FIELD OF THE INVENTION

The invention described herein relates to polypeptides comprising at least one amino acid sequence having significant identity with (homology to) human Factor VIII or biologically active portion(s) thereof, related molecules (such as nucleic acids encoding such polypeptides), compositions (such as pharmaceutical formulations) comprising such polypeptides, and methods of making and using such polypeptides and related biological molecules.

BACKGROUND OF THE INVENTION Coagulation Factor VIII ("Factor VIII" or "FVIII") is an essential clotting factor. The lack of normal FVIII causes Hemophilia A, an inherited bleeding disorder. Factor VIII participates in the intrinsic pathway of blood coagulation. Specifically, Factor VIII is a cofactor for Factor IXa which converts Factor X to activated Factor Xa (in the presence of Ca ⁺² and phospholipids).

The Factor VIII gene produces two alternatively spliced transcripts. Transcript variant 1 encodes a large glycoprotein, isoform a, which circulates in plasma and associates with von Willebrand factor ("vWF") in a noncovalent complex. This protein undergoes multiple cleavage events. Transcript variant 2 encodes a putative small protein, isoform b, which consists primarily of the phospholipid binding domain of factor VIIIc. This binding domain is essential for coagulant activity. The structure of FVIII is well known (see, e.g., Thompson, Semin Thromb Hemost. 2003 Feb;29(l) : 11-22).

Upon activation by thrombin, (Factor Ha), Factor Villa (FVIIIa) dissociates from the FVIII:vWF complex to interact with Factor IXa causing a well characterized chain of events that leads to the production of more thrombin. Thrombin cleaves fibrinogen into fibrin which polymerizes and crosslinks (using Factor XIII) into a blood clot. FVIIIa is proteolytically inactivated in this process by activated Protein C ("aPC") and Factor IXa and quickly cleared from the blood stream.

FVIII concentrated from donated blood plasma or alternatively recombinant FVIII can be given to hemophiliacs to restore hemostasis. Thus, FVIII is also known as Antihemophilic factor. FVIII has a circulatory half life of about 10-12 hours in vivo and it is therefore highly desirable to provide FVIII analogues with a prolonged half life in order to decrease the frequency of the therapeutic/prophylactic IV FVIII infusions.

WO2009140598 discloses a concept including hybrid molecules comprising FVIII and GPIIb/IIIa specific antibodies. This concept may, however, result in inhibition of the ability of the GPIIb/IIIa receptors to bind fibrinogen and the ability to form a primary clot will thus be decreased upon administration of such hybrid FVIII molecules. There is thus a need in the art for biologically active Factor VIII molecules with a capacity to bind to platelets with high affinity. There is furthermore a need in the art for biologically active Factor VIII molecules with a capacity to bind to platelets while essentially retaining the capacity of the platelets to aggregate and thus form a primary clot.

SUMMARY OF THE INVENTION

The present invention provides targeting molecules, e.g. Factor VIII (FVIII) molecules comprising a non-inhibitory GPIIb/IIIa specific antibody as well as related molecules, compositions, methods of making such molecules, and methods of using such molecules and compositions (e.g., in the treatment or regulation of hemophilia A).

These and other aspects, features, and advantages of the invention will be more fully de- scribed herein.

DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates potential methods by which a scFv may be attached chemically to, or expressed in cis as part of the polypeptide with, a FVIII or FVIII analog amino acid sequence, in order to obtain a modified FVIII molecule. Specifically shown are glycoconju- gation, chemical conjugation, and fusion protein approaches to the production of such a molecule. These methods can similarly be used, where suitable, to generate other FVIII molecules of the invention.

Figure 2 illustrates platelet aggregation. The bars show aggregation in SFLLRN (10 μM) ac- tivated platelets pre-treated with either F8-500 AP3-LC-HC scFV-Δa3 (AP3-N8) or Reo- Pro®. Data is shown as mean±sem of duplicate determinations in which SFLLRN (10 μM) alone was set as 100 %.

Figure 3 illustrates the final targeting vector Figure 4 illustrates binding of AP3-N8 2097 (A) and AP3-N8 MZ1 (C) to human platelets in a dose-dependent manner. The binding of both compounds can be almost completely competed with excess AP3-LC-HC scFV-FLAG (SEQ ID NO 22) .

DESCRIPTION OF THE INVENTION

In the context of this invention, a Factor VIII protein (or FVIII sprotein) is a protein comprising an amino acid sequence exhibiting at least about 70% amino acid sequence identity to an amino acid sequence selected from human factor VIII (SEQ ID NO: 1) or the mature portion thereof (i.e., residues 20-2351 thereof), or the thrombin activated portion thereof, or a B domain deleted/truncated version thereof as shown below.

Factor VIII (SEQ ID NO:1)

MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSF PFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL LVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PGLIGC HRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQ HDGMEAYVKVDSCPEEPQLRM KNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREA IQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI FKYKWTVTVE DGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLH EVAYWYILSIGAQTDFLSVFFS GYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKN TGDYYE DSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMP KIQNVSSS DLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMV FTPESGL QLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDS QLDTTLFG KKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPAL LTKDNA LFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR MLMDKNAT ALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNS LNSGQ GPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNL HENNTHN QEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQ DFRSLNDS TNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQ FRLPLEET ELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANR SPLPIAKV SSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMT GDQREVGS LGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLV EGSLLQGT EGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEK TAFKKK DTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTL QSDQEEID YDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQ SGSVPQ FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSL ISYEEDQR QGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLV CHTNTL NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAING YIMDTLP GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLP SKAGIWR VECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSG SINAWS TKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTL MVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQIT ASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF LISSS QDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRM EVLGCEAQDL

Y

Factor VIII B-domain-deleted (SEQ ID NO:2)

MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSF PFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL LVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PGLIGC HRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQ HDGMEAYVKVDSCPEEPQLRM KNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREA IQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI FKYKWTVTVE DGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQ TDFLSVFFS GYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKN TGDYYE DSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKK EDFDIYDE DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGS FTQPLY RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVK PNETKTYF WKVQHHMAPTKDEFDCKAW AYFSDVDLEKDVHSGLIGPLLVCHTNTLN PAHG RQVTVQEFALFFTI FDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW YLLSMG SNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA GMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLL APMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLH LQGRSN AWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQN GKVKV FQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

Factor VIII B-domain-truncated ("N8") (SEQ ID NO:3)

MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSF PFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL LVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PGLIGC HRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQ HDGMEAYVKVDSCPEEPQLRM KNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREA IQHESGI LGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI FKYKWTVTVE DGPTKSDPRCLTRYYSSFVNMERDLASG LIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQ TDFLSVFFS GYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKN TGDYYE DSYEDISAYLLSKNNAIEPRSFSQNSRHPSQNPPVLKRHQREITRTTLQSDQEEIDYDDT ISVEMKKE DFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVF QEFTDG SFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAE PRKN FVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGR QVTVQE FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMA QDQRIRW YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLI GEHLHAG MSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDL LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGI KHNIFNPP IIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATW SPSKARLH LQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQW TLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

In a more particular aspect, the invention relates to targeted FVIII derivatives that exhibit at least about 75%, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity to SEQ ID NO : 1 or the mature portion, or the thrombin activated portion thereof, or a B domain deleted/truncated version thereof.

In an even further particular aspect, the invention relates to targeted FVIII derivatives that exhibit at least about 90% identity (such as at least about 93% identity) to SEQ ID NO: 1, SEQ ID NO: 2, or the mature portion or the thrombin activated portion thereof, or a B domain deleted/truncated version thereof.

In another aspect, the invention provides targeted FVIII derivatives that exhibit at least about 95% identity to SEQ ID NO: 1 or the mature portion or ht ethrombin activated portion thereof. In more particular aspects, the invention provides targeted FVIII derivatives that exhibit at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or at least about 99.7%, at least about 99.8%, or even at least about 99.9% identity to SEQ ID NO: 1 or the mature portion thereof, or the thrombin activated portion thereof, or a B domain deleted/truncated version thereof. Stated another way, in one aspect the invention provides targeted FVIII derivatives characterized by comprising an amino acid sequence in at least 1, typically at least 2-3, frequently at least 1-5, and commonly at least in which 1-20 (such as 1-15, 1-12, 1-10, 1-7, 1-3, 2-20, 2-15, 2-12, 2-10, 2-7, 2-5, 3-20, 3-15, 3-12, 3-10, 3-7, 3-5, 4-20, 4-15, 4-12, 4-10, 4-7, 5-20, 5-15, 5-12, 5-10, or 5-7) amino acid residues are deleted, inserted, and/or substituted with respect to SEQ ID NO: 1, or the mature portion or the thrombin activated portion thereof, or a B domain deleted/truncated portion thereof.

Commonly, at least about 50%, at least about 60%, at least about 65%, at least about 70%, such as about 75% or more, about 80% or more, about 85% or more, about 90% or more, or even about 95% or more (such as at least about 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%) of the substitutions in the FVII "analog" sequence can be characterized as "conservative substitutions." Conservative substitutions can be defined by substitutions within the classes of amino acids reflected in one or more of the following three amino acid classification tables:

Table 1 - Amino Acid Residue Classes for Conservative Substitutions

Table 2 - Alternative Conservative Amino Acid Residue Substitution Groups

Table 3 - Further Alternative Physical and Functional Classifications of Amino Acid Residues

Even more conservative amino acid residue substitution groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Additional groups of amino acids can also be formulated using the principles described in, e.g., Creighton (1984) PROTEINS : STRUCTURE AND MOLECULAR PROPERTIES (2d Ed. 1993), W. H. Freeman and Company. In some instances it can be useful to further characterize substitutions based on two or more of such features (e.g., substitution with a "small polar" residue, such as a Thr residue, can represent a highly conservative substitution in an appropriate context).

The terms "targeted", "targeting" and the like are used to indicate that the FVIII molecule binds to one or more biological molecules (typically other proteins, and frequently cellular receptors) and/or cells with greater affinity and/or avidity than wild-type FVIII (or that are not typically bound by wild-type FVIII). As already noted, targeted molecules provided by the invention can exhibit increased stability as compared to a corre- sponding unmodified protein (a protein that lacks a "targeting factor," which usually is an amino acid sequence (a targeting domain) or molecule (a targeting moiety), but is otherwise identical to the targeted FVIII protein). In a particular aspect, targeted molecules that exhibit this or other desirable/modified properties are provided wherein one or more biological activities of FVIII are not significantly diminished (e.g., Factor IX binding). Typically, the amount of inhibition of such activity is about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 7% or less, about 5% or less, or about 3% or less). Assays for FVIII biological activity are known in the art (see, e.g., Mikaelsson et al., Semin Hematol. 2001 Apr;38(2 Suppl 4): 13-23).

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 the following point mutations: Y1680F, Y1680R, Y1680N, Y1680C, and E1682T.

Side group: The Factor VIII molecules according to the present invention may be conjugated with a side group that is not a non-inhibitory GPIIb/IIIa antibody. In this connec- tion, a "side group" is to be understood as a covalent attachment of any moiety that is not naturally part of Factor VIII molecule. Preferably said side chain is providing the Factor VIII molecule with a prolonged circulatory half-life. Conjugation of a side chain may be in the form of a fusion protein, and/or chemical conjugation and/or enzymatic conjugation processes. The side chain according to the present invention is typically selected from one or more of the list consisting of: hydrophilic polymers, fatty acids and derivates thereof (sometimes referred to as "albumin binders"), albumin, transferrin, elastin like peptides, isolated Fc domains, fragments of vWF, antibodies as well as fragments thereof comprising antigen binding sequences . Examples of suitable antibodies include antibodies, or fragments thereof, with the capacity to bind to blood components with a relatively long circulatory half life such as e.g. erythrocytes, platelets, fibrinogen etc. or binding to the vessel wall, e.g. collagen. Without being bound by theory it is envisaged that the reason why it functions more efficiently to attach side groups to Factor VIII molecules with reduced vWF binding capacity rather than attaching side groups to Factor VIII molecules with normal vWF binding ca- pacity 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. In connection with FVIII molecules according to the present invention, such molecules may furthermore be targeted to the platelets in a more efficient way if the FVIII part of the molecule has a reduced capacity to bind vWF.

Hydrophilic polymer: The modifying group/hydrophilic polymer according to the present invention is preferably non-naturally occurring. In one example, the "non-naturally occur- ring modifying group" is a polymeric modifying group, in which at least one polymeric moiety is non-naturally occurring. In another example, the non-naturally occurring modifying group is a modified carbohydrate. The locus of functionalization with the modifying group is selected such that it does not prevent the "modified sugar" from being added enzymatically to a polypeptide. "Modified sugar" also refers to any glycosyl mimetic moi- ety that is functionalized with a modifying group and which is a substrate for a natural or modified enzyme, such as a glycosy transferase.

The polymeric modifying group added to a polypeptide can alter a property of such polypeptide, for example, its bioavailability, biological activity or its half-life in the body. Ex- emplary 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. The term "water-soluble" refers to moieties that have some detectable degree of solubility in water. Methods to detect and/or quantify water solubility are well known in the art. Exemplary water-soluble polymers according to the invention include peptides, saccha- rides, poly(ethers), poly(amines), poly(carboxylic acids) and the like. Peptides can have mixed sequences and be composed of a single amino acid, e.g., poly(lysine). An exemplary polysaccharide is poly(sialic acid). An exemplary poly(ether) is poly(ethylene glycol), e.g., m-PEG. Poly(ethylene imine) is an exemplary polyamine, and poly(acrylic) acid is a representative poly(carboxylic acid).

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.

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. Patent No. 5,932,462, which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.

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. Patent No. 5,629,384, which is incorporated by reference herein in its entirety, as well as copolymers, terpolymers, and mixtures thereof.

Although the molecular weight of each chain of the polymer backbone can vary, it is typi- cally 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.

Albumin binder conjugates/side groups

It is known that the in vivo properties of such proteins can be improved by the use of al- bumin 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.

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.

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 moi- ety as compared to its point of attachment to the peptide. 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.

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

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, oxima- tion. 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). 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.

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. The albumin binding moiety may be, or may comprise a fatty acid or fatty diacid or a derivative or either thereof. 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. 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. The nomenclature is as is usual in the art, for example - COOH, as well as HOOC-, refers to carboxy; -C5H4- to phenylen; -CO-, as well as -OC-, to carbonyl (O=C<); and CgH5-0- to phenoxy. 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.

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-(CH2)2-O-(CH ₂ )2-O-CH ₂ -CO-). 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.

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.

The amino acid glutamic acid (GIu) 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 GIu residue, if present. The amino group of GIu 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 GIu, if present. This way of inclusion of GIu is occasionally briefly referred to as "gamma-Glu".

N- and O-linked oligosaccharides: 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). 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). Methods of conuugating polypeptides with various polymeric side groups is described e.g. in WO0331464.

"Glycoprotein Ilb/IHa" or "GPIIb/HIa" is an integrin found on platelets. It is a receptor for fibrinogen and aids in platelet activation. The complex is formed via calcium-dependent association of GPIIb and GPIIIa, a required step in normal platelet aggregation and endothelial adherence. Platelet activation leads to a conformational change in platelet GPIIb/IIIa receptors that induces binding to fibrinogen. A non-inhibitory GPIIb/IIIa antibody according to the invention should not be directed to the fibrinogen recognizing part which comprises the ligand binding pocket in the globular head of the integrin. Which parts of the GPIIb/IIIa that are directly involved in the RGD recognition and hence fibrinogen binding is not completely understood. However, in GPIIIa amino acids 109-171 are described to be engaged in the interaction (D'Souza et al., Science 242; 91-93, 1988) whereas in GPIIb the amino acids of importance are thought to comprise amino acids 294-314 (D'Souza et al., JBC 265:6 ;3440-46, 1990) or amino acids 145-224 (Kamata et al., JBC 271 : 18610-15, 1996, Tozer et al., Blood 93:918-24 1999, Basani et al., Blood 95.180-88, 2000). The non-inhibitory AP3 antibody according to the present invention is thought to have the capacity to bind to an epitope situated within amino acids 348-421 of GPIIIa and consequently does not interfere with fibrinogen binding (Kouns et al. Blood 15;78(12) :3215-23, 1991).

The term "GPIIb/IIIa antibody", "targeting factor" as used herein, is intended to refer to immunoglobulin molecules and fragments thereof that have the ability to specifically bind to the GPIIb/IIIa integrin. Said GPIIb/IIIa antibodies furthermore bind to the GPIIb/IIIa receptor in an essentially non-inhibitory fashion, meaning that the ability of the platelets to bind fibrinogen and to aggregate to form the primary clot is not significantly decreased (such as e.g. less than 20%, 15%, 10%, 5%, or 1% decreased compared to addition of a reference antibody) upon binding of the GPIIb/IIIa antibody. Platelet aggregation was measured by monitoring the change in light transmission through a suspension of isolated platelets. This method was first described essentially by Gustav von Born in the 1960s (Born, Nature 1962) and is today one of the most used methods for evaluation of platelet function. In brief, the method measures the capability of light to transverse through a suspension of platelets. This suspension of platelets might either be platelet rich plasma or isolated platelets. The sample is illuminated and the amount of light going through the sample is measured. Upon activation the GPIIb/IIIa change its conformation to a fibrinogen high-binding state and in the presence of fibrinogen the platelets will start to form aggregates. This is registered as an increase in light transmission since more light will go through a sample with few large aggregates than many single platelets.

Full-length antibodies comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHl, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to car- boxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. Thus, within the definition of an antibody is also one or more fragments of an antibody that retain the ability to specifically bind to GPIIb/IIIa.

It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 : 544-546 ), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as sin- gle chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426: and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antibody".

Other forms of single chain antibodies, such as diabodies are also encompassed. Diabod- ies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., HoI- liger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). It is understood that protein X may have one or more antigenic determinants comprising (1) peptide antigenic determinants which consist of single peptide chains within protein X, (2) conformational antigenic determinants which consist of more than one spatially contiguous peptide chains whose respective amino acid sequences are located disjointedly along the protein X polypeptide sequence; and (3) post-translational antigenic determinants which consist, either in whole or part, of mo- lecular structures covalently attached to protein X after translation, such as carbohydrate groups, or the like.

In another preferred embodiment, the non-inhibitory GPIIb/IIIa antibody is an AP3, Tab or a SZ22 antibody or fragment thereof.

The terms "human antibody", "human antibodies", as used herein, means antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.

The term "epitope" as used herein means any antigenic determinant on an antigen to which the antibody binds. Epitopic determinants usually consist of chemically active sur- face groupings of molecules such as amino acids or sugar side chains and usually have spe-cific three dimensional structural characteristics, as well as specific charge characteristics.

In general, the GPIIb/IIIa antibody can be bound to the FVIII or FVIII analog amino acid sequence in any suitable manner. Thus, for example, the antibody can be expressed as part of a "fusion protein" with the FVIII or FVIII analog amino acid sequence or separately attached via chemical and/or enzymatic methods. In this latter respect, the antibody can be bound to the FVIII or FVIII analog sequence by a sortase- or carboxypepti- dase-mediated transacylation (see, e.g., Stennicke, International Patent Publication WO2006/013202 A2; Hoess et al., International Patent Application WO206/015879 Al; Zhang et al., Protein Exp. Purif. (2004) 36, 292-299 for description of relevant methods and principles).

In one aspect, the GPIIb/IIIa antibody is bound to the FVIII or FVIII analog amino acid sequence (i.e., is "engineered") such that it can be cleaved from the FVIII or FVIII analog sequence under, e.g. by insertion into the B-domain.

In one aspect, the invention provides FVIII molecules that comprise two or more GPIIb/IIIa antibodies or that comprise a single antibody that specifically binds to two or more targets. For example, in one aspect, the invention provides FVIII molecules that comprise a bispecific or multispecific antibody or antibody fragment. In one aspect, the antibody is a multispecific antibody molecule (full-length antibody or antibody fragment) in which one target of the antibody (such as one "arm" of a bispecific antibody) specifically binds to a portion of the B-domain that is included in the FVIII molecule and an- other portion binds to a target associated with platelets and/or megakaryocytes. Examples of B-domain-specific antibodies are known in the art (see, e.g., Lavigne-Lissalde et al., THROMBOSIS AND HAEMOSTASIS, Volume: 98, Issue: 1, Pages: 138-147, 2007.

From the aspect of the foregoing paragraph it should be clear that a GPIIb/IIIa antibody can be associated with a FVIII or FVIII analog amino acid sequence-containing portion of the FVIII molecule by other suitable means other than covalent bonding including by non-covalent protein-protein, protein-moiety, or moiety-moiety interactions. In other aspects, as described elsewhere herein, a/the GPIIb/IIIa antibody is bound to FVIII or FVIII analog amino acid sequence by at least one covalent bond. In one aspect, the GPIIb/IIIa antibody is bound to the FVIII or FVIII analog amino acid sequence via an amide bond (e.g., in the case of a "fusion protein" of a FVIII amino acid sequence and the GPIIb/IIIa antibody). It also should be clear that the bond to the amino acid sequence can be direct or indirect. For example, the bond can comprise a suitable linker, which may be a chemical moiety (examples of which are described in, e.g., US Patent Publica- tion No. 20030236190) or an amino acid sequence, such as a flexible Gly(X)Ser(Y) linker. In another aspect, the linkage is through a glycan that is associated with an amino acid of the amino acid sequence. Even such glycan linkages can comprise additional linkage elements, e.g., where the glycan itself is derivatized with an element that binds to the targeting molecule. It will be more generally appreciated that the various aspects of the invention can be combined in any suitable manner. Thus, for example, in one facet the invention provides a FVIII molecule that comprises a bispecific GPIIb/IIIa antibody molecule portion which binds to the B-domain of the FVIII molecule and a cleavage site (which may be the natu- ral thrombin cleavage site) that permits the B-domain to be removed from the FVIII molecule, releasing the targeting antibody molecule portion from the active FVIII molecule.

The biologic activity of an FVIII molecule of the invention can also be readily tested using routine and known methods. For example, FVIII molecule can be incubated with resting platelets, the bound platelets by purified, by, e.g., gel-filtration or differential centrifuga- tion, and the FVIII molecule activated with thrombin; upon which catalytic efficiency and demonstration of activity can be visualized by standard chromogenic assays (e.g. COAT- EST) or a clot assay. Prolongation of in vivo half-life similarly can be assessed using standard methods. For example, hemophilia A mice (FVIH ^null/nu ") can be monitored for specific FVIII activity by several different methods, including, clot assays (FVIH ^null/nu " mice) or FX activity assays. It should be noted that the method of this invention, while described with reference to human Factor VIII, can be applied to Factor VIII proteins from other mammals, such as dogs, mice, etc. Such proteins are known in the art and the application of the methods of the invention to such homologs of FVIII and other species requires no more than routine experimentation.

The FVIII molecules of the invention can be provided in a dosage that is similar to or somewhat less than the amount of FVIII typically administered to a subject or patient for the relevant desired physiologic effect. In general, FVIII formulations known in the art also can be used in the preparation of pharmaceutical compositions comprising the FVIII molecules of the present invention.

The invention also provides a nucleic acid that encodes a fusion protein FVIII molecule according to the present invention. The nucleic acid can be any suitable type of nucleic acid that encodes such a molecule (e.g., a ssDNA, dsDNA, or an RNA, which may comprise various suitable modifications known to those of skill in the art such as a phos- phothioate backbone). A nucleic acid may further include expression elements, such as promoters, enhancers, polyA sequences, and the like. Such a nucleic acid can also be incorporated into a suitable vector comprising even further elements, such as resistance genes and the like, which may be any suitable type of vector (e.g., a viral vector, such as an adeno-viral, pox viral, or adeno-associated-viral vector or a plasmid vector). Such nucleic acids and vectors can further be incorporated into suitable host cells for expression or maintenance, which typically will be mammalian cells, such as COS or HEK cells. In this and other respects, the invention provides a method of producing FVIII molecules of the invention and potential FVIII molecules of the invention.

In a preferred embodiment, the FVIII molecule according to the invention furthermore has modulated, preferably reduced vWF binding capacity, preferably by comprising an amino acid substitution in position 1680, such as e.g. one of the following substitutions: Y1680F, Y1680R, Y1680N, or Y1680C. In other preferred embodiments, the FVIII molecule according to the invention comprise amino acid mutations that may result in e.g. modulated binding to e.g. LPR, various receptors, other coagulation factors, cell surfaces, etc.

In yet another embodiment, the Factor VIII molecule according to the invention is cova- lently attached to the non-inhibitory GPIIb/IIIa antibody via a linker. Preferably, the linker comprises an N-linked or an O-linked glycan on the FVIII molecule. According to a particularly preferred embodiment, the glycan is placed in the B domain of the Factor VIII molecule according to the invention. The B-domain is preferably a truncated B-domain.

In a particular preferred embodiment the non-inhibitory GPIIb/IIIa antibody and FVIII are linked through a linker connecting the O-linked glycan of the B-domain of FVIII and an N-linked glycan of the non-inhibitory GPIIb/IIIa antibody. In a further preferred embodiment, the non-inhibitory GPIIb/IIIa antibody is a full length antibody. In a preffered embodiment this full length antibody is an AP3-antibody, SEQ 1 and 2, or SEQ 1 and 3.

In a further preferred embodiment the N-linked glycan of the non-inhibitory GPIIb/IIIa antibody is part of the constant region of the antibody. In another preferred embodiment the N-linked glycan of the non-inhibitory GPIIb/IIIa antibody is part of the light chain of the antibody. The linker may comprise a polyethylene glycol polymer.

In a further preferred embodiment the non-inhibitory GPIIb/IIIa antibody and FVIII are linked through a linker connecting an intact O-linked glycan of the B-domain of FVIII and an intact N-linked glycan of the non-inhibitory GPIIb/IIIa antibody. In a different preferred embodiment the non-inhibitory GPIIb/IIIa antibody and FVIII are linked through a linker connecting an O-linked glycan of the B-domain of FVIII nd the N- terminus of the non-inhibitory GPIIb/IIIa antibody. In a preferred embodiment, the non- inhibitory GPIIb/IIIa antibody is an AP3 Fab-fragment.

In another preferred embodiment the non-inhibitory GPIIb/IIIa antibody and FVIII are linked through a linker connecting an O-linked glycan of the B-domain of FVIII and a Cys residue of the non-inhibitory GPIIb/IIIa antibody. In a preferred embodiment, the non- inhibitory GPIIb/IIIa antibody is an AP3 ScFv.

In yet another preferred embodiment the non-inhibitory GPIIb/IIIa antibody and FVIII are linked through a linker connecting an O-linked glycan of the B-domain of FVIII and one or more Lys residues of the non-inhibitory GPIIb/IIIa antibody. In a preferred embodiment, the non-inhibitory GPIIb/IIIa antibody is an AP3 full length antibody (SEQ 1 and 2 or SEQ 1 and 3).

In yet another preferred embodiment, the non-inhibitory GPIIb/IIIa antibody is fused to the B-domain of a B domain truncated Factor VIII molecule according to the invention. The present invention therefore also comprises nucleic acids and vectors encoding such molecules, as well as host cells comprising such nucleic acids and/or vectors.

In yet another preferred embodiment, the A3 domain of the VIII molecule according to the invention is replaced with the non-inhibitory GPIIb/IIIa antibody.

According to a particularly preferred embodiment, the FVIII molecule according to the invention comprises the sequence as set forth in SEQ ID NO 3, and the linker comprises an O-linked glycan placed in the B domain.

Another aspect of the invention relates to a method of producing a FVIII molecule ac- cording to the invention, said method comprising expressing a nucleic acid according to the invention. Alternatively, the method according to the invention comprises conjugation of the FVIII molecule with the GPIIb/IIIa antibody.

Yet another aspect relates to a pharmaceutical composition comprising a FVIII molecule according to the invention. In yet another aspect, the invention relates to use of a FVIII molecule according to the invention for producing a medicament for treatment of haemophilia A.

In a final aspect, the present invention relates to a method of treating hemophilia A in a mammalian host comprising administering to the host a therapeutically effective amount of a molecule according to the invention

Construction

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each ref- erence were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or ele- ments is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illumi- nate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. This invention includes all modifications and equivalents of the subject matter recited in the claims and/or aspects included herein as permitted by applicable law.

EXAMPLES

EXAMPLE 1

FVIII frameworks and fusion partners

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

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. 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 4), 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.

F8-500-Δa3 consists of F8-500 without the a3 region. In F8-500-Δa3 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-Δa3. The a3 region is important for binding of FVIII to vWF and therefore, the affinity of F8-500-Δa3 for vWF is reduced compared to wild-type FVIII.

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 5). F8-500-Δa3-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-Δa3-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 6).

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.

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 pro- teins

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 synthetis DNA into FVIII cDNA.

Fusions in the B-domain of F8-500 take place between aa750 and aal638. Restriction sites Avrll, Nrul, Agel and MIuI within or flanking the B-domain are used for insertion of the fusion partner encoding DNA.

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) 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 Notl sites. Subsequently, fusion partner DNA is inserted. 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 Agel and SacII sites or between the Avrll and SacII sites.

EXAMPLE 3

Full length AP3 mlgGl ab

The variable regions of the heavy and light chain of the anti-GPIIa/IIIB antibody, AP3 were amplified from RNA isolated from hybridoma cells expressing the AP3 antibody, using the SMART ^™ RACE cDNA Amplification Kit (Clontech, Ca, USA). The primers used for the amplification of the variable regions of the two AP3 chains were:

Heavy chain: Universal Primer Mix A (Clontech, CA) :

Long (0.4 μM) :

5'-ctaatacgactcactatagggcAAGCAGTGGTATCACGCAGAGT-3' (SEQ ID NO 7)

Short (2 μM) :

5'-ctaatacgactcactatagggc-3' (SEQ ID NO 8)

Primer 69 (10 μM)

5'-gctctagactaacactcattcctgttgaagctcttg-3' (SEQ ID NO 9)

Light chain Universal Primer Mix A (Clontech :

Long (0.4 μM) :

5'-ctaatacgactcactatagggcAAGCAGTGGTATCACGCAGAGT-3' (SEQ ID NO 10)

Short (2 μM) : 5'-ctaatacgactcactatagggc-3' (SEQ ID NO 11)

Primer 312 (10 μM) 5'-gtctaccacaacacacgtgac-3' (SEQ ID NO 12) The variable regions were cloned into the pCR4 vector using the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing (Cat. No. K287520, Invitrogen, CA, USA). The heavy chain variable region was subsequently subcloned into the EcoRI/BamHI sites of a pTT5 based expression vector containing a murine IgGl framework to generate an AP3 mlgGl heavy chain. The amino acids sequence of the AP3 mlgGl heavy chain is shown below. The AP3 IgK light chain was amplified from the pCR4 vector using the primer AP-3 LC kll Sense ( 5'- G ACTTTTTG- TATGAATTCCTCACCATGAGGTGC-3'; SEQ ID NO 13) and a M 13 Reverse primer. The PCR fragment was subcloned into the EcoRI site of an empty pTT5 based expression vector. The amino acids sequence of the AP3 mlgK light chain protein is shown below. The two vectors encoding the full length AP3 ab were transiently expressed in Hek293 6E cells. Transfections were carried out using 293fectin as transfection agent (cat. no 12347-019, Invitrogen, Ca, USA) following the in- structions supplied by the manufacturer. Transfections were left for 5 days before harvest.

A potential problematic cystein at position 39 (position 34 according to the Kabat numbering system) was identified in the Light chain of the AP3 antibody. The cystein residue was successfully mutated to a serine through site directed mutagenesis using the QuikChange Site Directed Mutagenesis Kit (Cat no 200518, Stratagene, CA, USA) and the following two primers:

AP3 LC C39S S 5'-caacacttacttgtcctggttcctgcag-3' (SEQ ID NO 14)

AP3 LC C39S AS 5'-ctgcaggaaccaggacaagtaagtgttg-3' (SEQ ID NO 15)

The sequence of the resulting protein is shown below. A full length AP3 mlgGl ab containing the C39S mutation could be expressed by combining the two vector constructs expressing SEQ ID NO 17 and SEQ ID NO 19. EXAMPLE 4

Purification of AP3 FL mlgGl wt and AP3 FL mlgGl wt HC LC C39S.

Purification of the AP3 FL mlgGl wt and AP3 FL mlgGl wt HC LC C39S proteins (EXAMPLE 3) were conducted by a 1-step process composed of affinity chroma- tography using a Protein A MabSelect SuRe resin (GE Healthcare, cat. no. 17- 5438-01). The purification was conducted using an AktaExplorer chromatography system (GE Healthcare, cat. no. 18-1112-41). The buffer systems used for the purification step was an equilibration buffer composed of Tris, 3 M NaCI, pH 8.5 and an elution buffer composed of 10 mM Formic acid pH 3.5. The supernatant was adjusted to 3 M NaCI and pH 8.5 prior to application to the MabSelect SuRe column. The column was washed with 15 column volumes of equilibration buffer and the protein was eluted isocratically in approx. 1 column volume of elution buffer. The AP3 FL mlgGl wt HC LC C39S was analyzed using SDS- PAGE/Coomassie and SEC-HPLC, showing that a pure and homogenous protein of approx. 150 kDa (approx. 50 kDa heavy chain component and approx. 25 kDa light chain component) was obtained from the purification with a purity of >98% as measured by SEC-HPLC. To measure the final protein concentration, a Nano- Drop spectrophotometer (Thermo Scientific) was used together with an extinction coefficient of 1.56.

Characterisation of the antibody was performed by LC-MS analysis of the reduced intact mAb. The heavy chain was shown to contain GOF and GIF glycans. The light chain was shown to be glycosylated with biantennary glycans with one sialic acid (G2FS) as the major structure. Treatment with sialidase gave the expected shift in mass values of 291 amu confirming the presence of a sialic acid

Binding of the full length AP3 FL mlgGl wt antibody to resting platelets was confirmed by FACS analysis.

SEQ ID NO 17: AP3 mlgGl HC

QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGY NK

YNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSV TVS

SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAV LQSD LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIF PP KPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSE L PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKA PQ VYTIPPPKEQMAKDKVSLTC MITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGK

SEQ ID NO 18: AP3 mlgK LC

DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMS NLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVS IF PPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS TLTLTKDEYERHNNYTCEATHKTSTSPIVKSFNRNEC

SEQ ID NO 19: AP3 IgK LC C39S DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLSWFLQRPGQSPQLLIYRMSNLA S GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVS IF PPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS TLTLTKDEYERHNNYTCEATHKTSTSPIVKSFNRNEC

EXAMPLE 5

AP3 scFV constructs

Based on the DNA sequences encoding the variable regions of the light and heavy chains of the AP3 antibody, two single chain antibody formats of AP3 ab (AP3 LC-HC scFV and AP3 HC-LC scFV) were ordered from MWG biotech, Ger- many. The amino acids sequence of the two scFV protein is indicated in SEQ ID NO 22 and SEQ ID NO 23. The construction included a 15 aa (G ₄ S) ₃ ) linker region introduced between the two variable regions in order to allow correct pairing of the V _H and the V _L fragments. A Flag tag was included in the C-terminus of both proteins for purification purposes.

The genes encoding the two single chain formats of the AP3 ab were subsequently subcloned into the HindIII site of a pTT5 based expression vector. The two construct encoding the two single-chain AP3 antibodies were transiently ex- pressed in Hek293 6E cells, using 293fectin as transfection agent (cat. no 12347- 019, Invitrogen, Ca, USA) following the instructions supplied by the manufacturer. Transfections were left for 5 days before harvest.

The binding of the two single-chain AP3 antibodies AP3-LC-HC scFV-FLAG, AP3- HC-LC scFV-FLAG to GPIIa/IIIB of resting platelets was confirmed by FACS analysis.

The potential problematic cystein at position 39 (position 34 according to the Ka- bat numbering system) identified in the Light chain of the AP3 antibody was mutated to a serine in the AP3 LC-HC scFV construct. This was performed using the QuikChange Site Directed Mutagenesis Kit (Cat no 200518, Stratagene, CA, USA) and following the instructions supplied by the manufacturer and using the following two primers

AP3 LC C39S S 5'-caacacttacttgtcctggttcctgcag-3' (SEQ ID NO 20)

AP3 LC C39S AS 5'-ctgcaggaaccaggacaagtaagtgttg-3' (SEQ ID NO 21)

The sequence of the resulting AP3 LC-HC scFV-FLAG C39S protein is shown in SEQ ID NO 24.

SEQ ID NO 22: AP3-LC-HC scFV-FLAG

DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMS NLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRGGGGSGGG GSGGGGSQVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIY PGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWG QGTSVTVSSDYKDDDDK*

SEQ ID NO 23: AP3-HC-LC scFV-FLAG QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNK YNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVS SGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSRS LLHSNGNTYLCWFLQ RPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTF G SGTKLEIKRDYKDDDDK*

SEQ ID NO 24: AP3-LC-HC scFV-FLAG C39S

DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLSWFLQRPGQSPQLLIYRMS NLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRGGGGSGGG GSGGGGSQVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIY PGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWG QGTSVTVSSDYKDDDDK*

Binding of the single chain antibody AP3 LC-HC scFV-FLAG C39S to resting plate- lets was confirmed by FACS analysis.

EXAMPLE 6

AP3 scFV-Cys constructs

In order to facilitate the conjugation of AP3 LC-HC scFV C39S to FVIII, a free Cys residue was introduced in the scFV by site directed mutagenesis. Two constructs were made. In one construct an unpaired Cystein residue was introduced in the

C-terminus of the AP3 LC-HC scFV protein by site directed mutagenesis through use of the QuikChange Site Directed Mutagenesis Kit (Cat no 200518,

Stratagene, CA, USA), following the instructions supplied by the manufacturer and using the following two primers:

AP3 scFV Cys S 5'-cgacgacgacaagtgctgaaagcttcgtacg-3' (SEQ ID NO 25)

AP3 scFV Cys AS

5'-cgtacgaagctttcagcacttgtcgtcgtcg-3' (SEQ ID NO 26) In another construct an unpaired Cystein was introduced by mutating a serine at position 248 in AP3 LC-HC scFV to cystein. The potential problematic cystein at position 39 (position 34 according to the Kabat numbering system) identified in the Light chain of the AP3 antibody was subsequently mutated to a serine using the QuikChange Site Directed Mutagenesis Kit (Cat no 200518, Stratagene, CA, USA), following the instructions supplied by the manufacturer and using the following two primer sets:

Primer set A AP3 scFV LC-HC S248C S

5'-gtgaccgtgagctgcgactacaaggac-3' (SEQ ID NO 27)

AP3 scFV LC-HC S248C AS

5'-gtccttgtagtcgcagctcacggtcac-3' (SEQ ID NO 28)

Primer set B AP3 LC C39S S 5'-caacacttacttgtcctggttcctgcag-3' (SEQ ID NO 29)

AP3 LC C39S AS

5'-ctgcaggaaccaggacaagtaagtgttg-3' (SEQ ID NO 30)

All AP3 scFV constructs were transiently expressed in Hek293 6E cells. Transfec- tions were carried out using 293fectin as transfection agent (cat. no 12347-019, Invitrogen, Ca, USA) following the instructions supplied by the manufacturer. Transfections were left for 5 days before harvest.

All scFV fragments were purified according to the following procedure :

EXAMPLE 7

Purification and characterization of AP3 LC-HC scFV proteins. Purification of AP3 LC-HC scFV proteins (EXAMPLES 5 and 6) were conducted using a 2-step process composed of affinity chromatography using an anti-FLAG M2 affinity gel (Sigma, cat. no. A2220) followed by a Superdex 75pg gelfiltration column to remove aggregates and other high-Mw contaminates if these were ob- served (GE Healthcare, cat.no. 17-1068-01). The purification was conducted using an AktaExplorer chromatography system (GE Healthcare, cat. no. 18-1112- 41). The buffer systems used for the first purification step was an equilibration buffer composed of 20 mM Hepes, 150 mM NaCI, 0.01% Tween-80 (v/v), pH 7.5 and an elution buffer composed of 100 mM Glycine pH 3.5/NaOH. The super- natant was either first adjusted to pH 6.7 with 0.5 M Hepes pH 10.5 or applied directly onto a pre-equilibrated anti-FLAG M2 affinity column. The column was washed with 10 column volumes of equilibration buffer and the protein was eluted isocratically in approx. 2 column volumes of elution buffer. The eluted protein was diluted 1 : 1 in 100 mM Hepes, 150 mM NaCI, pH 7.5 and analyzed using SDS-PAGE/Coomassie and SEC-HPLC. If a pure (>75%) and homogenous protein of approx. 28 kDa was obtained from the first purification step, no further purification was conducted. If not, then the second gelfiltration step was performed using 2OmM Tris, IM NaCI, pH 7.5. Between 2-3.5% load was applied and the fractions containing the eluted protein was analyzed using SDS-PAGE/Coomassie and SEC-HPLC. Based on the analyses, a pool was prepared containing a pure (>80%) and homogenous protein. To measure the final protein concentration, a NanoDrop spectrophotometer (Thermo Scientific) was used together with an extinction coefficient of 1.79.

SEQ ID NO 31: AP3 LC-HC scFV INS257C FLAG

DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMS NLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRGGGGSGGG GSGGGGSQVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIY PGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWG QGTSVTVSSDYKDDDDKC

SEQ ID NO 32: AP3 LC-HC scFV C39S S248C FLAG DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLSWFLQRPGQSPQLLIYRMSNLA S GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRGGGGSGGG GSGGGGSQVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIY PGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWG QGTSVTVSCDYKDDDDK

EXAMPLE 8

AP3 Fab construct

A truncated version of AP3 heavy chain was generated by introduction of a stop codon in the construct encoding SEQ ID NO 17 using the QuikChange Site Directed Mutagenesis Kit (Cat no 200518, Stratagene, CA, USA) and the following two primers:

JP433 AP3 HC Fab S Cagggattgtggttgaaagccttgcatatg (SEQ ID NO 33)

JP434 AP3 HC Fab S Catatgcaaggctttcaaccacaatccctg (SEQ ID NO 34)

The sequence of the resulting protein is shown in SEQ 22.

A functional Fab fragment of the AP3 antibody were expressed by combining the two constructs expressing SEQ ID NO 19 and SEQ ID NO 35. The two chains were transiently expressed in Hek293 6E cells. Transfections were carried out us- ing 293fectin as transfection agent (cat. no 12347-019, Invitrogen, Ca, USA) following the instructions supplied by the manufacturer. Transfections were left for 5 days before harvest.

Purification and characterization of AP3 Fab LC C39S protein. Purification of the AP3 Fab LC C39S protein was conducted using a 2-step process composed of a cation-exchange using a Source 3OS (GE Healhcare, cat. no. 17-1273-01) followed by a Superdex 75pg gelfiltration column (GE Healthcare, cat.no. 17-1068-01). The purification was conducted using an AktaExplorer chromatography system (GE Healthcare, cat. no. 17-1068-01). The buffer systems used for the first purification step was an equilibration buffer composed of 10 mM Na-acetate pH 5.0 and an elution buffer composed of 10 mM Na-acetate, 1 M NaCI pH 5.0. The harvest was adjusted to < 3 mS/cm with the addition of milliQ prior to pH adjustment to pH 5.0 with 0.5 M HCI and applied to the pre- equilibrated Source 3OS column. The column was washed with 15 column volumes of equilibration buffer. The protein was eluted by a linear gradient of equli- bration buffer and elution buffer over 20 column volumes. The protein eluted in approximately 8 column volumes. The second gelfiltration purification step was performed using 20 mM Na-phosphate, 150 mM NaCI, pH 7.2. Between 2-3.5% load was applied and the protein was collected in 5-7% of a column volume. The AP3 Fab LC C39S protein was analyzed using SDS-PAGE/Coomassie and SEC- HPLC, showing that a pure and homogenous protein of approx. 50 kDa was obtained from the purification with an approximate purity of 91.9% as measured by SEC-HPLC. To measure the final protein concentration, a NanoDrop spectrophotometer (Thermo Scientific) was used together with an extinction coefficient of 1.67.

SEQ ID NO 35: AP3 Fab HC QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNK YNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVS SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQS D LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG

EXAMPLE 9

FVIII- AP3 scFV fusions

AP3 LC-HC scFV or AP3 HC-LC scFV was fused to a B-domain deleted and a3- domain deleted FVIII variant using the Agel and SacII restriction enzymes. The B-domain deleted and a3-domain deleted FVIII lacks aa 751-1637 and aa 1649- 1685. The AP3 LC-HC scFV or AP3 HC-LC scFV was inserted between aa R1648 and Q1686. The AP3 coding sequence was amplified by PCR using primers harboring sites recognizable for the respective restriction enzymes, Agel and SacII. Partial restriction digestion of PCR products were performed as the DNA has an endogenous Agel site.

As the furin site (RHQR) is situated N-terminal the AP3 scFV, the AP3 scFV will after processing constitute the N-terminal of FVIII light chain. The thrombin site R1688-R1689 is also conserved and upon thrombin activation of these FVIII variants the AP3 scFVs is deliberated from FVIII.

Primers:

AP3-HC-LC-Da3-AgeI Aacccaccggtcttgaaacgccatcaacggcaggtccagctgcagcagagc (SEQ ID NO 36)

APS-HC-LC-DaS-SacII Gaaagctccgcgggctctgccgcttgatttccagcttgg (SEQ ID NO 37)

AP3-LC-HC-Da3-AgeI Aacccaccggtcttgaaacgccatcaacgggacatcgtgatgacccaggct (SEQ ID NO 38)

APS-LC-HC-DaS-SacII

Gaaagctccgcgggctctggctgctcacggtcacggagg (SEQ ID NO 39)

EXAMPLE 10

Transient ekspression of FVIII frameworks and fusion proteins

HKBIl cells at a density of 0.9 - 1.1 x 10 ⁶ are transfected with a complex of plasmid 0.7 mg/l and the transfection agent, 293Fectin (Invitrogen) 1.4 ml/1. 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 tempera- ture 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 ⁰ C and 5 % CO ₂ . The cell culture harvest is filtered on a 0.22 μm membrane filter.

EXAMPLE 11 General procedure for purification of FVIII frameworks and fusion proteins

A column was packed with the resin VIIISelect (GE Healthcare), with the dimensions 1.6cm in diameter and 4cm in bed height giving 8ml_, and was equilibrated with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% Tween80+250mM NaCI, pH7.3 at 500cm/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 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% Tween80+1.5M NaCI, pH7.3. The bound FVIII was eluted isocratic at 90cm/h with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% Tween80 + 2.5 M NaCI + 6.5M Propylenglycol, pH7.3. The fractions containing FVIII were pooled and diluted 1 : 10 with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO, pH7.3 and applied to a column packed with F25-Sepharose (Thim et al., Haemophilia, 2009). The column dimension was 1.6cm in diameter and 2cm in bed height giving 4ml_ in column volume. The column was equilibrated at 180cm/h with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO + 15OmM NaCI + IM Glycerol, pH7.3 prior to application. After application the column was washed first with equilibration buffer and then 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO + 65OmM NaCI, pH7.3. The bound FVIII was isocratic eluted with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% Tween80 + 2.5M NaCI + 50%(v/v) Ethylenglycol, pH7.3 at 30cm/h. The fractions containing FVIII were pooled and diluted 1 : 15 with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO, 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 0.5cm in diameter and 5cm in bed height giving ImL in column volume. The column was equilibrated at 300cm/h with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO + 5OmM NaCI + IM 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 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO + IM NaCI + IM Glycerol, pH7.3. The fractions containing FVIII were pooled and the pool was stored at -80° until use.

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 Healtcare) charged with 2 column volumes of IM NiSO ₄ . The column dimension was 0.5cm in diameter and 5cm bed height giving ImL column volume. The column was equilibrated with 3OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO + 1.5M NaCI, pH7.3 at 180cm/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 vol- umes to 25OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO + 1.5M NaCI, pH7.3. The fractions containing FVIII were pooled and diluted 1 :30 with 2OmM Imidazol + 1OmM CaCI ₂ + 0.01% TweenδO, pH7.3. The final purification step (Poros 50HQ) was performed as described above.

EXAMPLE 12

Purification and characterization of FVIII- AP3 scFV fusions

Purification of the said AP3 F8 fusion protein (EXAMPLE 9) was conducted using a

3-step process com-posed of an immunoaffinity chromatography step based on the VlllSelect resin (GE Healhcare, cat. no. 17-5455-02) followed by a second immunoaffinity chromatography step based on the antibody F25 coupled to CNBr-Sepharose FF (Thim L, Vandahl B, Karlsson J, Klausen NK, Pedersen J, Krogh TN, Kjalke M, Petersen JM, Johnsen LB, Bolt G, Nørby PL, Steenstrup TD (2009) Purification and characterization of a new recombinant factor VIII (N8) Haemophilia 16: 349-59) and finally an anion exchange chromatography step based on the resin Poros 50HQ (Applied Biosystems cat. no. 1-2559-07). The purification was conducted using an AktaExplorer chromatography system (GE Health-care, cat. no. 17-1068-01). The buffer systems used for the first purification step was an equilibration buffer composed of 2OmM imidazole, 1OmM CaCI ₂ , 0.02% TweenδO, 25OmM NaCI, pH 7.3, a wash buffer composed of 2OmM Imidazole, 1OmM CaCI ₂ , 0.02% TweenδO, 1.5M NaCI, pH 7.3 and an elution buffer composed of 2OmM imidazole, 1OmM CaCI ₂ , 0.02% TweenδO, IM Ammoniumace- tate, 6.5M 1,2-propanediol, pH 7.3. The harvest was diluted 2x with an dilution buffer composed of 2OmM Imidazole, 1OmM CaCI ₂ , 0.02% tweenδO, pH 7.3 and applied to the pre-equilibrated VlllSelect column. The column was washed with 6 column volumes of equilibration buffer and 6 column volumes of wash buffer. The protein was eluted isocratically in approx. 3 column volumes. Prior to the second immunoaffinity purification step, the elution pool from the first purification step was diluted 1Ox using the above mentioned dilution buffer. The buffer systems used for the second purification step was an equilibration buffer composed of 20 mM Imidazole, 10 mM CaCI ₂ , 0.02% Tween 80, 150 mM NaCI, pH 7.3, a wash buffer composed of 2OmM Imidazole, 1OmM CaCI2, 0.02%Tween80, 1.5M NaCI, pH 7.3 and an elution buffer composed of 0.5 M Imidazole, 10 mM CaCI ₂ , 0.02% Tween 80, 150 mM NaCI, pH 7.3. Following application of the sample to the pre-equilibrated F25-Sepharose FF column, the column was washed with 6 column volumes of equilibration buffer and with 6 column volumes of wash buffer. The protein was eluted isocratically in approx. 1.5 column volumes. Prior to the third anion exchange purification step, the elution pool from the second purification step was diluted 15x using the above mentioned dilution buffer. The buffer systems used for the third purification step was an equilibration buffer composed of 2OmM imidazole, 1OmM CaCI ₂ , 0.02% TweenδO, 5OmM NaCI, IM Glycerol, pH 7.2 and an elution buffer composed of 2OmM imidazole, 1OmM CaCI ₂ , 0.02% TweenδO, IM NaCI, IM Glycerol, pH 7.3. Following application of the sample to the pre-equilibrated Poros HQ50 column, the column was washed with 8 column volumes of equilibration buffer. The protein was eluted from the column using a linear gradient from 0 - 100 % over 5 column volumes followed by 10 column volumes of 100 % elution buffer. The protein eluted early on the gradient, typically between 10 - 30 % of elution buffer. Yields were followed using a chromogenic FVIII assay COATEST® Factor VIII (Chromogenix, COATEST SP FVIII cat. no. 82 4086 63) and a SpectraMax spectrophotometer (Molecular Devices, cat. no. M3). The protein quality was analyzed using SDS- PAGE/SilverStain and RP-HPLC, showing that a pure and homogenous protein preparation composed of light chain, heavy chain and single chain. The final protein concentration was determined based on the RP-HPLC analyses.

SEQ ID NO 40: F8-500 AP3-LC-HC scFV -Δa3

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDH LFNIA KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PG LIGCHRKSVYWHVIGMGTTPEVHSIFLEG HTFLVRNHRQAS LEISPITFLTAQTLLMDLG QFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDD NSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY K KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLY SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IG PLLICYKESVDQRGNQIMSDKRNVI LFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNSRHPSQNPPVLKRHQRDIVMTQAAPSVPVTPGESVSISCRSSRSLLH S NGNTYLCWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYY C MQHLEYPFTFGSGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGTSVKISCKA SGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNKYNENFKGKATLTADTSSSTAYMQL SSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVSSQSPRSFQKKTRHYFIAAVERLW DYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEV E DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS W YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAG M STLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV DS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITA SSY FTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWV HQIALRM EVLGCEAQDLY

SEQ ID NO 41: F8-500 AP3-HC-LC scFV -Δa3

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDH LFNIA KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PG LIGCHRKSVYWHVIGMGTTPEVHSIFLEG HTFLVRNHRQAS LEISPITFLTAQTLLMDLG QFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDD NSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY K KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLY SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IG PLLICYKESVDQRGNQIMSDKRNVI LFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTL FP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNSRHPSQNPPVLKRHQRQVQLQQSGAELVRPGTSVKISCKASGYTFTN YWLGWVKQRPGHGLEWIGDIYPGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSED SAVYFCAREYGNYDYAMDSWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPV TPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSG T AFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRQSPRSFQKKTRHYFIAAVERL WD YGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE D NIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDE F DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWY F TENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS TL FLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKV DLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS G IKHNIFN PPIIARYIRLHPTHYSIRSTLRMELMGCDLN SCSMPLGMESKAISDAQITASSYFT NMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY

SEQ ID NO 42: F8-500 AP3-HC-LC scFV

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDH LFNIA KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PG LIGCHRKSVYWHVIGMGTTPEVHSIFLEG HTFLVRNHRQAS LEISPITFLTAQTLLMDLG QFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDD NSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY K KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLY SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDG PTKSDPRCLTRYYSSFVNMERD LASG LIG PLLICYKESVDQRGNQIMSDKRNVI LFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTL FP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNSRHPSQVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRP GHGLEWIGDIYPGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYG NYDYAMDSWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCR SSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVE A EDVGVYYCMQHLEYPFTFGSGTKLEIKRSQNPPVLKRHQREITRTTLQSDQEEIDYDDTI SV EMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQF KKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLI SYE EDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLI GPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTF K ENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKM A LYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD F QITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSS L YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHY SIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLH LQGRSNA WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQN GKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

SEQ ID NO 43: F8-500 AP3-LC-HC scFV

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDH LFNIA KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREG SLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSL PGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LG QFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDD NSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY K KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLY SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDG PTKSDPRCLTRYYSSFVNMERD LASG LIG PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQ A SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTL FP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNSRHPSDIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFL Q RPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTF G SGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLG WVKQRPGHGLEWIGDIYPGGGYNKYNENFKGKATLTADTSSSTAYMQLSSLTSEDSAVYF CAREYGNYDYAMDSWGQGTSVTVSSSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVE MKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFK KVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLIS YEE DQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKE NYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMA L YNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF Q ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL Y ISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYS IRS TLRMELMGCDLN SCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQG RSNAW RPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNG KVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

EXAMPLE 13

Step 1 :

A reagent of the general formula

reactive wherein non-exclusive examples for the reactive group are groups comprising maleimide, azide, alkynes, aldehydes and a non-exclusive example for the spacer is a PEG moiety with an average molecular weight of e.g. 3, kDa, 5 kDa, 10 kDa, or 20 kDa may be reacted with a platelet binding protein such as e.g. Abciximab or AP3 or proteins derived from Abciximab or AP3 such as but not exclusive e.g. single chain variants or FAB- fragments, leading to a compound with the general formula of intermediate Bl. platelet binding

intermediate B1

The attachment point of the spacer at the platelet binding protein may depend on the type of reactive group which had been used to assemble a compound of the general formula of intermediate Bl. If e.g. the reactive group had been an aldehyde, one possibility could be that the spacer has been attached to one of the /V-termini of the platelet binding protein by reductive alkylation in the presence of NaCNBH ₃ . If e.g. the reactive group had been a maleimide, one possibility could be that the spacer has been attach to a free Cystein in the platelet binding protein. The Cystein may be liberated prior reaction by treatment with a suitable reagent such as but not exclusively e.g. and enzyme or tris(carboxyethyl)posphine hydrochloride.

Step 2:

Sialic acids may be removed from glycans of FVIII by reaction with a sialidase. The compound with the general formula of intermediate Bl may be reacted in the presence of a suitable enzyme such as e.g. ST3-Gal-I - when reacted with an O-glycan of FVIII - or ST3-GAIIII - when reacted with a /V-glycan of FVIII - to give a compound of the general structure of product:

platelet binding grou I Intermediates

Intermediate Example 1

/V-((3-(ω-(4-formylbenzoylamino) 10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ- neuraminic acid :

Step l :

/V-((3-(ω-(9H-Fluoren-9-ylmethoxycarbonylamino)10 kDa PEGyl)propionylamino)acetyl)- O ² -[5']cytidylyl-ξ-neuraminic acid :

Λ/-(aminoacetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid (18 mg, 0.029 mmol) was dissolved in a buffer consisting of 50 mM TRIS which had been adjusted to pH 8.9 (4 ml). The current pH was checked and was adjusted to pH 8.9 by addition of 0.1 N hydrochloric acid. THF (16 ml) was added. Approximately half of the final amount of 3-(ω-(9H-fluoren- 9-ylmethoxycarbonylamino)10 kDa PEGyl)propionic acid N-hydroxysuccinimidyl ester (commercially available at for example at Rapp Polymere GmbH, 200 mg in total, 0.019 mmol) was added. The reaction mixture was stirred at room temperature. After 1 h, the second half of 3-(ω-(9H-fluoren-9-ylmethoxycarbonylamino)10 kDa PEGyl)propionic acid N-hydroxysuccinimidyl ester was added. The reaction mixture was stirred for 16 h at room temperature. The THF was removed in vacuo with a bath temperature of 25 ⁰ C. The remaining mixture was filtered and subjected to a size exclusion chromatography, using a G25 gel with a bed size of 26 mm in diameter and 10 cm in length at a flow of 7 ml/min, utilizing a buffer of 25 mM ammonium hydrogencarbonate. The fractions containing the desired compound were pooled and lyophilized to give 453 mg of material containing Λ/-((3-(ω-(9H-fluoren-9ylmethoxycarbonylamino)10 kDa

PEGyI) propionyla mi no)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid.

The ¹ H-NMR-SPeCtOJiTi performed in DMSOd ₆ showed the presence of the cytidylyl moiety as well as the fluorenyl-9-ylmethoxycarbonyl moiety. The material was stored in the freezer.

This reaction was repeated with 1 g of 3-(ω-(9H-fluoren-9-ylmethoxycarbonylamino)10 kDa PEGyl)propionic acid N-hydroxysuccinimidyl ester and 90 mg of O ² -[5']cytidylyl-ξ- neuraminic acid. The purification was performed on a HPLC C4-column with a diameter of 2 cm, using a gradient of 30-50% of a mixture consisting of a 5 mM aqueous solution of ammonium hydrogencarbonate in acetontrile in a 50 mM solution of ammonium hydrognecarbonate in water. The fractions containing the desired compound were collected and lyophilized to give 288 mg of the desired compound. The ¹ H-NMR spectrum corresponded to the ¹ H-NMR spectrum found in the experiment described above.

Step 2:

Λ/-((3-(ω-AminolO kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid

/V-((3-(ω-(9H-Fluoren-9-ylmethoxycarbonylamino)10 kDa PEGyI) propiony Ia mi no)acetyl)- O ² -[5']cytidylyl-ξ-neuraminic acid (453 mg) were dissolved in N,N-dimethylformamide (12 ml). Piperidine (1.25 ml) was added. The clear solution was stirred for 20 min at room temperature. Ether (200 ml) was added. The mixture was left at room temperature for 1.5 h, in order to let the formed precipitation grow old. The precipitation was isolated by nitration. It was dissolved in dichloromethane (4 ml). Ethyldiisopropylamine (1 ml) was added. Ether (250 ml) was added. The mixture was left at room temperature for 16 h in order to let the formed precipitation grow oled. The precipitation was isolated by filtration and dried in vacuo. The ¹ H-NMR spectrum in DMSOd ₆ showed no signals of a 9H-fluoren-9-ylmethoxycarbonyl group, whereas signals assigned to a cytidylyl moiety could be found. The material was stored in the freezer.

Step 3 :

4-Formylbenzoic acid 2,5-dioxopyrrolidin-l-yl ester

Triethylamine (2.04 ml, 14.65 mmol) and 2-succinimido-l,l,3,3-tetramethyluronium tetrafluoroborate (TSTU, 4.44 g, 14.65 mmol) were successively added to a solution of 4- formylbenzoic acid (2.0 g, 13.3 mmol) in Λ/,Λ/-dimethylformamide (30 ml). The reaction mixture was stirred at room temperature for 16 h. It was diluted with ethyl acetate (150 ml) and washed with a 10% aqueous solution of sodium hydrogen sulphate (100 ml). The aqueous phase was extracted with ethyl acetate (2 x 30 ml). The combined organic layers were washed with a mixture of brine (50 ml) and water (50 ml). The combined organic layers were dried over magnesium sulphate. The solvent was removed in vacuo. The crude product was recrystallized from ethyl acetate to give 1.89 g of 4-formylbenzoic acid 2,5-dioxopyrrolidin-l-yl ester. 1H-NMR (CDCI ₃ ). δ 2.95 (s, 4 H); 8.04 (d, 2 H), 8.32 (d, 2 H); 10.15 (s, 1 H).

Step 4:

Λ/-((3-(ω-AminolO kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid (42 mg, 0.004 mmol) was dissolved in dichloromethane (2 ml). Ethyldiisopropylamine (0.002 ml, 0.012 mmol) was added to the solution. A solution of 4-formylbenzoic acid 2,5-dioxopyrrolidin-l-yl ester (19.32 mg, 0.078 mmol) was in dichloromethane (0.5 ml) was added. The reaction mixture was stirred for 16 h at room temperature. The solvent was removed in vacuo with a bath temperature of 25 ⁰ C. The residue was suspended in a

25 mM aqueous solution of ammonium hydrogencarbonate (15 ml). The non-soluble material was removed by filtration. It was divided into 5 parts. Each of them were subjected to a size exclusion chromatography using a G25 on a column with diameter of

26 mm and a length of 10 cm with a flow of 7 ml/min utilizing a buffer of 25 mM ammonium hydrogencarbonate. All the fractions containing the desired material were combined and lyophilized. The ¹ H-NMR spectrum in DMSO-d6 showed the presence of both the aldehyde moiety and the cytidylyl moiety. The obtained material was kept in the freezer.

Intermediate Example 2:

Λ/-((3-(ω-(3-(2,5-dioxo-2,5-dihydropyrrol-l-yl)propiony lamino)10 kDa

PEGyI) propionyla mi no)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid

Step 1 :

3-(2,5-Dioxo-2,5-dihydropyrrol-l-yl)propionic acid 2,5-dioxopyrrolidiny-l-yl ester

3-Maleimidopropionic acid (1.0 g, 5.9 mmol) was dissolved in tetrahydrofuran (20 ml). 2- Succinimido-l,l,3,3-tetramethyluronium tetrafluoroborate (TSTU, 2.14 g, 7.1 mmol) and ethyldiisopropylamine (1.24 ml, 7.1 mmol) were added subsequently. N ₁ N-

Dimethylformamide (5 ml) was added. The reaction mixture was stirred at room temperature, while it was turning sluggish. The mixture was stirred for 2 min. N ₁ N-

Dimethylformamide (5 ml) was added. The mixture was stirred for 2.5 h at room temperature. It was diluted with dichloromethane (150 ml) and was washed subsequently with a 10% aqueous solution of sodium hydrogensulphate (150 ml), a saturated aqueous solution of sodium hydrogencarbonate (150 ml) and water (150 ml).

It was dried over magnesium sulphate. The solvent was removed in vacuo. The crude product was recrystallized from ethyl acetate to give 1.20 g of 3-(2,5-dioxo-2,5- dihydropyrrol-l-yl)propionic acid 2,5-dioxopyrrolidin-l-yl ester.

MS: m/z = 289, required for [M + Na] ⁺ : 289

¹ H-NMR (CDCI ₃ ) δ 2.82 (m, 4 H); 3.02 (t, 2 H); 3.94 (t, 2 H), 6.73 (s, 2 H).

Step 2: Λ/-((3-(ω-AminolO kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid (100 mg, 0.009 mmol) was dissolved in a mixture of tetrahydrofuran (2 ml) and dichloromethane (10 ml). A solution of 3-(2,5-dioxo-2,5-dihydropyrrol-l-yl)propionic acid 2,5-dioxopyrrolidiny-l-yl ester (50 mg, 0.18 mmol) in dichloromethane (3 ml) was added. Ethyldiisopropylamine (0.005 ml, 0.028 mmol) was added. The reaction mixture was stirred at room temperature fro 16 h. Dichloromethane (2 ml) and ethyldiisopropylamine (0.5 ml) were added. Amionomethylated polystyrene resin (commercially available at e.g. Novabiochem, loading 0.85 mmol/g, 438 mg, 0.372 mmol) was added. The mixture was slowly stirred at room temperature for 1 h. The resin was removed by filtration. The solvent was removed in vacuo with a bath temperature of 25 ⁰ C. The residue was dissolved in dichloromethane (4 ml). Ether (200 ml) was added. The mixture was left at room temperature for 2 h in order to let the formed precipitation grow old. The precipitation was isolated by filtration and dried in vacuo to give 38 mg of the title compound. The ¹ H-NMR spectrum in DMSOd ₆ showed the presence of a maleimide group.

Intermediate Example 3

Attachment of Λ/-((3-(ω-(3-(2,5-dioxo-2,5-dihydropyrrol-l-yl)propionylam ino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid to Cys 248 of AP3 scFV C34S S248C

AP3 scFv LC-HC C34S S248C

A solution of Tris(2-carboxyethyl)phosphine hydrochloride (0.40 mg) in a buffer (0.40 ml)consisting of 20 mM imidazole, 10 mM CaCI ₂ , 0.02% TweenδO, 1 M glycerol which had been adjusted to pH 7.35 was added to a solution with a concentration of 0.53 mg/ml of AP3 scFv LC-HC C39S S248C with a Flag tag at its C-terminus and an extrac Cys attached to the Cysteine at position 248 via a S-S bridge in a solution of 100 mM HEPES and 150 mM NaCI which had been adjusted to pH 7.5 with (4 ml, 2.12 mg, 76 nmol). The reaction mixture was gently shaken at 20 ⁰ C. It was divided into two parts. Each of them were added to a PD-IO column (GE-Healthcare), using a buffer of 25 mM HEPES which had been adjusted to pH 7.0. The eluates of the column (each of them 3.5 ml) were combined.

A solution of Λ/-((3-(ω-(3-(2,5-dioxo-2,5-dihydropyrrol-l-yl)propionylam ino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid (3.3 mg, 305 nmol) in a buffer of 25 mM HEPES, which had been adjusted to pH 7.0 (0.43 ml) was added to the solution of the protein. The reaction mixture was gently shaken at 20 ⁰ C for 4 h. It was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. It was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 10 min. The mixture was kept in the freezer until purification.

For purification the mixture was thawed. It was subjected to a size exclusion chromatography utilizing a Superdex 200 gel with a bed size of 16 mm in diameter and 60 cm in length at a flow of 1 ml/min and a buffer of 25 mM TRIS and 150 mM NaCI which had been adjusted to pH 8.0. The fractions containing the desired product were pooled to give 1.61 mg of the desired protein. The SDS-PAGE gel was in accordance with the expectation. It was found that the material contained a lot of aggregated protein.

Intermediate Example 4

Conjugation of Λ/-((3-(ω-(4-formylbenzoylamino)10 kDa PEGyl)propionylamino)acetyl)- O ² -[5']cytidylyl-ξ-neuraminic acid to one N-terminus of Abxicimab

A solution of commercially available Abciximab (ReoPro, 10 mg, 215 nmol, in a 2 mg/ml solution the commercial buffer) was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. Buffer consisting of 25 mM HEPES, which had been adjusted to pH 7.4

(5 ml) was added. An ultracentrifugation was performed at 4000 rpm at 10 ⁰ C for 10 min. Buffer consisting of 25 mM HEPES, which had been adjusted to pH 7.4 (10 ml) was added. An ultracentrifugation was performed at 4000 rpm at 10 ⁰ C for 10 min. Buffer consisting of 25 mM HEPES, which had been adjusted to pH 7.4 (10 ml) was added. An ultracentrifugation was performed at 4000 rpm at 10 ⁰ C for 10 min. The remaining solution of 0.65 ml was placed in a plastic reactor. Buffer consisting of 25 mM HEPES, which had been adjusted to pH 7.4 (3.85 ml) was added. A solution of Λ/-((3-(ω-(4- formylbenzoylamino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid (14 mg, 1290 nmol) in a buffer consisting of 25 mM HEPES which had been adjusted to pH 7.0 (1.5 ml) was added. The reaction mixture was gently shaken at 300 rpm for 3 min at 20 ⁰ C. A freshly prepared 1.0 M solution of sodium cyanoborohydride in water (0.025 ml) was added. The reaction mixture was gently shaken at 300 rpm at 20 ⁰ C. After 1 h, another portion of the solution of sodium cyanoborohydride (0.025 ml) was added. After 1 h, another portion of the solution of sodium cyanoborohydrice (0.025 ml) was added. After 1 h, another portion of the solution of sodium cyanoborhydride was added. The solution was gently shaken at 300 rpm at 20 ⁰ C for 16 h. The reaction mixture was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. It was subjected to an ultracentrifiugation at 4000 rpm for 10 min at 18 oC. The remaining solution of 0.360 ml was filtered and subjected to a size exclusion chromatography on a Superdex200 gel with a bed size of 016 mm X 60 cm at a flow of 1 ml/min utilizing a buffer of 25 mM TRIS, 150 mM NaCI, which had been adjusted to pH 8.0 as eluent. Fractions containing the desired product were combined into two batches. Each of those batches were placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. They were subjected to an ultracentrifugation at 4000 rpm at 10 °Cfor 10 min, yielding 2.67 mg and 3.27 mg respectively. For quantification a molar absorbance of 10.94 was used on a Nanodrop ® spectrophotometer. The analysis of the product by SDS-PAGE were in accordance with the expectation for a conjugate of Λ/-((3-(ω-(4- formylbenzoylamino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid to one N-terminus of Abxicimab.

Intermediate Example 5

Reaction of Λ/-((3-(ω-(4-formylbenzoylamino)10 kDa PEGyl)propionylamino)acetyl)-O ² - [5']cytidylyl-ξ-neuraminic acid with AP3-FAB-fragment at one of its N-termini

A 0.34 mg/ml solution of AP3 C39S FAB fragment in a PGS buffer pH 7.2 (1.7 mg, 35 nmol) was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. A buffer consisting of 25 mM HEPES, 25 mM NaCI pH 7.0 (7 ml) was added. The mixture was submitted to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 15 min. Buffer consisting of 25 mM HEPES, 25 mM NaCI pH 7.0 (10 ml) was added. The mixture was submitted to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 15 min. Buffer consisting of 25 mM HEPES, 25 mM NaCI pH 7.0 (10 ml) was added. The mixture was submitted to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 15 min. Approximately 0.480 ml of solution was left. A solution of N-((3-(ω-(4-formylbenzoylamino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid (2.31 mg, 212 nmol) in a buffer consisting of 25 mM HEPES, 25 mM NaCI pH 7.0 (0.37 ml) was added. The mixture was gently shaken at 20 ⁰ C for 5 min. A I M solution of sodium cyanoborohydride in water (0.0045 ml) was added. The reaction mixture was shaken at 20 ⁰ C for 1 h. A 1 M solution of sodium cyanoborohydride in water (0.0045 ml) was added. The reaction mixture was shaken at 20 ⁰ C for 1 h. A 1 M solution of sodium cyanoborohydride in water (0.0045 ml) was added. The reaction mixture was shaken at 20 ⁰ C for 18 h. The reaction mixture was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. A buffer consisting of 25 mM TRIS, 25 mM NaCI pH 8.00 (2.5 ml) was added. The mixture was subjected to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 4 min. A buffer consisting of 25 mM TRIS, 25 mM NaCI pH 8.00 (2.2 ml) was added. The mixture was subjected to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 22 min. A buffer consisting of 25 mM TRIS, 25 mM NaCI pH 8.00 (3.2 ml) was added. The mixture was subjected to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 12 min. Buffer consisting of 25 mM TRIS, 25 mM NaCI pH 8.00 (2.2 ml) was added to the mixture of approximately 0.120 ml. This mixture was divided into two equal parts. Each of which were added to a spin Q column (VIVAPURE Q MINI M, Sartorius, product no. : VS- 1X01QM24) which had been equilibrated with a buffer consisting of 25 mM TRIS, 25 mM NaCI pH 8.00. Each column was subjected to an ultracentrifugation at 2000 rpm at room temperature for 5 min. Buffer consisting of 25 mM TRIS, 25 mM NaCI pH 8.00 (0.400 ml) was added to each of the spin columns. Each column was subjected to an ultracentrifugation at 2000 rpm at room temperature for 5 min. Buffer consisting of 25 mM TRIS, 500 mM NaCI pH 8.00 (0.400 ml) was added to each of the spin columns. Each column was subjected to an ultracentrifugation at 2000 rpm at room temperature for 5 min. The filtrates were combined and placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. They were subjected to an ultracentrifugation at 4000 rpm at 18 ⁰ C for 8 min. The obtained solution was kept at -80 ⁰ C until purification.

The solution was thawed and subjected to a size exclusion chromatography on a Superdex 75 column with a bed size of 016 mm x 600 mm at a flow of 0.80 ml/min, utilizing a buffer of 25 mM TRIS, 150 mM NaCI at pH 8.00. The fractions containing the desired product - as judged by SDS-PAGE - were pooled and placed in an Amicoun ultracentrifugation device with a cut off of 10 kDa. They were subjected to an ultracentrifugation at 4000 rpm at 18 ⁰ C for 18 min. The solution of 0.280 ml was frozen to -80 ⁰ C as soon as possible. Quantification on a Nanodrop® apparatus using a molar absorbance o f 13.05 showed a concentration of 0.23 mg/ml. The product showed the expected bands in a SDS-PAGE by silver staining. No unreacted PEG was identified on a SDS-PAGE using a staining procedure as described in Kurfϋrst, M. M. Analytical Biochemistry 200, 244-248. 1992.

EXAMPLE 14 Conjugation of AP3 scFv LC-HC C39S S248C to FVIII

AP3 scFv LC-HC C34S (Ser in B-domain residue)

A solution of B-domain deleted FVIII which has a residual B-domain sequence of SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO 4) at the C-terminus of the heavy chain (1 mg, 5.64 mmol) in a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 150 mM NaCI, 0.02% TweenδO and 1 M glycerol, which had been adjusted to pH 7.35 (0.018 ml) was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. A solution of the attachment product of Λ/-((3-(ω-(3-(2,5-dioxo-2,5-dihydropyrrol-l-yl)propionylam ino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid to Cys 248 of AP3 scFV C34S S248C (Intermediate example 3/EXAMPLE 13, 1.29 mg, 34 nmol) in a solution of 25 mM TRIS and 150 mM NaCI which had been adjusted to pH 8.0 (0.680 ml) was added and a buffer consisting of 20 mM histidine, 10 mM CaCI ₂ , 10% glycerol, 0.02% TweenδO, 500 mM NaCI which had been adjusted to pH 6.07 (3 ml) were added subsequently. The mixture was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 20 min. The remaining volume was 0.800 ml or 1.25 mg/ml for FVIII. A solution of Sialidase from A. Urifaciens (0.43 mg/ml, 302 U/mg, 0.0049 ml, 0.645 U) and a solution of ST3-Gal-I (2.5 mg/ml, 0.105 mg, 0.042 ml) were added subsequently. The reaction mixture was gently shaken at 32 ⁰ C for 1 min and thereafter left at 32 ⁰ C for 18 h. It was kept in the freezer until purification.

The reaction mixture was thawed. It was divided into two parts, each of which were subjected to a size exclusion chromatography using a Superose 6 gel with a bed size of 10 mm in diameter and 300 mm in length at a flow of 0.30 ml/min and a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 0.02% TweenδO, 150 mM NaCI, and 1 M glycerol, which had been adjusted to pH 7.35 as eluent. All fractions of both runs containing the desired product were pooled. They were placed in an Amicon ultracentrifugation device with a cut off of 10 kDa and subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 18 min.

A solution of CMP-Λ/-acetylneuraminic acid (CMP Neu/Vac, 1.5 mg, 2597 nmol) in a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 0.02% TweenδO, 150 mM NaCI, and 1 M glycerol, which had been adjusted to pH 7.35 (0.100 ml) and a 0.33 mg/ml solution of ST3Gal-III (0.10 ml, 0.033 mg) were added subsequently. The reaction mixture was gently shaken at 300 rpm and thereafter kept in the freezer until purification.

The reaction mixture was divided into two parts. Each of those was filtered through a 0.00045 mm filter. They were applicated to a sepharose column with a bed size of 5 mm in diameter and 5 cm in length to which a F25 antibody had been attached after activation with CNBr. F25 is a known antibody for FVIII. After application, the column was washed for 3 CV with a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 0.02% TweenδO, 150 mM NaCI, and 1 M glycerol, which had been adjusted to pH 7.35 at a flow of 0.6 ml/min. Thereafter it was washed for 3 CV with a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 0.02% TweenδO, and 650 mM NaCI, which had been adjusted to pH 7.35 at a flow of 0.6 ml/min. Finally, the compound was eluted within 6 CVwith a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 0.02% TweenδO, 2.5 M NaCI in a 50%v/v ethylene glycol/water solution, which had been adjusted to pH 7.35 at a flow of 0.1 ml/min. The fractions of both runs containing the desired product were pooled. They were placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. They were subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 14 min. The remaining solution was subjected to a size exclusion chromatography on a Superose 6 material with a bed size of 10 mm in diameter and 30 cm in length with a flow of 0.50 ml/min utilizing a buffer consisting of 10 mM Histidine, 1.7 mM CaCI ₂ , 0.01% TweenδO, 0.3 M NaCI, 8.8 mM sucrose which had been adjusted to pH 7. The fractions containing the desired compound in a suitable purity were pooled and place in an Amicon ultracentrifugation device with a cut off of 10 kDa. They were subjected to an ultracentrifugation at 4000 rpm at 9 ⁰ C for 12 min. Using a molar absorbance of 14.46, the yield was found to be 0.0176 mg of a conjugate of AP3 scFv LC-HC C39S S248C to FVIII. The analyses by SDS- PAGE gel under non-reduced conditions were in accordance with the expectation. From the form of the peaks in the chromatograpies it was concluded that the material contained a lot of aggregated protein.

EXAMPLE 15

Conjugation of Abciximab to FVIII at the O-glycan

residue)

A buffer consisting of 20 mM histidine, 10 mM CaCI ₂ , 150 mM NaCI, 0.02% TweenδO and 1 M glycerol which had been adjusted to pH 7.35 (2.5 ml) was added to a solution of of B-domain deleted FVIII which has a residual B-domain sequence of SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO 4) at the C-terminus of the heavy chain (5.7 mg/ml, 1 mg, 5.6 nmol) in a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 150 mM NaCI, 0.02% TweenδO and 1 M glycerol which had been adjusted to pH 7.35. A solution of the conjugate of of Λ/-((3-(ω-(4-formylbenzoylamino)10 kDa PEGyl)propionylamino)acetyl)-O ² -[5']cytidylyl-ξ-neuraminic acid to one N-terminus of Abxicimab (Intermediate example 4, EXAMPLE 13, 2.3 mg, 39.5 nmol) in a buffer consisting of 25 mM TRIS, 150 mM NaCI, which had been adjusted to pH 8.0 (0.323 ml) was added. The solution was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. It was subjected to a ultracentrifugation at 4000 rpm at 10 ⁰ C for 20 min. Buffer consisting of 20 mM histidine, 10 mM CaCI ₂ , 150 mM NaCI, 0.02% TweenδO and 1 M glycerol which had been adjusted to pH 7.35 (2 ml) was added. The solution was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 30 min, leaving a solution with a volume of 1.4 ml. A solution of sialidase of A. Urifaciens (0.4 mg/ml, 242 U/mg, 0.0066 ml) and a solution of ST3Gal-I (2.5 mg/ml, 0.042 ml) were added subsequently. The reaction mixture was gently shaken at 32 ⁰ C for 15 min. After that the reaction mixture was left standing at 32 ⁰ C for 20.5 h. The reaction mixture was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. It was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 15 min. The remaining solution of 0.300 ml was subjected to a size exclusion chromatography, using Superose 6 material with a bed size of 010 mm x 300 mm at a flow of 0.5 ml/min and using a buffer consisting of 10 mM Histidine, 1.7 mM CaCI ₂ , 0.01% TweenδO, 0.3 M NaCI, 8.8 mM sucrose which had been adjusted to pH 7 as eluent. The fractions, containing the desired product were pooled and placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. Buffer, consisting of 20 mM histidine, 10 mM CaCI2, 10% glycerol, 0.02% TweenδO, 500 mM NaCI which had been adjusted to pH 6.07 (2.5 ml) was added. The solution was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 15 min. Buffer, consisting of 20 mM histidine, 10 mM CaCI2, 10% glycerol, 0.02% TweenδO, 500 mM NaCI which had been adjusted to pH 6.07 (1.5 ml) was added. The solution was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 15 min. The remaining solution of 0.220 ml was placed in a plactic reactor. A solution of commercially available CMP NeuNAc (1.73 mg, 2800 nmol) in buffer consisting of 20 mM histidine, 10 mM CaCI2, 10% glycerol, 0.02% TweenδO, 500 mM NaCI which had been adjusted to pH 6.07 (0.173 ml) was added. The reaction mixture was gently shaken at 300 rpm at 32 ⁰ C for 1 h. It was subjected to a size exclusion chromatography using using Superose 6 material with a bed size of 010 mm x 300 mm at a flow of 0.5 ml/min and using a buffer consisting of 10 mM Histidine, 1.7 mM CaCI ₂ , 0.01% TweenδO, 0.3 M NaCI, 8.8 mM sucrose which had been adjusted to pH 7 as eluent. The fractions, containing the desired product were pooled an placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. The pool was subjected to an ultracentrifugation at 4000 rpm at 10 ⁰ C for 5 min to give a solution of 0.275 ml of 0.0358 mg of a conjugation product of Abciximab to FVIII at the O-glycan. For quantification a molar absorbance of 13.15 was used on an Nanodrop ® spectrofphotometer. The SDS-PAGE analysis was in accordance with the expectation for a SDS-PAGE of a conjugation product of Abciximab to FVIII at the O-glycan.

EXAMPLE 16

Conjugation of AP3-FAB fragment to BDD-FVIII

The product of the reaction of intermediate example 5 was placed in an Amicon ultracentrifugation device with a cut off of 10 kDa. A buffer consisting of 20 mM histidine, 10 mM CaCI2, 10% glycerol, 0.02% Tween80, 500 mM NaCI at pH 6.07 (4 ml) was added. The solution was subjected to an ultracentrifugation at 4000 rpm, at 20 ⁰ C for 12 min. A solution of B-domain deleted FVIII which has a residual B-domain sequence of SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO 4) at the C-terminus of the heavy chain (0.5 mg, 2.8 nmol) in a buffer consisting of 20 mM imidazole, 10 mM CaCI ₂ , 150 mM NaCI, 0.02% TweenδO and 1 M glycerol, which had been adjusted to pH 7.35 (0.088 ml) was added to the remaining 0.600 ml. The solution was subjected to an ultracentrifugation at 4000 rpm at 20 ⁰ C for 30 min. The remaining 0.125 ml were placed in an Eppendorf vial. A solution of sialidase of A. Urifaciens with His6 TAG (0.4 mg/ml, 0.0033 ml) and a solution of ST3Gal-I (2.5 mg/ml, 0.021 ml) were added subsequently. The reaction mixture was gently shaken at 300 rpm at 32 ⁰ C. After 20 min, the shaking was stopped, and the reaction mixture was left at 32 ⁰ C for 19 h. It was filtered and subjected to a size exclusion chromatography using Superose 6 material with a bed size of 010 mm x 300 mm, using a buffer consisting of 10 mM Histidine, 1.7 mM CaCI ₂ , 0.01% TweenδO, 0.3 M NaCI, 8.8 mM sucrose which had been adjusted to pH 7 as eluent at a flow of 0.500 ml/min. The fractions containing the desired compound - as judged by SDS-PAGE - were pooled. The pool was transferred into an Amicon ultracentrifugation device with a cut off of 10 kDa. It was subjected to an ultracentrifugation at 4000 rpm at 20 ⁰ C. The concentration of the desired product in the remaining 0.270 ml was determined to be 0.29 mg/ml on a Nanodrop apparatus, using a molar absorption of 13.61. The results of SDS-PAGE under both reducing and non-reducing conditions of the isolated product were in accordance with the presence of a conjugate of the FAB-fragment of AP3 with FVIII.

EXAMPLE 17

Coupling of N8-(O)-PEG10kD-CHO to AP3-ONH2 to obtain AP3-N8 conjugate, compound MZl

Intermediate example A: Introduction of a hydroxylamine handle on the N-glycans of AP3 antibody

1st step: Galactosidation of AP3 antibody N-αlvcans: The N-glycans of AP3 FL mlgG are mostly of the complex, biantennary type. The majority of the biantennary N-glycans carry two N-acetyl glucosamine moieties, or one N-acetyl glucosamine moiety and one galactose moiety as the ultimate monosaccharide moiety (GOF, GIF). The light chain has a biantennary glycan with one sialic acid and one galactose moiety as the ultimate monosaccharides. The GOF and GIF glycans were converted to the G2F form by (bovine) βl,4- galactosyltransferase catalyzed transfer of galactose from galactose -UDP. Briefly, to the AP3 FL mlgG antibody in solution in 5OmM MES buffer pH6.4 (3mg, 800μl) was added 5OmM MES buffer pH6.4 (500μl), galactose-UDP (500μl of a 12.2mg/ml solution in 1OmM phosphate buffer pH7.4 containing 14OmM NaCI, 3mM KCI) and manganese chloride (lOOμl of a 12.6mg/ml solution in water). The reaction was started by addition of βl,4-galactosyltransferase (lOOμl of a lOU/ml enzyme solution in 10OmM HEPES pH7.5 buffer). The reaction mixture was incubated for overnight at 25°C.

2nd step: Introduction of a hydroxylamine handle on the N-glycans of AP3 antibody

To the galactosylated antibody obtained in the first step (3mg, 2ml) was added GSC-ONH2 (5.8mg in 190μl 1OmM phosphate buffer pH7.4 containing 14OmM NaCI, 3mM KCI). The reaction was started by addition of ST3Gal III (80μl, 0.4mg, 48OmU). The reaction mixture was incubated at 32°C for 21h.

GSC-ONH2: 5'-(2-(12-((aminoxymethylcarbonyl)amino)-4-7-10- trioxadodecanoyl)-aminoethanoyl)-neuraminic acid cytidine monophosphate

Any unmodified galactose moiety was subsequently capped by adding NAN-CMP in solution in PBS buffer (4.5mg, lOOμl). The reaction mixture was incubated for Ih at 25°C.

The reaction mixture was then filtered (0.45μ filter, Gelman GHP) and buffer shifted to 2OmM phosphate buffer pH7.2 containing 15OmM NaCI (by ultra filtration, Millipore Centrifugal Filter Units Amicon Ultra, 0.5ml device, 1OkD cut off).

The purification was run on a HiTrap Protein A HP column (ImI) using an Akta

Purifier 10 system (GE Healthcare). The loading and washing buffer was 2OmM phosphate buffer pH7.2 containing 15OmM NaCI, the elution buffer was 1OmM formic acid adjusted to pH3.5 with 1OM sodium hydroxide. The flow was 0.5ml/min, the fraction volume 0.5ml (fractionation in a deep well microtiter plate). Prior to fractionation, 0.1M Tris buffer pH9 (15μl) was added in the microtiter plate wells to avoid keeping the product at acidic pH. Relevant fractions were pooled, upconcentrated and buffer shifted to buffer A (by ultra filtration, Millipore Centrifugal Filter Units Amicon Ultra, 0.5ml device, 1OkD cut off), giving the product ("AP3-ONH2") with an overall protein recovery of 50% (1.5mg, 0.91mg/ml).

In order to find out if the hydroxylamine moiety had been successfully introduced, an aliquot of the product was pegylated, using PEG20kD-CHO (60 molar equivalents of PEG20kD-CHO in solution in buffer A). An SDS polyacrylamide gel electrophoresis analysis using NuPage 4-12% Bis-Tris gel (Invitrogen) under reducing conditions (200V, 40min) was done. The gel was coomassie blue stained using SimplyBlue SafeStain (Invitrogen). Standard proteins were from Invitrogen (Novex Sharp Unstained Stds (3.5-26OkD). The gel showed the disappearance of the light chain (the light chain of the unpegylated, N-glycan modified AP3 appearing at about 32kD), and only a faint band corresponding to the remaining heavy chain at about 55kD) was present. Four new bands had appeared at about 70, 100, 130, 17OkD, assumed to correspond to respectively pegylated LC glycan, monopegylated Fc glycans, di- pegylated Fc glycans and an undefined product.

Thus, hydroxylamine moieties have been introduced on AP3. Intermediate example B: Introduction of an aldehyde handle on the N8 O-glycans:

1st step: desialylation of N8 and ST3Gall catalyzed transfer of GSC-PEGlOkD- CHO on O-αlvcans f looti :

GSC-PEGlOkD-CHO: /V-((3-(ω-(4-formylbenzoylamino)10 kDa PE- Gyl)propionylamino)acetyl)-0 ² -[5']cytidylyl-ξ-neuraminic acid.

To a solution of N8 in 2OmM imidazole buffer pH7.3, 1OmM CaCI2, 0.02% Tween 80, IM glycerol, 0.5M NaCI (263μl, 1.5mg) was added a solution of sialidase (sialidase from Arthrobacter ureafaciens) (7μl, 68OmU), followed by addition of a solution of GSC-PEGlOkD-CHO in PBS buffer (68μl, 1.85mg) and a solution of His-ST3Gal I in 5OmM Tris, 10OmM NaCI, pH8 (108μl, 0.27mg). The reaction mixture was incubated at 23°C for 24h.

After dilution in 2OmM imidazole buffer pH7.3, 1OmM CaCI2, 0.02% Tween 80, IM glycerol to lower the ion concentration, the mixture was purified by anion exchange (MonoQ 5/50GL column, GE Healthcare), using an Akta purifier 10 system (GE Healthcare) : The mixture was loaded on the MonoQ 5/50 GL column equilibrated with 20 mM Imidazol pH7.3, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol. The elution buffer B was 20 mM Imidazol pH7.3, 10 mM CaCI ₂ , 0.02% Tween 80, 1.5M NaCI, 1 M glycerol. The elution program was: 0 to 20%B over 5CV, 20%B over lOCV, 100%B over lOCV, with a flow of 0.5ml/min. The product was eluted with 100% elution buffer. The relevant fractions were pooled. Yield 1.2 mg, 80% protein recovery.

2nd step: Capping :

To a solution of the product obtained in the first step was upconcentrated, (1.13mg protein, 400μl), a solution of NAN-CMP in PBS buffer (40μl, 1.05mg) was added. The reaction was started by addition of ST3Gal III (llOμl, llOμg, 0.13U). The reaction mixture was incubated at 32°C for Ih.

The reaction mixture was then purified on MonoQ, using a slightly modified elution program (compared to purification method iin stepl): The elution program was: 0 to 13%B over 5CV, 13%B over lOCV, 100%B over lOCV, with a flow of 0.5ml/min. The product ("N8-(O)-PEG10kD-CHO" (NB: this product is a mixture of des-O-glycan and (O)-PEG-CHO-N8) was eluted with 100% elution buffer. The relevant fractions were pooled. Yield 0.96mg, 80% protein recovery, ie overall protein recovery from N8: 64%.

An SDS polyacrylamide gel electrophoresis analysis using NuPage 7% Tris acetate gel (Invitrogen) under reducing conditions (150V, 70min) was run. The gel was coomassie blue stained using SimplyBlue SafeStain (Invitrogen).

Standard proteins were from Invitrogen (HiMark Unstained HMW). The pattern obtained was as expected : 3 bands of MW about 84kD, 93kD and 12OkD, which could correspond to respectively the light chain, the heavy chain, and the pegylated heavy chain. The bands at 93 and 12OkD have about the same intensity: this was expected, since only about 50% of N8 carries an O-glycan on its B domain. Thus, the aldehyde handle has been successfully introduced on N8.

Coupling of N8-(O)-PEG10kD-CHO to AP3-ONH2 N8-(O)-PEG10kD-CHO prepared according to intermediate example B was buffer shifted to 5OmM imidazol buffer pH6.2 containing 1OmM CaCI2, 0.02% Tween 80, 0.5M NaCI and upconcentrated by ultrafiltration (Millipore Centrifugal Filter Units Amicon Ultra, 0.5ml device, 5OkD cut off) to a concentration of about 16mg/ml. Likewise, AP3-ONH2 prepared according to intermediate example A was buffer shifted to 5OmM imidazol buffer pH6.2 containing 1OmM CaCI2, 0.02% Tween 80, 0.5M NaCI and upconcentrated by ultrafiltration (Millipore Centrifugal Filter Units Amicon Ultra, 0.5ml device, 1OkD cut off) to a concentration of about 34.7mg/ml.

To the upconcentrated AP3-ONH2 solution above (45μl, 1.56mg) was added the upconcentrated solution of N8-(O)-PEG10kD-CHO (21.5μl, 244μg), followed by the addition of an aqueous solution of aniline (5μl, 180μg). The mixture was incubated at 25°C overnight. The unreacted hydroxylamine moieties were quenched by addition of acetone (1.8μl, 1.4mg, about 600 molar equivalents). The reaction mixture was incubated 30min at 25°C.

The reaction mixture was diluted to 3ml with 2OmM imidazole buffer pH7.3, 1OmM CaCI2, 0.02% Tween 80, IM glycerol, 25mM NaCI, and filtered (0.45μ Gelman GHP filter), and purified by ion exchange on MonoQ column 5/50 GL(GE Healthcare). The loading and washout buffer (buffer A) was 2OmM imidazole buffer pH7.3, 1OmM CaCI2, 0.02% Tween 80, IM glycerol, 25mM NaCI, the elution buffer (buffer B) was 2OmM imidazole buffer pH7.3, 1OmM CaCI2, 0.02% Tween 80, IM glycerol, IM NaCI. The elution program consisted in 4 steps: 0 to 20%B over 4CV, 20%B over lOCV, 20 to 100%B over 16CV, 100% B over 5CV. The flow was 0.5ml/min, the temperature was 15C. ImI fractions were recovered in the first two steps, 0.5ml fractions in the last two steps.

AP3-ONH2 eluted first at roughly 20%B, the remaining material eluted between 30 and 65%B as one major peak and several minor, not fully resolved peaks. After upconcentration (by ultrafiltration, 5OkD cut off), the fractions were analyzed by gel electrophoresis using NuPage 7% Tris acetate gel (Invitrogen) (150V, 70min). The gel was coomassie blue stained using SimplyBlue SafeStain (Invitrogen). Standard proteins were from Invitrogen (HiMark Unstained HMW). The main peak from the material eluting between 30 and 65%B (peak top elutes at 52%B) corresponds to the conjugated material. The unreduced conjugated material shows three bands on the gel at MW 84kD, 285kD, and >500kD (very faint band). They are assumed to correspond respectively to the (N8) light chain of N8-(O)-PEG10kD-AP3, the (N8) heavy chain of N8-(O)-PEG10kD-AP3 (for the control samples of unreduced N8-(O)- PEGlOkD-CHO: the heavy chain appears at about HOkD, and the control samples of the unreduced AP3-ONH2 appears at about 176kD), and to an unidentified compound.

The reduced conjugated material shows three bands on the gel, at MW 84kD, 145kD and 17OkD, assumed to correspond to respectively (N8) light chain of N8-

(O)-PEG10kD-AP3 and N8 pegylated heavy chain conjugated to the light chain of AP3, and N8 pegylated heavy chain conjugated to the heavy chain of AP3.

The amount of recovered conjugated material was 129μg, 53% yield from N8-

PEGlOkD-CHO.

EXAMPLE 18

Disodium (5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-

3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-oxido-phosphor yl]oxy-4- hydroxy-5-[[2-[3-(2-pyridyldisulfanyl)propanoylamino]acetyl] amino]-6- [(2R)-l,2,3-trihydroxypropyl]tetrahydropyran-2-carboxylate, compound

IM

S,S'-2,2'-Dithiodipyridine (1.59 mmol, 350 mg) and 3-mercaptopropionic acid (0.477 mmol, 50.1 mg, 41.5 microliter) were mixed in tetrahydrofuran (2 ml). The mixture was stirred for 45 minutes. /V-Hydroxysuccinimide (0.477 mmol, 54.9 mg) and /V,/V'-diisopropylcarbodimide (0.954 mmol, 148 microliter) were added to the mixture which was subsequently stirred for 3 h.

Disodium (5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-3 ,4- dihydroxy-tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy- 4-hydroxy-5-[2- aminoacetylamino]-6-[(2R)-l,2,3-trihydroxypropyl]tetrahydrop yran-2- carboxylate (0.159 mmol, 100 mg) was dissolved in water (500 microliter). pH was adjusted to 8.5 by addition of 500 mM aq. Na ₂ HPO ₄ / NaH ₂ PO ₄ (aq., sat.). The final volume was adjusted to 2 ml by addition of water.

The two solutions (NHS ester in tetrahydrofuran and nucleotide in water) were mixed. The resulting mixture was gently agitated for 3 h. The volume was adjusted to 20 ml by addition of water. The crude compound was purified using reversed phase HPLC (0-30 vol% acetonitrile in water, 10 % 500 mM aq.

NH ₄ HCO ₃ , Ci8 column). The selected fractions were pooled. Sodium hydroxide (1

M, 156 microliter) was added. The resulting mixture was lyophilised.

Purity and identity were determined by analytical HPLC and LCMS ([M H- H] ⁺ :

827.4)

EXAMPLE 19

2-[2-[4-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]piperazi n-l-yl]-2- oxo-ethoxy] acetic acid, compound 2M

6-hydroxy-9-[2-(piperazine-l-carbonyl)phenyl]xanthen-3-one hydrochloride

(5.72 mmol, 2.5 g) (prepared as described in J. Am. Chem. Soc, 2007, 129, 8400-8401) was suspended in a mixture of sat. aq. sodium bicarbonate (50 ml) and tetrahydrofuran (50 ml). The mixture was stirred for 10 minutes. Diglycolic anhydride (8.58 mmol, 1.0 g) was added. After 3 h, additional diglycolic anhydride (500 mg) was added. The mixture was stirred for 20 h. The mixture was acidified with fuming hydrochloric acid to pH 1. Dichloromethane (100 ml) and hydrochloric acid (1 M, 100 ml) were added. Brine (100 ml) and solid sodium chloride were added. A solid was observed in between the two phases. The phases were separated. The aq. phase was extracted with DCM (3x100 ml). The combined org. phases were dried (Na ₂ SO ₄ ), filtered, and concentrated in vacuo. The solid formed in between the phases was isolated by filtration. The solid was suspended in hydrochloric acid (1 M) on a filter. The solids were continuously extracted with hydrochloric acid (1 M). The filtered extract was combined with the aqueous extracts and the resulting solution was purified using reversed phase HPLC (Ci ₈ column, 10-35 vol% aceonitrile in water, 0.1 % trifluoroacetic acid). The selected fractions were pooled and lyophilised. LCMS: 517.2776 [M-I-H] ⁺

EXAMPLE 20

2-[[2-[2-[4-[2-(3-hydroxy-6-oxo-xanthen-9-yl) benzoyl] piperazin-1-yl]-

2-oxo-ethoxy]acetyl]-[2-[2-[2-[3-(2- pyridyldisulfanyl)propanoylamino]ethoxy]ethoxy]ethyl]amino]a cetic acid, compound 3M

Trityl polystyrene resin (200 mg, 1.5 mmol/g, Pepchem) was placed in a fritted syringe. Thionyl chloride / dichloromethane (1 : 1, 5 ml) was added. The mixture was shaken for Vi h. The resin was washed with dichloromethane. A mixture of bromoacetic acid (0.9 mmol, 125 mg) and /V,/V'-diisopropyl- ethylamine (0.9 mmol, 156 microliter) in dichloromethane (3 ml) was added. After 5 minutes, more /V,/V'-diisopropyl-ethylamine (0.9 mmol, 156 microliter) was added. The mixture was shaken for Vi h. The resin was drained and an identical mixture of bromoacetic acid and /V,/V'-diisopropyl-ethylamine in dichloromethane was added to the resin. The mixture was shaken for IVi h. The resin was washed with dichloromethane (5x), methanol, and /V- methylpyrrolidone. During the methanol-wash vigorous shaking was done in order to suppress any formation of lumps.

A solution of l,8-diamino-3,6-dioxaoctane (7.5 mmol, 1100 microliter) in /V- methylpyrrolidone (3.75 ml) was added. The mixture was shaken for 3 h. The resin was washed. A solution of 2-acetyldimedone (3.0 mmol, 546 mg) in /V- methylpyrrolidone (3 ml) was added to the resin. The mixture was shaken over night. The resin was washed with /V-methylpyrrolidone. A mixture of 2-[2-[4-[2- (3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]piperazin-l-yl]-2-oxo- ethoxy]acetic acid, compound 2M (155 mg, 0.3 mmol), l-hydroxy-7-azabenzotriazole (0.9 mmol) and /V,/V'-diisopropylcarbodiimide (0.9 mmol, 140 microliter) in /V- methylpyrrolidone (1.8 ml) was added to the resin. The mixture was shaken over night. The resin was washed with /V-methylpyrrolidone (3-4 times). The resin was shaken in a mixture of hydrazine and /V-methylpyrrolidone (5 vol% hydrazine mono hydrate) for 2x5 minutes. The syringe was drained. The resin was washed with /V-methylpyrrolidone.

A mixture of S,S'-2,2'-dithiodipyridine (7.5 mmol, 1650 mg) and 3- mercaptopropionic acid (3.0 mmol, 261 microliter) in /V-methylpyrrolidone (3 ml) was incubated for Vi h. /V,/V'-diisopropylcarbodiimide (1.5 mmol, 233 microliter) was added. The resulting mixture was added to the resin. The mixture was shaken for IVi h. The resin was washed with /V-methylpyrrolidone and dichloromethane.

The resin was treated with a mixture of trifluoroacetic acid and water (95 : 5) for 15 min. The filtrate was triturated with diethyl ether/n-heptane. The crude compound was dissolved in acetic acid (2 ¹ /_ ml). Water (15 ml) and a little acetonitrile were added . The mixture was filtered and purified using reversed phase HPLC (Ci ₈ column, 20-50 % acetonitrile in water, 0.1 % TFA). LCMS : 902.2807 [M-I-H] ⁺

EXAMPLE 21

Diammonium (4S,5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo- pyrimidin-l-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-o xido- phosphoryl]oxy-4-hydroxy-5-[[2-[[2-[[2-[2-[4-[2-(3-hydroxy-6 -oxo- xanthen-9-yl) benzoyl] piperazin-l-yl]-2-oxo-ethoxy]acetyl]-[2-[2-[2-[3-

(2- pyridyldisul- fanyl)propanoylamino]ethoxy]ethoxy]ethyl]amino]acetyl]amino] acetyl] amino]-6-[(lR _/ 2R)-l _/ 2 _/ 3-trihydroxypropyl]tetrahydropyran-2- car boxy late, compound 4M

2-[[2-[2-[4-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]pipe razin-l-yl]-2-oxo- ethoxy]acetyl]-[2-[2-[2-[3-(2- pyridyldisulfanyl)propanoylamino]ethoxy]ethoxy]ethyl]amino]a cetic acid, compound 3M (0.3 mmol, 270 mg) was dissolved in tetrahydrofuran (2 ml). /V- Hydroxysuccinimide (0.9 mmol, 104 mg) and /V,/V'-diisopropylcarbodiimide (1.5 mmol, 233 microliter) were added to the mixture which was subsequently stirred for IV ₂ h.

Disodium (5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-3 ,4- dihydroxy-tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy- 4-hydroxy-5-[2- aminoacetylamino]-6-[(2R)-l,2,3-trihydroxypropyl]tetrahydrop yran-2- carboxylate (0.45 mmol, 303 mg) was dissolved in water (500 microliter). pH was adjusted to 8.5 by addition of 500 mM aq. Na ₂ HPO ₄ / NaH ₂ PO ₄ (aq., sat.). The final volume was adjusted to 2 ml by addition of water.

The two solutions were mixed. The resulting pH was 7.5-8.0. The resulting mixture was stirred for 20 h. The mixture was diluted to 15 ml with water, filtered, and purified using reversed phase HPLC (0-35 % acetonitrile in water, 10 % 500 mM NH ₄ HCO ₃ aq.). The selected fractions were pooled and lyophilised. LCMS: 1513.9 [M-I-H] ⁺ , 757.5 [M-I-H] ²⁺

EXAMPLE 22

Oregon Green 488 labelling of F8-500 AP3-LC-HC scFv -Δa3 via rø-glycan, compound 5M

VIII fusion protein

Fusion protein (F8-500 AP3-LC-HC scFv -Δa3 (SEQ ID NO 40), 11 microgram) in aqueous buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 500 mM NaCI, pH 7.3; 1.2 ml) was mixed with from recombinant sialidase from Clostridium perfringens (0.1 U). The final volume was 1.2 ml. The mixture was left at 25 degrees Celsius for 30 minutes. The mixture was diluted to 10 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, pH 7.3). The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program : 10 CV eq 0 % Buffer B, 2 CV wash out unbound sample (0 % Buffer B), 10 CV 0 - 100 % (Buffer B), 5 CV 100 % Buffer B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

The selected fractions were pooled and mixed with disodium (2R,5R,6R)-2- [[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-3,4-dihydro xy- tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy-5-[6-[[4-c arboxy-3-(2,7- difluoro-3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]amino]hexanoyl amino]-4- hydroxy-6-[(2R)-l,2,3-trihydroxypropyl]tetrahydropyran-2-car boxylate and sialyl transferase (rat rec. ST3GalIII). The final concentrations were: Factor VIII: 0.026 micromolar, fluorescence labelling reagent: 39 micromolar, ST3GalIII: 177 U/l. Final volume: 2.1 ml. The mixture was incubated at 32 degrees Celsius for 4 hours. Disodium (2R,5R,6R)-5-acetamido-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo- pyrimidin-l-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-o xido- phosphoryl]oxy-4-hydroxy-6-[(2R)-l,2,3-trihydroxypropyl]tetr ahydropyran-2- carboxylate was added to the mixture (final concentration : 54 micromolar). The mixture was incubated at 32 degrees Celsius for 3 hours. The mixture was diluted to 25 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, no NaCI, pH 7.3). The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program : 10 CV eq , 5 CV wash out unbound sample, 10 CV 0 - 25 % buffer B, 10 CV 25-28 % (buffer B), 25 CV 28 - 100 % buffer B, and 10 CV 100 % buffer B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

SDS-PAGE analysis (reduced, 7 % NuPAGE Tris acetate, 1.0 mm, Invitrogen, 150 V, 70 min) confirmed the incorporation of the fluorophor in the protein. EXAMPLE 23

Λ/,O-glycan-asialo-BDD Factor VIII, compound 6M

BDD factor VIII (1 mg) in aqueous buffer (20 mM Imidazol, 10 mM CaCI2, 0.02% Tween 80, 1 M glycerol, 500 mM NaCI, pH 7.3) was mixed with recombinant sialidase from Arthrobacter Ureafaciens (Biotechnol. Appl. Biochem., 2005, 41 , 225-231). The final concentrations were: factor VIII : 5.7 mg/ml and sialidase: 1.5 U/ml. The mixture was left at 25 degrees Celsius for 30 minutes. The mixture was diluted to 20 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 25 mM NaCI, pH 7.3). The mixture was loaded onto a pre- conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program : 0.1 CV eq 5 % Buffer B, 2 CV wash out unbound sample (5 % Buffer B), 10 CV 5 - 100 % (Buffer B), 5 CV 100 % Buffer B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

EXAMPLE 24

3-(2-pyridyldisulfanyl)propanoylamino handle conjugated to BDD FVIII via O-glycan, compound 7M

Factor VIII

Asialo BDD factor VIII, compound 6 (3.95 mg) in buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 500 mM NaCI, pH 7.3) was mixed with disodium (5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-3 ,4- dihydroxy-tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy- 4-hydroxy-5-[[2- [3-(2-pyridyldisulfanyl) propanoylamino]acetyl]amino]-6-[(2R)- 1,2,3- trihydroxypropyl]tetrahydropyran-2-carboxylate and sialyl transferase (ST3GalI). The final concentrations were: factor VIII: 1.16 mg/ml, sialyltranferase: 0.22 mg/ml, nucleotide: 250 micromolar. Final volume: 3.4 ml. The mixture was left at 32 degrees Celsius for 20 hours. Disodium (2R,5R,6R)-5-acetamido-2- [[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-3,4-dihydro xy- tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy-4-hydroxy- 6-[(2R)- 1,2,3- trihydroxypropyl]tetrahydropyran-2-carboxylate was added to the mixture (final concentration : 54 micromolar). The mixture was incubated at 32 degrees Celsius for 30 minutes. The mixture was diluted to 25 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 25 mM NaCI, pH 7.3).

The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program: 1 CV eq 0 % Buffer B, 2 CV wash out unbound sample (0 % Buffer B), 10 CV 0 - 20 % Buffer B, 10 CV 20 % Buffer B, 10 CV 100 % Buffer B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

The isolated protein was mixed with sialyl tranferase (ST3GalIII) and disodium (2R,5R,6R)-5-acetamido-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-py rimidin-l-yl)- 3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl] oxy-4-hydroxy- 6-[(2R)-l,2,3-trihydroxypropyl]tetrahydropyran-2-carboxylate . The final concen- trations were: factor VIII : 1.12 mg/ml, sialyltranferase: 0.13 mg/ml, nucleotide: 54 micromolar. Final volume: 3.2 ml. The mixture was left at 32 degrees Celsius for 1 hour.

The mixture was diluted to 40 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 25 mM NaCI, pH 7.3). The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program : 5 CV wash out unbound sample (0 % Buffer B), 5 CV 0 - 20 % Buffer B, 15 CV 20 % Buffer B, 10 CV 100 % Buffer B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3. Incubation of the product with PEG 30 kDa thiol prior to SDS-PAGE analysis resulted in presence of a band with MW higher that FVIII under non-reduced conditions but not under reduced conditions.

EXAMPLE 25

Fluorescent 2-pyridyldisulfanyl handle conjugated to AP3 full length antibody via JV-glycan, compound 8M

AP3 mlgGl wt HC LC C39S full length antibody (1.5 mg) in aqueous buffer (50 % 100 mM HEPES. HCI, 150 mM NaCI, pH 7.5, 33 % 100 mM Glycine. HCI, pH 3.5, 17 % 20 mM HEPES. HCI, 150 mM NaCI, 0,01 % TweenδO, pH 7.5 - final pH 7.2) was mixed with diammonium (4S,5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo- pyrimidin-l-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-o xido- phosphoryl]oxy-4-hydroxy-5-[[2-[[2-[[2-[2-[4-[2-(3-hydroxy-6 -oxo-xanthen-9- yl)benzoyl]piperazin-l-yl]-2-oxo-ethoxy]acetyl]-[2-[2-[2-[3- (2- pyridyldisulfanyl)propanoylamino]ethoxy] ethoxy] ethyl]amino]acetyl]amino]acetyl]amino]-6-[(lR,2R)-l,2,3-trih ydroxypropyl] tet- rahydropyran-2-carboxylate, compound 4M and sialyl tranferase (ST3GalIII). The final concentrations were: Antibody: 1.4 mg/ml, sialyltranferase: 0.12 mg/ml, nucleotide: 190 micromolar. Final volume: 650 microliter. The mixture was left at 32 degrees Celsius for 19 hours. The sample was transferred to a Millipore Amicon Ultra Centrifugal device, MWCO 10.000 Da. The protein was washed several times with buffer (Histidine, 1.5 mg/ml, CaCI ₂ , 0.25 mg/ml, Tween 80, 0.1 mg/ml, NaCI, 18 mg/ml, Sucrose 3 mg/ml, pH 7.0. The solution was concentrated to 100 microliter in said buffer. The absorbance at 280 nm and 500 nm were determined to be SDS-PAGE analysis (reduced and non-reduced, 7 % NuPAGE Tris acetate, 1.0 mm, Invitrogen, 150 V, 70 min) confirmed the incorporation of the fluorophor in antibody light chain.

EXAMPLE 26

Fluorescent 2-pyridyldisulfanyl handle conjugated to BDD FVIII via O- glycan, compound 9M

Asialo BDD factor VIII, compound 6M (1.86 mg) in buffer (20 mM Imidazol, 10 mM CaCI2, 0.02% Tween 80, 1 M glycerol, 500 mM NaCI, pH 7.3) was mixed with diammonium (4S,5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l- yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-oxido-phospho ryl]oxy-4- hydroxy-5-[[2-[[2-[[2-[2-[4-[2-(3-hydroxy-6-oxo-xanthen-9- yl)benzoyl]piperazin-l-yl]-2-oxo-ethoxy]acetyl]-[2-[2-[2-[3- (2- pyridyldisulfanyl)propanoylamino]ethoxy]ethoxy]ethyl] amino]acetyl]amino] ace- tyl]amino]-6-[(lR,2R)-l,2,3-trihydroxypropyl]tetrahydropyran -2-carboxylate, compound 4M and sialyl transferase (ST3GalI). The final concentrations were: factor VIII : 1.5 mg/ml, sialyltranferase: 0.3 mg/ml, nucleotide: 95 micromolar. Final volume: 1.25 ml. The mixture was left at 32 degrees Celsius for 15 hours. Disodium (2R,5R,6R)-5-acetamido-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo- pyrimidin-l-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxy-o xido- phosphoryl]oxy-4-hydroxy-6-[(2R)-l,2,3-trihydroxypropyl]tetr ahydropyran-2- carboxylate (final concentration : 2.5 mM) and ST3GalIII were added to the mixture. The mixture was incubated at 32 degrees Celsius for 30 minutes. The mixture was diluted to 25 ml with buffer (20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 150 mM NaCI, pH 7.3).

The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program: 1 CV eq 0 % Buffer B, 2 CV wash out unbound sample (0 % Buffer B), 10 CV 0 - 20 % Buffer B, 10 CV 20 % Buffer B, 10 CV 100 % Buffer B. Imidazol Buffer A: 20 mM Imida- zol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

SDS-PAGE analysis (reduced and non-reduced, 7 % NuPAGE Tris acetate, 1.0 mm, Invitrogen, 150 V, 70 min) confirmed the incorporation of the fluorophor in BDD FVIII heavy chain.

EXAMPLE 27

3-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy]ethoxy]p ropanoyl handle conjugated to AP3 full length antibody, random positions, compound IO M (random position)

AP3 mlgGl wt Hc Lc C39S full length antibody (1.3 mg) in buffer (50 % 100 mM

HEPES. HCI, 150 mM NaCI, pH 7.5, 33 % 100 mM Glycine. HCI, pH 3.5, 17 % 20 mM HEPES. HCI, 150 mM NaCI, 0,01 % TweenδO, pH 7.5 - final pH 7.2) was placed in a Millipore Amicon Ultra Centrifugal device, MWCO 10.000 Da. The pro- tein was washed several times with buffer (20 mM HEPES, pH 7.3). The solution was concentrated to 65 microliter in said buffer.

(2,5-dioxopyrrolidin-l-yl) 3-[2-[2-[2-(2-acetylsulfanylethoxy)ethoxy]ethoxy] ethoxy]propanoate (Pierce/Thermo Scientific, 2.2 mg, 5.2 micromol) was dissolved in buffer (20 mM HEPES, pH 7.3, 100 microliter). 4 microliter of this solution (209 nmol) was added to the protein solution. The resulting mixture was incubated for 1 hour. The solution was placed in a Millipore Amicon Ultra Centrifugal device, MWCO 10.000 Da. The protein was washed several times with buffer (20 mM HEPES, pH 7.3).

EXAMPLE 28

N -glycan-asialo-BDD Factor VIII, compound HM

BDD factor VIII (7 mg) in aqueous buffer (20 mM Imidazol, 10 mM CaCI2, 0.02% Tween 80, 1 M glycerol, 500 mM NaCI, pH 7.3) was mixed with recombinant sialidase from recombinant sialidase from Clostridium perfringens (4 U) . The final volume was 4 ml. The mixture was left at 25 degrees Celsius for 30 minutes. The mixture was diluted to 5 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 25 mM NaCI, pH 7.3). The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program : 5 CV eq 0 % Buffer B, 2 CV wash out unbound sample (0 % Buffer B), 25 CV 0 - 70 % (Buffer B), 10 CV 70-100 % (Buffer B), 5 CV 100 % Buffer B. Buffer A: Histidine (1.5 mg/ml), CaCI ₂ (0.25 mg/ml), Tween 80 (0.1 mg/ml), NaCI (2.9 mg/ml), and sucrose (3 mg/ml), pH 7.0. Buffer B: Histidine (1.5 mg/ml), CaCI2 (0.25 mg/ml), Tween 80 (0.1 mg/ml), NaCI (58 mg/ml), and sucrose (3 mg/ml), pH 7.0

EXAMPLE 29

3-(2-pyridyldisulfanyl)propanoylamino handle conjugated to fluorescence labelled BDD FVIII via /V-glycan, compound 12M

/V-glycan-asialo-BDD Factor VIII, compound HM (3 mg) in buffer (Histidine (1.5 mg/ml), CaCI2 (0.25 mg/ml), Tween 80 (0.1 mg/ml), NaCI (30 mg/ml), and sucrose (3 mg/ml), pH 7.0) was mixed with disodium (5R,6R)-2-[[(2R,3S,4R,5R)- 5-(4-amino-2-oxo-pyrimidin-l-yl)-3,4-dihydroxy-tetrahydrofur an-2-yl]methoxy- oxido-phosphoryl]oxy-4-hydroxy-5-[[2-[3-(2- pyridyldisulfanyl)propanoylamino]acetyl]amino]-6-[(2R)- 1,2,3- trihydroxypropyl]tetrahydropyran-2-carboxylate, compound IM, disodium (2R,5R,6R)-2-[[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl )-3,4-dihydroxy- tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy-5-[6-[[4-c arboxy-3-(2,7- difluoro-3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]amino]hexanoyl amino]-4- hydroxy-6-[(2R)-l,2,3-trihydroxypropyl]tetrahydropyran-2-car boxylate and sialyl transferase (rat rec. ST3GalIII). The final concentrations were: factor VIII : 0.85 mg/ml, sialyltranferase: 271 U/l, Oregon Green 488 nucleotide: 14 micro- molar, compound IM : 15 micromolar. Final volume: 3.5 ml. The mixture was left at 32 degrees Celsius for 18 hours. Disodium (2R,5R,6R)-5-acetamido-2- [[(2R,3S,4R,5R)-5-(4-amino-2-oxo-pyrimidin-l-yl)-3,4-dihydro xy- tetrahydrofuran-2-yl]methoxy-oxido-phosphoryl]oxy-4-hydroxy- 6-[(2R)- 1,2,3- trihydroxypropyl]tetrahydropyran-2-carboxylate was added to the mixture (final concentration : 54 micromolar). The mixture was incubated at 32 degrees Celsius for 30 minutes.

The mixture was diluted to 14 ml with buffer (Histidine (1.5 mg/ml), CaCI ₂ (0.25 mg/ml), Tween 80 (0.1 mg/ml), NaCI (2.9 mg/ml), and sucrose (3 mg/ml), pH 7.0). The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program: 5 CV eq, 2 wash out unbound sample, 25 CV 0 - 70 %, 10 CV 70-100 % buffer B, and 5 CV 100 % B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

SDS-PAGE analysis (reduced and non-reduced, 7 % NuPAGE Tris acetate, 1.0 mm, Invitrogen, 150 V, 70 min) confirmed the incorporation of the fluorophor in BDD FVIII heavy and light chains. Incubation of the product with PEG 30 kDa thiol prior to SDS-PAGE analysis resulted in presence of a fluorescent band with MW higher that FVIII under non-reduced conditions but not under reduced conditions.

EXAMPLE 30

Conjugate between 3-(2-pyridyldisulfanyl) propanoylamino handle conjugated to fluorescence labelled BDD FVIII via /V-glycan, compound 12M and AP3 scFv LC-HC, compound 13M

AP3 scFv in a 1 : 1 mixture of glycine buffer (100 mM, pH 3.5) and HEPES buffer (100 mM, pH 7.5) was mixed with 3-(2-pyridyldisulfanyl) propanoylamino handle conjugated to fluorescence labelled BDD FVIII via N-glycan, compound 12M in aqueous buffer (20 mM Imidazol, 10 mM CaCI2, 0.02% Tween 80, 1 M glycerol, 500 mM NaCI, pH 7.3). The final concentrations were: compound 12M : 0.25 mg/ml, ScFv: 2 micromolar, 0.06 mg/ml. Final volume: 2.1 ml. The mixture was left at 23 degrees Celsius for 3 hours. Solutions of Cysteine (1 mg/ml) in buffer (2.3 microliter) and Cystine (1 mg/ml) in buffer (3.5 microliter) were added. The mixture was left at 23 degrees Celsius for 15 minutes. The mixture was diluted to 40 ml with buffer (20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M glycerol, 25 mM NaCI, pH 7.3). The mixture was loaded onto a pre-conditioned (with buffer A) MonoQ 5/50 GL column (GE Healthcare) and eluted using the following program : 5 CV eq 0 % Buffer B, 2 CV wash out unbound sample (0 % Buffer B), 25 CV 0 - 70 % (Buffer B), 50 CV 70-100 % (Buffer B), 5 CV 100 % Buffer B. Imidazol Buffer A: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. Imidazol Buffer B: 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3.

The selected fractions were transferred to a Millipore Amicon Ultra Centrifugal device, MWCO 50.000 Da and the total volume was reduced to 0.5 ml. The mixture was loaded onto onto a pre-conditioned (with buffer) Superose 6 10/300 GL column (GE Healthcare) and eluted using the following buffer: Histidine (1.5 mg/ml), CaCI ₂ (0.25 mg/ml), Tween 80 (0.1 mg/ml), NaCI (18 mg/ml), and su- crose (3 mg/ml), pH 7.0.

SDS-PAGE analysis (non-reduced as well as thrombin digested/non-reduced, 7 % NuPAGE Tris acetate, 1.0 mm, Invitrogen, 150 V, 70 min) showed the presence of fluorescent bands with higher MW than the starting material. The protein was shown not bind to platelets which could be rationalised by the fact that Cys-34 is found in a CDR region, hence effecting binding affinity.

EXAMPLE 31

Conjugate between fluorescent 3-(2-pyridyldisulfanyl) handle conjugated AP3 mlgGl wt HC LC C39S full length antibody via JV-glycan, compound 8M and BDD-FVIII via handle on O-glycan, compound 7M

FVIII

AP3 mlgGl wt Hc Lc C39S full length antibody conjugated to a handle, e.g., compound 8M in buffer is mixed with a solution of tris(carboxyethyl)phosphine in buffer. The buffer is exchanged in order to remove excess trialkylphosphine after selective reduction of the disulfide bond. A solution of a bis-maleimide, e.g., bis- maleimide PEG 6000 from Rapp Polymere Gmbh, cat. No. : 11 6000-45, in buffer is added. The buffer is exchanged after completion of the reaction.

FVIII conjugated to a 3-(2-pyridyldisulfanyl)propanoylamino handle, e.g., compound 7M, in buffer is mixed with a solution of tris(carboxyethyl)phosphine in buffer. The buffer is exchanged in order to remove excess trialkylphosphine after selective reduction of the disulfide bond.

The two solutions of modified proteins (FVIII and AP3 Ab) are mixed. The formation of the desired conjugate is monitored by SDS-PAGE analysis. Purification is accomplished using a suitable type of chromatography, e.g., ion-exchange or anti-FVIII affinity chromatography. EXAMPLE 32

Conjugate between fluorescent 3-(2-pyridyldisulfanyl) handle conjugated AP3 mlgGl wt HC LC C39S full length antibody via JV-glycan, compound 8M and BDD-FVIII via handle on O-glycan, compound 7M

FVIII

AP3 mlgGl wt Hc Lc C39S full length antibody conjugated to a handle, e.g., compound 8M in buffer is mixed with a solution of a dithiol, e.g., 3,6-dioxa-l,8- octanedithiol, in buffer. The buffer is exchanged after completion of the reaction. A solution of 3-(2-pyridyldisulfanyl)propanoylamino handle conjugated to FVIII, e.g., compound 7M, is added. The formation of the desired conjugate is monitored by SDS-PAGE analysis. Purification is accomplished using a suitable type of chromatography, e.g., ion-exchange or anti-FVIII affinity chromatography.

EXAMPLE 33

Conjugate between fluorescent 3-(2-pyridyldisulfanyl) handle conjugated AP3 mlgGl wt HC LC C39S full length antibody via JV-glycan, compound 8M and Cys mutant of FVIII

Cys mutant

A surface accessible Cys mutant of FVIII is conjugated to AP3 antibody or similar (or fragment thereof) using the same methods as described in above. The Cys- mutant of FVIII is, if necessary, treated with reduction agent in order to remove substituents covalently bound to the mutated Cystein, thus resulting in the generation / ensuring the presence of a thiol. The formed protein is conjugated to a suitable reagent of the type described, e.g., Fluorescent 2-pyridyldisulfanyl handle conjugated to AP3 full length antibody via /V-glycan, compound 8M. The formation of the desired conjugate is monitored by SDS-PAGE analysis. Purification is accomplished using a suitable type of chromatography, e.g., ion-exchange or anti-FVIII affinity chromatography.

Example 34

Preparation of FVIII modified with Oregon Green 488 on the N-glycans and dPEG12-SH on the O-glycan

Step 1 : Preparation of PySS-dPEGi ₂ -GSC (compound Jl)

J1

Glycyl sialic acid cytosine 5'-monophosphate ester (GSC, dimethylamine salt, MW 673, 85% pure, 96 mg, 153 μmol) was dissolved in 100 mM TRIS buffer pH 8.4 (650 μl_) followed by acetonitrile (650μl_) and stirred to observe a two-phase system. SPDP-dPEGi ₂ -NHS-ester (Quanta Biodesign, prod no 10378, 1.2 eq, 183 μmol, 170 mg) was dissolved in 650 μl_ of THF, followed by 100 mM TRIS, pH 8.4, (650 μl_) mild white hazy precipitate was observed and this NHS-ester solution was added to the GSC solution. The solution was slightly hazy at pH 8.4. The solution was stirred at room temperature for 7 h and the resulting mixture was frozen and lyophilized to get the crude compound. The white powder obtained after lyophilisation was purified by preparative HPLC using neat water as equilibration buffer and a linear gradient of neat acetonitrile for elution. Fractions containing the desired product were pooled and lyophilized to yield 100 mg of the target compound, which was homogeneous by analytical HPLC and identified by mass spectrometry.

Step 2: Coupling of PySS-dPEGi ₂ -GSC to the O-glycan of wt BDD FVIII (compound 32) To wt BDD FVIII ("N8", 5.7 mg/ml; 5 mg, 875μl) was added sialidase A. urifa- ciens (0.44 mg/ml, 50 μl) and PySS-dPEGi ₂ -GSC (Jl), 1 mg/ml in buffer 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 200 mM NaCI, 1 M glycerol, pH 7.3, 100 ul was used) and His-ST3Gal-I (2.5 mg/ml, 375 μl). The reaction mixture was incubated at 32°C overnight for a period of 17 h. It was then diluted with a 25 ml buffer 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3 to lower the conductivity. The mixture was then loaded to a MonoQ 5/50 GL column equilibrated with 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. The product was eluted using a two-step-gradient with elution buffer 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3. FVIII-unrelated material was eluted with 20% elution buffer, while the product was eluted with 100% elution-buffer. Fractions containing the desired product (32) were pooled. Yield 4.1 mg, 1.17 mg/ml.

Step 3: Labelling with Oregon Green and capping of the N-glycans (J3)

The modified protein prepared according to Step 2 (32) was mixed with MBP- ST3Gal-III (1.2 U/ml , 300 μl) and Oregon Green 488-GSC (2 mg was dissolved in 800 μl buffer 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 200 mM NaCI, 1 M glycerol, pH 7.3; 200 μl, 0.5 mg, 20 eq. was used) and incubated in the dark at 32 C overnight. Subsequently, CMP-NAN (9 mg in 250 μl buffer 20 mM Imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 200 mM NaCI, 1 M glycerol, pH 7.3) was added to the reaction mixture and incubated for further 1 hour at 32°C. It was then diluted with 30 ml 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3 to lower the conductivity. Purification was carried out by AIEX as outlined in Step 2. Yield : 1.54 mg, 0.51 mg/ml.

Step 4: Reduction with TCEP

The modified protein prepared according to Step 3 (J3) was mixed with triscar- boxylethyl phosphine (TCEP, 700 eq., 1.69 mg; 6 mg TCEP was dissolved in buffer 1 ml, 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 200 mM NaCI, 1 M glycerol, pH 7.3, 282 μl was used) and incubated for 30 min at 5 C in the dark. It was then diluted with 30 ml 20 mM imidazol, 10 mM CaCI ₂ , 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3 to lower the conductivity. Purification was carried out by AIEX as outlined in Step 2. Yield : 610 μg, 610 μg/ml. The product was further purified by SEC (column : Superdex 200 10/300 GL) using buffer histidine (1.5 mg/ml), CaCI ₂ (0.25 mg/ml), Tween 80 (0.1 mg/ml), NaCI (18 mg/ml), sucrose (3 mg/ml) as mobile phase. Fractions containing the desired product (J4) were pooled. Yield : 420 μg, 120 μg/ml. Data from SDS PAGE gel analysis of the product were essentially identical to N8 and gel fluorescence imaging showed that the HC and LC both contained fluorescence label. The product was subjected to a test reaction using a 30 kDa PEG-maleimide. SDS PAGE analysis of this reaction showed bands with increased MW and disappearance of the FVIII HC band, thus indicating the presence of of the free thiol group.

This example shows that FVIII can be modified on the O-glycan with a thiol group.

EXAMPLE 35

Preparation of full length AP3 mlgG antibody modfied with -SSPy groups (J5)

Full length AP3 mlgGl wt LC C39S (3.0 mg/ml, 400 μl, 1200 μg, 8 nmol, pH 7,2, in a mixture of buffers 50 % 100 mM HEPES HCI, 150 mM NaCI, pH 7.5, 33 % 100 mM Glycine HCI, pH 3.5, and 17 % 20 mM HEPES HCI, 150 mM NaCI, 0,01 % TweenδO, pH 7.5) was buffer exchanged to HEPES 100 mM, pH 7.5 by concentrating to 100 μl (15 min, 12.000 g, Millipore Amicon Ultra 10 kDa), diluting to 900 μl and then concentrate to 100 ul (12 min, 12.000 g) x 6. Final volume was ca. 80 μl. It was then diluted to ca. 260 μl using HEPES 100 mM pH 7.5. Subsequently, SPDP-dPEGi ₂ -NHS-ester (Quanta Biodesign, prod no. 10378, 1.8 mg was dissolved in 400 μl ml HEPES 100 mM, pH 7.5; from this, 20 eq., 144 μg, 32 μl was used) was added to the buffer exchanged AP3 FL mlgG. The reaction mixture was incubated at r.t. overnight. Reduced and un-reduced SDS PAGE gel analysis showed that the bands corresponding to FL antibody and the individ- ual heavy and light chains were increased in molecular weight, as well as being broader in appearence, thus showing that the desired modication had taken place. The protein was buffer exchanged to HEPES 100 mM, pH 7.5 as described above. Yield 765 μg, 8.5 mg/ml.

This example shows that AP3 FL mlgG can be modified with an acylation agent containing thiol sensitive -SSPy conjugation group. EXAMPLE 36

Preparation of a conjugate between FVIII and AP3 FL mlgG

AP3 mlgGl ClPEGi ₂ -SSPy (35, 8.5 μg/μl, 0.057 nmol/μl, 31 μl, 1.76 nmol) was mixed with 2500 μl of the FVIII derivative 34 (300 μg, 1.76 nmol, 120 μg/ml). The mixture was sterile-filtered, wrapped in alu foil and left at r.t. overnight after which SDS PAGE gel analysis showed that products with MW corresponding to the desired adduct(s) between wt BDD FVIII and AP3 FL mlgG had formed. Gel fluorescence imaging showed that these bands contained fluorescence label, showing that the originated from the FVIII HC or LC chains. Gel analysis was also performed in the presence of excess N-ethylmaleimide to verify that the conjugation did not happen during SDS sample preparation. The mixture was allowed to react for an additional 3 days, after which it was diluted with 30 ml 20 mM imi- dazol, 10 mM CaCI2, 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3 and loaded to a MonoQ 5/50 GL column equilibrated with 20 mM Imidazol, 10 mM CaCI2, 0.02% Tween 80, 25 mM NaCI, 1 M glycerol, pH 7.3. The product was eluted using a linear gradient with elution buffer 20 mM Imidazol, 10 mM CaCI2, 0.02% Tween 80, 1 M NaCI, 1 M glycerol, pH 7.3. In this manner, the free AP3 FL mlgG was separated from free BDD FVIII and the conjugate. Reduced and non-reduced SDS PAGE analysis showed that a few fractions contained the de- sired product. The non-reduced gel indicated a disappearance of the FVIII HC band while showing high MW bands corresponding to conjugates between AP3 FL mlgG and FVIII. Western blot with mlgG specific Ab showed that these bands contained the AP3 FL antibody. The reduced gel showed the presence of both FVIII and AP3 heavy and light chains.

This example shows that AP3 FL mlgG and BDD FVIII can be conjugated via a disulfide linkage formed by the reaction between a thiol group on the AP3 FL mlgG and an -SSPy group on the BDD FVIII.

EXAMPLE 37

FVIII:C in cell culture harvests measured by chromogenic assay

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 (Chro- mogenix) as follows: rFVIII samples and a FVIII standard (Coagulation reference, Tech- noclone) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCI, 1 % BSA, pH 7.3, with preservative). Fifty μl 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 CaCI ₂ from the Coatest SP kit were mixed 5: 1 :3 (vol :vol :vol) and 75 μl of this added to the wells. After 15 min incubation at room temperature, 50 μl of the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions were incubated 5 min at room temperature before 25 μl 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 concentra- tion determined by ELISA.

EXAMPLE 38

Specific activity of AP3-FVIII fusion-proteins in clinically relevant FVIII activity assays

FVIII activity of purified proteins and conjugates (N8 control, F8-500 AP3-LC-HC scFV-Δa3 (SEQ ID NO 40), F8-500 AP3-LC-HC scFV (SEQ ID NO 43) and F8-500 AP3-HC-LC scFV (SEQ ID NO 42)) was evaluated in standard FVIII :C assays, either chromogenic assays or in a one-stage clotting assay. Chromogenic FVIII activity of purified proteins was assessed using the the Coatest SP assay (Chromogenix, Lexington, MA, USA). The assay was preformed according to manufacturer's instructions with a few minor modifications and run in a 96-well plate format. Briefly, diluted FVIII samples and FVIII reference material were incubated with a mixture of factor IXa/factor X reagent, phospholipid re- agent and CaCI ₂ from the Coatest SP kit. After 15 min incubation at room temperature the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions incubated 10 min at room temperature before the reaction was stopped with 20% citric acid. The absorbance at 415 nm was measured on a Spectramax microtiter plate reader (Molecular Devices) with absorbance at 620 nm used as reference wavelength. A recombinant FVIII calibrated against the 7th WHO/NIBSC recombinant FVIII standard was used as reference material. See table 4

EXAMPLE 39

FVIII: C in purified samples measured by one-stage clot assay

The FVIII clotting activity of the FVIII compounds was determined by diluting the concentrated FVIII samples and reference material first in buffer and then in FVIII-deficient plasma with von Willebrand Factor (Siemens, Deerfield, IL, USA) according to SSC recommendations. Each FVIII sample was measured in 4 different concentrations. The FVIII clotting activity was measured on an ACL 9000 instrument using the single factor program (FVIII:Cd) where FVIII samples were mixed with APTT reagent (Synthasil, ILS, Bedford, MA, USA), 25 mM CaCI2 and FVIII-deficient plasma. A recombinant FVIII calibrated against the 7th WHO/NIBSC recombinant FVIII standard was used as reference material. S

Table 4

As show in table 4 AP3-FVIII fusion proteins and conjugates have similar FVIII activities in FVIII Chromogenic- and Clotting activity assays as the N8 starting material. EXAMPLE 40

Thrombin generation supported by AP3-N8 fusion-proteins specifically bound to plate- lets

Global Coagulation tests such as Thrombin Generation Test (TGT) have been suggested to be more physiologically relevant measurements of factor VIII (FVIII) activity than clotting and chromogenic assays especially when platelets are included in the assays. In order to assess the effect of AP3-N8 on platelets the FVIII compounds (F8-500 AP3-LC-HC scFV-Δa3 and N8 control) were tested in a system that mimic of human hemophilia A. Mimics of hemophilia A were prepared by substituting FVIII-deficient plasma (>1% FVIII activity) with washed platelets from individual normal blood donors. The coagulation process is initiated with tissue factor and calcium and the thrombin generation can be followed by including fluorogenic thrombin substrate in the assay that can be detected even as the fibrin clot forms. Thus in mimic of hemophilia A the amount of thrombin generated, and the speed of thrombin generation will be dependent on the amount of FVIII present in the TGT. The TGT method used here is modified from the Platelet Rich Plasma (PRP) assay described by Hemker (1 ). Briefly, Platelets isolated from citrated blood collected from individual normal healthy donors and purified through a series of wash steps according to the procedure of Mustard (2). *Washed platelets were added to FVIII-deficient plasma (George King, Overland Park, KA, USA) to a concentration of 150,000 platelets/μl. 80μL of this FVIII-deficient PRP (100,000 platelets/μl final) was mixed with 10μl of Innovin (final dilution 1 :200,000) in a microtitter plate and pre-incubated for 10 min at 37 ^° C before 10 μl FVIII compound or buffer, and 20 μl fluorogenic substrate (Z-G Iy-G ly-Arg-AMC, Bachem, final concentration 417 nM) mixed with CaCI2 (final concentration 16.7 mM) was added. Emission at 460 nm after excitation at

390nm was measured continuously for 2 hours in Fluoroskan Ascent plate reader (Thermo Electron Corporation). The fluorescence signal was corrected for α2-macroglobulin bound thrombin activity and converted to thrombin concentration using a calibrator and Trombino- scope software (Synapse BV).

In order to demonstrate that AP3 convey a specific platelet binding to N8 that would allow the FVIII molecule to be carried with the platelet in circulation and not just localize to the platelet at the time of activation a the TGT assay described above was performed with the following additional steps: ^* once the washed platelets had been obtained the platelets were incubated for 30 min at room temperature with either AP3-N8-fusion protein or N8 (20 U/ml) in order to allow the N8 compounds to bind to the platelets. This incubation was follow by two additional wash steps to remove all N8 not specifically bound to the resting platelets. Following these two additional wash steps the platelets were added to FVIII deficient plasma, calcium and fluorogenic substrate and FVIII activity in the form of thrombin generation was measured as described. Parameters obtained from the software are recorded as "Lag time" which describes the time to start of thrombin generation, "Peak" which described the maximum rate of thrombin generation in nM and "Time to peak". A strong clot is characterized by fast early onset and high rates of thrombin formation.

Table 5

The results show strong thrombin generation in the experiment where platelets were incubated with the AP3-N8-fusion protein whereas the platelets incubated with N8 showed barely more than background (hemophilia A) levels of thrombin generation. Thus, while the N8 control could be washed away from the platelets the AP3-N8-fusion protein stayed bound to the platelets thereby directly demonstrating a FVIII activity carried by the platelets.

EXAMPLE 41 Analysis of fusion protein binding to purified GPIIIa

Binding interaction analysis was obtained by Surface Plasmon Resonance in a Biacore T-100 instrument. Capture of purified GPIIbIIIa (Enzyme Research Laboratories) at a fixed concentration was obtained by direct immobilization to a CM5 chip to a level of 1000-4000 RU in 10 mM sodium acetate pH 4.5-5.0. Two-fold dilutions of the FVIII derivatives from 5-0.31 nM were tested for binding to the immobilized GPIIbIIIa. Running and dilution buffer: 10 mM HEPES, 150 mM NaCI, 5 mM CaCI2, 0.005% p20, pH 7.4. All FVIII derivatives were dialysed and diluted in running buffer. Regeneration was obtained by 10 mM Glycine, pH 1.7. Determination of kinetic and binding constants (k _on , k _Off ) was obtained assuming a 1 : 1 interaction of the FVIII derivative and GPIIbIIIa using the Biacore TlOO evaluation software. Results are shown in the table below. Table 6: Surface Plasmon Resonance (SPR) analysis. Binding to GPIIbIIIa. Kinetic constants.

n.d. observed dissociation rate too low for accurate determination of k _off

EXAMPLE 42

Analysis of FVIII fusions/conjugates binding to platelets

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. 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).

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. Internalization of the fu- sion 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 43

Test of FVIII-AP3 fusions and conjugates binding to platelets

Platelet binding of F8-500 AP3-LC-HC scFV-Δa3 (SEQ ID NO 40) (AP3-N8 2097) and full length AP3 IgG chemically coupled to N8 (MZ1 in example 18) were tested by flow cytometry. Washed platelets from human peripheral blood were prepared and incubated with AP3-N8 in dark at room temperature for 30 min (~ 300.000 platelets per sample). As a platelet marker, peridinin chlorophyll protein (PerCP)-labelled anti CD42a was added to the samples along with the AP3-N8 constructs. After incubation, the cells were washed with buffer (20 mM Hepes, 150 mM NaCI, 1 mg/ml BSA, 5 mM CaCI ₂ ) to remove unbound antibody, and a phycoerythrin-Cy7 (PE-Cy7) anti-FVI Il monoclonal antibody was added (10 μg/ml). After 30 min incubation, the cells were washed with buffer, and the samples were analyzed on a BD LSRFortessa™ flow cytometer with the forward and side scatter detectors in log mode ( at least 5.000 events per sample were analyzed). Doses of AP3-N8 from ~ 0.025 - 51.2 nM (25.6 nM for MZ1 ) were analyzed to assess dose-dependency of the binding. Three donors were tested in individual experiments for AP3-N8 2097 and one donor for the MZ1 construct. The specificity of the AP3-N8 binding was assessed by adding excess unlabelled AP3-LC- HC scFV-FLAG (SEQ ID NO 22) to the samples (up to 50- fold excess). Thus, the data show AP3-specific binding of the constructs to the GPIIb/llla receptor on platelets. Experi- ments using a similar AP3-N8 construct, where the N8 moiety was directly labelled with Oregon Green gave comparable results, supporting specific binding of the AP3-N8 construct to the platelets (n = 5).

Internalization of the AP3-N8 can be assessed by first incubating platelets with fluorescently labelled AP3-N8 and measuring the fluorescence by flow cytometry and thereafter adding an antibody, which quenches the signal from surface-bound (i.e. not internalized) AP3-N8. When re-analyzing the samples by flow cytometry, only the internalized AP3-N8 will be detected.

EXAMPLE 44

Testing of anti-aggregatory effect of F8-500 AP3-LC-HC scFV-Δa3 (SEQ ID NO 40) on platelets

The possible anti-aggregatory effect of F8-500 AP3-LC-HC scFV-Δa3 (SEQ ID NO 40) was measured by monitoring the change in light transmission through a suspension of iso- lated platelets. This method was first described essentially by Gustav von Born in the 1960s (Born, Nature, 194:927-29 1962) and is today one of the most used methods for evaluation of platelet function. In brief, the method measures the capability of light to transverse through a suspension of platelets. This suspension of platelets might either be platelet rich plasma or isolated platelets. Upon activation the GPIIb/llla changes its conformation to a fibrinogen high-binding state and in the presence of fibrinogen the platelets will start to form aggregates. This is registered as an increase in light transmission since more light will go through a sample with few large aggregates than many single platelets. The inhibitory effect of an antibody is generally examined by the ability of the antibody to decrease platelet aggregatory response to an activator (e.g. ADP or thrombin) measured for example by change in light transmission (Coller et al. JCI 72(1 ):325-38, 1983).

The term "isolated human platelets" in this example refer to platelets derived from human whole blood kept in an isotonic buffer. The platelets were isolated from heparinized human venous human blood from healthy volunteers that was mixed with acid-citrate-dextrose (ACD) solution (6:1 , v/v) containing 85 mM Na-citrat, 71 mM citric acid and 1 11 mM glucose. The blood was centrifuged at 22Og for 20 minutes to obtain platelet-rich plasma (PRP). Ace- tylsalicylic acid (100 μM) and apyrase 0.5 U/ml were added to prevent activation of the platelets by eicosanoids and adenine nucleotides during the preparation procedure. The platelets in the PRP were spun down by a 20 minutes centrifugation at 48Og and the supernatant was removed. The platelets were gently resuspended in calcium free Hepes solution (pH 7.4) composed of 145 mM NaCI, 5 mM KCI, 1 mM MgSO4, 10 mM glucose, 10 mM Hepes and 1 U/ml apyrase. The platelet suspensions were kept in plastic tubes at room temperature and [Ca ²⁺ ] was adjusted to 1 mM and the temperature was adjusted to 37°C in each experiment.

The platelet samples were kept in siliconized glass cyvettes with continuous stirring (1200 rpm) at 37°C while measuring light transmission using the Platelet Aggregation Profiler® (PAP)-8E instrument (Bio/Data Corporation, Horsham, PA). The samples were incubated with either F8-500 AP3-LC-HC scFV-Δa3 (SEQ ID NO 40) (30 nM), AP3-LC-HC scFV- FLAG (SEQ ID NO 22) (30 nM) or abciximab (ReoPro®) (30 nM) for 3 minutes before acti- vated with a protease activated receptor-1 (PAR-1 ) activating peptide (amino acid sequence SFLLRN) (10 μM). The results show no significant difference in platelet aggregation in AP3- N8 treated platelets compared to control (Table 7). Furthermore, AP3-LC-HC scFV-FLAG was tested for antiaggregatory properties at 30 nM showing no indication for an inhibitory effect. However, the anti-aggregatory abciximab (ReoPro®) (30 nM) effectively inhibited platelet aggregation by almost 40 % in SFLLRN activated platelets (Table 7). The difference between F8-500 AP3-LC-HC scFV-Δa3 and ReoPro® in respect to inhibition of platelet aggregation was even more pronounced when increasing the concentration. Figure 2 show platelet aggregation when using F8-500 AP3-LC-HC scFV-Δa3 (100 nM) or ReoPro® (100 nM). This higher concentration results in an augmented inhibition with ReoPro® whereas the increased concentration F8-500 AP3-LC-HC scFV-Δa3 do not influence on platelet aggregation.

Table 7. Platelet aggregation

Data shown as % of control (SFLLRN 10 μM) ±SD and n=3 in all groups.

EXAMPLE 45

Preparation and use of a mouse strain with a humanised IgB3 receptor

A vector containing the Human ITGB3 cDNA {from exon 2 (G26) to exon 15 (including partial 3'UTR from cDNA provided)} is engineered by inserting human ITGB3 intron 5 (647 bp) between sequences corresponding to human exons 5 and 6. A transcriptional STOP cassette is inserted immediately downstream of the human partial 3'UTR. The engineered human ITGB3 cDNA is inserted at its corresponding position in mouse exon 1. In order to optimize processing of the human ITGB3 protein in mouse cells, the human- ized allele will express a fusion protein between mouse Itgb3 signal peptide (encoded within exon 1) and human ITGB3 mature protein. Selection marker (Puro) is flanked by F3 sites and inserted downstream of human ITGB3 3'UTR. Humanized allele after FIp- mediated removal of selection marker. The human Itgb3 protein will be expressed under the control of the mouse Itgb3 promoter. The insertion of the engineered human ITGB3 cDNA, including its 3'UTR region, into mouse exon 1 should lead to the inactivation of the mouse Itgb3 gene. The confirmed sequence of the final targeting vector is shown in figure 3.

Generation of heterozygous targeted C57BL/6 ES cells The C57BL/6N ES cell line is grown on a mitotically inactivated feeder layer comprised of mouse embryonic fibroblasts in DMEM High Glucose medium containing 20% FBS and 1200 u/mL Leukemia Inhibitory Factor. IxIO ⁷ cells and 30 μg of linearized DNA vector are electroporated at 240 V and 500 μF. Clone selection is based on Puromycin selection (1 μg/mL) started on d2 and counterselection with Gancyclovir (2 μM) started on d5 after electroporation. ES clones are isolated on d8 and analyzed by Southern Blotting after ex- pansion.

Generation of hererozygous animals:

After administration of hormones, superovulated Balb/c females are mated with Balb/c males. Blastocysts is isolated from the uterus at dpc 3.5. For microinjection, blastocysts are placed in a drop of DMEM with 15% FCS under mineral oil. A flat tip, piezo actuated microinjection-pipette with an internal diameter of 12 - 15 micrometer is used to inject 10-15 targeted C57BL/6 N.tac ES cells into each blastocyst. After recovery, 8 injected blastocysts are transferred to each uterine horn of 2.5 days post coitum, pseudopregnant NMRI females. Chimerism is measured in chimeras (GO) by coat colour contribution of ES cells to the Balb/c host (black/white). Highly chimeric mice are bred to strain C57BL/6 females. Germline transmission was identified by the presence of black, strain C57BL/6, offspring (Gl).

Generation of homozygous animals Offspring Gl was propagated either by breeding of heterozygous mice or by in vitro fertilization. From the initial attemps at mating and IVF, genotypic distributions was determined as outlined in table 8.

Table 8. Genotyping of humanized GPIIIb mice.

EXAMPLE 46

Pharmacokinetics of GPIIIa-targeted fusion proteins in GPIIIa transgenic mice

The AP3 antibody binds to the human GPIIb/IIIa (integrin αllβ3) receptor on platelets, but it does not recognize murine GPIIb/IIIa, preventing the use of wild type mice for pharmacokinetic (PK) analyses. The PK profile of an AP3-FVIII fusion or conjugate can be analyzed in transgenic mice expressing human GPIIIa (bred at Taconic M&B), which associates with murine GPIIb enabling the binding of AP3 to the receptor. The GPIIIa transgenic mice will receive a single i.v. injec- tion of AP3-FVIII fusion or conjugate and blood will be collected from the pre- orbital plexus at time-points up to 288 hours after injection. About three samples will be collected from each mouse during the study and 2-4 samples collected for each time point. The blood is stabilized and diluted in appropriate buffer. The injected AP3-FVIII fusion or conjugate (free and/or plate bound) can be quantified by means of ELISA or flow cytometry either using antibodies against N8 or directly labelled AP3-FVIII fusion or conjugate.

EXAMPLE 47

Duration of effect of GPIIIa-targeted fusion proteins in GPIIIa transgenic mice In order to evaluate efficacy and duration of action of effect of a GPIIIa-targeted fusion protein in an animal disease model a mouse strain with a humanised IgB3 receptor is generated. The generated mouse strain may be cross breed with other (transgenic) mice strains this includes but are not limited to mice lacking the coagulation factor VIII or IX. The mice may be breed by natural mating or using IVF transfer (e.g. G.Vergara Theriogenology 1997; 47, 1245-1252). Alternative the humanized platelets may be transferred to other mice by the means of bone marrow transplantation. The bone marrow cells are isolated from the humanised mice for example by the method described in Shi et al. (Blood. 2008; 112, 2713-2721) and injected into appropriated prepared recipient mice ie. mice lacking factor VIII or IX. The efficacy can be tested in the above men- tioned animals models by measuring the ability to reduce the bleeding in a tail bleeding test (Holmberg et al., J Thromb Haemost 2009; 7: 1517-22) or the ability to form a clot in FeCI ₃ injury model (Moller & Tranholm, Haemophilia 2010; 16, e216-e222). The duration of action can be tested by the efficacy of the drug after prolonged in the above mentioned models.