, and M428L (residue numbering according to the EU index).

[0013] Other fusion partners are presumed to delay in vivo clearance of FVIII by interaction with immunoglobulins. A non-limiting example of fusion partners that assumingly protracts FVIII by interaction with immunoglobulins is Fc receptors such as Fc.hoarfrost.RI (CD64) or FcRn or parts of these proteins. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with immunoglobulins are shown in Table 3.

[0014] Some fusion partners are presumed to delay in vivo clearance of FVIII by reducing the interaction with clearance receptors. A non-limiting example of fusion partners that assumingly protracts FVIII by reducing the interaction with clearance receptors is members of the low-density lipoprotein receptor family Fc receptors such as low-density lipoprotein receptor-related protein or parts of these proteins for instance for instance cluster of repeat 5 (CR5), 6 (CR6), and/or 7 (CR7). Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by reducing the interaction with clearance receptors are shown in Table 4.

[0015] Other fusion partners are presumed to delay in vivo clearance of FVIII by interaction with platelets. A non-limiting example of fusion partners that assumingly protracts FVIII by interaction with platelets is single-chain (SC) antibodies binding to proteins on the platelet surface such as GPIIIa. In SC antibodies, the polypeptide sequence derived from immunoglobulin heavy chain can be situated N-terminal to the polypeptide sequence derived from immunoglobulin light chain. This order is referred to as HC-LC. The polypeptide sequence derived from immunoglobulin heavy chain can also be situated C-terminal to the polypeptide sequence derived from immunoglobulin light chain. The latter order is referred to as LC-HC. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with platelets are shown in Table 5.

[0016] Some fusion partners are presumed to delay in vivo clearance of FVIII by interaction with serum albumin. Non-limiting examples of fusion partners that assumingly protracts FVIII by interaction with serum albumin are single-chain anti-serum albumin antibodies (SC anti-HSA) and albumin-binding polypeptides such as the ABD035 polypeptide. The albumin-binding polypeptides can be repeated several times, for instance 4 repetitions as present in 4XABD035 fusion partner. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with serum albumin are shown in Table 6.

[0017] Other fusion partners are presumed to delay in vivo clearance of FVIII by shielding. A non-limiting example of fusion partners that assumingly protracts FVIII by shielding is polypeptides with stretches of non-hydrophobic amino acids such as Sequence A (seq A) or repletion of elastin-like polypeptide (ELP) for instance ELP60. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by shielding are shown in Table 7.

[0018] Some fusion partners are presumed to delay in vivo clearance of FVIII by modulating the affinity to vWF. A non-limiting example of fusion partners that assumingly protracts FVIII by modulating the affinity to vWF is the a3 region of FVIII (amino acid 1649-1689 of wild-type human FVIII) or parts of the a3 region, thus adding one or more extra a3 regions to FVIII. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by modulating the affinity to vWF are shown in Table 8.

[0019] Some fusion partners are presumed to delay in vivo clearance of FVIII by mechanisms remaining to be determined Non-limiting examples of fusion partners that assumingly protracts FVIII by mechanisms remaining to be determined are growth hormone binding protein (GHBP), parts of coagulation factor IX (FIX), parts of vWF, vWF binding protein, parts of chorion gonadotropin, and parts of coagulation factor X (FX). Non limiting examples of fusion partners derived from FIX are amino acid 298-342 of human FIX (FIX298-342) and amino acid 47-125 of human FIX (FIX47-125). Non-limiting examples of fusion partners derived from vWF are amino acid 1-272 of human vWF (vWF1-272), amino acid 1-1390 of human vWF (vWF1-1390, and amino acid 497-716 of human vWF (vWF497-716). A non-limiting example of fusion partners derived from chorion gonadotropin is the C-terminal 28 amino acids of the beta-chain of human chorion gonadotropin (hCG C-terminus). A non-limiting example of fusion partners derived from FX is the activation peptide of human FX (F10AP). Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by mechanisms remaining to be determined are shown in Table 9.

[0020] Some fusion partners are presumed to delay in vivo clearance of FVIII by modulating the affinity to lipids. A non-limiting example of fusion partners that assumingly protracts FVIII by modulating the affinity to lipids is the C2 domain of FVIII, thus adding one or more extra C2 domains to FVIII. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by modulating the affinity to lipids are shown in Table 10

[0021] Fc receptor: 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: Fc.gamma.RI (CD64), Fc.gamma.RII (CD32), and FC.gamma.RIII (CD16). According to the present invention, the Fc receptors fused to Factor VIII molecules may be of full length or partial length, as well as any variants thereof (variants include amino acid substitutions, deletions and additions). If they are of partial length it follows that the ability to bind antibodies should be retained.

[0022] Von Willebrandt Factor (vWF): vWF is a large mono-/multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (.alpha.-granules of platelets), and subendothelial connective tissue. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites.

[0023] Factor VIII is bound to vWF while inactive in circulation; Factor VIII degrades rapidly or is cleared when not bound to vWF. It thus follows that reduction or abolishment of vWF binding capacity in FVIII has thus far been considered as a highly undesirable approach in obtaining Factor FVIII variants with prolonged circulatory half life.

[0024] The term "reduced capacity to bind vWF" is herein meant to encompass Factor VIII variants, wherein the capacity to bind vWF is decreased by at least 50%, preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, and most preferably about 100%. FVIII binding to vWF may be measured either by an ELISA like assay or as direct binding to immobilized vWF using surface plasmon resonance. The region in Factor VIII responsible for binding to vWF is the region spanning residues 1670-1684 as disclosed in EP0319315. It is envisaged that Factor VIII point and/or deltion mutatants involving this area will modify the ability to bind to vWF. Examples of particularly preferred point mutations according to the present invention include variants comprising one or more of the following point mutations: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

[0025] Factor VIII molecules: FVIII/Factor VIII is a large, complex glycoprotein that primarily is produced by hepatocytes. FVIII consists of 2351 amino acids, including 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. FVIII circulates in plasma as two chains, separated at the B-A3 border. The chains are connected by bivalent metal ion-bindings. The A1-A2-B chain is termed the heavy chain (HC) while the A3-C1-C2 is termed the light chain (LC).

[0026] Endogenous Factor VIII molecules circulate in vivo as a pool of molecules with B domains of various sizes. What probably occurs in vivo is a gradual enzymatic removal of the B domain resulting in a pool of molecules with B-domains of various sizes. It is generally believed that cleavage at position 740, by which the last part of the B-domain is removed, occurs in connection with thrombin activation. However, it cannot be ruled out that a Factor VIII variant in which e.g. the cleavage site at position 740 has been impaired may be active.

[0027] "Factor VIII" or "FVIII" as used herein refers to a human plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation. "Native FVIII" is the full length human FVIII molecule as shown in SEQ ID NO. 1 (amino acid 1-2332). The B-domain is spanning amino acids 741-1648 in SEQ ID NO 1.

TABLE-US-00001 SEQ ID NO 1: ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYK KTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASH PVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVL KENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEK TQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHR QASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSC PEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQAS RPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTV EDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGN QIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS NIMHSINGYFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALL KVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQ KQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTP HGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDM VFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNL AAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDN ALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILES DTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKE GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQL VSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFL TNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNF MKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSK KGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQ FRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGA ITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQD NSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQRE VGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDL FPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATE SSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTIL SLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRH QREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQK KTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKA WAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDT LPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAL YNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQ TPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYR GNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR YLRIHPQSWVHQIALRMEVLGCEAQDLY

[0028] The factor VIII molecules according to the present invention may be B domain truncated Factor FVIII molecules wherein the remaining domains correspond closely to the sequence as set forth in amino acid no 1-740 and 1649-2332 in SEQ ID NO 1 although there may also e.g. be one or more alterations within the vWF binding region between residues 1670-1684. However, B domain truncated molecules according to the invention may differ slight from the sequence set forth in SEQ ID NO 1, meaning that the remaining domains (i.e. the three A-domains and the two C-domains) may differ slightly e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or about 1%, 2%, 3%, 4% or 5% from the amino acid sequence as set forth in SEQ ID NO 1 (amino acids 1-740 and 1649-2332) due to the fact that mutations can be introduced in order to reduce e.g. vWF binding capacity. Furthermore, it is plausible that amino acid modifications (substitutions, deletions, etc.) are introduced other places in the molecule in order to modify the binding capacity of Factor VIII with various other components such as e.g. LRP, various receptors, other coagulation factors, cell surfaces, introduction and/or abolishment of glycosylation sites, etc.

[0029] Factor VIII molecules according to the present invention have Factor VIII activity, meaning the ability to function in the coagulation cascade in a manner functionally similar or equivalent to FVIII, induce the formation of FXa via interaction with FIXa on an activated platelet, and support the formation of a blood clot. The activity can be assessed in vitro by techniques well known in the art such as e.g. clot analysis, endogenous thrombin potential analysis, etc. Factor VIII molecules according to the present invention have FVIII activity being at least about 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and 100% or even more than 100% of that of native human FVIII.

[0030] B domain: The B-domain in Factor VIII spans amino acids 741-1648 in SEQ ID NO 1. The B-domain is cleaved at several different sites, generating large heterogeneity in circulating plasma FVIII molecules. The exact function of the heavily glycosylated B-domain is unknown. What is known is that the domain is dispensable for FVIII activity in the coagulation cascade. This apparent lack of function is supported by the fact that B domain deleted/truncated FVIII appears to have in vivo properties identical to those seen for full length native FVIII. That being said there are indications that the B-domain may reduce the association with the cell membrane, at least under serum free conditions.

[0031] B domain truncated/deleted Factor VIII molecule: 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 B domain-deleted FVIII can be produced from two different strategies. Either the heavy chain without the B-domain and the light chain are synthesized individually as two different polypeptide chains (two-chain strategy) or the B-domain deleted 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.

[0032] In a B domain-deleted FVIII precursor polypeptide, the heavy and light chain moieties are normally separated by a linker To minimize the risk of introducing immunogenic epitopes in the B domain-deleted FVIII, the sequence of the linker is preferable derived from the FVIII B-domain. As a minimum, the linker must comprise a recognition site for the protease that separates the B domain-deleted FVIII precursor polypeptide into the heavy and light chain. In the B domain of full length FVIII, amino acid 1644-1648 constitutes this recognition site. The thrombin site leading to removal of the linker on activation of B domain-deleted FVIII is located in the heavy chain. Thus, the size and amino acid sequence of the linker is unlikely to influence its removal from the remaining FVIII molecule by thrombin activation. Deletion of the B domain is an advantage for production of FVIII. Nevertheless, parts of the B domain can be included in the linker without reducing the productivity. The negative effect of the B domain on productivity has not been attributed to any specific size or sequence of the B domain.

[0033] The truncated B-domain may contain several O-glycosylation sites. However, according to a preferred embodiment, the molecule comprises only one, alternatively two, three or four O-linked oligosaccharides in the truncated B-domain.

[0034] According to a preferred embodiment, the truncated B domain comprises only one potential O-glycosylation sites and a side chain, such as e.g. PEG or an albumin binder is covalently conjugated to this O-glycosylation site.

[0035] The O-linked oligosaccharides in the B-domain truncated molecules according to the invention may be attached to O-glycosylation sites that were either artificially created by recombinant means and/or by exposure of "hidden" O-glycosylation sites by truncation of the B-domain. In both cases, such molecules may be made by designing a B-domain truncated Factor VIII amino acid sequence and subsequently subjecting the amino acid sequence to an in silico analysis predicting the probability of O-glycosylation sites in the truncated B-domain. Molecules with a relatively high probability of having such glycosylation sites can be synthesized in a suitable host cell followed by analysis of the glycosylation pattern and subsequent selection of molecules having O-linked glycosylation in the truncated B-domain.

[0036] The Factor VIII molecule also contains a number of N-linked oligosaccharides and each of these may likewise serve as an anchor for attachment of a hydrophobic side group.

[0037] Suitable host cells for producing recombinant factor VIII fusion proteins according to the invention are preferably of mammalian origin in order to ensure that the molecule is glycosylated. In practicing the present invention, the cells are mammalian cells, more preferably an established mammalian cell line, including, without limitation, CHO, COS-1, baby hamster kidney (BHK), and HEK293 cell lines. Other suitable cell lines include, without limitation, Rat Hep I, Rat Hep II, TCMK, Human lung, DUKX cells (CHO cell line) Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of myelomas with other cells. In some embodiments, the cells may be mutant or recombinant cells, such as, e.g., cells that express a qualitatively or quantitatively different spectrum of enzymes that catalyze post-translational modification of proteins (e.g., glycosylation enzymes such as glycosyl transferases and/or glycosidases, or processing enzymes such as propeptides) than the cell type from which they were derived. DUKX cells (CHO cell line) are especially preferred.

[0038] Currently preferred cells are HEK293, COS, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) and myeloma cells, in particular Chinese Hamster Ovary (CHO) cells.

[0039] The length of the B domain in the wt FVIII molecule is about 908 amino acids. The length of the truncated B domain in molecules according to the present invention may vary from about 10 to about 800 amino acids, such as e.g. from about 10 amino acids to about 700 acids, such as e.g. about 12-500 amino acids, 12-400 amino acids, 12-300 amino acids, 12-200 amino acids, 15-100 amino acids, 15-75 amino acids, 15-50 amino acids, 15-45 amino acids, 20-45 amino acids, 20-40 amino acids, or 20-30 amino acids. The truncated B-domain may comprise fragments of the heavy chain and/or the light chain and/or an artificially introduced sequence that is not found in the wt FVIII molecule. The terms "B-domain truncated" and "B-domain deleted" may be used interchangeably herein.

[0040] Modified circulatory half life: Molecules according to the present invention have a modified circulatory half life compared to the wild type Factor VIII molecule, preferably an increased circulatory half life. Circulatory half life is preferably increased at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably at least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and most preferably at least 250% or 300%. Even more preferably, such molecules have a circulatory half life that is increased at least 400%, 500%, 600%, or even 700%.

[0041] Hydrophilic polymer: The side group according to the present invention is preferably a non-naturally occurring hydrophilic polymer comprising at least one non-naturally occurring polymeric moiety. In another example, the non-naturally occurring modifying group is a modified carbohydrate.

[0042] Exemplary hydrophilic polymers according to the invention include water soluble polymers that can be linear or branched and can include one or more independently selected polymeric moieties, such as poly(alkylene glycol) and derivatives thereof. The polymeric modifying group according to the invention may include a water-soluble polymer, e.g. poly(ethylene glycol) and derivatived thereof (PEG, m-PEG), poly(propylene glycol) and derivatives thereof (PPG, m-PPG) and the like.

[0043] The polymer backbone of the water-soluble polymer according to the invention can be poly(ethylene glycol) (i.e. PEG). The term PEG in connection with the present invention includes poly(ethylene glycol) in any of its forms, including alkoxy PEG, difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein.

[0044] The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine or cysteine. In one example, the branched poly(ethylene glycol) can be represented in general form as R(-PEG-OH)m in which R represents the core moiety, such as glycerol or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in U.S. Pat. No. 5,932,462, which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.

[0045] Many other polymers are also suitable for the invention. Polymer backbones that are non-peptidic and water-soluble, are particularly useful in the invention. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly([alpha]-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such as described in U.S. Pat. No. 5,629,384, which is incorporated by reference herein in its entirety, as well as copolymers, terpolymers, and mixtures thereof.

[0046] Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 100 Da to about 160,000 Da, such as e.g. from about 5,000 Da to about 100,000 Da. More specifically, the size of each conjugated hydrophilic polymer according to the present invention may vary from about 500 Da to about 80,000 Da, such as e.g. about 1000 Da to about 80,000 Da; about 2000 Da to about 70,000 Da; about 5000 to about 70,000 Da; about 5000 to about 60,000 Da; about 10,000 to about 70,000 Da; about 20,000 to about 60,000 Da; about 30,000 to about 60,000 Da; about 30,000 to about 50,000 Da; or about 30,000 to about 40,000 Da. It should be understood that these sizes represent estimates rather than exact measures. According to a preferred embodiment, the molecules according to the invention are conjugated with a heterogenous population of hydrophilic polymers, such as e.g. PEG of a size of e.g. 10,000, 40,000, or 80,000 Da+/-about 5000, about 4000, about 3000, about 2000, or about 1000 Da.

[0047] Side chain/side group: The Factor VIII molecules according to the present invention may also be conjugated with side groups other than hydrophilic polymers. The side chain according to the present invention may e.g. be selected from one or more of the list consisting of: fatty acids and derivates thereof (sometimes referred to as "albumin binders"), peptides, etc., The hydrophobic side groups according to the invention are preferably biologically degradable. There may furthermore be more than one hydrophobic side group conjugated to an individual Factor VIII molecule, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. It furthermore follows that it is possible to conjugate Factor VIII molecules according to the invention with one or more hydrophobic side groups as well as one or more hydrophobic side groups, peptidic side groups, etc.

[0048] An individual Factor VIII molecule according to the invention may thus comprise side groups of e.g. both hydrophilic and hydrophobic/peptidic nature. It is nevertheless associated with additional efforts to conjugate the molecule with different types of side groups and such solutions may thus turn out to be relatively impractical in practice. The molecules according to the present invention thus preferably comprise only one type of side chains (e.g. hydrophilic/hydrophobic side chains).

[0049] Conjugation of Factor VIII with hydrophobic side groups has thus far, however, not been considered being an attractive alternative to conjugation with hydrophilic groups. One explanation may be that laborious and/or harsh chemical methods employing organic solvents would have been expected to be required. Another explanation may be that the usually relatively small hydrophobic molecules cannot be expected to be efficient in binding a conjugated (large) Factor VIII molecule to e.g. albumin and thereby possibly shielding the molecule from clearance.

Albumin Binder Conjugates

[0050] It is known that the in vivo properties of such proteins can be improved by the use of albumin binding side chains. Such side chains, or albumin binders, can be attached to the protein prior to administration and can, for example, stabilise the protein in vivo or improve or extend the in vivo half-life of the protein.

[0051] The albumin binder may thereby promote the circulation of the derivative with the blood stream. The albumin binder may have the effect of extending or protracting the time of action of the protein that it is bound to it, due to the fact that the complexes of the peptide derivative and albumin are only slowly disintegrated to release the active pharmaceutical ingredient. Thus, a preferred substituent, or side chain, as a whole may be referred to as an albumin binding moiety.

[0052] The albumin binder (albumin binding moiety) may comprise a portion which is particularly relevant for the albumin binding and thereby the protraction of circulation in the blood stream, which portion may accordingly be referred to as a protracting moiety. The protracting moiety is preferably at, or near, the opposite end of the albumin binding moiety as compared to its point of attachment to the peptide.

[0053] In a preferred embodiment, the albumin binder is, or comprises, a side chain that is capable of forming non-covalent complexes with albumin. The albumin binder may bind albumin non-covalently and/or reversibly. The albumin binder may bind albumin specifically. As is clear from the methods described below, the albumin binder may bind to cyclodextrin. The albumin binder may bind cyclodextrin non-covalently and/or reversibly. The albumin binder may bind cyclodextrin specifically.

[0054] An albumin binder as described herein is generally a hydrophobic group.

[0055] The other portion of the albumin binding moiety, i.e. the portion in-between the protracting moiety and the point of attachment to the peptide, may be referred to as a linker moiety, linker, spacer, or the like. However, the presence of such a linker is optional, and hence the albumin binding moiety may be identical to the protracting moiety.

[0056] In particular embodiments, the albumin binding moiety and/or the protracting moiety is lipophilic, and/or negatively charged at physiological pH (7.4).

[0057] The albumin binding moiety and/or the protracting moiety may be covalently attached to an amino group of the peptide by conjugation chemistry such as by alkylation, acylation, or amide formation; or to a hydroxyl group, such as by esterification, alkylation, oximation.

[0058] In a preferred embodiment, an active ester of the albumin binding moiety and/or the protracting moiety is covalently linked to an amino group of a sialic acid residue or a sialic acid derivative, under formation of an amide bond (this process being referred to as acylation).

[0059] Unless otherwise stated, when reference is made to an acylation of a protein, it is understood to be to an amino-group linked to a sialic acid residue on on glycoprotein.

[0060] For the present purposes, the terms "albumin binding moiety", "protracting moiety", and "linker" include the un-reacted as well as the reacted forms of these molecules. Whether or not one or the other form is meant is clear from the context in which the term is used.

[0061] The albumin binding moiety may be, or may comprise a fatty acid or fatty diacid or a derivative or either thereof.

[0062] The term "fatty acid" refers to aliphatic monocarboxylic acids having from 4 to 28 carbon atoms, such as 16 carbon atoms. It is preferably unbranched, and/or even numbered, and it may be saturated or unsaturated.

[0063] The term "fatty diacid" refers to fatty acids as defined above but with an additional carboxylic acid group in the omega position. Thus, fatty diacids are dicarboxylic acids.

[0064] The nomenclature is as is usual in the art, for example --COOH, as well as HOOC--, refers to carboxy; --C.sub.6H.sub.4-- to phenylen; --CO--, as well as --OC--, to carbonyl (O.dbd.C<); and C.sub.6H.sub.5--O-- to phenoxy.

[0065] In a preferred embodiment the linker moiety, if present, has from 2 to 80 C-atoms, preferably from 5 to 70 C-atoms. In additional preferred embodiments, the linker moiety, if present, has from 4 to 20 hetero atoms, preferably from 2 to 40 hetero atoms, more preferably from 3 to 30 hetero atoms. Particularly preferred examples of hetero atoms are N-, and O-atoms. H-atoms are not hetero atoms.

[0066] In another embodiment, the linker comprises at least one OEG molecule, and/or at least one glutamic acid residue, or rather the corresponding radicals (OEG designates 8-amino-3,6-dioxaoctanic acid, i.e. this radical: --NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--CO--).

[0067] In one preferred embodiment, the linker moiety comprises a di-carboxyl residue linked to a sialic acid residue by an amide bond. In preferred examples, the di-carboxyl residue has from 2-30 C-atoms, preferably 4-20 C-atoms, more preferably 4-10 C-atoms. In additional preferred examples, the di-carboxyl residue has from 0-10 hetero-atoms, preferably 0-5 hetero-atoms.

[0068] In another preferred example, the linker moiety comprises a group containing both an amino and a distal carboxyl-group linked to a sialic acid residue by an amide bond through its distal carboxyl groups. In one preferred embodiment the this group is an OEG group.

[0069] The amino acid glutamic acid (Glu) comprises two carboxylic acid groups. Its gamma-carboxy group is preferably used for forming an amide bond with an amino group of a sialic acid residue or a sialic acid derivative, or with an amino group of an OEG molecule, if present, or with the amino group of another Glu residue, if present. The amino group of Glu in turn forms an amide bond with the carboxy group of the protracting moiety, or with the carboxy group of an OEG molecule, if present, or with the gamma-carboxy group of another Glu, if present. This way of inclusion of Glu is occasionally briefly referred to as "gamma-Glu".

[0070] Without being bound by theory it is envisaged that the reason why it may be advantageous to attach hydrophobic side groups to Factor VIII molecules with reduced vWF binding capacity rather than attaching such side groups to Factor VIII molecules with normal vWF binding capacity is that the relative size of the side group is relatively small in the large Factor VIII/vWF complex. It is hypothesized that a relatively large side group functions more efficiently in shielding the free Factor VIII from clearance. It is further hypothesized that the half life of FVIII is related to that of vWF. FVIII molecules with reduced ability to bind vWF most likely have exposed clerance epitopes which would normally have been shielded by vWF. By attaching side groups it is thus hypothesized that this "clearance shielding" can be regained. In other cases, attachment of side groups such as e.g. antibody fragments may function by e.g. attaching the molecule to proteins, cells, or platelets having a relatively long circulatory half life.

[0071] O-linked oligosaccharide: Both N-glycans and O-glycans are attached to proteins by the cells producing the protein. The cellular N-glycosylation machinery recognizes and glycosylates N-glycosylation signals (N-X-S/T motifs) in the amino acid chain, as the nascent protein is translocated from the ribosome to the endoplasmic reticulum (Kiely et al. 1976; Glabe et al. 1980).

[0072] Likewise, O-glycans are attached to specific O-glycosylation sites in the amino acid chain, but the motifs triggering O-glycosylation are much more heterogenous than the N-glycosylation signals, and our ability to predict O-glycosylation sites in amino acid sequences is still inadequate (Julenius et al. 2004). The construction of artificial O-glycosylation sites it is thus associated with some uncertainty.

[0073] Linkage of hydrophobic side groups: Conjugation of Factor FVIII molecules with hydrophobic side groups may be made using chemical methods. However, a number of advantages are potentially associated with employment of an enzymatic approach. According to a preferred enzymatic method according to the present invention, [hydrophobic protractor group]-sialyl-CMP substrate can be prepared chemically. This substrate can be transferred enzymatically to glycans present on Factor FVIII using a sialyltransferase enzyme. The inventors of the present invention have surprisingly demonstrated that this enzymatic approach can be performed without addition of any organic solvents that may. A number of disadvantages are associated with use of organic solvents, e.g. loss of biological activity, environmental concerns, additional steps to be taken to ensure that the organic solvents are completely removed, etc. It may also be possible to avoid addition of cyclodextrin--cyclodextrin is a detergent which may also contribute to loss of biological function. In the process of enzymatic conjugation, it may be an advantage to add glycerol, e.g. 5-30% glycerol, preferably 10-20% glycerol. The presence of glycerol seem to stabilize the Factor VIII molecule e.g. in the freeze/thaw process and it may also prevent formation of Factor VIII crystals. Glycerol present during enzymatic conjugation does thus not need to be removed after the conjugation process has been completed.

[0074] Sialyltransferase: Sialyltransferases are enzymes that transfer sialic acid to nascent oligosaccharide. Each sialyltransferase is specific for a particular sugar substrate. Sialyltransferases add sialic acid to the terminal portions of the sialylated glycolipids (gangliosides) or to the N- or O-linked sugar chains of glycoproteins. There are about twenty different sialyltransferases which can be distinguished on the basis of the acceptor structure on which they act and on the type of sugar linkage they form. Preferred sialyltransferases according to the present invention are ST3Gal-I (specific for O-glycans) and ST3Gal-III (specific for N-glycans). It is thus possible to engineer the structure of the conjugated Factor VIII molecules according to the present invention by e.g. selection of a specific sialyltransferase and/or engineering of a Factor VIII molecule with a particular glycosylation pattern.

[0075] Pharmaceutical composition: A pharmaceutical composition is herein preferably meant to encompass compositions comprising Factor VIIII molecules according to the present invention suitable for parenteral administration, such as e.g. ready-to-use sterile aqueous compositions or dry sterile compositions that can be reconstituted in e.g. water or an aqueous buffer. The compositions according to the invention may comprise various pharmaceutically acceptable excipients, stabilizers, etc.

[0076] Additional ingredients in such compositions may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the FVIII compound in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the FVIII compound of the invention may also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.

[0077] The term "treatment", as used herein, refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health. Thus, said treatment may be prophylactic, palliative, symptomatic and/or curative.

[0078] In a first aspect, the present invention relates to a recombinant Factor VIII molecule, wherein said Factor VIII molecule is a fusion protein comprising a Factor VIII molecule and a fusion partner. In a first embodiment, the fusion partner is replacing the a3-domain of the Factor VIII molecule. The small a3-domain of Factor VIII may thus be fully or partly replaced by the fusion partner. In a second embodiment, the fusion partner is inserted into the B-domain of Factor VIII. The B-domain may be a full length B-domain but in a preferred embodiment, the B domain is significantly truncated. There will thus be at least three, four, five, six, seven, eight, nine or ten amino acids originating from the B-domain at both the N- and the C-terminal ends of the fusion partner. In another preferred embodiment, the fusion partner is inserted in or at the C-terminal end of the C2 domain in Factor FVIII.

[0079] In a third embodiment, the Factor VIII molecule according to the invention is fused to an antibody binding molecule such as e.g. an Fc receptor. Examples of antibody binding molecules are listed in the tables below. In a fourth embodiment, the Factor VIII molecule is fused to a molecule having the capability of binding to human serum albumin. In another embodiment, the Factor VIII is fused to transferrin. Examples thereof are also provided below. In a fifth embodiment, the Factor VIII molecule is fused with a molecule having the capability of binding to platelets--specific examples of such molecules are likewise provided below. In yet another embodiment, the Factor VIII molecule is fused to a molecule with the capacity of binding to a Factor VIII clearance receptor.

[0080] In a sixth embodiment, the Factor VIII molecule according to the invention has reduced vWF binding capacity. Preferably, such Factor VIII molecules comprise a mutation (substitution, deletion or addition of amino acids) within the area spanning amino acids 1670-1684 in SEQ ID NO:1. Most preferably, such Factor VIII molecules comprise one of the following point mutations: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

[0081] In a seventh embodiment, the molecule according to the invention is conjugated with a side group. This side group may be selected from one or more of the list consisting of: hydrophilic polymers, peptides and hydrophobic side groups. Preferably, the side group is a PEG group, a fatty acid derivative, or a polypeptide. Preferably, such side groups are attached to the molecule using enzymatic approaches, such as e.g. the technology disclosed in WO0331464, wherein O-linked and/or N-linked glycans are used as linkers.

[0082] Another aspect of the present invention relates to FVIII molecule fused to a fusion partner, wherein the fusion partner is replacing the A3-domain of the Factor VIII molecule.

[0083] In one embodiment, the fusion partner is albumin. In another embodiment, the fusion partner is an Fc receptor. In another embodiment, the Fc receptor is Fc.gamma.RI. In another embodiment, the fusion partner is an Fc domain. In another embodiment, the Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor. In another embodiment, the fusion protein is conjugated with a side group. In another embodiment, the side group is linked to the fusion protein via an N-linked and/or an O-linked glycan. In another embodiment, the side group is linked to an N-linked and/or an O-linked glycan via a sialic acid. In another embodiment, the side group is selected from one or more of the list consisting of hydrophilic polymers, peptides, and hydrophobic side groups. In another embodiment, the FVIII molecule is a B domain truncated molecule wherein the B domain comprises the sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein comprises a side group linked to the O-linked glycan in the truncated B domain that comprises the amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the Factor VIII molecule has reduced vWF binding capacity. In another embodiment, the Factor VIII molecule having reduced vWF binding capacity comprises a mutation selected from the list consisting of: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

[0084] Another aspect of the present invention relates to a FVIII molecule fused to a fusion partner, wherein the fusion partner is inserted into the B-domain of Factor VIII.

[0085] In one embodiment, the fusion partner is albumin. In another embodiment, the fusion partner is an Fc receptor. In another embodiment, the Fc receptor is Fc.gamma.RI. In another embodiment, the fusion partner is an Fc domain. In another embodiment, the Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor. In another embodiment, the fusion protein is conjugated with a side group. In another embodiment, the side group is linked to the fusion protein via an N-linked and/or an O-linked glycan. In another embodiment, the side group is linked to an N-linked and/or an O-linked glycan via a sialic acid. In another embodiment, the side group is selected from one or more of the list consisting of hydrophilic polymers, peptides, and hydrophobic side groups. In another embodiment, the FVIII molecule is a B domain truncated molecule wherein the B domain comprises the sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein comprises a side group linked to the O-linked glycan in the truncated B domain that comprises the amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the Factor VIII molecule has reduced vWF binding capacity. In another embodiment, the Factor VIII molecule having reduced vWF binding capacity comprises a mutation selected from the list consisting of: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

[0086] Another aspect of the present invention relates to a FVIII molecule fused to a fusion partner, wherein the fusion partner is inserted in the C-terminal end of the C2 domain in Factor FVIII.

[0087] In one embodiment, the fusion partner is albumin. In another embodiment, the fusion partner is an Fc receptor. In another embodiment, the Fc receptor is Fc.gamma.RI. In another embodiment, the fusion partner is an Fc domain. In another embodiment, the Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor. In another embodiment, the fusion protein is conjugated with a side group. In another embodiment, the side group is linked to the fusion protein via an N-linked and/or an O-linked glycan. In another embodiment, the side group is linked to an N-linked and/or an O-linked glycan via a sialic acid. In another embodiment, the side group is selected from one or more of the list consisting of hydrophilic polymers, peptides, and hydrophobic side groups. In another embodiment, the FVIII molecule is a B domain truncated molecule wherein the B domain comprises the sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein comprises a side group linked to the O-linked glycan in the truncated B domain that comprises the amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the Factor VIII molecule has reduced vWF binding capacity. In another embodiment, the Factor VIII molecule having reduced vWF binding capacity comprises a mutation selected from the list consisting of: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

[0088] Another aspect relates to a method of making a molecule according to the present invention, wherein said method comprises incubating a host cell encoding said molecule under appropriate conditions. Accordingly, the present invention also relates to nucleic acids molecules as well as expression vectors and host cells comprising nucleic acid sequences that encode a molecule according to the present invention. Molecules obtained by or obtainable by such methods are likewise an aspect of the present invention.

[0089] Another aspect relates to use of a molecule according to the present invention as a medicine.

[0090] Another aspect relates to use of a molecule according to the present invention for treatment of haemophilia, preferably haemophilia A.

[0091] Another aspect relates to a pharmaceutical composition comprising a molecule according to the invention and optionally one or more pharmaceutically acceptable excipients.

[0092] Another aspect of the invention relates to a method of treatment of a hemophilic disease comprising administering to a patient in need thereof a therapeutically effective amount of a molecule according to the present invention.

EXAMPLES

Example 1

FVIII Frameworks and Fusion Partners

[0093] The fusion proteins of the present invention consist of a FVIII protein (FVIII part) joined to a polypeptide (fusion partner) from another protein.

[0094] The FVIII part of the fusion protein can be any protein with FVIII activity. The FVIII part can be a B domain-deleted/truncated (BDD) FVIII protein, in which parts of the FVIII B domain has been removed from the protein. Non-limiting examples of FVIII frameworks that can constitute the FVIII part of fusion proteins is shown in Table 1. F8-500 is a BDD human FVIII protein. Starting at the N-terminus, F8-500 consists of FVIIIs signal peptide (amino acid -19 to -1) followed by FVIII HC without the B domain (amino acid 1-740), a 21 amino acid linker (SFSQNSRHPSQNPPVLKRHQR) (SEQ ID NO: 2), and FVIII LC (amino acid 1649-2332 of wild-type human FVIII. The sequence of the 21 amino acid linker is derived from the B domain of FVIII and consists of amino acid 741-750 and 1638-1648 of full-length wild-type human FVIII.

[0095] F8-500-.DELTA.a3 consists of F8-500 without the a3 region. In F8-500-.right brkt-bot.a3 amino acid 1647-1687 of wild-type human FVIII is eliminated from F8-500. Thereby, the furin site at amino acid 1645-1648 is destroyed. A combined furin and thrombin site is, however, created by the R1645-H1646-P1688-R1689 amino acid stretch in F8-500-.DELTA.a. The a3 region is important for binding of FVIII to vWF and therefore, the affinity of F8-500-.DELTA.a3 for vWF is reduced compared to wild-type FVIII.

[0096] F8-500-His consists of F8-500 with a His tag inserted in the linker of F8-500. Thus the linker sequence of F8-500-His is SFSQNSRHPSHHHHHHSQNPPVLKRHQR (SEQ ID NO: 3).

[0097] F8-500-.DELTA.a3-His consists of F8-500 without the a3 region but with a His tag inserted in the linker of F8-500. Thus, in F8-500-.DELTA.a3-His amino acid 1647-1687 of wild-type human FVIII has been eliminated from F8-500 and the linker sequence is SFSQNSRHPSHHHHHHSQNPPVLKRHQR (SEQ ID NO: 4).

[0098] F8-500-Y1680F and F8-500-Y1680C consist of F8-500 in which amino acid 1680 of full-length wild-type human FVIII has been changed from tyrosine to phenylalanine and cysteine, respectively. Both these amino acid replacements reduce the affinity of FVIII to vWF factor. Furthermore, the Y1680C amino acid replacement introduces a free cysteine that can be used as a handle for conjugating protracting moieties to the fusion protein.

[0099] The fusion partner can be joined to several positions on the FVIII part of the fusion protein. Non-limiting examples of positions on FVIII for joining to the fusion partner are in the B domain or the B-domain-derived linker between the FVIII HC and LC, at the position of a3, and at the C-terminus of FVIII LC.

Example 2

Construction of Expression Vectors Encoding FVIII Frameworks and Fusion Proteins

[0100] The fusions between FVIII and fusions partners all involves PCR for amplifying the fusion partner. Restriction sites are added to the ends of the PCR primers used. Restriction enzymes are used for cloning of fusion partner cDNA or synthetic DNA into FVIII cDNA.

[0101] Fusions in the B-domain of F8-500 takes place between aa750 and aa1638. Restriction sites AvrII, NruI, AgeI and MluI within or flanking the B-domain are used for insertion of the fusion partner encoding DNA.

[0102] For fusions at the carboxy terminus of FVIII light chain, the F8-500 coding construct is modified. The internal BamHI site (aa 604-606) is eliminated by site-directed mutagenesis and DNA encoding the flexible (GGGS).sub.6 linker is inserted 3' to the coding region. A new BamHI site is introduced in the 3' end of the linker-coding DNA in order to ease cloning of C-terminal fusion partners between BamHI and NotI sites. Subsequently, fusion partner DNA is inserted. The fusions proteins derived from this construct is referred to as F8-500-C2-linked-(GGGS)6-X in Table 2-12 Similar to the (GGGS)6 linker a minimal GS-linker (BamHI restriction site) was inserted in the 3' end of the F8-500 coding region. The BamHI restriction site (GGATCC) form the two codons of GS (Glycine-Serine). The fusions proteins derived from this construct is referred to as F8-500-C2-linked-GS-X in Table 2-12. Fusions to the C-terminus of F8-500 without any linker were made by PCR amplifying the fusions with extended primers harboring the last 109 bp of the F8-500 coding region in the 5'end and a NotI restriction site in the 3'end of the PCR product. The XbaI restriction site is present 104-109 bp from the F8-500 stop codon. XbaI and NotI restriction enzymes were used for cloning the fusion partners without linkers. The fusions proteins derived from these latter construct is referred to as F8-500-C2-linked-X in Table 2-12.

[0103] For insertion of the fusion partner coding DNA at a3 positions thus replacing a3 with the fusion partner in the encoded protein, the SacII restriction site is introduced 3' to the coding region of a3. Thus, fusion partner coding DNA can be introduced by insertion between the AgeI and SacII sites or between the AvrII and SacII sites.

TABLE-US-00002 hFc (SEQ ID NO: 5) HTCPPCPAPEAEGEPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK mFc (SEQ ID NO: 6) KPCPPCKCPAPNAEGEPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVLTA QTQTHREDYQSTLRVVSALPIQHQDWMSGKEFKCKVNNKALPAPIERTISKPKGSVRAPQVYVL PPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEK KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Human Serum Albumin (HSA) (SEQ ID NO: 7) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCD KSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGK ASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLEC ADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLV EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKR MPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEG KKLVAASQAALGL Transferrin (SEQ ID NO: 8) VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDA GLVYDAYLAPNNLIKPVVAEFYGSKEDPQTFYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAG WNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAF KCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYL GYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIA KIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDLTW DNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLN LCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDG TRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLL FRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP hFc_RI (CD64) (SEQ ID NO: 9) QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDS GEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAF KFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVIKELFPAPVLNASVTSPLLEGNLVT LSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLIKRSP ELELQVLGLQLPTP FcRn (SEQ ID NO: 10) AESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNSLRGEAEPCGAWVWENQVSWYWE KETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGT WGGDWPEALAISQRWQQQDKAANKELTFLLFSCPHRLREHLERGRGNLEWKEPPSMRLKARPS SPGFSVLTCSAFSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQ HAGLAQPLRVELESPAKSS FcRn-H166K (SEQ ID NO: 11) AESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNSLRGEAEPCGAWVWENQVSWYWE KETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGT WGGDWPEALAISQRWQQQDKAANKELTFLLFSCPHRLREKLERGRGNLEWKEPPSMRLKARPS SPGFSVLTCSAFSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQ HAGLAQPLRVELESPAKSS LRP-CR5-6 (SEQ ID NO: 12) TCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDND NDCGDNSDEAGCSH LRP-CR6-7 (SEQ ID NO: 13) TCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHWTCDGDN DCGDYSDETHANCTNQATR LRP-CR6 (SEQ ID NO: 14) TCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSH SC anti-GPIIIa-1-HC-LC (SEQ ID NO: 15) MDILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFMGLIYYGTNLVDGVPSRFSGS GSGADYSLTISSLDSEDFADYYCVQYAQLPYTFGGGTKLEKLGGGGSGGGGSGGGGSNSVQLQ QSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPANGYTKYDPKFQGKA TITADTSSNTAYLQLSSLTSEDTAVYYCVRPLYDYYAMDYWGQGTSVTVSS Linker-SC anti-GPIIIa-1-HC-LC (SEQ ID NO: 16) MDILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFMGLIYYGTNLVDGVPSRFSGS GSGADYSLTISSLDSEDFADYYCVQYAQLPYTFGGGTKLEKLGGGGSGGGGSGGGGSNSVQLQ QSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPANGYTKYDPKFQGKA TITADTSSNTAYLQLSSLTSEDTAVYYCVRPLYDYYAMDYWGQGTSVTVSSGGGGSGGGGSGG GGS SC anti-GPIIIa-2-HC-LC (SEQ ID NO: 17) QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNKYNEN FKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVSSGGGGSG GGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRM SNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKR SC anti-GPIIIa-2-LC-HC (SEQ ID NO: 18) DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMSNLASGVPDR FSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRGGGGSGGGGSGGGGSQV QLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNKYNENFK GKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVSS ABD035 (SEQ ID NO: 19) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP Sequence A (SEQ ID NO: 20) GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG ELP80 (SEQ ID NO: 21) GVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVG VPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGV PGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVP GVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPG AGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGV GVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGG VPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGV PGVGVPGA Extra a3 (SEQ ID NO: 22) EITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR GHBP (SEQ ID NO: 23) FSGSEATAAILSRAPWSLQSVNPGLKTNSSKEPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQL FYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQP DPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILT TSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQMSQ FIX298-342 (SEQ ID NO: 24) IFLKFGSGYVSGWARVFHKGRSALVLQYLRVPLVDRATCLRSTKF FIX47-125 (SEQ ID NO: 25) DDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV CSCTEGYRLAENQKSCE vWF1-272 (SEQ ID NO: 26) SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALER CPCFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGEC QYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVIKRPMKDETHF EVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDP VDFGNSWKVSSQCADTR vWF1-1390 (SEQ ID NO: 27) SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALER CPCFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGEC

QYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVIKRPMKDETHF EVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDP VDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLD VCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRY NSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGK KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLL DLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSEL RRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLK KKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPD MAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSI HVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQ APNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLV LQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSV LQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAV VILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTM VTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGL RPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVIL HNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEV RFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEW TVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKV vWF497-716-R545A (SEQ ID NO: 28) EDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLAISQKWVRVAVVEYHD GSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEP QRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVS YLCDLAPEAPPPTLPPDMAQVTVG vWF binding protein (SEQ ID NO: 29) NPELKDFNEEEQLKCDLELNKLENQILMLGKTFYQNYRDDVESLYSKLDLIMGYKDEERANKK AVNKRMLENKKEDLETIIDEFFSDIDKTRPNNIPVLEDEKQEEKNHKNMAQLKSDTEAAKSDESK RSKRSKRSLNTQNHKPASQEVSEQQKAEYDKRAEERKARFLDNQKIKKTPVVSLEYDFEHKQRI DNENDKKLVVSAPTKKPTSPTTYTETTTQVPMPTVERQTQQQIIYNAPKQLAGLNGESHDFITTH QSPTTSNHTHNN hCG C-terminus (SEQ ID NO: 30) SSSSKAPPPSLPSPSRLPGPSDTPILPQ F10AP (SEQ ID NO: 31) SVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNL

Example 3

Transient Expression of FVIII Frameworks and Fusion Proteins

[0104] HKB11 cells at a density of 0.9-1.1.times.10.sup.6 are transfected with a complex of plasmid 0.7 mg/l and the transfection agent, 293Fectin (Invitrogen) 1.4 ml/l. The transfection complex is prepared by diluting the plasmid and the transfection separately in OPTIMEM (Invitrogen), mixing of the two solutions, and incubation of the mixture at room temperature for 20 minutes. The complex mixture is added to the cell suspension and the suspension is incubated in shaker incubator for 5 days at 36.5.degree. C. and 5% CO.sub.2. The cell culture harvest is filtered on a 0.22 .mu.m membrane filter. FVIII frameworks and fusion proteins are purified from the cell culture harvest as described in Example 5.

Example 4

Stable Cell Expressing Fusion Protein

[0105] Serum-free adapted CHO-DUKX-B11 cells were transfected with an expression plasmid encoding the F8-500-albumin-.DELTA.a3 fusion protein. Transfected cells were selected with the dihydrofolate reductase system and cloned by limiting dilution. Clones were screened for FVIII production by ELISA and chromogenic activity assay. The clone JsJH009 was seleceted for upscaling. The cells were transferred to a bioreactor. The F8-500-albumin-.DELTA.a3 fusion was purified from cell culture harvests as described in Example 5.

Example 5

Purification of FVIII Frameworks and Fusion Proteins

[0106] A column was packed with the resin VIIISelect (GE Healthcare), with the dimensions 1.6 cm in diameter and 4 cm in bed height giving 8 mL, and was equilibrated with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+250 mM NaCl, pH7.3 at 500 cm/h. The culture filtrate prepared as described in Example 3 was applied to the column, and the column was subsequently washed with first equilibration buffer and then 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+1.5M NaCl, pH7.3. The bound FVIII was eluted isocratic at 90 cm/h with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+2.5 M NaCl+6.5M Propylenglycol, pH7.3. The fractions containing FVIII were pooled and diluted 1:10 with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80, pH7.3 and applied to a column packed with F25-Sepharose (Thim et al., Haemophilia, 2009). The column dimension was 1.6 cm in diameter and 2 cm in bed height giving 4 mL in column volume. The column was equilibrated at 180 cm/h with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+150 mM NaCl+1M Glycerol, pH7.3 prior to application. After application the column was washed first with equilibration buffer and then 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+650 mM NaCl, pH7.3. The bound FVIII was isocratic eluted with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+2.5M NaCl+50% (v/v) Ethylenglycol, pH7.3 at 30 cm/h. The fractions containing FVIII were pooled and diluted 1:15 with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80, pH7.3, except FVIII-variants with deletions of the a3 domain which were diluted 1:45 in the same buffer. The diluted pool was applied to a column packed with Poros 50HQ (PerSeptive Biosystem), with the column dimensions 0.5 cm in diameter and 5 cm in bed height giving 1 mL in column volume. The column was equilibrated at 300 cm/h with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+50 mM NaCl+1M Glycerol, pH7.3 prior to application. The column was washed with equilibration buffer before the elution using a linear gradient over 5 column volumes from equilibration buffer to 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+1M NaCl+1M Glycerol, pH7.3. The fractions containing FVIII were pooled and the pool was stored at -80.degree. until use. A table of yields over a typical purification is shown in Table 11.

[0107] The FVIII-variants with HIS-tag was purified essentially as described above, however the second purification step (F25-sepharose) was exchanged to Chelating Sepharose FF (GE Healthcare) charged with 2 column volumes of 1M NiSO4. The column dimension was 0.5 cm in diameter and 5 cm bed height giving 1 mL column volume. The column was equilibrated with 30 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+1.5M NaCl, pH7.3 at 180 cm/h prior to application. After application the column was washed with 30 column volumes of equilibration buffer prior to elution using a linear gradient over 5 column volumes to 250 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80+1.5M NaCl, pH7.3. The fractions containing FVIII were pooled and diluted 1:30 with 20 mM Imidazol+10 mM CaCl.sub.2+0.01% Tween80, pH7.3. The final purification step (Poros 50HQ) was performed as described above.

Example 6

FVIII:C in Cell Culture Harvests Measured by Chromogenic Assay

[0108] The FVIII activity (FVIII:C) of the rFVIII compound in cell culture harvest (supernatant fraction) was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: rFVIII samples and a FVIII standard (Coagulation reference, Technoclone) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty .mu.l of samples, standards, and buffer negative control were added to 96-well microtiter plates (Spectraplates MB, Perkin Elmer). All samples were tested diluted 1:100, 1:400, 1:1600, and 1:6400. The factor IXa/factor X reagent, the phospholipid reagent and CaCl.sub.2 from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 .mu.l of this added to the wells. After 15 min incubation at room temperature, 50 .mu.l of the factor Xa substrate 5-2765/thrombin inhibitor I-2581 mix was added and the reactions were incubated 5 min at room temperature before 25 .mu.l 1 M citric acid, pH 3, was added. The absorbance at 405 nm was measured on an Envision microtiter plate reader (Perkin Elmer) with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration. The specific activity was calculated by dividing the activity of the samples with the protein concentration determined by ELISA. The results are shown in Table 1-10.

Example 7

Purification of FVIII Frameworks and Fusion Proteins

[0109] F8-500-albumin-.DELTA.a3 purified as described in Example 5 (13 mg, 6.5 mg/ml) in a buffer consisting of: imidazol (20 mM), calcium chloride (10 mM), Tween 80 (0.02%), sodium chloride (500 mM), and glycerol (1 M) in water (pH 7.3, was thawed.

[0110] Nine microgram sialidase from Arthrobacter ureafaciens (1.9 U, in 7 microliter buffer), 280 microgram sialyl tranferase (in 112 microliter, His-ST3Gal-I, 2.5 mg/ml, EC 2.4.99.4, WO 2006102652), and 59 milligram cytidine monophospate N-5'-PEG-glycerol-neuraminic acid (in 290 microliter, see WO2007/056191) were added, and pH was adjusted to 6.9 using HCl (1M). The final volume was 2.7 mL. The resulting mixture was then left for 22 hours at 22-25 degrees Celsius (room temperature).

[0111] After glycopegylation, the mixture was diluted to 50 ml with Buffer A (Tris (25 mM), calcium chloride (10 mM), Tween80 (0.02%), and glycerol (1 M) in water, pH 7.5). The deluted mixture was loaded onto a Source30Q column (GE Healthcare Bio-Sciences, Hillerod, Denmark, column volume 1.1 mL). The immobilised material was then washed with Buffer A (4 column volumes) and subsequently eluted from the column using a gradient of 0-100% Buffer B (Tris (25 mM), calcium chloride (10 mM), Tween 80 (0.02%), sodium chloride (0.7 M), and glycerol (1 M) in water, pH 7.5). Gradient: 8 CV 5-10% Buffer B, 13.5 CV 10-100% Buffer B, and 3 CV 100% Buffer B.

[0112] The early eluting 2.5 mL peak fraction was mixed with 1.2 milligram cytidine monophospate N-5'acetyl-neuraminic acid (in 12 microliter) and 62 microgram sialyltransferase (in 52 microliter, MBP-SBD-ST3Gal-III, EC 2.4.99.6, see WO 2006102652).

[0113] The mixture was left for 22 hours at 22-25 degrees Celsius (room temperature), and subsequently loaded onto a Superdex 200 pg column (GE Healthcare Bio-Sciences, Hillerod, Denmark; column volume 120 ml). Product was then eluted using a buffer consisting of: L-histidine (1.5 g/L), L-methionine (55 mg/L), calcium chloride (250 mg/L), Tween 80 (0.1 g/L), sodium chloride (18 g/L), and sucrose (1.5 g/L) in water, pH 6.9. The first product-containing fractions (12 mL) were isolated and pooled; product concentration was approximately 0.06 mg/mL.

[0114] Finally, the product was concentrated by centrifugation in an Amicon Centriprep YM-50 (cut-off: 50 kDa). The volume after concentration was 1.7 mL, containing 0.35 mg/mL glycopegylated F8-500-albumin-.DELTA.a3 (purity >90%). This compound is referred to in Table 12 as 40K-PEG-O-F8-500-albumin-.DELTA.a3.

Example 8

FVIII:C in Purified Samples Measured by Chromogenic Assay

[0115] The FVIII activity (FVIII:C) of the purified rFVIII compound (isolated as disclosed in Example 5) was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: rFVIII samples and a FVIII standard (e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty .mu.l of samples, standards, and buffer negative control were added to 96-well microtiter plates (Nunc) in duplicates. The factor IXa/factor X reagent, the phospholipid reagent and CaCl.sub.2 from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 .mu.l of this added to the wells. After 15 min incubation at room temperature 50 .mu.l of the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions incubated 10 min at room temperature before 25 .mu.l 1 M citric acid, pH 3, was added. The absorbance at 415 nm was measured on a Spectramax microtiter plate reader (Molecular Devices) with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration. The specific activity was calculated by dividing the activity of the samples with the protein concentration determined by HPLC. For HPLC, the concentration of the sample was determined by integrating the area under the peak in the chromatogram corresponding to the light chain and compare with the area of the same peak in a parallel analysis of a wild-type rFVIII, where the concentration was determined by amino acid analyses. The results are shown in Table 1-10.

Example 9

FVIII:C in Purified Samples Measured by One-Stage Clot Assay

[0116] FVIII:C of the rFVIII compounds was further evaluated in a one-stage FVIII clot assay as follows: rFVIII samples and a FVIII standard (e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC) were diluted in HBS/BSA buffer (20 mM hepes, 150 mM NaCl, pH 7.4 with 1% BSA) to approximately 10 U/ml followed by 10-fold dilution in FVIII-deficient plasma containing VWF (Dade Behring). The samples were subsequently diluted in HBS/BSA buffer. The APTT clot time was measured on an ACL300R or an ACL5000 instrument (Instrumentation Laboratory) using the single factor program. FVIII-deficient plasma with VWF (Dade Behring) was used as assay plasma and SynthASil, (HemosIL.TM., Instrumentation Laboratory) as aPTT reagent. In the clot instrument, the diluted sample or standard is mixed with FVIII-deficient plasma, aPTT reagents at 37.degree. C. Calcium chloride is assed and time until clot formation is determined by turbidity. The FVIII:C in the sample is calculated based on a standard curve of the clot formation times of the dilutions of the FVIII standard. The results are shown in Table 1-10.

Example 10

Pharmacokinetics of FVIII Frameworks and Fusion Proteins in FVIII- and VWF-Deficient Mice

[0117] The pharmacokinetics of rFVIII variants were evaluated in FVIII-deficient mice (FVIII exon 16 knock out (KO) mice with C57Bl/6 background, bred at Taconic M&B) or in vWF-deficient mice (vWF exon 4+5 KO mice with C57Bl/6 background bred at Charles River, Germany) The vWF-KO mice had 13% of normal FVIII:C (see example 6), while the FVIII-KO mice had no detectable FVIII:C. A mixture of male and female (approximately 1:1) with an approximate weight of 25 grams and age range of 16-28 weeks were used. The mice received a single i.v. injections of rFVIII (280 IU/kg) in the tail vein. Blood was taken from the orbital plexus at time points up to 64 hours after dosing using non-coated capillary glass tubes. Three samples were taken from each mouse, and 2 to 4 samples were collected at each time point. Blood was immediately stabilized with sodium citrate and diluted in four volumes FVIII Coatest SP buffer (see example 6) before 5 min centrifugation at 4000.times.g. Plasma obtained from diluted blood was frozen on dry ice and kept at -80.degree. C. The FVIII:C was determined in a chromogenic assay as described in example 6 Pharmacokinetic analysis was carried out by non-compartmental methods (NCA) using WinNonlin Pro version 4.1 software. Results are shown in Table 12. The fold prolongation of fusion protein is calculated by dividing the half-life of the fusion protein with that of the FVIII framework without the fusion partner.

Example 11

Analysis of Fusion Protein Binding to Platelets

[0118] Platelet-binding of a fusion protein can be tested by flow cytometry. Peripheral blood platelets may be purified, or whole blood can be used. The platelets may be activated or resting. The platelets are incubated with fusion protein for 15-30 min. The fusion protein may be directly labelled with a fluorophore or detected using a fluorescently labelled secondary antibody.

[0119] A fluorescently labelled platelet specific antibody not interfering with binding of the fusion protein can be added to assess whether the particles binding the fusion protein are indeed platelets. After incubation, the cells are washed to remove fusion protein, and the samples are analyzed on a flow cytometer. The flow cytometer detects unlabelled cells and fluorescently labelled molecules binding to cells and thus can be used to specifically analyze to which extent fusion protein is bound to platelets (or other cells).

[0120] The specificity of binding can be assessed e.g. by adding a surplus of unlabelled antibody (when using directly labelled fusion protein). Binding of the FVIII moiety to the platelets can be assessed e.g. by adding a surplus of annexin V or FVIII.

[0121] Internalization of the fusion protein by the resting platelet may be assessed e.g. by incubating platelets with directly labelled fusion protein followed by incubation with an antibody, which quenches the signal from surface-bound (i.e. not internalized) fusion protein. Only the internalized fusion protein will then be detected by flow cytometry. It may hypothesized that activated platelets will release internalized fusion-protein at the site of clot formation.

Example 12

Pharmacokinetics of GPIIIa-Targeted Fusion Proteins in GPIIIa Transgenic Mice

[0122] The GPIIIa-targeted fusion protein binds to the human GPIIb/IIIa (integrin a2b3) receptor on platelets, but it may not recognize murine GPIIb/IIIa, preventing the use of wild type mice for pharmacokinetic analyses. The pharmacokinetic profile of GPIIIa-targeted fusion proteins can be analyzed in transgenic mice expressing human GPIIIa, which associates with murine GPIIb enabling the binding of fusion protein to the receptor. Fusion protein will be injected intravenously to GPIIIa transgenic mice and blood collected at various time-points after injection (e.g. 0.5, 24, 72, 288 hours). The injected fusion protein (free and/or platelet-bound) may be quantified by means of an ELISA or the fusion protein may be radioactively or fluorescently labelled and detected.

TABLE-US-00003 TABLE 1 BDD FVIII frameworks that can constitute the FVIII part of fusion proteins Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500 F8-500 n.a n.a 17.9/17.0 F8-500-.DELTA.a3 F8-500-.DELTA.a3 n.a n.a 13.6/13.8 F8-500-His F8-500-His n.a. n.a. 12.1/8.1 F8-500-.DELTA.a3-His F8-500-.DELTA.a3- n.a. n.a. n.d./n.d. His F8-500-Y1680F F8-500- n.a. n.a. 16.6**/n.d Y1680F F8-500-Y1680C F8-500- n.a. n.a. 19.7/n.d. Y1680C *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00004 TABLE 2 Fusion proteins interacting with FcRn Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-hFc(IgG1) F8-500 hFc In B domain 19.9**/n.d. 14.1**/n.d. F8-500-hFc(IgG1)-His F8-500-His hFc In B domain F8-500-Y1680F- F8-500- hFc In B domain hFc(IgG1) Y1680F F8-500-Y1680C- F8-500- hFc In B domain hFc(IgG1) Y1680C F8-500- F8-500-.DELTA.a3 hFc At a3s position 6.0**/n.d. hFc(IgG1)-.DELTA.a3 28.5**/n.d. F8-500-C2 linked- F8-500 hFC At C-terminus 16.7**/n.d. (GGGS)6-hFc(IgG1) of LC F8-500-C2 linked-GS- F8-500 hFC At C-terminus 14.4**/n.d. hFc(IgG1) of LC F8-500-C2 linked- F8-500 hFC At C-terminus 13.4**/n.d. hFc(IgG1) of LC (F8-500-hFC-LC) F8-500-Y1680F-C2- F8-500- hFC At C-terminus linked-hFc(IgG1) Y1680F of LC (F8-500-Y1680F-hFC- LC) F8-500-Y1680C-C2- F8-500- hFC At C-terminus linked-hFc(IgG1) Y1680C of LC (F8-500-Y1680C-hFC- LC) F8-500-mFc(IgG2A) F8-500 mFc In B domain 18.5**/n.d. 13.9**/n.d. F8-500-mFc(IgG2A)- F8-500-His mFc In B domain His F8-500-Y1680F- F8-500- mFc In B domain mFc(IgG2A) Y1680F F8-500-Y1680C- F8-500- mFc In B domain mFc(IgG2A) Y1680C F8-500- F8-500-.DELTA.a3 mFc At a3s position mFc(IgG2A)-.DELTA.a3 F8-500-C2 linked- F8-500 mFC At C-terminus 15.5**/n.d. (GGGS)6- of LC mFc(IgG2A) F8-500-C2 linked-GS- F8-500 mFC At C-terminus 15.2**/n.d. mFc(IgG2A) of LC F8-500-C2 linked- F8-500 mFC At C-terminus 12.9**/n.d mFc(IgG2A) of LC F8-500-Y1680F-C2- F8-500- mFC At C-terminus linked-mFc(IgG2A) Y1680F of LC F8-500-Y1680C-C2- F8-500- mFC At C-terminus linked-mFc(IgG2A) Y1680C of LC F8-500-albumin F8-500 HSA In B domain 14.7/n.d. F8-500-albumin-His F8-500-His HSA In B domain .sup. 8.1/6.2 F8-500-Albumin- F8-500 HSA In B domain 12.3**/n.d. S727P F8-500-Y1680F- F8-500- HSA In B domain albumin Y1680F F8-500-Y1680C- F8-500- HSA In B domain albumin Y1680C F8-500-albumin-.DELTA.a3 F8-500-.DELTA.a3 HSA At a3s position 2.0/n.d. F8-500-C2 linked- F8-500 HSA At C-terminus 22.7**/n.d. (GGGS)6-albumin of LC F8-500-C2 linked-GS- F8-500 HSA At C-terminus 17.7**/n.d. albumin of LC F8-500-C2 linked- F8-500 HSA At C-terminus albumin of LC (F8-500-albumin-LC) F8-500-Y1680F-C2- F8-500- HSA At C-terminus linked-albumin Y1680F of LC (F8-500-Y1680F- albumin-LC) F8-500-Y1680C-C2- F8-500- HSA At C-terminus linked-albumin Y1680C of LC (F8-500-Y1680C- albumin-LC) F8-500-transferrin F8-500 Transferrin In B domain F8-500-transferrin-His F8-500-His Transferrin In B domain F8-500-Y1680F- F8-500- Transferrin In B domain transferrin Y1680F F8-500-Y1680C- F8-500- Transferrin In B domain transferrin Y1680C F8-500- F8-500-.DELTA.a3 Transferrin At a3s position transferrin-.DELTA.a3 F8-500-C2 linked- F8-500 Transferrin At C-terminus transferrin of LC (F8-500-transferrin- LC) F8-500-Y1680F-C2- F8-500- Transferrin At C-terminus linked- transferrin Y1680F of LC (F8-500-Y1680F- transferrin-LC) F8-500-Y1680C-C2- F8-500- Transferrin At C-terminus linked-transferrin Y1680C of LC (F8-500-Y1680C- transferrin-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00005 TABLE 3 Fusion proteins interacting with immunoglobulins Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-hFc.gamma.RI F8-500 hFc.gamma.RI (CD64) In B domain .sup. 22.2/20.0 F8-500-hFc.gamma.RI-His F8-500-His hFc.gamma.RI (CD64) In B domain .sup. 11.6/4.9 F8-500-Y1680F- F8-500- hFc.gamma.RI (CD64) In B domain hFc.gamma.RI Y1680F F8-500-Y1680C- F8-500- hFc.gamma.RI (CD64) In B domain hFc.gamma.RI Y1680C F8-500- F8-500-.DELTA.a3 hFc.gamma.RI (CD64) At a3s position 5.3/n.d. hFc.gamma.RI-.DELTA.a3 F8-500-C2-linked- F8-500 hFc.gamma.RI (CD64) At C-terminus hFc.gamma.RI of LC (F8-500-hFc.gamma.RI-LC) F8-500-Y1680F-C2- F8-500- hFc.gamma.RI (CD64) At C-terminus linked-hFc.gamma.RI Y1680F of LC (F8-Y1680F-500- hFc.gamma.RI-LC) F8-500-Y1680C-C2- F8-500- hFc.gamma.RI (CD64) At C-terminus linked- hFc.gamma.RI Y1680C of LC (F8-500-Y1680C- hFc.gamma.RI-LC) F8-500-FcRn F8-500 FcRn In B domain 27.2**/n.d. F8-500-FcRn-H166K F8-500 FcRn In B domain 20.8**/n.d. *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00006 TABLE 4 Fusion proteins with the capability of reducing interaction with clearance receptors Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-LRP-CR5-6 F8-500 LRP CR5-6 In B domain 14.2/n.d. F8-500-LRP-CR5-6- F8-500- LRP CR5-6 In B domain His His F8-500-LRP-CR6-7 F8-500 LRP CR6-7 In B domain 9.5**/n.d. F8-500-LRP-CR6 F8-500 LRP CR6 In B domain 8.9**/n.d. *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00007 TABLE 5 Fusion proteins binding to platelets Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-anti-GPIIIa- F8-500 SC anti-GPIIIa-1- In B domain 29.3**/n.d. 1-HC-LC HC-LC F8-500-linker- anti- F8-500 SC anti-GPIIIa-1- In B domain 40.3**/n.d. GPIIIa-1-HC-LC HC-LC F8-500-SC anti- F8-500 SC anti-GPIIIa-2- In B domain 22.5**/n.d. GPIIIa-2-HC-LC HC-LC F8-500-SC anti- F8-500-His SC anti-GPIIIa-2- In B domain -- GPIIIa-2-HC-LC-His HC-LC F8-500-linker-SC F8-500-His SC anti-GPIIIa-2- In B domain 45.9**/n.d. anti-GPIIIa-2-HC-LC- HC-LC His F8-500- a3-SC anti- F8-500- SC anti-GPIIIa-2- In B domain -- GPIIIa-2-HC-LC-His a3-His HC-LC F8-500-Y1680F-SC F8-500- SC anti-GPIIIa-2- In B domain anti-GPIIIa-2-HC-LC Y1680F HC-LC F8-500-Y1680C-SC F8-500- SC anti-GPIIIa-2- In B domain anti-GPIIIa-2-HC-LC Y1680C HC-LC F8-500-SC anti- F8-500- a3 SC anti-GPIIIa-2- At a3s position 26.2**/n.d. GPIIIa-2-HC-LC-.DELTA.a3 HC-LC F8-500-C2-linked-SC F8-500 SC anti-GPIIIa-2- At C-terminus anti-GPIIIa-2-HC-LC HC-LC of LC (F8-500-SC anti- GPIIIa-2-HC-LC-LC) F8-500-Y1680F-C2- F8-500- SC anti-GPIIIa-2- At C-terminus linked-SC anti-GPIIIa- Y1680F HC-LC of LC 2-HC-LC (F8-500-Y1680F-SC anti-GPIIIa-2-HC-LC- LC) F8-500-Y1680C-C2- F8-500- SC anti-GPIIIa-2- At C-terminus linked-SC anti-GPIIIa- Y1680C HC-LC of LC 2-HC-LC (F8-500-Y1680C-SC anti-GPIIIa-2-HC-LC- LC) F8-500-SC anti- F8-500 SC anti-GPIIIa-2- In B domain 28.3**/n.d. GPIIIa-2-LC-HC LC-HC F8-500-SC anti- F8-500 SC anti-GPIIIa-2- In B domain 95.4**/n.d. GPIIIa-2-LC-HC LC-HC F8-500-linker-SC F8-500-His SC anti-GPIIIa-2- In B domain 47.5**/n.d. anti-GPIIIa-2-LC-HC- LC-HC His F8-500-.DELTA.a3-SC anti- F8-500- SC anti-GPIIIa-2- In B domain 81.4**/n.d. GPIIIa-2-LC-HC-His .DELTA.a3-His LC-HC F8-500-Y1680F-SC F8-500- SC anti-GPIIIa-2- In B domain anti-GPIIIa-2-LC-HC Y1680F LC-HC F8-500-Y1680C- SC F8-500- SC anti-GPIIIa-2 In B domain anti-GPIIIa-2-LC-HC Y1680C (LC-HC) F8-500-SC anti- F8-500-.DELTA.a3 SC anti-GPIIIa-2- At a3s position 29.2**/n.d. GPIIIa-2-LC-HC-.DELTA.a3 LC-HC F8-500-C2-linked- F8-500 SC anti-GPIIIa-2- At C-terminus 22.4**/n.d. (GGGS)6-SC anti- LC-HC of LC GPIIIa-2-LC-HC (F8-500-SC anti- GPIIIa-2-LC-HC-LC) F8-500-Y1680F-C2- F8-500- SC anti-GPIIIa-2- At C-terminus linked-SC anti-GPIIIa- Y1680F LC-HC of LC 2-LC-HC (F8-500-Y1680F-SC anti-GPIIIa-2-LC-HC- LC) F8-500-Y1680C-C2- F8-500- SC anti-GPIIIa-2- At C-terminus 16.6**/n.d. linked-GS-SC anti- Y1680C LC-HC of LC GPIIIa-2-LC-HC (F8-500-y1680C-SC anti-GPIIIa-2-LC-HC- LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00008 TABLE 6 Fusion proteins interacting with serum albumin Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-ABD035 F8-500 Albumin binding In B domain peptide ABD035 F8-500-ABD035-His F8-500-His Albumin binding In B domain 8.3/n.d. peptide ABD035 F8-500-Y1680F- F8-500- Albumin binding In B domain ABD035 Y1680F peptide ABD035 F8-500-Y1680C- F8-500- Albumin binding In B domain ABD035 Y1680C peptide ABD035 F8-500- F8-500- a3 Albumin binding At a3s position 4.0/0.5 ABD035-.DELTA.a3 peptide ABD035 F8-500-C2-linked- F8-500 Albumin binding At C-terminus ABD035 peptide ABD035 of LC (F8-500-ABD035- LC) F8-500-Y1680F-C2- F8-500- Albumin binding At C-terminus linked ABD035 Y1680F peptide ABD035 of LC (F8-500-Y1680F- ABD035-LC) F8-500-Y1680C-C2- F8-500- Albumin binding At C-terminus linked ABD035 Y1680C peptide ABD035 of LC (F8-500-Y1680C- ABD035-LC) F8-500-4XABD035 F8-500 4 X Albumin In B domain 14.8/n.d. binding peptide ABD035 F8-500-4XABD035- F8-500-His 4 X Albumin In B domain His binding peptide ABD035 F8-500-Y1680F-4X F8-500- 4 X Albumin In B domain ABD035 Y1680F binding peptide ABD035 F8-500-Y1680C-4X F8-500- 4 X Albumin In B domain ABD035 Y1680C binding peptide ABD035 F8-500- F8-500-.DELTA.a3 4 X Albumin At a3s position 4X ABD035-.DELTA.a3 binding peptide ABD035 F8-500-C2-linked-4X F8-500 4 X Albumin At C-terminus ABD035 binding peptide of LC (F8-500-4X ABD035 ABD035-LC) F8-500-Y1680F-C2- F8-500- 4 X Albumin At C-terminus linked-4X ABD035 Y1680F binding peptide of LC (F8-500-Y1680F-4X ABD035 ABD035-LC) F8-500-Y1680C-C2- F8-500- 4 X Albumin At C-terminus linked-4X ABD035 Y1680C binding peptide of LC (F8-500-Y1680C-4X ABD035 ABD035-LC) F8-500-SC anti-HSA F8-500 SC anti-HSA In B domain F8-500-SC anti-HSA- F8-500-His SC anti-HSA In B domain His F8-500-Y1680F-SC F8-500- SC anti-HSA In B domain anti-HSA Y1680F F8-500-Y1680C-SC F8-500- SC anti-HSA In B domain anti-HSA Y1680C F8-500- F8-500-.DELTA.a3 SC anti-HSA At a3s position SC anti-HSA-.DELTA.a3 F8-500-C2-linked-SC F8-500 SC anti-HSA At C-terminus anti-HSA of LC (F8-500-SC anti- HSA-LC) F8-500-Y1680F-C2- F8-500- SC anti-HSA At C-terminus linked-SC anti-HSA Y1680F of LC (F8-500-Y1680F-SC anti-HSA-LC) F8-500-Y1680C-C2- F8-500- SC anti-HSA At C-terminus linked-SC anti-HSA Y1680C of LC (F8-500-Y1680C-SC anti-HSA-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00009 TABLE 7 Fusion proteins shielding the molecule from clearance receptors Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-Seq A F8-500 Sequence A In B domain F8-500-Seq A-His F8-500-His Sequence A In B domain 19.2**/n.d. F8-500-Y1680F-Seq F8-500- Sequence A In B domain A Y1680F F8-500-Y1680C-Seq F8-500- Sequence A In B domain A Y1680C F8-500- F8-500-.DELTA.a3 Sequence A At a3s position Seq A-.DELTA.a3 F8-500-C2-linked- F8-500 Sequence A At C-terminus Seq A of LC (F8-500-Seq A-LC) F8-500-Y1680F-C2- F8-500- Sequence A At C-terminus linked Seq A Y1680F of LC (F8-500-Y1680F-Seq A-LC) F8-500-Y1680C-C2- F8-500- Sequence A At C-terminus linked Seq A Y1680C of LC (F8-500-Y1680C-Seq A-LC) F8-500-ELP80 F8-500 ELP80 In B domain F8-500-ELP80-His F8-500-His ELP80 In B domain 13.8**/n.d. F8-500-Y1680F- F8-500- ELP80 In B domain ELP80 Y1680F F8-500-Y1680C- F8-500- ELP80 In B domain ELP80 Y1680C F8-500- F8-500-.DELTA.a3 ELP80 At a3s position ELP80-.DELTA.a3 F8-500-C2-linked- F8-500 ELP80 At C-terminus ELP80 of LC F8-500-Y1680F-C2- F8-500- ELP80 At C-terminus linked-ELP80 Y1680F of LC F8-500-Y1680C-C2- F8-500- ELP80 At C-terminus linked ELP80 Y1680C of LC *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00010 TABLE 8 Fusion proteins with modulated affinity to vWF Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500- Extra a3 F8-500 Extra a3 In B domain 10.0**/n.d. F8-500- Extra a3-His F8-500-His Extra a3 In B domain 7.7**/n.d. F8-500-Y1680F- F8-500- Extra a3 In B domain Extra a3 Y1680F F8-500-Y1680C- F8-500- Extra a3 In B domain Extra a3 Y1680C F8-500- F8-500-.DELTA.a3 Extra a3 At a3s position Extra a3-.DELTA.a3 F8-500-C2-linked- F8-500 Extra a3 At C-terminus Extra a3 of LC (F8-500-Extra a3-LC) F8-500-Y1680F-C2- F8-500- Extra a3 At C-terminus linked- Extra a3 Y1680F of LC (F8-500-Y1680F- Extra a3-LC) F8-500-Y1680C-C2- F8-500- Extra a3 At C-terminus linked Extra a3 Y1680C of LC (F8-500-Y1680C- Extra a3-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00011 TABLE 9 Other fusion proteins Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-GHBP F8-500 Growth hormone In B domain 22.8**/n.d. binding protein F8-GHBP-His F8-500-His Growth hormone In B domain .sup. 7.6/4.5 binding protein F8-500-FIX298-342 F8-500 AS 298-342 of FIX In B domain 12.3**/n.d. F8-500-FIX47-125 F8-500 AS 47-125 of FIX In B domain 18.0/n.d. F8-500-FIX47-125- F8-500-His AS 47-125 of FIX In B domain .sup. 14.4/13.3 His F8-500-vWF1-272 F8-500 AS 1-272 of vWF In B domain 44.5**/n.d. F8-500-vWF1-1390 F8-500 AS 1-1390 of vWF In B domain 16.0**/n.d. F8-500-vWF497-716- F8-500 AS 497-716 of In B domain 11.5/8.8. R545A vWF F8-500-vWF497-716- F8-500-His AS 497-716 of In B domain 256.9**/n.d. R545A-His vWF F8-500-vWF binding F8-500 vWF-binding In B domain 19.8**/n.d. protein protein F8-500-hCG C- F8-500-His 28 C-terminal AS In B domain 11.4/n.d. terminus-His of hCG b-chain F8-500- F8-500-.DELTA.a3 28 C-terminal AS At a3s position 18.2/n.d. hCG-C-terminus- of hCG b-chain .DELTA.a3 F8-500- F8-500 Activation peptide In B domain 22.5**/n.d. F10AP of hFX F8-500- F8-500-His Activation peptide In B domain 16.8/n.d. F10AP-His of hFX F8-500-Y1680F- F8-500- Activation peptide In B domain F10AP Y1680F of hFX F8-500-Y1680C- F8-500- Activation peptide In B domain F10AP Y1680C of hFX F8-500- F8-500-.DELTA.a3 Activation peptide At a3s position F10AP-.DELTA.a3 of hFX F8-500-C2-linked- F8-500 Activation peptide At C-terminus F10AP of hFX of LC (F8-500-F10AP-LC) F8-500-Y1680F-C2- F8-500- Activation peptide At C-terminus linked-F10AP Y1680F of hFX of LC (F8-500-Y1680F- F10AP-LC) F8-500-Y1680C-C2- F8-500- Activation peptide At C-terminus linked- F10AP Y1680F of hFX of LC (F8-500-Y1680C- F10AP-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00012 TABLE 10 Fusion proteins with modulated affinity to lipids Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500- Extra C2 F8-500 Extra C2 In B domain F8-500- Extra C2-His F8-500-His Extra C2 In B domain . F8-500-Y1680F- F8-500- Extra C2 In B domain Extra C2 Y1680F F8-500-Y1680C- F8-500- Extra C2 In B domain Extra C2 Y1680C F8-500- F8-500-.DELTA.a3 Extra C2 At a3s position Extra C2-.DELTA.a3 F8-500-C2 linked- F8-500 Extra C2 At C-terminus 8.3**/n.d. (GGGS)6-extra C2 of LC F8-500-C2-linked- F8-500 Extra C2 At C-terminus 7.7**/n.d. GS-Extra C2 of LC F8-500-Y1680F-C2- F8-500- Extra C2 At C-terminus linked-Extra C2 Y1680F of LC F8-500-Y1680C-C2- F8-500- Extra C2 At C-terminus linked-Extra C2 Y1680C of LC *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE-US-00013 TABLE 11 Typical yields of the purification process described in Example 5 Yield/% Sample Vol./mL Activity/U*mL.sup.-1 Step Total Culture filtrate 1650 17 100 100 Step1: Flow thru 1650 3.5 21 -- Step1: Eluate pool 14 1021 51 51 Step2: Flow thru 133 19 17 -- Step2: Eluate pool 8 820 45 23 Step3: Flow thru 117 0.9 2 -- Step3: Eluate pool 1.5 3000 69 16

TABLE-US-00014 TABLE 12 In vivo half-lifes of FVIII frameworks and fusion proteins in FVIII- or vWF deficient mice Half-life Fold Half-life Fold in FVIII- prolongation in vWF- prolongation deficient in FVIII- deficient in vWF- Name mice (h)* deficient mice* mice (h)* deficient mice* F8-500 4.5/8.6 n.a. n.d./0.3 n.a. F8-500-.DELTA.a3 0.3/0.3 n.a. n.d. n.a. F8-500-His n.d. n.a. 0.1/0.1 n.a. F8-500-.DELTA.a3-His n.d. n.a. n.d. n.a. F8-500-Y1680F 0.5/n.d. n.a. n.d. n.a. F8-500-Y1680C n.d. n.a. n.d. n.a. F8-500-hFc(IgG1)-.DELTA.a3 0.7/n.d. 2.3/n.d. n.d. n.d F8-500-C2 linked- 9.9/n.d. 2.2./n.d n.d n.d (GGGS)6-hFc(IgG1) F8-500-C2 linked- 15.0/n.d. 3.5/n.d n.d./1.1 n.d/4 (GGGS)6-mFc(IgG2A) F8-500-albumin 7.8/9.1 1.7/1.1 n.d. n.d. F8-500-albumin-His 6.7/6.0 ?/? 1.6/0.6 16/6 F8-500-albumin-.DELTA.a3 1.4/n.d. 4.7/n.d. n.d. n.d. 40K-PEG-O-F8-500- 12.0/n.d. 40/n.d. n.d. n.d. albumin-.DELTA.a3 F8-500-C2 linked- n.d/9.6 n.d./1.1 n.d. n.d (GGGS)6-albumin F8-500-C2 linked-GS- 8.0/n.d. 1.8./n.d n.d./0.8 n.d/3 albumin F8-500-hFc.gamma.RI 9.2/9.7 2.0/1.1 n.d. n.d. F8-500-hFc.gamma.RI-His 10/n.d. 2.2/n.d. 1.7/1.2 .sup. 17/12 F8-500-hFc.gamma.RI-.DELTA.a3 0.2/n.d. 0.7/n.d. n.d. n.d. F8-500-LRP-CR5-6 6.2/6.4 1.4/0.7 n.d. n.d F8-500-ABD035-His 10.3/--.sup. ?/? n.d./0.8 n.d/8 F8-GHBP-His n.d n.d. 0.1/0.2 .sup. 1/2 F8-500-FIX47-125-His 5.3/5.3 ?/? n.d. n.d. F8-500-F10AP-His n.d n.d. 1.0/0.8 10/8 *Chromogenic assay/ELISA

Sequence CWU 1

1

3112332PRThomo sapiens 1Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830 Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950 955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065 Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080 Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095 Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125 Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140 Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155 Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170 Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185 Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200 Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215 Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230 Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260 His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320 Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335 Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350 Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365 Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380 Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395 Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410 Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425 Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440 Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455 Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470 Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515 Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545 Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560 Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575 Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590 Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605 Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620 Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635 Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650 Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665 Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680 Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695 Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710 Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770 Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815 Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830 Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1835 1840 1845 Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1850 1855 1860 Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875 Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890 Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905 Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915 1920 Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925 1930 1935 Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950 His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1970 1975 1980 Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995 Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010 Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025 Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035 2040 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 2045 2050 2055 Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070 Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 2075 2080 2085 Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090 2095 2100 Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115 Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130 Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145 Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160 Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 2170 2175 Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2180 2185 2190 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205 Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 2210 2215 2220 Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235 Lys Val Thr Gly Val Thr Thr

Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250 Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265 His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290 2295 Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310 Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315 2320 2325 Gln Asp Leu Tyr 2330 221PRTArtificial SequenceSynthetic 2Ser Phe Ser Gln Asn Ser Arg His Pro Ser Gln Asn Pro Pro Val Leu 1 5 10 15 Lys Arg His Gln Arg 20 328PRTArtificial SequenceSynthetic 3Ser Phe Ser Gln Asn Ser Arg His Pro Ser His His His His His His 1 5 10 15 Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 20 25 428PRTArtificial SequenceSynthetic 4Ser Phe Ser Gln Asn Ser Arg His Pro Ser His His His His His His 1 5 10 15 Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg 20 25 5224PRTArtificial SequenceSynthetic 5His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Glu Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 6226PRTArtificial SequenceSynthetic 6Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala Glu Gly Glu 1 5 10 15 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 20 25 30 Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp 35 40 45 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val Leu 50 55 60 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Gln Ser Thr Leu Arg 65 70 75 80 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 85 90 95 Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 115 120 125 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 130 135 140 Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 145 150 155 160 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 165 170 175 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu 180 185 190 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 195 200 205 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 210 215 220 Gly Lys 225 7585PRThomo sapiens 7Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 8678PRThomo sapiens 8Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala 1 5 10 15 Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro Ser 20 25 30 Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys 35 40 45 Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala 50 55 60 Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val 65 70 75 80 Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Ala 85 90 95 Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu Arg 100 105 110 Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn 115 120 125 Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys Pro 130 135 140 Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro Cys 145 150 155 160 Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly Cys 165 170 175 Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe Lys 180 185 190 Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His Ser Thr 195 200 205 Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu Leu 210 215 220 Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp Cys 225 230 235 240 His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser Met Gly 245 250 255 Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln Glu His 260 265 270 Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro His 275 280 285 Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu Lys Val 290 295 300 Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val Thr 305 310 315 320 Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro Thr Asp 325 330 335 Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg Leu 340 345 350 Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys Val 355 360 365 Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly Glu 370 375 380 Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly Lys 385 390 395 400 Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp Asn 405 410 415 Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Val Lys 420 425 430 Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys Ser 435 440 445 Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met Gly 450 455 460 Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe Ser 465 470 475 480 Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys Leu 485 490 495 Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys Glu Gly 500 505 510 Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys Gly Asp 515 520 525 Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly Gly Lys 530 535 540 Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Glu Leu 545 550 555 560 Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn Cys 565 570 575 His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys Asp Lys 580 585 590 Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu Phe Gly 595 600 605 Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser Glu 610 615 620 Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys Leu 625 630 635 640 His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val Lys 645 650 655 Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu Ala 660 665 670 Cys Thr Phe Arg Arg Pro 675 9273PRThomo sapiens 9Gln Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val 1 5 10 15 Ser Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His 20 25 30 Leu Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr 35 40 45 Gln Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp 50 55 60 Ser Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro 65 70 75 80 Ile Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser 85 90 95 Arg Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp 100 105 110 Lys Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala 115 120 125 Phe Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn 130 135 140 Ile Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg 145 150 155 160 Tyr Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala 165 170 175 Pro Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu 180 185 190 Val Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu 195 200 205 Gln Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg 210 215 220 Asn Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser 225 230 235 240 Gly Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys 245 250 255 Arg Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr 260 265 270 Pro 10274PRThomo sapiens 10Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 1 5 10 15 Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro 20 25 30 Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys 35 40 45 Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu 50 55 60 Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80 Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys 85 90 95 Glu Leu Gly Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu 100 105

110 Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly 115 120 125 Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln 130 135 140 Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro 145 150 155 160 His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp 165 170 175 Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser Ser Pro Gly 180 185 190 Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu 195 200 205 Gln Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gln Gly 210 215 220 Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu 225 230 235 240 Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His 245 250 255 Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Ser Pro Ala Lys 260 265 270 Ser Ser 11274PRTArtificial SequenceSynthetic 11Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser 1 5 10 15 Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro 20 25 30 Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys 35 40 45 Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu 50 55 60 Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys 65 70 75 80 Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys 85 90 95 Glu Leu Gly Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu 100 105 110 Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly 115 120 125 Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln 130 135 140 Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro 145 150 155 160 His Arg Leu Arg Glu Lys Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp 165 170 175 Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser Ser Pro Gly 180 185 190 Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu 195 200 205 Gln Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gln Gly 210 215 220 Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu 225 230 235 240 Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His 245 250 255 Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Ser Pro Ala Lys 260 265 270 Ser Ser 1279PRTArtificial SequenceSynthetic 12Thr Cys Pro Pro Asn Gln Phe Ser Cys Ala Ser Gly Arg Cys Ile Pro 1 5 10 15 Ile Ser Trp Thr Cys Asp Leu Asp Asp Asp Cys Gly Asp Arg Ser Asp 20 25 30 Glu Ser Ala Ser Cys Ala Tyr Pro Thr Cys Phe Pro Leu Thr Gln Phe 35 40 45 Thr Cys Asn Asn Gly Arg Cys Ile Asn Ile Asn Trp Arg Cys Asp Asn 50 55 60 Asp Asn Asp Cys Gly Asp Asn Ser Asp Glu Ala Gly Cys Ser His 65 70 75 1383PRTArtificial SequenceSynthetic 13Thr Cys Phe Pro Leu Thr Gln Phe Thr Cys Asn Asn Gly Arg Cys Ile 1 5 10 15 Asn Ile Asn Trp Arg Cys Asp Asn Asp Asn Asp Cys Gly Asp Asn Ser 20 25 30 Asp Glu Ala Gly Cys Ser His Ser Cys Ser Ser Thr Gln Phe Lys Cys 35 40 45 Asn Ser Gly Arg Cys Ile Pro Glu His Trp Thr Cys Asp Gly Asp Asn 50 55 60 Asp Cys Gly Asp Tyr Ser Asp Glu Thr His Ala Asn Cys Thr Asn Gln 65 70 75 80 Ala Thr Arg 1439PRTArtificial SequenceSynthetic 14Thr Cys Phe Pro Leu Thr Gln Phe Thr Cys Asn Asn Gly Arg Cys Ile 1 5 10 15 Asn Ile Asn Trp Arg Cys Asp Asn Asp Asn Asp Cys Gly Asp Asn Ser 20 25 30 Asp Glu Ala Gly Cys Ser His 35 15243PRTArtificial SequenceSynthetic 15Met Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu 1 5 10 15 Gly Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser 20 25 30 Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Met Gly Leu 35 40 45 Ile Tyr Tyr Gly Thr Asn Leu Val Asp Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp 65 70 75 80 Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Leu Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Lys Leu Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ser Val Gln Leu 115 120 125 Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu 130 135 140 Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Val His Trp 145 150 155 160 Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp 165 170 175 Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala 180 185 190 Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser 195 200 205 Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Pro Leu 210 215 220 Tyr Asp Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 225 230 235 240 Val Ser Ser 16258PRTArtificial SequenceSynthetic 16Met Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu 1 5 10 15 Gly Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser 20 25 30 Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Met Gly Leu 35 40 45 Ile Tyr Tyr Gly Thr Asn Leu Val Asp Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp 65 70 75 80 Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Leu Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Lys Leu Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ser Val Gln Leu 115 120 125 Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu 130 135 140 Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Val His Trp 145 150 155 160 Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp 165 170 175 Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala 180 185 190 Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser 195 200 205 Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Pro Leu 210 215 220 Tyr Asp Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 225 230 235 240 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser 17248PRTArtificial SequenceSynthetic 17Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Asn Lys Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ala Ala Pro 130 135 140 Ser Val Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser 145 150 155 160 Ser Arg Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Cys Trp Phe 165 170 175 Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser 180 185 190 Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly 210 215 220 Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Ser 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Arg 245 18248PRTArtificial SequenceSynthetic 18Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Cys Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 130 135 140 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 145 150 155 160 Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 165 170 175 Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Asn Lys Tyr Asn Glu Asn Phe 180 185 190 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 195 200 205 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 210 215 220 Ala Arg Glu Tyr Gly Asn Tyr Asp Tyr Ala Met Asp Ser Trp Gly Gln 225 230 235 240 Gly Thr Ser Val Thr Val Ser Ser 245 1946PRTArtificial SequenceSynthetic 19Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly 1 5 10 15 Val Ser Asp Phe Tyr Lys Arg Leu Ile Asn Lys Ala Lys Thr Val Glu 20 25 30 Gly Val Glu Ala Leu Lys Leu His Ile Leu Ala Ala Leu Pro 35 40 45 20865PRTArtificial SequenceSynthetic 20Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu 1 5 10 15 Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu 20 25 30 Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 35 40 45 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 50 55 60 Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 65 70 75 80 Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 85 90 95 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala 100 105 110 Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro 115 120 125 Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 130 135 140 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala 145 150 155 160 Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu 165 170 175 Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 180 185 190 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 195 200 205 Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 210 215 220 Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 225 230 235 240 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 245 250 255 Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 260 265 270 Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 275 280 285 Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu 290 295 300 Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly 305 310 315 320 Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 325 330 335 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 340 345 350 Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu 355 360 365 Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 370 375 380 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 385 390 395 400 Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 405 410 415 Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 420 425 430 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 435 440 445 Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 450 455 460 Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 485 490 495 Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 500 505 510 Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 515 520 525 Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 530 535 540 Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro 545 550 555 560 Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 565 570

575 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 580 585 590 Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 595 600 605 Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 610 615 620 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 625 630 635 640 Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 645 650 655 Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 660 665 670 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 675 680 685 Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr 690 695 700 Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 705 710 715 720 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu 725 730 735 Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro 740 745 750 Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 755 760 765 Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro 770 775 780 Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr 785 790 795 800 Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 805 810 815 Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 820 825 830 Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 835 840 845 Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 850 855 860 Gly 865 21400PRTArtificial SequenceSynthetic 21Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly 1 5 10 15 Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 20 25 30 Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 35 40 45 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly 50 55 60 Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 85 90 95 Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 100 105 110 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro 115 120 125 Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly 130 135 140 Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly 145 150 155 160 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 165 170 175 Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val 180 185 190 Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro 195 200 205 Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 210 215 220 Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val 225 230 235 240 Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 245 250 255 Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 260 265 270 Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro 275 280 285 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val 340 345 350 Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro 355 360 365 Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 370 375 380 Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala 385 390 395 400 2241PRTArtificial SequenceSynthetic 22Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1 5 10 15 Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 20 25 30 Asp Glu Asp Glu Asn Gln Ser Pro Arg 35 40 23238PRTArtificial SequenceSynthetic 23Phe Ser Gly Ser Glu Ala Thr Ala Ala Ile Leu Ser Arg Ala Pro Trp 1 5 10 15 Ser Leu Gln Ser Val Asn Pro Gly Leu Lys Thr Asn Ser Ser Lys Glu 20 25 30 Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe Ser Cys 35 40 45 His Trp Thr Asp Glu Val His His Gly Thr Lys Asn Leu Gly Pro Ile 50 55 60 Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln Glu Trp 65 70 75 80 Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys Tyr Phe 85 90 95 Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys Leu Thr 100 105 110 Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser Val Asp Glu Ile 115 120 125 Val Gln Pro Asp Pro Pro Ile Ala Leu Asn Trp Thr Leu Leu Asn Val 130 135 140 Ser Leu Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu Ala Pro 145 150 155 160 Arg Asn Ala Asp Ile Gln Lys Gly Trp Met Val Leu Glu Tyr Glu Leu 165 170 175 Gln Tyr Lys Glu Val Asn Glu Thr Lys Trp Lys Met Met Asp Pro Ile 180 185 190 Leu Thr Thr Ser Val Pro Val Tyr Ser Leu Lys Val Asp Lys Glu Tyr 195 200 205 Glu Val Arg Val Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr Gly Glu 210 215 220 Phe Ser Glu Val Leu Tyr Val Thr Leu Pro Gln Met Ser Gln 225 230 235 2445PRTArtificial SequenceSynthetic 24Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp Ala Arg Val 1 5 10 15 Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro 20 25 30 Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe 35 40 45 2580PRTArtificial SequenceSynthetic 25Asp Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser 1 5 10 15 Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe 20 25 30 Glu Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly 35 40 45 Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys 50 55 60 Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu 65 70 75 80 26272PRTArtificial SequenceSynthetic 26Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 271390PRThomo sapiens 27Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430 Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445 Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460 Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu 465 470 475 480 Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val 485 490 495 Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu 500 505 510 Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala 515 520 525 Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu 530 535 540 Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 545 550 555 560 Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu 565 570 575 Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala 580 585 590 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys 595 600 605 Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu Leu Met Ala Ser 610 615 620 Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly 625 630 635 640 Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His 645 650 655 Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn 660 665 670 Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp 675 680 685 Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro 690 695 700 Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly Pro Gly Leu Leu 705 710 715 720 Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val 725 730 735 Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn 740 745 750 Arg Ser Lys Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly 755 760 765 Gln Asp Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr 770 775 780 Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln 785 790 795 800 Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly 805 810 815 Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly 820 825 830 Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro 835 840 845 Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro 850 855 860 Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly 865 870 875 880 Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg 885 890 895 Glu Ala Pro Asp Leu Val Leu Gln Arg Cys

Cys Ser Gly Glu Gly Leu 900 905 910 Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu Asp 915 920 925 Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr Phe 930 935 940 Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile 945 950 955 960 Gly Pro Arg Leu Thr Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr 965 970 975 Thr Ile Asp Val Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu 980 985 990 Ser Leu Val Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly 995 1000 1005 Asp Ala Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His 1010 1015 1020 Gly Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr 1025 1030 1035 Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg 1040 1045 1050 Ser Asn Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr 1055 1060 1065 Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser 1070 1075 1080 Asn Val Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val 1085 1090 1095 Thr Leu Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val 1100 1105 1110 Arg Ile Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp 1115 1120 1125 Val Trp Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro 1130 1135 1140 Asp Gly Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg 1145 1150 1155 Gly Leu Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val 1160 1165 1170 Glu Glu Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr 1175 1180 1185 Gly Ser Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe 1190 1195 1200 Lys Leu Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu 1205 1210 1215 Gln Asp Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly 1220 1225 1230 Ala Arg Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala 1235 1240 1245 Leu Ser Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly 1250 1255 1260 Arg Leu Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn 1265 1270 1275 Val Tyr Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly 1280 1285 1290 His Ile Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln 1295 1300 1305 Leu Ser Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly 1310 1315 1320 Ile Cys Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly 1325 1330 1335 Thr Val Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val 1340 1345 1350 Gln Arg Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys 1355 1360 1365 Leu Val Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu 1370 1375 1380 Phe Ala Glu Cys His Lys Val 1385 1390 28220PRTArtificial SequenceSynthetic 28Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu 1 5 10 15 Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala 20 25 30 Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu 35 40 45 Ala Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 50 55 60 Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu 65 70 75 80 Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala 85 90 95 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys 100 105 110 Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu Leu Met Ala Ser 115 120 125 Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly 130 135 140 Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His 145 150 155 160 Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn 165 170 175 Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp 180 185 190 Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro 195 200 205 Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly 210 215 220 29269PRThomo sapiens 29Asn Pro Glu Leu Lys Asp Phe Asn Glu Glu Glu Gln Leu Lys Cys Asp 1 5 10 15 Leu Glu Leu Asn Lys Leu Glu Asn Gln Ile Leu Met Leu Gly Lys Thr 20 25 30 Phe Tyr Gln Asn Tyr Arg Asp Asp Val Glu Ser Leu Tyr Ser Lys Leu 35 40 45 Asp Leu Ile Met Gly Tyr Lys Asp Glu Glu Arg Ala Asn Lys Lys Ala 50 55 60 Val Asn Lys Arg Met Leu Glu Asn Lys Lys Glu Asp Leu Glu Thr Ile 65 70 75 80 Ile Asp Glu Phe Phe Ser Asp Ile Asp Lys Thr Arg Pro Asn Asn Ile 85 90 95 Pro Val Leu Glu Asp Glu Lys Gln Glu Glu Lys Asn His Lys Asn Met 100 105 110 Ala Gln Leu Lys Ser Asp Thr Glu Ala Ala Lys Ser Asp Glu Ser Lys 115 120 125 Arg Ser Lys Arg Ser Lys Arg Ser Leu Asn Thr Gln Asn His Lys Pro 130 135 140 Ala Ser Gln Glu Val Ser Glu Gln Gln Lys Ala Glu Tyr Asp Lys Arg 145 150 155 160 Ala Glu Glu Arg Lys Ala Arg Phe Leu Asp Asn Gln Lys Ile Lys Lys 165 170 175 Thr Pro Val Val Ser Leu Glu Tyr Asp Phe Glu His Lys Gln Arg Ile 180 185 190 Asp Asn Glu Asn Asp Lys Lys Leu Val Val Ser Ala Pro Thr Lys Lys 195 200 205 Pro Thr Ser Pro Thr Thr Tyr Thr Glu Thr Thr Thr Gln Val Pro Met 210 215 220 Pro Thr Val Glu Arg Gln Thr Gln Gln Gln Ile Ile Tyr Asn Ala Pro 225 230 235 240 Lys Gln Leu Ala Gly Leu Asn Gly Glu Ser His Asp Phe Thr Thr Thr 245 250 255 His Gln Ser Pro Thr Thr Ser Asn His Thr His Asn Asn 260 265 3028PRTArtificial SequenceSynthetic 30Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 1 5 10 15 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 20 25 3150PRTArtificial SequenceSynthetic 31Ser Val Ala Gln Ala Thr Ser Ser Ser Gly Glu Ala Pro Asp Ser Ile 1 5 10 15 Thr Trp Lys Pro Tyr Asp Ala Ala Asp Leu Asp Pro Thr Glu Asn Pro 20 25 30 Phe Asp Leu Leu Asp Phe Asn Gln Thr Gln Pro Glu Arg Gly Asp Asn 35 40 45 Asn Leu 50

Claims

1. A FVIII molecule fused to a fusion partner, wherein the fusion partner replaces the A3-domain of the Factor VIII molecule, is inserted into the B-domain of Factor VIII, or is inserted in the C-terminal end of the C2 domain in Factor FVIII.

2. The FVIII molecule according to claim 1, wherein the fusion partner is albumin.

3. The FVIII molecule according to claim 2, wherein said molecule is conjugated with a side group.

4. The FVIII molecule according to claim 1, wherein said FVIII molecule is a B domain truncated variant comprising a B domain having the sequence as set forth in SEQ ID NO: 2, and wherein the fusion partner is an Fc domain that is inserted in the C-terminal end of the C2 domain in Factor FVIII.

5. The FVIII molecule according to claim 4, wherein said Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor.

6. The FVIII molecule according to claim 1, wherein the Factor VIII molecule is fused to an Fc receptor.

7. The FVIII molecule according to claim 6, wherein said Fc receptor is Fc.gamma.RI.

8. The FVIII molecule according to claim 1, wherein the Factor VIII molecule has reduced vWF binding capacity.

9. The FVIII molecule according to claim 1, wherein the FVIII fusion protein is conjugated with a side group.

10. The FVIII molecule according to claim 9, wherein the side group is selected from one or more of the list group consisting of: hydrophilic polymers, peptides and hydrophobic side groups.

11. A method of making a molecule according to claim 1, wherein said method comprises incubating a host cell encoding said molecule under appropriate conditions.

12. A method for treatmenting of haemophilia comprising administering an effective amount of the molecule of claim 1 to a subject in need thereof.

13. A pharmaceutical composition comprising a molecule according to claim 1.