MUTEINS OF CLOTTING FACTOR Vlll

Abstract

The present disclosure provides muteins of FVIII to which a biocompatible polymer may be attached to increase the circulatory half-life of the muteins, as well as conjugates of such muteins and biocompatible polymers.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/948,186, filed Mar. 5, 2014, the contents of which are incorporated herein by reference in its entirety.

[0002] REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted concurrently herewith in computer readable form (CRF) via EFS-Web as file name PC72098A_SEQLIST_ST25.txt is incorporated herein by reference. The electronic copy of the Sequence Listing was created on Mar. 1, 2015, with a size of 69,421 bytes.

BACKGROUND OF THE INVENTION

[0004] Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the gene encoding clotting Factor VIII (FVIII). Because the gene is carried on the X chromosome, the disorder occurs almost exclusively in males with an incidence of approximately 1 in 5000 male births. Untreated, HA can result in uncontrolled bleeding resulting in death. Even if affected individuals survive bleeding episodes, bleeding into the joints can cause incapacitating joint damage, and spontaneous intracerebral bleeding can result in disabling neurological impairment.

[0005] Before the advent of FVIII replacement therapies, the disorder was highly lethal with most affected boys dying before the age of 20 from spontaneous bleeding or bleeding caused by trauma. After the discovery of plasma cryoprecipitate, containing FVIII and other clotting factors, and development of plasma-derived preparations of FVIII, the life expectancy and quality of life of HA patients improved dramatically. Although the number and severity of bleeding episodes was reduced by treatment with these products, viral contamination of the plasma supply with HIV and hepatitis viruses eventually caused devastating illnesses of their own. Eventually, the discovery of the FVIII gene and development of recombinantly produced FVIII preparations essentially eliminated the risk of viral contamination of FVIII replacement therapy.

[0006] Despite the availability of abundant supplies of recombinant FVIII for nearly two decades, replacement therapy for many HA patients remains sub-optimal. Due to the short half-life of FVIII (about 8-12 hours), severely affected HA patients require FVIII replacement every other day to maintain sufficient FVIII concentration to prophylactically prevent spontaneous bleeding. Although FVIII can be administered less frequently to stop bleeding caused by trauma, such on demand therapy is not effective to prevent spontaneous bleeding into the joints or brain which can also cause death or severe disability. Unfortunately, the frequent FVIII infusions required for prophylactic therapy is accompanied by its own problems. First, the therapy is very expensive. Second, it is associated with side effects such as septic arthritis. Third, it often requires placement of a central venous access device, which can be a source of infection and thrombosis. Fourth, patient compliance is difficult. And, fifth, frequent infusions of FVIII can cause development of antibody inhibitors to the clotting factor.

[0007] In light of the challenges associated with providing optimal prophylactic FVIII replacement therapy, there is a need in the art for ways of extending FVIII half-life after infusion. By increasing FVIII half-life, HA patients could administer the drug less frequently than is currently possible while maintaining sufficient FVIII concentration to prevent spontaneous bleeding.

[0008] Previous strategies to increase FVIII half-life have only achieved modest increases compared to wild type FVIII, but such improvements are not sufficient. Accordingly, there is a particular need in the art for new forms of FVIII having sufficiently long half-life that patients could maintain prophylaxis against spontaneous bleeding by administering the drug less frequently than is now possible.

SUMMARY OF THE INVENTION

[0009] As described further below, applicants have invented novel FVIII variants having improved properties. Among other uses, these variants can be used for attaching moieties such as biocompatible polymers to increase circulatory half-life compared to unmodified FVIII. The FVIII variants of the disclosure are therefore expected to be useful in improved methods of treating HA.

[0010] Accordingly, the present disclosure provides a modified FVIII protein comprising at least one cysteine substitution mutation at a position corresponding to one or more of the following amino acids of SEQ ID NO:1: 59, 239, 333, 336, 379, 481, 484, 486, 488, 489, 490, 492, 493, 495, 496, 497, 499, 500, 501, 507, 555, 562, 568, 571, 582, 1680, 1778, 1793, 1794, 1797, 1798, 1799, 1800, 1801, 1806, 1810, 1811, 1814, 1816, 1818, 1891, 2035, 2068, 2092, 2093, 2094, 2095, 2118, 2125, 2183, 2186, 2191, 2196, 2204, 2206, and 2212.

[0011] In some embodiments, the modified FVIII proteins further includes at least one additional substitution mutation other than cysteine selected from the group R336A, R562A, K1968A, and Y1680F.

[0012] Other embodiments include nucleic acids encoding the modified FVIII proteins of the disclosure, which may be cloned into expression vectors and then used to transfect or transform host cells for the purpose of expressing said modified FVIII proteins.

[0013] By virtue of these mutations, modified FVIII proteins of the disclosure can have improved functions compared to unmodified FVIII, such as higher expression, greater procoagulant activity, reduced immunogenicity, greater stability, reduced susceptibility to degradation, greater resistance to proteases, greater resistance to oxidation, improved ability to be activated by thrombin or other coagulation factors, improved ability to combine with other coagulation factors and components into the tenase complex, increased association or binding to vWF, improved shelf-life, reduced binding by inhibitory antibodies, reduced interaction with the low density lipoprotein receptor related protein, reduced interaction with low density lipoprotein receptor, reduced interaction with cell surface heparin sulphate proteoglycans, increased circulatory half-life, and improved pharmacokinetics.

[0014] In other embodiments, the modified FVIII proteins of the disclosure can have a moiety which may include a biocompatible polymer attached, for example, to a substituted cysteine, or elsewhere, directly or via a linker. Moieties and linkers can be attached covalently, such as to the cysteine thiol group, and in some embodiments through a reactive group in the moiety. Examples of moieties include a small organic molecule, macromolecule, antibody, antibody fragment, antigen binding domain, antibody Fc region, protein of immunologic origin, protein of immunologic function, intact clotting factor, functional fragment of a clotting factor, enzyme, nucleic acid, DNA, RNA, organometallic compound, lipid, fatty acyl chain, phospholipid, glycolipid, protein, peptide, amino acid, carbohydrate, monosaccharide, disaccharide, hydrophobic compound, hydrophilic compound, organic acid, and an organic base.

[0015] According to some embodiments, the moiety is a biocompatible polymer, such as polyethylene glycol (PEG), hydroxyalkyl starch, hydroxyethyl starch (HES), polysialic acid (PSA), a zwitterionic brush polymer, and a poly-phosphorylcholine branched polymer. Biocompatible polymers can be attached, for example, to a substituted cysteine in FVIII or elsewhere in FVIII, directly or via a linker. Biocompatible polymers or linkers can be attached covalently to FVIII, such as to a cysteine thiol group, and in some embodiments via a reactive group in the polymer or linker, which in some embodiments is an electrophile and in other embodiments is a nucleophile.

[0016] As a result of attaching moieties, including for example, a biocompatible polymer, the circulatory half-life of modified FVIII can be increased, for example, at least about 2 times, 5 times, 10 times or even more, compared to unmodified FVIII.

[0017] Other embodiments include methods of treating or preventing uncontrolled bleeding in subjects suffering from a deficiency of FVIII activity by administering an effective dose of a composition containing a modified FVIII protein of the disclosure to which is attached a moiety, including for example, a biocompatible polymer. In some embodiments, the biocompatible polymer is polyethylene glycol (PEG), hydroxyalkyl starch, hydroxyethyl starch (HES), polysialic acid (PSA), a zwitterionic brush polymer, and a poly-phosphorylcholine branched polymer, but other biocompatible polymers can be effective.

[0018] As a result of increasing the circulatory half-life of modified FVIII proteins, it is possible to achieve prophylaxis with substantially reduced frequency of administration. Thus, for example, effective prophylaxis against uncontrolled bleeding may be achieved with administrations occurring not more frequently than once per every 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, and 3 weeks, or even longer periods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 provides the amino acid sequence of the single chain form of mature human FVIII, including the B domain (SEQ ID NO:1). The mature protein lacks a 19 amino acid signal sequence present in full length FVIII that is removed prior to secretion. The amino acids constituting that portion of the B domain missing from the B domain deleted version of FVIII illustrated in FIG. 2 are underlined. Positions of cysteine substitution mutations of the disclosure are identified by bold and underlined font. Unless otherwise indicated, numbering of FVIII amino acids described herein is based on their position in SEQ ID NO:1.

[0020] FIG. 2 provides the amino acid sequence of the single chain form of a mature partially B domain deleted version of FVIII (BDD FVIII) (SEQ ID NO:2) into which the cysteine substitutions of the disclosure were introduced and tested as described in the Examples. The mature protein lacks a 19 amino acid signal sequence removed prior to secretion. As in FIG. 1, positions of cysteine substitution mutations of the disclosure are identified by bold and underlined font.

[0021] FIG. 3A and FIG. 3B respectively provide the amino acid sequences of the heavy and light chains for the two-chain form of the BDD FVIII illustrated in FIG. 2. In certain cells expressing the single chain form of the protein, intracellular processing cleaves the protein into a heavy chain shown in FIG. 3A (SEQ ID NO:3) and a light chain shown in FIG. 3B (SEQ ID NO:4). The chains are held together non-covalently.

[0022] FIG. 4 provides the results of an experiment to test thrombin cleavage of selected FVIII muteins containing cysteine substitution mutations and unmutated BDD FVIII. The image is of a silver stained denaturing SDS polyacrylamide gel showing the protein bands corresponding to different proteolytic cleavage products. On the left is a protein molecular weight marker. BDD FVIII stands for B domain deleted FVIII, the positive control. The numbers (2093, 2094, 2186, and 2206) identify the position of the cysteine substitution relative to mature human FVIII of SEQ ID NO:1 (also Table 1 and FIG. 1). The plus sign (+) indicates that thrombin was added to the reaction. The minus sign (-) indicates no thrombin added. When no thrombin was added, FVIII proteins resolved to show uncleaved single chain form as well as the heavy and light chains that when non-covalently bound constitute the inactive heterodimeric form of the protein. After incubation with thrombin, the single chain and heavy and light chains disappear having been converted to expected lower molecular weight cleavage products (indicated as light chain cleaved, A1, A2, and two bands corresponding to A2 cleaved. A band corresponding to thrombin is also present.

[0023] FIG. 5 provides data comparing the in vivo procoagulant effect of unmodified BDD FVIII with that of three PEGylated FVIII muteins comprising a cysteine substitution at position 2094. Mice phenotypic for Hemophilia A were administered BDD FVIII or one of the muteins. At 5 min, 24 hr and 48 hr thereafter, the distal ends of the tails were transected and the total blood loss over 10 minutes determined. Compared to unmodified BDD FVIII, the duration of the procoagulant effect of each of the muteins was longer.

DETAILED DESCRIPTION

Hemophilia A

[0024] Hemophilia A (HA) is a genetic disorder characterized by a deficiency in functional clotting Factor VIII (FVIII). Numerous mutations have been characterized that result in too little normal FVIII being produced, or that result in production of an altered protein with diminished or no activity. A list of exemplary mutations may be found at the website http://www.uniprot.org/uniprot/P00451. The incidence of HA in the general population is about 1 in 5000. Because it is an X-linked recessive disorder, HA mostly affects males.

[0025] Disease severity is a function of how much functional FVIII is present. In severe HA FVIII concentrations are less than 1% of normal. Patients with severe HA can suffer spontaneous bleeding. In moderate HA FVIII concentration is about 1-5% of normal. Patients with moderate HA may experience bleeding after mild trauma. Finally, in mild HA FVIII concentrations are about 5-25% normal. White et al., Thromb Haemost 85:560 (2001), which is incorporated by reference. Patients with mild HA may experience greater than normal bleeding with surgery or after experiencing major trauma. Depending on severity, HA may manifest itself with excessive uncontrolled bleeding, spontaneous or injury-induced bleeding into the brain, which can be fatal, bleeding into the joints (hemarthrosis), which can be debilitating, and soft tissue hematomas.

Factor VIII (FVIII)

[0026] Wild type FVIII precursor is a glycoprotein 2351 amino acids long, including a 19 amino acid long signal peptide. Mature uncleaved FVIII is a single chain protein 2332 amino acids long. The amino acid sequence of mature human FVIII (lacking the signal peptide) is set forth in FIG. 1 (SEQ ID NO:1). Unless otherwise noted, this sequence serves as the reference for all numbering of amino acid residues used herein. The nucleic acid sequence encoding human FVIII is provided as SEQ ID NO:6, in which nucleotides 172-7227 encode full length FVIII including the signal peptide. Sequence analysis demonstrates that FVIII contains three so-called A domains, a B domain, and two so-called C domains. The A domains are weakly homologous to each other (about 30%) and to the A domains found in coagulation Factor V (FV) and in ceruloplasmin. The C domains are structurally related to the C domains of FV, whereas the B domain has no known homology to any other protein. FVIII additionally includes so-called acidic regions, which are short spacer regions containing Asp and Glu residues, positioned after domains A1, A2 and B. The domain structure of mature FVIII can be represented as A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2 and A3 are the A domains, B is the B domain, C1 and C2 are the C domains and a1, a2 and a3 are the acidic regions.

[0027] With reference to FIG. 1, the FVIII domains in some embodiments correspond to the following amino acid ranges: A1 domain (1-336); a1 acidic region (337-372); A2 domain (373-710); a2 acid region (711-740); B domain (741-1648); a3 acidic region (1649-1689); A3 domain (1690-2019); C1 domain (2020-2172); C2 domain (2173-2332).

[0028] After expression in liver (in hepatocytes or sinusoidal cells), and possibly spleen, lung and kidney, mature FVIII is translocated into the endoplasmic reticulum (ER) where it undergoes N-linked glycosylation, predominantly in the A1 and B domains. The protein then travels to the Golgi apparatus where it is further modified. Among the modifications occurring within the Golgi, N-linked oligosaccharides are converted to more complex carbohydrate structures. In addition, FVIII undergoes 0-linked glycosylation and sulfation at specific tyrosine residues in acidic regions. Sulfation at certain sites reportedly can affect function of FVIII as a cofactor for thrombosis or binding to von Willebrand Factor (vWF). Fay, P. J., Blood Reviews 18:1-15 (2004), which is incorporated by reference.

[0029] Before secretion from cells, mature FVIII undergoes intracellular cleavage within the Golgi to form the circulating inactive two-chain complex comprising a FVIII heavy chain (about MW 200 kD, consisting of A1-a1-A2-a2-B) and FVIII light chain (about MW 73 kD, consisting of a3-A3-C1-C2) non-covalently bound to each other. Cleavage occurs at the junction between the B and A3 domains, and at various sites within the B domain. Because cleavage within the B domain is heterogeneous, the resulting size of the heavy chain, which includes the A1 and A2 domains, is variable depending on how much B domain is removed by cleavage. Interestingly, the B domain is not required for the procoagulant activity of FVIII. By contrast, the size of the light chain, which includes A3, C1 and C2 domains is typically constant. Although heterodimeric FVIII includes two polypeptide chains (heavy and light), amino acid numbering in each refers to the numbering of amino acid residues in the mature single-chain FVIII protein sequence of SEQ ID NO:1. The heavy and light chains bind each other non-covalently through a metal ion-dependent linkage. Presence of a copper ion has been found to substantially increase inter-chain binding, but other ions, such as Ca.sup.+2 and Mn.sup.+2 may also influence FVIII activity.

[0030] In circulation, heterodimeric FVIII tightly binds non-covalently with another protein, von Willebrand Factor (vWF), which influences FVIII in at least two ways. First, vWF may prevent premature binding of FVIII to activated Factor IX (FIXa) and lipid membrane in the tenase complex, where FVIII acts as a FIXa cofactor. And second, vWF protects FVIII from proteolytic inactivation thereby increasing its circulatory half-life. Lenting et al, Blood. 92(11): 3983-96 (1998); Lenting et al, J. Thromb. Haemost. 5(7): 1353-60 (2007); each of which are incorporated by reference. In particular, binding by vWF may protect FVIII against cleavage by the membrane-dependent proteases activated protein C (APC) and activated Factor X (FXa). Two peptide regions in FVIII are implicated in vWF binding, one at the amino-terminal end of the intact light chain and one at the carboxy-terminal end (residues 2303-2332). Cleavage of the FVIII light chain at Arg 1689, which occurs during activation, markedly reduces vWF binding, permitting activated FVIII (FVIIIa) to participate in the tenase complex.

[0031] Circulating FVIII is activated by thrombin cleavage in the heavy chain at Arg 372 (at the a1-A2 junction) and Arg 740 (at the a2-B junction) and near the amino-terminus of the light chain at Arg 1689 to form a heterotrimer of A1-a1 (about 50 kDa), A2-a2 (about 43 kDa) and A3-C1-C2 (about 73 kDa) (amino acid numbering is with respect to mature single chain FVIII protein sequence of SEQ ID NO:1). FXa also activates FVIII, cleaving at Arg 336, Arg 372 and Arg 740, but thrombin is likely the physiologically relevant activator. After activation, FVIIIa dissociates from vWF and can be incorporated in the tenase (Xase) complex with activated Factor IX (FIXa), calcium, and phospholipid where FVIIIa functions as a cofactor for FIXa, enhancing the ability of FIXa to cleave and activate FX, forming FXa, which in turn can generate thrombin. Thrombin then cleaves fibrinogen to form fibrin monomers which then polymerize. Thrombin also activates Factor XIII (forming FXIIIa) which crosslinks fibrin polymers, stabilizing them and rendering them insoluble.

[0032] Inactivation of FVIIIa is believed to occur by several mechanisms. For example, several enzymes, including APC, FXa, FIXa and plasmin can cleave the heavy chain at position Arg 336, preceding the carboxy-terminal acidic region of the A1 domain. This cleavage releases a1 causing the A2 domain to disassociate more rapidly from the FVIIIa heterotrimer resulting in inactivation. APC can also cleave at position Arg 562 which results in loss of FIXa binding to the A2 domain, which may further destabilize the activated heterotrimer. Interestingly, spontaneous dissociation of the A2 domain from A1 and A3-C1-C2 may also result in inactivation, which may be accelerated by binding of the A2 domain to the LDL receptor related protein. Studies have also shown that interaction between FVIII and low density lipoprotein-receptor-related protein (LRP) may contribute to uptake and transport of FVIII to intracellular degradation pathways. Heparin sulphate proteoglycans (HSPG) may facilitate interaction between FVIII and LRP. LRP binds to the A2 domain in activated FVIII (Arg 484-Phe 509) and to at least three sites within the FVIII light chain (Lys 1804-Phe 1838, Lys 2065-Lys 2092, and Ser 2173-Tyr 2332). Interaction with the light chain regions, however, is completely inhibited by vWF, suggesting another mechanism by which vWF can protect FVIII from clearance.

[0033] Additional information about FVIII biology can be found in the references Fay, Blood Reviews, 18:1-15 (2004); Fay, Int. J. Hema., 83:103-8 (2006); Lenting, et al., Blood, 92(11):3983-96 (1998); and Lenting, et al., Haemophilia, 16:6-15 (2010), each of which is incorporated by reference.

[0034] As used herein, the term Factor VIII (or FVIII) refers to a protein or polypeptide having at least some procoagulant activity of wild type FVIII (whether obtained from plasma or made recombinantly) or, if initially inactive, can acquire such activity after being activated, for example and without limitation, by proteolytic cleavage. FVIII activity includes the ability to at least partially complement the procoagulant function of endogenous FVIII absent from Hemophilia A plasma or normal plasma immunodepleted of FVIII. FVIII activity also includes serving (once activated, for example, by thrombin or another protease forming FVIIIa) as a cofactor for FIXa in the tenase complex to catalyze the conversion of FX to FXa. Other procoagulant activities are possible. Assays for testing whether a protein has FVIII activity are familiar to those of ordinary skill in the art. Non-limiting examples of such assays include the single stage assay based on measuring activated partial thomboplastin time (aPTT) and the two-stage chromogenic assay. Other assays for detecting and measuring FVIII activity are possible.

[0035] FVIII includes full length FVIII (i.e., possessing a signal peptide sequence) from human or other species (for example and without limitation chimpanzee, pig, dog, rat and mouse), chimeric versions of FVIII constructed by combining one or more FVIII domains present in human FVIII with domains present in FVIII proteins from other species, and mature FVIII lacking the signal peptide sequence found in full length FVIII. A non-limiting example of mature human FVIII protein is the amino acid sequence of SEQ ID NO:1 (illustrated in FIG. 1). FVIII also includes full length FVIII lacking the methionine (Met) residue normally present at the amino terminus, mature FVIII possessing a Met at the amino terminus, or full-length or mature FVIII lacking the tyrosine (Tyr) normally found at the carboxy terminus of the protein. FVIII also includes Factor Villa, Factor VIII:C, and Factor VIII in association with von Willebrand factor (vWF) or FVIII disassociated from vWF.

[0036] FVIII additionally includes FVIII proteins from which the B domain has been completely or partially deleted, so-called B domain deleted FVIII (BDD FVIII). Complete deletion would remove amino acids 741-1648 as shown in FIG. 1. A non-limiting example of a human partial B domain deleted FVIII protein is the amino acid sequence of SEQ ID NO:2 (illustrated in FIG. 2), which is the amino acid sequence of the drug ReFacto.RTM.. The nucleic acid sequence of a cDNA encoding the protein illustrated in FIG. 2 is provided in SEQ ID NO:7, which additionally encodes a 19 amino acid signal peptide from the full length FVIII protein at the amino-terminus and a 12 amino acid spacer and FLAG epitope at the carboxy-terminus.

[0037] The BDD embodiment of FIG. 2 includes 3 amino acids from the B domain amino-terminus (corresponding to amino acid positions 741 to 743 in SEQ ID NO:1) and 11 amino acids from the B domain carboxy-terminus (corresponding to amino acid positions 1638 to 1648 in SEQ ID NO:1) together forming the sequence SFSQNPPVLKRHQR (SEQ ID NO:5) corresponding to amino acid positions 741 to 754 in SEQ ID NO:2 and SEQ ID NO:3. Thus, this embodiment lacks amino acids 744-1637 as shown in FIG. 1. According to other non-limiting embodiments of BDD FVIII, as shown in FIG. 1, deletions may start with any of amino acids 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, and end at any of amino acids 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, or 1648. In certain embodiments, B domain deleted FVIII lacks amino acids 740-1633, 741-1634, 742-1635, 743-1636, 744-1637, 745-1638, 746-1639, 747-1640, 748-1641, 749-1642, 750-1643, 751-1644, 747-1638, 771-1666, 868-1562, 982-1562, 760-1639, 797-1562, 741-1646, 747-1560, 741-1648, 816-1598, 741-1648, as shown in FIG. 1. Other partial B domain deleted embodiments are also possible, including some in which certain B domain amino acids are replaced with a peptide linker or non-peptide chemical linker.

[0038] Although the embodiments of FIG. 1 and FIG. 2 are shown as single chain polypeptides, it should be understood that FVIII as used herein also includes inactive heterodimers that can result from proteolytic cleavage of the single chain form (intracellularly after expression or by some other means), as well as the active (e.g., FVIIIa) heterotrimeric or other activated forms of the these proteins resulting from extracellular cleavage by thrombin, FXa or some other protease. In a non-limiting example, the BDD FVIII protein illustrated in FIG. 2 can be processed intracellularly and secreted as an inactive heterodimer comprising a heavy chain having the amino acid sequence of SEQ ID NO:3 (shown in FIG. 3A) and a light chain having the amino acid sequence of SEQ ID NO:4 (shown in FIG. 3B). In some embodiments, the heavy chain only, the light chain only, or both the heavy and the light chains in the heterodimer are truncated by one or more amino acids compared to the corresponding amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4 respectively. Subunits within inactive FVIII heterodimers or activated FVIIIa heterotrimers can be held together non-covalently, as occurs naturally, or through peptide or non-peptide linkers added using recombinant or chemical means.

[0039] In other embodiments, FVIII includes proteins having FVIII activity and an amino acid sequence at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Percentage amino acid sequence identity can readily be determined using the Basic Local Alignment Search Tool (BLAST) algorithm as implemented at the website of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi), or some other sequence comparison algorithm familiar to those of ordinary skill in the art. In other embodiments, FVIII includes proteins having FVIII activity and a heavy chain with an amino acid sequence at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3 and/or a light chain with an amino acid sequence at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4.

[0040] As used herein, the term FVIII includes fragments of FVIII having FVIII activity and deletion mutations (including but not limited to that of the B domain) of FVIII having FVIII activity. FVIII also encompasses fusion proteins having FVIII activity, which can be constructed by fusing in frame at least one peptide or polypeptide to the amino-terminus and/or carboxy terminus of the FVIII protein (or fragment or deletion thereof), or by inserting at least one peptide or polypeptide in frame within the FVIII amino acid sequence at a location that does not abolish FVIII activity.

[0041] FVIII also encompasses naturally or non-naturally occurring variants or mutants of FVIII, including FVIII proteins comprising one or more amino acid substitutions, deletions or additions, each as compared to the wild type sequence. The amino acids can be the standard amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), or non-standard amino acids, such as pyrrolysine, selenocysteine, ornithine or others.

[0042] FVIII also encompasses one or more types of chemical derivatives or modifications of FVIII. Chemical modifications include but are not limited to post-translational modifications, including for example, N-linked or O-linked glycosylation, sulfation, or phosphorylation. Chemical modifications also include non-natural modifications, such as conjugation with a biocompatible polymer of the disclosure, or conjugation with any other chemical. Chemical modifications can be made enzymatically or using non-enzymatic chemical reactants. In some embodiments, modifications can occur in cells, such as mammalian cells, such as CHO cells, expressing FVIII. These modifications can occur as a result of ordinary cellular processes as FVIII is expressed and, in some embodiments, secreted from the cells. In other embodiments, cells can be engineered to express enzymes intended to modify FVIII in specified ways. In yet other embodiments, FVIII can be chemically derivatized or modified using reagents and methods familiar to those of ordinary skill in the art.

[0043] In other embodiments, the term FVIII refers to the FVIII muteins described in greater detail below, either alone or in the context of or in combination with any other FVIII embodiment disclosed herein or that may be familiar to one of ordinary skill in the art.

Modified FVIII Proteins

[0044] The present disclosure provides muteins of FVIII (sometimes referred to as modified FVIII proteins) comprising substitutions (alone and in combination with other substitutions or mutations as described herein) of one or more amino acids present in the wild type protein with cysteine. FVIII amino acid positions that may be substituted with cysteine are listed in Table 1, where the numbering of the mutation positions is in reference to the amino acid sequence of SEQ ID NO:1, which is the amino acid sequence of mature human wild type FVIII protein (see also FIG. 1). Importantly, muteins of the disclosure should not be construed as being limited by SEQ ID NO:1. Rather, also included within the scope of the disclosure are cysteine substitutions at correspondingn amino acids in other FVIII proteins (as that term is defined herein) that correspond to the positions in SEQ ID NO:1 that are specified by number in Table 1 and FIG. 1. FIG. 2, for example, shows the amino acid sequence of a particular human B-domain deleted (BDD) FVIII protein and identifies in bold underline font the cysteine substitution positions corresponding to those listed in Table 1. Those of ordinary skill in the art will readily be able to determine in the amino acid sequence of any other FVIII embodiment which positions correspond to those listed in Table 1. For example, one of ordinary skill can use computer software implementing amino acid sequence comparison algorithms, such as the Basic Local Alignment Search Tool (BLAST), or other algorithms, to compare SEQ ID NO:1 or SEQ ID NO:2 (or subsequences of these amino acid sequences) to any other FVIII protein amino acid sequence to find areas of similarity overall and correspondence between particular amino acids. The cysteine substitution mutations of the present disclosure are useful alone or as attachment sites for different kinds of moieties.

[0045] Without further modification, the cysteine substitution mutations are useful for a variety of purposes. For example, and without limitation, compared to unmutated FVIII the FVIII muteins of the present disclosure may be expressed at higher levels, have greater procoagulant activity, reduced immunogenicity, greater stability, reduced susceptibility to degradation, greater resistance to proteases (such as APC and FXa), greater resistance to oxidation, improved ability to be activated by thrombin or other coagulation factors, improved ability to combine with other coagulation factors and components into the tenase complex, increased association or binding to vWF, improved shelf-life, reduced binding by inhibitory antibodies, reduced interaction with the low density lipoprotein receptor related protein, reduced interaction with low density lipoprotein receptor, reduced interaction with cell surface heparin sulphate proteoglycans, increased circulatory half-life or have improved pharmacokinetics. Identification of FVIII muteins having these beneficial properties, and others, is within the skill of those ordinarily skilled in the art. Certain FVIII muteins having higher expression and/or greater procoagulant activity compared to unmutated BDD FVIII are identified in Table 1.

[0046] In other embodiments, the cysteine substitution mutations of the disclosure can serve as attachment sites for moieties where the combination of the FVIII mutein and moiety has improved performance or function and/or reduced side effects compared to FVIII muteins lacking the attached moieties. For example, and without limitation, compared to unmutated FVIII, FVIII muteins bearing moieties may have greater procoagulant activity, reduced immunogenicity, greater stability, reduced susceptibility to degradation, greater resistance to proteases (such as APC and FXa), greater resistance to oxidation, improved ability to be activated by thrombin or other coagulation factors, improved ability to combine with other coagulation factors and components into the tenase complex, increased association or binding to vWF, improved shelf-life, reduced binding by inhibitory antibodies, reduced interaction with the low density lipoprotein receptor related protein, reduced interaction with low density lipoprotein receptor, reduced interaction with cell surface heparin sulphate proteoglycans, increased circulatory half-life or have improved pharmacokinetics. Identification of combinations of FVIII muteins and moieties having these beneficial properties, and others, is within the skill of those ordinarily skilled in the art.

[0047] According to certain embodiments, moieties that can be attached directly or indirectly to the cysteine substitution mutations of the disclosure include, without limitation, naturally occurring or synthetic small organic molecules, macromolecules, antibodies, antibody fragments, antigen binding domains, antibody Fc regions, other proteins of immunologic origin or function, complete clotting factors, functional fragments of clotting factors, enzymes, nucleic acids, DNA, RNA, organometallic compounds, lipids, fatty acyl chains, phospholipids, glycolipids, proteins, peptides, amino acids, carbohydrates, monosaccharides, disaccharides, hydrophobic compounds, hydrophilic compounds, organic acids, organic bases, and others.

[0048] In other embodiments, moieties can be attached to the FVIII muteins of the present disclosure on sites other than the cysteine substitution mutations disclosed herein. For example, without limitation, moieties can be attached to amines exposed on the surface of the FVIII mutein, exposed glycosylation sites, exposed tyrosines, and other sites.

[0049] According to certain embodiments attaching a moiety, such as a biocompatible polymer, to one or more of the cysteine substitution mutations of the disclosure is useful for increasing circulatory half-life.

[0050] Without wishing to be bound by any particular theory of operation, it is believed that attaching a biocompatible polymer or other moiety to the cysteine mutations of the disclosure may be effective to increase circulatory half-life through a variety of mechanisms. One such mechanism includes eliminating or obscuring FVIII epitopes to which inhibitory antibodies are generated and bind. Regions of FVIII that interact with inhibitory antibodies include amino acids 66-75, 288-297, 306-315, 378-387, 484-509, 636-645, 648-657, 1779-1818, 2181-2243, and 2248-2319. Particular residues implicated in inhibitory antibody binding include 484, 487, 489, and 492. Another mechanism includes preventing FVIII interaction with the low density lipoprotein receptor related protein (LRP). Regions of FVIII that interact with LRP include amino acids 484-509, 1811-1818, and 2092-2093. Another mechanism includes preventing FVIII interaction with low density lipoprotein (LDL) receptor. Another mechanism includes preventing FVIII interaction with cell surface heparin sulphate proteoglycans (HSPG). A region of FVIII that interacts with heparins includes amino acids 558-566. Another mechanism includes eliminating or obscuring FVIII proteolytic cleavage sites, such as that of APC or other proteases. Particular FVIII amino acids that are involved in cleavage by APC include 336 and 562. Another possible mechanism is to increase or decrease binding by vWF. Other mechanisms by which attaching a biocompatible polymer to the cysteine mutations of the disclosure can increase circulatory half-life are possible, and the particular mechanism or mechanisms by which such improvement in half-life occurs is not intended to be limiting in any way.

[0051] FVIII mutations of the present disclosure are set forth in Table 1 along with data regarding expression levels and procoagulant activity compared to unmutated BDD FVIII. Expression was estimated by Western analysis using an antibody to FVIII. It is therefore possible that certain substitutions interfered with antibody binding such that actual expression levels may in some instances be greater than indicated.

[0052] Many of the cysteine substitution mutations tested demonstrated substantial levels of expression and procoagulant activity compared with unmutated BDD FVIII. In some cases, the mutations demonstrated expression levels or activity that was even greater than unmutated BDD FVIII (see, e.g., positions 495, 1806, 336, 1778 and 2093). The fact that many of the cysteine substitutions express well and possess substantial procoagulant activity is surprising because mutations in proteins are often associated with substantial or even complete abrogation of protein function. Even those cysteine mutations having low apparent expression and/or activity, however, may have sufficient expression or activity to usefully serve as procoagulants in the methods of the disclosure.

TABLE-US-00001 TABLE 1 FVIII cysteine substitutions in mature FVIII amino Mutation acid sequence ID no. (SEQ ID NO: 1) 1 F59C 2 N239C 3 P333C 4 R336C 5 P379C 6 T481C 7 R484C 8 L486C 9 S488C 10 R489C 11 R490C 12 P492C 13 K493C 14 V495C 15 K496C 16 H497C 17 K499C 18 D500C 19 F5C1C 20 E5C7C 21 Y555C 22 R562C 23 S568C 24 R571C 25 N582C 26 Y1680C 27 Q1778C 28 E1793C 29 E1794C 30 R1797C 31 Q1798C 32 G1799C 33 A1800C 34 E1801C 35 F1806C 36 N1810C 37 E1811C 38 T1814C 39 F1816C 40 K1818C 41 F1891C 42 F2035C 43 F2068C 44 K2092C 45 F2093C 46 S2094C 47 S2095C 48 N2118C 49 V2125C 50 K2183C 51 S2186C 52 T2191C 53 F2196C 54 S2204C 55 S2206C 56 L2212C

[0053] In certain embodiments of the disclosure, FVIII muteins comprise a single cysteine substitution mutation selected from Table 1. In related embodiments, a biocompatible polymer is conjugated to the cysteine so as to increase the circulating half-life of the FVIII so modified. In other embodiments, FVIII muteins comprise at least two cysteine substitution mutations from Table 1. In related embodiments, identical or different biocompatible polymers are conjugated to each cysteine to increase the circulating half-life of the FVIII so modified. Combinations of multiple cysteine mutations from Table 1 greater than two are also possible.

[0054] When two or more cysteine mutations from Table 1 are combined in the same FVIII mutein, they can be combined in different permutations, as explained in the following non-limiting examples where the mutation number refers to the mutation identification number in the left-most column of Table 1. Thus, in some embodiments, mutation 1 is combined with one or more of the other mutations numbered 2-56. In other embodiments, mutation 2 is combined with one or more of the other mutations numbered 1 or 3-56. In other embodiments, mutation 3 is combined with one or more of the other mutations numbered 1-2 or 4-56. In other embodiments, mutation 4 is combined with one or more of the other mutations numbered 1-3 or 5-56. In other embodiments, mutation 5 is combined with one or more of the other mutations numbered 1-4 or 6-56. In other embodiments, mutation 6 is combined with one or more of the other mutations numbered 1-5 or 7-56. In other embodiments, mutation 7 is combined with one or more of the other mutations numbered 1-6 or 8-56. In other embodiments, mutation 8 is combined with one or more of the other mutations numbered 1-7 or 9-56. In other embodiments, mutation 9 is combined with one or more of the other mutations numbered 1-8 or 10-56. In other embodiments, mutation 10 is combined with one or more of the other mutations numbered 1-9 or 11-56. In other embodiments, mutation 11 is combined with one or more of the other mutations numbered 1-10 or 12-56. In other embodiments, mutation 12 is combined with one or more of the other mutations numbered 1-11 or 13-56. In other embodiments, mutation 13 is combined with one or more of the other mutations numbered 1-12 or 14-56. In other embodiments, mutation 14 is combined with one or more of the other mutations numbered 1-13 or 15-56. In other embodiments, mutation 15 is combined with one or more of the other mutations numbered 1-14 or 16-56. In other embodiments, mutation 16 is combined with one or more of the other mutations numbered 1-15 or 17-56. In other embodiments, mutation 17 is combined with one or more of the other mutations numbered 1-16 or 18-56. In other embodiments, mutation 18 is combined with one or more of the other mutations numbered 1-17 or 19-56. In other embodiments, mutation 19 is combined with one or more of the other mutations numbered 1-18 or 20-56. In other embodiments, mutation 20 is combined with one or more of the other mutations numbered 1-19 or 21-56. In other embodiments, mutation 21 is combined with one or more of the other mutations numbered 1-20 or 22-56. In other embodiments, mutation 22 is combined with one or more of the other mutations numbered 1-21 or 23-56. In other embodiments, mutation 23 is combined with one or more of the other mutations numbered 1-22 or 24-56. In other embodiments, mutation 24 is combined with one or more of the other mutations numbered 1-23 or 25-56. In other embodiments, mutation 25 is combined with one or more of the other mutations numbered 1-24 or 26-56. In other embodiments, mutation 26 is combined with one or more of the other mutations numbered 1-25 or 27-56. In other embodiments, mutation 27 is combined with one or more of the other mutations numbered 1-26 or 28-56. In other embodiments, mutation 28 is combined with one or more of the other mutations numbered 1-27 or 29-56. In other embodiments, mutation 29 is combined with one or more of the other mutations numbered 1-28 or 30-56. In other embodiments, mutation 30 is combined with one or more of the other mutations numbered 1-29 or 31-56. In other embodiments, mutation 31 is combined with one or more of the other mutations numbered 1-30 or 32-56. In other embodiments, mutation 32 is combined with one or more of the other mutations numbered 1-31 or 33-56. In other embodiments, mutation 33 is combined with one or more of the other mutations numbered 1-32 or 34-56. In other embodiments, mutation 34 is combined with one or more of the other mutations numbered 1-33 or 35-56. In other embodiments, mutation 35 is combined with one or more of the other mutations numbered 1-34 or 36-56. In other embodiments, mutation 36 is combined with one or more of the other mutations numbered 1-35 or 37-56. In other embodiments, mutation 37 is combined with one or more of the other mutations numbered 1-36 or 38-56. In other embodiments, mutation 38 is combined with one or more of the other mutations numbered 1-37 or 39-56. In other embodiments, mutation 39 is combined with one or more of the other mutations numbered 1-38 or 40-56. In other embodiments, mutation 40 is combined with one or more of the other mutations numbered 1-39 or 41-56. In other embodiments, mutation 41 is combined with one or more of the other mutations numbered 1-40 or 42-56. In other embodiments, mutation 42 is combined with one or more of the other mutations numbered 1-41 or 43-56. In other embodiments, mutation 43 is combined with one or more of the other mutations numbered 1-42 or 44-56. In other embodiments, mutation 44 is combined with one or more of the other mutations numbered 1-43 or 45-56. In other embodiments, mutation 45 is combined with one or more of the other mutations numbered 1-44 or 46-56. In other embodiments, mutation 46 is combined with one or more of the other mutations numbered 1-45 or 47-56. In other embodiments, mutation 47 is combined with one or more of the other mutations numbered 1-46 or 48-56. In other embodiments, mutation 48 is combined with one or more of the other mutations numbered 1-47 or 49-56. In other embodiments, mutation 49 is combined with one or more of the other mutations numbered 1-48 or 50-56. In other embodiments, mutation 50 is combined with one or more of the other mutations numbered 1-49 or 51-56. In other embodiments, mutation 51 is combined with one or more of the other mutations numbered 1-50 or 52-56. In other embodiments, mutation 52 is combined with one or more of the other mutations numbered 1-51 or 53-56. In other embodiments, mutation 53 is combined with one or more of the other mutations numbered 1-52 or 54-56. In other embodiments, mutation 54 is combined with one or more of the other mutations numbered 1-53 or 55-56. In other embodiments, mutation 55 is combined with one or more of the other mutations numbered 1-54 or 56. In other embodiments, mutation 56 is combined with one or more of the other mutations numbered 1-55.

[0055] In other embodiments, one or more of the cysteine mutations from Table 1 can be combined in a FVIII mutein with substitutions at different positions with amino acids other than cysteine. Thus, in certain embodiments, one or more of the cysteine mutations from Table 1 can be combined with one or more substitutions at other positions that reduce or eliminate binding by inhibitory antibodies, that reduce or eliminate interaction of FVIII with LRP, reduce or eliminate interaction of FVIII with LDL receptor, reduce or eliminate FVIII interaction with heparin or HSPGs, or that prevent cleavage of FVIII by APC or other proteases responsible for the short circulatory half-life of FVIII.

[0056] According to certain embodiments, any one or more of the cysteine mutations from Table 1 can be combined in FVIII with one or more substitution mutations selected from the group consisting of Y1680F, R336A, R562A, and K1968A. The Y1680F substitution is expected to abolish vWF binding, the R336A and R562A substitutions are expected to interfere with proteolytic cleavage of FVIII by APC or FXa, and the K1968A substitution is expected to increase the stability of activated FVIII. Thus, according to certain non-limiting exemplary embodiments, FVIII muteins comprise or consist of the following combinations of two mutations in FVIII (referring to the amino acid numbering of SEQ ID NO:1): K493C+R336A, K496C+R336A, G1799C+R336A, 52094C+R336A, 52186C+R336A, 52204C+R336A, 52206C+R336A, K493C+Y1680F, K496C+Y1680F, G1799C+Y1680F, 52094C+Y1680F, 52186C+Y1680F, 52204C +Y1680F, and 52206C+Y1680F. According to yet further non-limiting exemplary embodiments, FVIII muteins comprise or consist of the following combinations of three mutations in FVIII (referring to the amino acid numbering of SEQ ID NO:1): K493C+R336A+Y1680F, K496C+R336A+Y1680F, G1799C+R336A+Y1680F, 52094C+R336A+Y1680F, 52186C+R336A+Y1680F, 52204C+R336A+Y1680F, and S2206C+R336A+Y1680F. Additional combinations of substitution mutations in FVIII are also possible.

[0057] Mutations in FVIII, such as without limitation those listed in Table 1, can be made using site directed mutagenesis and other techniques familiar to those of ordinary skill in the art, and confirmed by sequencing.

[0058] Using tools and techniques of molecular biology familiar to those of ordinary skill in the art, nucleic acids encoding the FVIII muteins of the disclosure can be introduced into a variety of prokaryotic or eukaryotic expression vectors known in the art. Expression vectors can then be introduced into prokaryotic or eukaryotic host cells for eventual expression of the FVIII muteins. Suitable host cells are familiar to those of ordinary skill in the art and include without limitation mammalian cell lines, insect cell lines, plant cell lines, fungal cells (such as yeasts), and bacterial cells (such as E. coli.). Use of other host cells is also possible. Exemplary mammalian cell lines that can serve as host cells for expression of FVIII muteins includes CHO cells, COS-1 cells, BHK cells and HEK293 cells. Use of other mammalian cell lines as hosts is also possible. Host cells can be transiently or stably transfected or transformed with nucleic acids encoding the FVIII muteins of the disclosure, optionally inserted in expression vectors. In some embodiments, expression from such vectors can be constituitive or inducible.

[0059] Host cells can be grown under conditions supporting expression of the FVIII muteins of the disclosure. Muteins so produced can be purified from the host cells and/or growth media using techniques familiar to those of ordinary skill in the art. Thereafter, the purified FVIII muteins of the present disclosure can be treated chemically to attach one or more types of biocompatible polymers. The resulting conjugates can then be tested to determine whether the presence of the biocompatible polymer in the conjugates increases circulatory half-life of the modified FVIII mutein. Methods for measuring circulatory half-life of the conjugates are familiar to those of ordinary skill in the art.

[0060] In some embodiments, a conjugate of a FVIII mutein and a biocompatible polymer of the disclosure has a circulatory half-life that is increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000% or more compared to the unmutated FVIII protein used to generate the mutein or the unconjugated mutein lacking the biopolymer. In other embodiments, the circulatory half-life of a conjugate of a FVIII mutein and a biocompatible polymer of the disclosure is increased about 1.5 to 20 fold, 1.5 to 15 fold, 1.5 to 10 fold, 1.5 to 9 fold, 1.5 to 8 fold, 1.5 to 7 fold, 1.5 to 6 fold, 1.5 to 5 fold, 1.5 to 4 fold, 1.5 to 3 fold, or 1.5 to 2 fold compared to the unmutated FVIII protein used to generate the mutein or the unconjugated mutein lacking the biopolymer. In yet other embodiments, the circulatory half-life of a conjugate of a FVIII mutein and a biocompatible polymer of the disclosure is increased about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10 times or more compared to the unmutated FVIII protein used to generate the mutein or the unconjugated mutein lacking the biopolymer. In some embodiments, the circulatory half-life of a conjugate of a FVIII mutein and a biocompatible polymer of the disclosure is at least about 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 65 hours, 70 hours, 75 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 140 hours, 150 hours, one week, two weeks, three weeks, or more compared to the unmutated FVIII protein used to generate the mutein or the unconjugated mutein lacking the biopolymer. In any of the foregoing embodiments, the FVIII mutein can comprise any one or more of the cysteine substitution mutations identified in Table 1 and the biocompatible polymer to which said mutein is conjugated can be a polyethylene glycol (PEG), a hydroxyalkyl starch, such as hydroxyethyl starch (HES), or another biocompatible polymer.

Factor VIII Activity

[0061] The activity, or potency, of FVIII muteins and conjugates thereof in a patient sample can be measured using a variety of bioassays known in the art, including the one-stage method, the two-stage method and the chromogenic method, which is based on the two-stage method. Morfini et al, J Thomb Haemost 1:2283-9 (2003); Mikaelsson et al, Semin Thromb Hemost 28:257-64 (2002); Mackie et al, Int J Lab Hematol 35(1):1-13 (2013); Over, J., Scand J Haematol, 33(Suppl. 41):13-24 (1984); Gallimore et al, Blood Rev 5:117-27 (1991); Barrowcliffe et al, Semin Thromb Hemost 28(3):247-56 (2002); Barrowcliffe, T W, Scand J Haematol 33(Suppl 41):25-38 (1984); Rosen et al in Triplett D A, ed. Advances in Coagulation Testing, Skokie, Ill.: College of American Pathologists, 255-260 (1986); each of which is incorporated by reference.

[0062] In one embodiment, FVIII activity can be measured using the one-stage assay. The one-stage assay measures the ability of a patient plasma sample to shorten the activated partial thromboplastin time (aPTT) of FVIII deficient plasma confirmed to contain adequate von Willebrand factor. The depleted plasma is obtained either from a patient with Hemophilia A or by immunodepletion of FVIII. The FVIII deficient plasma and test sample are preincubated with the aPTT reagent which contains a contact activator (e.g., ellagic acid, kaolin, silica, cellite) and phospholipid. Calcium chloride is added and time to fibrin clot formation is measured. FVIII concentration in the test sample is assumed to be the rate-limiting determinant of the clotting time. The result is compared with a standard curve generated from samples containing known FVIII activities (e.g., serial dilutions of a standard reference plasma) and the relative FVIII activity in the test sample is calculated.

[0063] In another embodiment, FVIII activity can be measured in a patient sample using the two-stage FVIII-clotting assay. In the first stage of the assay, FXa and prothrombinase are produced using a reaction mixture containing excess phospholipid, calcium, Factor V (from bovine serum), and clotted human serum from a patient sample as a source of FIXa and FX. Alumina hydroxide is used to adsorb prothrombin and other activated and vitamin K dependent factors to prevent clotting. The amount of functional FVIII in the sample determines the rate of FXa generation. In the second stage of the assay, pooled normal plasma is added as a source of prothrombin and fibrinogen. Clotting time is inversely proportional to the amount of FVIII in the test sample, which can be calculated by comparison to a standard curve generated using normal human plasma or other positive control.

[0064] According to another embodiment FVIII activity can be measured using a chromogenic assay which also consists of two stages. In the first stage, a test sample obtained from a patient is added to a reaction mixture containing thrombin or prothrombin, FIXa, FX, calcium, and phospholipid to produce FVIIIa, which works in concert with FIXa to activate FX. Production of FXa is assumed to be proportional to the amount of functional FVIII present in the sample. In the second stage FXa activity is measured. A chromogenic FXa peptide substrate is added and incubated for a predetermined time, after which the amount of chromogen produced is detected photometrically at the wavelength characteristic for the chromogen (e.g., 405 nm for p-nitroaniline). Chromogen concentration is assumed to be proportional to FXa activity and to FVIII activity in the test sample. FVIII activity can then be calculated by comparison to a standard curve generated using normal human plasma or other positive control.

[0065] FVIII activity can also be measured using a Thrombin Generation Assay (TGA) or using thromboelastography (TEG). TGA measures the initiation phase, activation phase and inactivation phase of thrombin generation as a function of FVIII activity. Varadi et al., Haemophilia 10 (Suppl. 2):17-21 (2004); Hemker, et al., Pathophysiol Haemost Thromb 33:4-15 (2003); each of which is incorporated by reference. Thrombin generation can be determined using the Calibrated Automated Thrombography (CAT) system (Thrombinoscope BV, Maastricht, Netherlands) and TGA reagent. TEG, and a related methodology called rotational thromboelastogram (ROTEM), measures the visco-elastic properties of whole blood clot formation under low shear stress though time. Young, et al., Blood 121(11):1944-50 (2013); Ganter, et al., Anesth Analg 106(5):1366-75 (2008), which is incorporated by reference.

[0066] The assays described above can also be modified to permit comparative testing of the activity of the different FVIII muteins and conjugates of the disclosure. For example, instead of testing patient samples, plasma from Hemophilia A patients, or normal human plasma depleted of FVIII using antibodies, can be spiked with different amounts of FVIII muteins or conjugates thereof to be tested and the assays carried out as otherwise described.

Biocompatible Polymers

[0067] Biocompatible polymers are selected so that the conjugate resulting from the combination of a FVIII mutein and biocompatible polymer exhibits increased circulatory half-life compared to the same FVIII mutein lacking the biocompatible polymer. At the same time, however, the presence of the biocompatible polymer should not unacceptably increase immunogenicity of the modified FVIII mutein, nor have unacceptably high toxicity or side effects, nor unacceptably reduce the function of the modified mutein as a procoagulant. Methods for measuring circulatory half-life and assessing immunogenicity, toxicity, side effects, and procoagulant properties of FVIII muteins modified by the addition of a biocompatible polymer are within the knowledge of the ordinarily skilled artisan.

[0068] Biocompatible polymers useful for attaching to FVIII muteins of the present disclosure to increase circulatory half-life include linear and branched polymers. Exemplary biocompatible polymers include but are not limited to polyalkylene glycols, polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, polybutylene glycols (PBG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), polypeptides, polysaccharides, polyethers, polyamines, polycarboxylic acids, polysialic acid, polyethylene imine, polyacrylic acid, polyoxyethylated polyol, polyolefmic alcohol, polyvinylpyrrolidone, polyhydroxypropylmethacrylamide, poly(alpha)-hydroxy acid, polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline, poly-N-acryloylmorpholine, polyacryloylmorpholine, 2-(methacryloyloxy)ethyl phosphorylcholine (mPC) polymers, dextrans, colominic acids, biotin derivatives, polycarboxylates, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, heparin, albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, and alginic acid hydrolysates. Others are possible.

Polyethylene Glycol (PEG)

[0069] In certain embodiments, a biocompatible polymer useful for attaching to FVIII muteins of the present disclosure is polyethylene glycol, abbreviated PEG. As used herein, PEG includes any water soluble poly(ethylene oxide). In some embodiments, PEG comprises the structure --(OCH.sub.2CH.sub.2).sub.n-- where n=2 to 4000. PEG also includes CH.sub.2CH.sub.2--O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2-- and --(OCH.sub.2CH.sub.2).sub.nO--. PEG also refers to polymers comprising more than 50% OCH.sub.2CH.sub.2-repeating subunits and less than 50% of other types of subunits. PEG includes without limitation PEG polymers of varied structures, including without limitation, branched PEG, linear PEG, forked PEG and multi-armed PEG (star-PEG) configurations.

[0070] PEG may include various terminal or end capping groups, such as without limitation a hydroxyl group, an alkoxy group, a C.sub.1-C.sub.20 alkoxy group, a substituted alkoxy group, alkenoxy group, substituted alkenoxy group, alkynoxy group, substituted alkynoxy group, aryloxy group, and substituted aryloxy group. In some embodiments, PEG is methoxypolyethylene glycol (mPEG).

[0071] PEG includes PEG polymers of any molecular weight PEG. According to certain non-limiting exemplary embodiments, PEG polymers can range from 1 kD to 100 kD, 2 kD to 80 kD, 3 kD to 70 kD, 4 kD to 60 kD, 5 kD to 50 kD, and 5 kD to 40 kD. Other ranges are possible.

Hydroxyalkyl Starch

[0072] In certain embodiments, the biocompatible polymer is hydroxyalkyl starch (HAS), which is a derivative of starch. Starches are carbohydrate polymers of many glucose subunits (also called units or monomers) joined to each other via glycosidic bonds. Starches may be linear or branched depending on the types of glycosidic bonds that are present. Specific examples of starches are amylose, which is predominantly linear although may have a few branches, amylopectin and glycogen, both of which have many more branch points than amylose. Amylose can also adopt a helical structure.

[0073] In amylose, most or all of the glucose subunits are connected to each other via .alpha.-1,4-glycosidic bonds and relatively few or no glucose subunits are connected via .alpha.-1,6-glycosidic bonds which create branch points permitting new chains of glucose subunits connected via .alpha.-1,4-glycosidic bonds to form off the main chain. By contrast, amylopectin and glycogen comprise glucose subunits linked to each other via .alpha.-1,4-glycosidic bonds as well as .alpha.-1,6-glycosidic bonds resulting in many more branches compared to amylose. In amylopectin, branching occurs at .alpha.-1,6-glycosidic bonds occurring about every 20 to 30 glucose units. Glycogen is even more branched, with branching occurring at .alpha.-1,6-glycosidic bonds occurring about every 8 to 12 glucose units. Starches can comprise a wide ranging number of glucose subunits, numbering from the dozens to many thousands. In some non-limiting embodiments, starches can comprise about 10-300,000 glucose subunits, about 50 to 200,000 glucose subunits, about 50 to 100,000 glucose subunits, about 50 to 50,000 glucose subunits, about 100 to 10,000 glucose subunits, and other ranges.

[0074] Hydroxyalkyl starch can be made from any type of starch, including without limitation amylose, amylopectin or glycogen, as well as any other type of starch known in the art. Starches from which hydroxyalkyl starches can be prepared include, but are not limited to, cereal starches and potato starches. Cereal starches include, but are not limited to, rice starches, wheat starches such as einkorn starches, spelt starches, soft wheat starches, emmer starches, durum wheat starches, or kamut starches, corn starches, rye starches, oat starches, barley starches, triticale starches, spelt starches, and millet starches such as sorghum starches or teff starches. Starches from which hydroxyalkyl starches are prepared desirably have a high content of amylopectin relative to amylose. The amylopectin content of these starches can be, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% by weight, or be up to 96%, 97%, 98%, 99%, or 100% by weight. Exemplary starches having an especially high amylopectin content include certain potato starches such as waxy potato starches which can be extracted from essentially amylose-free potatoes that are either traditionally cultivated (e.g., the natural variety Eliane) or genetically modified amylopectin potato varieties, and starches of waxy varieties of cereals such as waxy corn or waxy rice.

[0075] Hydroxyalkyl starch can comprise any number of glucose subunits. For example, in some embodiments, hydroxyalkyl starch can include at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, or more glucose subunits, and ranges between any of the foregoing numbers of glucose subunits. According to certain other embodiments, hydroxyalkyl starch contains from 1 to 2000, 5 to 1000, 10 to 500, 20 to 250, 50 to 200, or 75 to 150 glucose subunits. Other ranges are also possible.

[0076] Without limitation, hydroxyalkyl starch includes starches wherein the glucose subunits are linked to each other via .alpha.-1,4-glycosidic bonds only, in which case the starch will be completely unbranched, or a combination of .alpha.-1,4-glycosidic bonds and .alpha.-1,6-glycosidic bonds, in which case the starch will exhibit some degree of branching. Thus, hydroxyalkyl starch can demonstrate any degree of branching, from no branching to very highly branched. The degree of branching can be expressed as the ratio of the average number of .alpha.-1,4-glycosidic bonds to the average number of .alpha.-1,6-glycosidic bonds in a starch. In some embodiments, this ratio can be about 1000:1, 500:1, 400:1, 300:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, or 4:1. For amylopectin, the branching ratio ranges from about 30:1 to about 20:1, whereas for glycogen, the branching ratio ranges from about 12:1 to about 8:1. Due to their branched structure, the hydroxyalkyl starches of the disclosure have a plurality of terminal glucose monomers. In some embodiments, the number of terminal glucose monomers ranges from 2 to about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more.

[0077] Hydroxyalkyl starch is a derivative of starch comprising substitution at any one or more of the hydroxyl groups of the C.sub.2, C.sub.3 or C.sub.6 carbon atoms of any one or more of the saccharide units in the starch with at least one hydroxyalkyl group. According to certain non-limiting embodiments, hydroxyalkyl starches of the disclosure include those having the following structure:

##STR00001##

[0078] Depicted in formula (I) is the terminal saccharide unit at the reducing end of the starch molecule in the non-oxidized, hemiacetal form which may, depending on solvent or other conditions, be in equilibrium with the aldehyde form. In reference to formula (I) the abbreviations HAS' and HAS'' refer to one or more additional saccharide units that, with the terminal saccharide unit, are present in the same starch molecule. In formula (I), R.sup.b and R.sup.c are each --[(CR.sup.1R.sup.2).sub.mO].sub.n--H, being the same or different from each other, and R.sup.a is either --[(CR.sup.1R.sup.2).sub.mO].sub.n--H (in which case it can be the same or different as R.sup.b and/or R.sup.c) or HAS''. In R.sup.a, R.sup.b and Re, R.sup.1 and R.sup.2 are independently --H or an alkyl group having 1 to 4 carbon atoms, m is independently 2 to 4, and n is independently 0 to 6, with R.sup.1 and R.sup.2 being the same or different from each other. When R.sup.a is --[(CR.sup.1R.sup.2).sub.mO].sub.n--H, HAS' is present representing additional saccharide unit(s) of the starch molecule. When R.sup.a is HAS'', HAS' is also present, both HAS' and HAS'' representing additional saccharide unit(s) of the starch molecule.

[0079] In some non-limiting embodiments of the disclosure, the hydroxyalkyl starch is hydroxyethyl starch (HES). Without limitation, HES can be defined using formula (I) in which R.sup.b and R.sup.c are each --(CH.sub.2--CH.sub.2--O).sub.n--H, being the same or different from each other, and R.sup.a is either --(CH.sub.2--CH.sub.2--O).sub.n--H (in which case it can be the same or different as R.sup.b and/or R.sup.c) or HAS''. In R.sup.a, R.sup.b and Re, n is independently 0 to 6. When R.sup.a is --(CH.sub.2--CH.sub.2--O).sub.n--H, HAS' is present representing additional saccharide unit(s) of the starch molecule. When R.sup.a is HAS'', HAS' is also present, both HAS' and HAS'' representing additional saccharide unit(s) of the starch molecule.

[0080] In some non-limiting embodiments of HES, HAS' is present, R.sup.b and R.sup.c are independently --H or --CH.sub.2--CH.sub.2--OH, and R.sup.a is independently --H, --CH.sub.2--CH.sub.2--OH, or HAS'', HAS' and HAS'' representing additional saccharide unit(s) of the starch molecule.

[0081] HAS or HES, as defined herein, are not limited to starch derivatives where only the terminal saccharide unit is derivatized by hydroxyalkyl groups, such as the groups --[(CR.sup.1R.sup.2).sub.mO].sub.n--H or --(CH.sub.2--CH.sub.2--O).sub.n--H described with respect to formula (I). Rather, HAS or HES of the disclosure include derivatized starch molecules where at least one hydroxy group present anywhere in the starch, whether in the reducing end terminal saccharide unit or some other saccharide unit in the starch, is modified with a hydroxyalkyl group, such as --[(CR'R.sup.2).sub.mO].sub.n--H in the case of HAS, or --(CH.sub.2--CH.sub.2--O).sub.n--H in the case of HES.

[0082] In certain embodiments of HAS or HES, hydroxyalkyl groups contain one hydroxyl group. In other embodiments, hydroxyalkyl groups can contain 2, 3, 4, 5, 6 or more hydroxyl groups. In yet other embodiments of HAS or HES, all hydroxyalkyl groups in the starch have the same number hydroxyl groups, for example one hydroxyl group, or hydroxyalkyl groups in the starch have different numbers of hydroxyl groups. According to some embodiments of HAS or HES, hydroxyalkyl groups may be monosubstituted or polysubstituted with heteroatoms or groups in addition to hydroxyl.

[0083] In addition, HAS or HES, as defined herein, are not limited to starch derivatives where each derivatized saccharide unit (whether the terminal residue at the reducing end or others) has the same substitutions of hydroxyalkyl moieties, but rather includes starch derivatives where substituted saccharides have distinct and different patterns of substitutions and substitutions with differently structured hydroxyalkyl moieties. According to certain embodiments, substitution with a hydroxyalkyl moiety occurs at the hydroxyl group bonded to any one or more of the C.sub.2, C.sub.3 or C.sub.6 carbon atoms of any one or more of the saccharide units in the starch, but substitution could occur at other hydroxyl groups as well. Typically, a plurality of saccharide units will be substituted with at least one hydroxyalkyl group. In such embodiments, the substituted hydroxyl groups may be substituted with the same type or different types of hydroxyalkyl groups, such as those identified below.

[0084] In some embodiments, hydroxyalkyl starch is an ether derivative of starch. According to other embodiments, hydroxyalkyl starch includes derivatives of starch in which any one or more of the hydroxyl groups of the C.sub.2, C.sub.3 or C.sub.6 carbon atoms of any one or more of the saccharide units in the starch is esterified. Without limitation, such esterified derivatives include unsubstituted mono- or dicarboxylic acids with 2-12 carbon atoms, 2-6 carbon atoms, or substituted derivatives thereof. In some embodiments, the ester derivatives are unsubstituted monocarboxylic acids with 2-6 carbon atoms. For other embodiments, the saccharide units are esterified with acetic acid, propionic acid, butyric acid and other mono- or dicarboxylic acids. In some embodiments esterified with dicarboxylic acids, the second carboxy group of the dicarboxylic acid is also esterified. In other embodiments containing mono- or dicarboxylic acids, the mono- or dicarboxylic acids are monosubstituted or polysubstituted. In non-limiting examples, the mono- or dicarboxylic acids are substituted with a halogen, such as fluorine, or an aryl group.

[0085] As used herein, the term alkyl appearing in the term hydroxyalkyl refers to a hydrocarbon chain, and may contain any number of carbon atoms, such as 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 011, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or more carbon atoms. Such hydrocarbon chains may be saturated, partially unsaturated, or unsaturated, and may be linear (i.e., straight chain), branched or cyclic. Exemplary alkyl groups include but are not limited to methyl, ethyl, propyl, butyl (e.g., n-butyl, i-butyl, and t-butyl), pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl.

[0086] Alkyl can also refer to cycloalkyl as well as cycloalkylene-containing alkyl groups. Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, and may include 3 to about 12 carbon atoms, such as 3 to about 8 carbon atoms. Cycloalkylene refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.

[0087] In some embodiments, the alkyl moiety of the hydroxyalkyl group can itself be further substituted with any atom or group of atoms compatible with the biological function of FVIII and that does not cause unacceptable immunogenicity, toxicity or other negative side effects. Alkyl groups can be substituted in any position by one or more substituents, for example by 1, 2, 3, 4, 5 or 6 substituents. If two or more substituents are present, each substituent may be the same as or different from the at least one other substituent. Non-limiting examples of atoms or groups of atoms that may be used as substituents to modify hydroxyalkyl groups of the disclosure include hydroxyl group, alkyl group, C.sub.3-8 cycloalkyl group (e.g., cyclopropyl or cyclobutyl groups), halogens (e.g., fluorine, chlorine, bromine, or iodine atoms), cyano group, alkoxy group (e.g., methoxy, ethoxy, or propyloxy groups), phenyl group, substituted phenyl group (on which ring substituents may be positioned at one or more of the ortho, meta, or para positions), aryl group, substituted aryl group, heteroaryl group, substituted heteroaryl group, heterocycle group or substituted heterocycle group.

[0088] Aryl means one or more aromatic rings, each of 5 or 6 core carbon atoms. Aryl includes multiple aryl rings that may be fused, as in naphthyl, or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. Heteroaryl is an aryl group containing from one to four heteroatoms, such as sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings. Heterocycle means one or more rings of 5-12 atoms, such as 5-7 atoms, that may be saturated, unsaturated, or partially unsaturated, with or without aromatic character, and having at least one ring atom that is not carbon. Exemplary heteroatoms include sulfur, oxygen, and nitrogen.

[0089] In certain non-limiting embodiments, the hydroxyalkyl group of the disclosure is selected from among hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl (including 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxyisopropyl and 2-hydroxyisopropyl), hydroxyethyl (including 1-hydroxyethyl and 2-hydroxyethyl), as well as hydroxymethyl groups. In some other embodiments, just one type of these hydroxyalkyl groups is present in a hydroxyalkyl starch, whereas in other embodiments, two or more types of these hydroxyalkyl groups are present in a hydroxyalkyl starch.

[0090] In some embodiments, the hydroxyalkyl starch is hydroxyethyl starch (HES) in which the starch is amylose, amylopectin or glycogen modified by substitution at one or more glucose hydroxyl groups with a hydroxyethyl group, which may be 1-hydroxyethyl and/or the 2-hydroxyethyl group. Thus, in some HES embodiments, all hydroxyethyl groups are 1-hydroxyethyl, whereas in other HES embodiments, all hydroxyethyl groups are 2-hydroxyethyl. In some other HES embodiments, hydroxyethyl groups include both 1-hydroxyethyl and 2-hydroxyethyl, in equal proportions to each other, or in different proportions to each other.

[0091] Hydroxyalkyl starches, such as hydroxyethyl starch, can be characterized in different ways. One way to characterize HAS, such as HES, is by the molecular weight of the carbohydrate polymers. As will be familiar to one of ordinary skill in the art of polymer chemistry, molecular weight can be defined and calculated in at least two ways. According to one embodiment, molecular weight is expressed as the number average molecular weight, abbreviated Mn. In another embodiment, molecular weight is expressed as the weight average molecular weight, abbreviated Mw. Additional detail regarding how these measures differ and how to calculate Mn and Mw are found, for example, in WO 2014/147173, which is incorporated by reference.

[0092] In some embodiments, the molecular weight of HAS, such as HES, expressed as number average molecular weight or as weight average molecular weight, is about 1 kilo Daltons (kDa), 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 105 kDa, 110 kDa, 115 kDa, 120 kDa, 125 kDa, 130 kDa, 135 kDa, 140 kDa, 145 kDa, 150 kDa, 155 kDa, 160 kDa, 165 kDa, 170 kDa, 175 kDa, 180 kDa, 185 kDa, 190 kDa, 195 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 1000 kDa, or higher, and ranges among and between these values. In other embodiments, HAS, such as HES, has a molecular weight (Mn or Mw) ranging from about 500 Da to about 300 kDa, about 1 kDa to about 200 kDa, about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 10 kDa to about 40 kDa. Other ranges are also possible.

[0093] Hydroxyalkyl starch, such as hydroxyethyl starch, can be monodisperse or polydisperse. Monodispersity means that the HAS, such as HES, is of uniform size, or very small deviation from the mean. Polydispersity means that the HAS, such as HES, is non-uniform, or has a relatively large deviation from the mean. In some embodiments, the deviation is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, or 30% of the mean molecular weight. In other embodiments, the deviation can be expressed as plus or minus (.+-.) 10 Daltons (Da), 50 Da, 100 Da, 150 Da, 200 Da, 250 Da, 300 Da, 350 Da, 400 Da, 450 Da, 500 Da, 650 Da, 700 Da, 750 Da, 800 Da, 850 Da, 900 Da, 950 Da, 1,000 Da, 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 11,000 Da, 12,000 Da, 13,000 Da, 14,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 40,000 Da, 45,000 Da, 50,000 Da, 55,000 Da, 60,000 Da, 65,000 Da, 70,000 Da, 75,000 Da, 80,000 Da, 85,000 Da, 90,000 Da, 95,000 Da, or 100,000 Da, from the mean molecular weight.

[0094] Another way to characterize HAS, such as HES, is the degree of molar substitution (MS), which is the average number of hydroxyalkyl groups, such as hydroxyethyl groups, per glucose monomer. In some embodiments, the molar substitution is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 hydroxyethyl groups per glucose, and ranges among and between these values. In some embodiments, the degree of molar substitution may vary from the mean by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, or more.

[0095] Yet another way to characterize HAS, such as HES, is to express the ratio at which the average glucose subunit in the polymer is substituted at the C2 and C6 positions, i.e., the C2/C6 ratio. In some embodiments, the C2/C6 ratio is about 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 100, 1000, or more, and ranges among and between these values.

[0096] The characteristics of mean molecular weight, degree of molar substitution and C2/C6 ratio can each be varied independently in particular preparations of hydroxyalkyl starch, such as hydroxyethyl starch, according to knowledge of those ordinarily skilled in the art. Non-limiting examples of HES embodiments characterized according to specific combinations of molecular weight and molar substitution include Mn.about.500 kDa and MS ranging from 0.5 to 1.5, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5; Mn.about.200 kDa and MS ranging from 0.5 to 1.5, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5; Mn.about.135 kDa and MS ranging from 0.5 to 1.5, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5; Mn.about.100 kDa and MS ranging from 0.5 to 1.5, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5; Mn 65 kDa and MS ranging from 0.5 to 1.5, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5. According to other non-limiting examples, each of the foregoing HES embodiments can additionally be characterized as having a C2/C6 ratio in the range of 1 to 50, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, HES conjugated to a FVIII mutein of the disclosure has a single set of characteristics (i.e., unique combination of molecular weight, molar substitution, and optionally C2/C6 ratio), whereas in other embodiments, HES with two or more sets of characteristics can be conjugated to the FVIII muteins of the disclosure. Techniques for determining the mean molecular weight, molar substitution and C2/C6 ratio are familiar to those of ordinary skill in the art.

Polysialic Acid and Other Polymers

[0097] In some other embodiments of biocompatible polymers, a biocompatible polymer is polysialic acid (PSA) including, without limitation, .alpha.-2,8-linked polysialic acid. The properties of such polymers are discussed further in G. Gregoriadis, et al., Improving the therapeutic efficiency of peptides and proteins: a role for polysialic acids, Int. J. Pharmaceutics, 300, 125-130 (2005), which is incorporated by reference. In certain non-limiting embodiments, PSA includes from 2 to about 80 sialic acid subunits. Other sizes are also possible. In some embodiments, PSA can be homopolymeric, consisting of sialic acid subunits linked only by .alpha.-2,8-linkages, whereas in other embodiments, PSA can be heteropolymeric, for example comprising sialic acid subunits linked by alternating .alpha.-2,8-linkages and .alpha.-2,9-linkages.

[0098] In yet other embodiments, a biocompatible polymer is a zwitterionic brush polymer, for example and without limitation, a poly-phosphorylcholine branched polymer. The properties of such polymers are discussed further in M. Chen et al., Lubrication at Physiological Pressures by Polyzwitterionic Brushes, Science 323, 1698-1701 (2009), which is incorporated by reference.

Conjugates of FVIII Muteins and Biocompatible Polymers

[0099] The disclosure provides conjugates comprising a mutein of FVIII and a biocompatible polymer. In certain embodiments, the FVIII muteins are FVIII proteins comprising one or more of the cysteine substitutions disclosed herein and the biocompatible polymer is polyethylene glycol (PEG) or hydroxyalkyl starch (HAS), for example hydroxyethyl starch (HES), or some other biocompatible polymer. According to some embodiments, the biocompatible polymer is covalently coupled to the thiol sulfur atom of the one or more substituted cysteines of a mutein of the disclosure. In some embodiments, the linkage is a direct one whereas in other embodiments the biocompatible polymer is attached indirectly through a spacer and/or a linker interposed between the thiol sulfur and the polymer, in which case the mutein and polymer are each bonded directly to the spacer and/or linker.

[0100] Linkers, also called cross-linkers, are molecules bearing one or more chemical reactive groups for attachment at certain sites on a biocompatible polymer, and/or to a mutein of the disclosure, for example to a thiol of a substituted cysteine. As will be appreciated by those of ordinary skill, after reactions have been carried out to effect attachment of a linker to a polymer, to a mutein, or to both a polymer and mutein, one or more atoms from the linker may remain as part of the modified polymer, modified mutein, or conjugate comprising the attached polymer and mutein. Linkers can optionally include a spacer which is an additional atom or atoms serving to separate reactive groups in a bifunctional (or higher, e.g., trifunctional) linker, or that separate a reactive group of a linker from an attachment site on a polymer or mutein, or that separate a polymer and mutein in a conjugate. Spacers can be any length and contain their own functional groups.

[0101] Conjugates can be made according to the knowledge of those ordinarily skilled in the art. In some embodiments, a biocompatible polymer is coupled to a linker with a reactive group capable of attaching to the thiol sulfur of a substituted cysteine. In a subsequent step, the polymer-linker combination is coupled to the cysteine. In other embodiments, a mutein is coupled to a linker via a cysteine thiol group where the linker has a reactive group capable of attaching to corresponding group of the polymer. In a subsequent step, the mutein-linker combination is coupled to the polymer. In some embodiments, a reactive group is first created chemically at a predetermined site on the polymer to enable coupling between the group and a linker in a subsequent reaction.

[0102] In some embodiments, the linker is bifunctional, meaning it has two reactive groups and optionally a spacer between them. One of the reactive groups is capable of reacting with a corresponding group on a polymer whereas the other reactive group is capable of reacting with the thiol of the substituted cysteine of a mutein. The bifunctional linker can be reacted with the polymer first, followed by reaction of the polymer-linker combination with the mutein. Alternatively, the bifunctional linker can be reacted with the mutein first, followed by reaction of the mutein-linker with the polymer. In some embodiments reaction conditions may be chosen so that the polymer, bifunctional linker and mutein can be reacted with each other simultaneously to form a conjugate. In some embodiments, a reactive group is first created chemically at a predetermined site on the polymer to enable coupling between the group and a linker in a subsequent reaction. In some embodiments, bifunctional linkers are homobifunctional, possessing two of the same type of reactive group. In other embodiments, bifunctional linkers are heterobifunctional, possessing different reactive groups. Use of linkers with more than two reactive groups is also possible, although use of protecting groups to prevent unwanted side reactions at certain reactive groups may be desirable.

[0103] In some embodiments, linkers and spacers can be designed with physiologically cleavable linkages. A physiologically cleavable linkage is one that reacts with water (i.e., is hydrolyzed) under physiological conditions, the rate depending on the particular atoms, bonds, substituents if present and conditions. Non-limiting examples include esters (e.g., carboxylate ester, phosphate ester, carbonate ester, orthoester, thiolester), carbamate, sulfate, phosphate anhydride, acetal, ketal, acyloxyalkyl ether, imine, hydrazone, amide, urethane, peptide and oligonucleotide linkages. Linkers and spacers can also be designed with enzymatically cleavable linkages, such as groups that can be recognized and cleaved by esterases, proteases, phosphatases, nucleases, and other enzymes. Linkers or spacers can be designed to include specific amino acid sequences cleavable by proteases recognizing such sites.

[0104] Methods and reagents for synthesizing linkers with particular reactive groups are within the knowledge of those ordinarily skilled in the art, as are methods and reagents for reacting polymers, linkers and muteins to form conjugates of the disclosure. For example, in some embodiments, before a FVIII mutein is reacted with a linker or polymer-linker combination, the cysteine thiol is first reduced using a reducing agent such as DTT, TCEP, or another suitable reducing agent familiar to those of ordinary skill in the art. In addition, potentially reactive side groups of a mutein (for example, a thiol of a native cysteine residue), polymer or linker, can be protected to prevent unwanted side reactions from occurring during later reaction steps required to produce the desired conjugate. Later, after the conjugate or a desired intermediate has been produced, such protecting groups can be removed according the knowledge of those ordinarily skilled in the art.

[0105] Typically, though not necessarily, conjugates are purified away from leaving groups and other impurities from the chemical reaction or reactions used to make modified polymers and/or muteins, as well as the final conjugate product. Non-limiting examples of a purification step include gel filtration, ultrafiltration, dialysis, and precipitation, but other methods can be used as well, such as ion exchange chromatography, immunoaffinity chromatography (e.g., with antibodies against FVIII), or other purification methods. In some embodiments, additional purification steps may be used, for example, to purify desired intermediates from undesired reactants before proceeding to the next reaction in a multistep reaction scheme for making conjugates of the disclosure.

[0106] According to certain non-limiting embodiments, a thiol reactive polymer useful for conjugating to a substituted cysteine can be produced by reacting a polymer having an electrophilic reactive group with a linker having a nucleophilic reactive group (for reacting with the polymer's electrophile) and a thiol reactive group (for reacting with the substituted cysteine's thiol). The polymer-electrophile can be represented by the formula POLY-W.sub.0,1-E, where POLY stands for a polymer such as PEG, HAS or HES, W stands for a spacer, which can be absent (0) or present (1), and E stands for an electrophile, i.e., electrophilic reactive group. The linker can be represented by the formula NU-Y-T, where NU stands for a nucleophile, Y stands for a group, moiety, or spacer interposed between NU and T, and T stands for a thiol-reactive group, or protected thiol (i.e., --S with additional atoms). After reacting the electrophilically active polymer with the linker, the resulting thiol reactive polymer can be represented by the formula POLY-W.sub.0,1--X--Y-T, where POLY, W, Y and T are defined as before, and X stands for the moiety formed by the reaction between the electrophile (E) and nucleophile (NU), not including any leaving group or groups that may have been formed during the reaction. Once synthesized, the thiol reactive polymer can be reacted with a FVIII mutein comprising a substituted cysteine (represented by the formula S-FVIII, where S is the cysteine thiol and FVIII stands for the remainder of the Factor VIII mutein) to produce a conjugate of the polymer and mutein. The conjugate can be represented by the formula POLY-W.sub.0,1-X--Y--Z-FVIII, where POLY, W and Y are defined as before, and Z stands for the moiety formed by the reaction between the thiol reactive group of the polymer, T, and the mutein's cysteine thiol, S, not including any leaving group(s).

[0107] In other embodiments, the position of electrophile and nucleophile can be reversed, such that the nucleophile is initially associated with the polymer (represented as POLY-W.sub.0,1-NU) and the electrophile is associated with the linker (represented as E-Y-T). In these embodiments, reaction of the nucleophilically active polymer with the linker can yield a similar thiol-reactive polymer as before (represented as POLY-W.sub.0,1--X--Y-T), but where X results from reaction of the differently situated nucleophile and electrophile.

[0108] Electrophile refers to an ion, atom, or collection of atoms that may be ionic, having an electrophilic center, i.e., a center that is electron seeking and capable of reacting with a nucleophile. In some embodiments, the electrophile is naturally present as part of the polymer without further modification. In other embodiments, the electrophile is added by chemically modifying the polymer, optionally including a spacer W. Where the electrophile is naturally part of the polymer, then no spacer is present.

[0109] Exemplary, non-limiting electrophiles include carboxylic acid or an activated carboxylic acid derivative, amide, carboxylic acid ester, carbonate ester, carbonic acid, acid halide, activated esters (e.g., N-hydroxysuccinimidyl (NHS) ester or 1-hydroxybenzotriazolyl ester), active carbonates (e.g. N-hydroxysuccinimidyl carbonate, para-nitrophenylcarbonate, and 1-hydroxybenzotriazolyl carbonate), acetal, hemi-acetal, aldehyde, aldehyde hydrate, active anhydrides such as acid anhydrides, aryl halide, ketone, isocyanate, isothiocyanate, imidoester, pentafluorophenyl (PFP), and others.

[0110] The spacer W, when present, can in certain non-limiting embodiments be C.sub.1-C.sub.10 alkyl (such as a methylene group), C.sub.1-C.sub.10 substituted alkyl, linear lower alkyl, branched lower alkyl, or other groups, including non-alkyl groups.

[0111] Nucleophile refers to an ion or atom or collection of atoms that may be ionic, having a nucleophilic center, i.e., a center that is seeking an electrophilic center and capable of reacting with an electrophile. In some embodiments, the nucleophile NU can be amino (primary, secondary or other), hydroxy, thiol, imino, or thioester groups, or others known to those of ordinary skill in the art.

[0112] According to certain non-limiting embodiments, NU can be one of the following groups: CH.sub.3--NH--, CH.sub.3--CH.sub.2--NH--, CH.sub.3--CH.sub.2--CH.sub.2--NH--, (CH.sub.3).sub.2-CH--NH--, H.sub.2N--, H.sub.2N--O--, H.sub.2N--NH--, H.sub.2N--NH--(C.dbd.O)--, H.sub.2N--NH--(C.dbd.O)--NH--, H.sub.2N--NH--(C.dbd.O)--O-- and H.sub.2N--NH--SO.sub.2--.

[0113] Group Y may be linear, branched or some other configuration. In some embodiments, Y groups can be alkyl (branched or non-branched), substituted alkyl, alkenyl, substituted alkenyl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, aryl, substituted aryl, heteroaryl group, substituted heteroaryl group, cycloalkyl, substituted cycloalkyl, alkylenecycloalkyl, substituted alkylenecycloalkyl, alkylene, substituted alkylene, cycloalkylene, or substituted cycloalkylene, and may include such groups as methylene (--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--), propylene (--CH.sub.2CH.sub.2CH.sub.2--), butylene (--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), pentylene (--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), 2-methylpropyl, and other groups as well.

[0114] The term "substituted" in the context of group Y refers to replacement of some atom, usually but not necessarily H, with a heteroatom or group. Groups (such as alkyl, etc.) can be substituted with multiple substituents, for example, 1, 2, 3, 4, 5 or 6 substituents. If two or more substituents are present, each substituent may be the same as or different from the at least one other substituent. Non-limiting examples of substituents includes groups such as alkyl, aryl, alkenyl, alkynyl, fluorine, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkoxycarbonyl, am inocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, tertiary amino, acylamino, including alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido, nitro, alkylthio, arylthio, amide, sulfate, alkylsulfmyl, sulfonate, sulfonamido, trifluoromethyl, cyano, azido, carboxymethylcarbamoyl (i.e., the group --C(.dbd.O)(--NH--CH.sub.2--COOH)), cycloalkyl (e.g., cyclopentyl or cyclohexyl), heterocycloalkyl (e.g., morpholino, piperazinyl or piperidinyl), alkylaryl, arylalkyl and heteroaryl, examples of the latter of which include but are not limited to benzodioxolyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzoimidazolyl, benzothiophenyl, methylenedioxyphenyl, napthyridinyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, purinyl, benzofuranyl, deazapurinyl, and indolizinyl groups.

[0115] If linear, Y can be different lengths, for example, ranging from about 1 to about 20 atoms, from about 2 to about 15 atoms, from about 2 to about 10 atoms, from about 1 to about 6 atoms, or some other length, including longer than 20 atoms.

[0116] Non-limiting examples of Y groups include the following structures: --CH.sub.2--, --CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C- H.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C- H.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C- H.sub.2--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--, --CH(CH.sub.3)--CH.sub.2--, --CH.sub.2--CH(CH.sub.3)--, --CH(CH.sub.3)--CH.sub.2--CH.sub.2--, --CH.sub.2--CH(CH.sub.3)--CH.sub.2--, --CH.sub.2--CH.sub.2--CH(CH.sub.3)--, --CH(CH.sub.3)--CH.sub.2--CH.sub.2-- CH.sub.2--, --CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--, --CH(CH.sub.3)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH(CH.sub.3)-- CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--, --CH(CH.sub.3)-- CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2-- CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(CH.sub.3)--, --CH.sub.2--C(CH.sub.3).sub.2--CH.sub.2--, --CH(CH.sub.3)--CH(CH.sub.3)--, --C(CH.sub.3).sub.2--C(CH.sub.3).sub.2--, --CH(CH.sub.2OH)--CH.sub.2--, --CH(CH.sub.2OH)--CH.sub.2--CH.sub.2--, --CH(CONH.sub.2)--CH.sub.2--, --CH(COOH)--CH.sub.2--, --CH(COOH)--CH.sub.2--CH.sub.2--, --CH(COOH)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH(CONH.sub.2)--C(CH.sub.3).sub.2--, --CH(CONH.sub.2)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH(OH)--CH.sub.2--, --CH.sub.2--CH(OH)--CH(OH)--CH.sub.2-- and --CH(COOH)--C(CH.sub.3).sub.2--, --CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH- .sub.2--CH.sub.2--, --CH(COOH)--CH.sub.2--CH.sub.2--C(.dbd.O)NH--CH--C(.dbd.O)(--NH--CH.sub.2- --COOH)--CH.sub.2--, --O--CH.sub.2--, --CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--, --CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--O--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--O--, --C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--, --CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--, --C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--, --C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--, --C(O)--O--CH.sub.2--, --CH.sub.2--C(O)--O--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--O--CH.sub.2--, --C(O)--O--CH.sub.2--CH.sub.2--, --NH--C(O)--CH.sub.2--, --CH.sub.2--NH--C(O)--CH.sub.2--, --CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--, --NH--C(O)--CH.sub.2--CH.sub.2--, --CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2, --CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2, --C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--, --O--C(O)--NH--CH.sub.2--, --O--C(O)--NH--CH.sub.2--CH.sub.2--, --NH--CH.sub.2--, --NH--CH.sub.2--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--NH--CH.sub.2--, --C(O)--CH.sub.2--, --C(O)--CH.sub.2--CH.sub.2--, --CH.sub.2--C(O)--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--CH.sub.2--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--C- H.sub.2--, a cycloalkylene group, or a substituted cycloalkylene group, --CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--C- H.sub.2--CH.sub.2--, --(CH.sub.2).sub.1,2,3,4,5--NH--C(O)--CH.sub.2CH.sub.2--, and combinations of two or more of any of the foregoing.

[0117] Group T stands for thiol-reactive groups or protected thiol, i.e., thiol coupled with a protecting group. Non-limiting examples of thiol-reactive groups include maleimide, vinyl sulfone, vinylsulfide, pyridyl disulfide, orthopyridyl disulfide, haloacetyl groups (e.g., containing an iodoacetyl or bromoacetyl group), iodoacetamide, thiol (--SH), thiolate (--S--), TNB-thiol, aziridine, oxirane, acryloyl derivatives, and arylating agents. Maleimide groups and haloacetyl groups react specifically with thiol groups to form stable thioether linkages. Pyridyl disulfides react with thiol groups to form disulfide bonds. Other thiol-reactive groups are possible.

[0118] In some embodiments, a thiol protecting group forms with --S a thioether. Non-limiting examples include alkyl thioethers, benzyl thioethers, allyl thioethers, triarylmethyl thioethers (e.g., including a trityl (Trt) group, such as --S-Trt). In other embodiments, a protecting group forms with --S a disulfide. Non-limiting examples include S-sulfonates (e.g., --S--SO.sub.3.sup.-, --S--SO.sub.2-aryl and --S--SO.sub.2-alkyl), S-tert-butyl (e.g., --S--S-tBu), S-(2-aminoethyl), S-2-pyridyl (e.g., --S--S-(2-pyridyl)), and linkers that are symmetrical disulfides where each half of the linker serves as protecting group for the other. Additional non-limiting examples of thiol protecting groups include monothio acetals, dithio acetals, aminothio acetals, thioesters, thiocarbonates, thiocarbamates, and sulfenyl derivatives. Some non-limiting examples of linkers with protected thiols include the following: H.sub.2N--CH.sub.2--CH.sub.2--S-Trt; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--S-Trt; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S-Trt; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S-Trt; H.sub.2N--CH.sub.2--CH.sub.2--S--S-tBu; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--S--S-tBu; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S--S-tBu; H.sub.2N--CH(COOH)--CH.sub.2--S-Trt; H.sub.2N--CH(COOH)--C(CH.sub.3).sub.2--S-Trt; H.sub.2N--CH.sub.2--CH.sub.2--S--S--CH.sub.2--CH.sub.2--NH.sub.2; H.sub.2N--CH(COOH)--CH.sub.2--CH.sub.2--C(.dbd.O)NH--CH--[C(.dbd.O)(--NH-- -CH.sub.2--COOH)]--CH.sub.2--S--S--CH.sub.2--CH--[C(.dbd.O)(--NH--CH.sub.2- --COOH)]--NH--C(.dbd.O)--CH.sub.2--CH.sub.2--CH(COOH)--NH.sub.2; and H.sub.2N--CH(COOH)--CH.sub.2--S--S--CH.sub.2--CH(COOH)--NH.sub.2.

[0119] When a linker with a protected thiol is employed to produce at thiol-reactive polymer, the protecting group must be removed to activate the polymer before it can be reacted with a thiol of a substituted cysteine. Reagents and conditions for deprotecting a thiol based on the nature of the protecting group is within the knowledge of the skilled artisan. According to a non-limiting example, a thiol protected by a disulfide can be activated by a reducing agent, such as sodium borohydride, dithiothreitol (DTT), dithioerythritol (DTE), or a phosphine, such as TCEP.

[0120] In some embodiments, the linker is a symmetrical disulfide having two identical nucleophilic groups for reaction with the electrophilic group of the polymer. Exemplary symmetrical linkers possess a central disulfide (--S--S--) bond where the sulfur atoms are each connected to identical Y groups and then nucleophiles NU. Non-limiting examples of symmetrical linkers are cystamine and cysteamine, but many others are possible. Reaction of an electrophilically active polymer with a symmetrical disulfide linker such as cystamine results in formation of a symmetrical disulfide polymer having identical polymer segments extending from each of the sulfur atoms of a central disulfide linkage. This arrangement can be represented by the formula POLY-W.sub.0,1-X--Y--S--S--Y--X--W.sub.0,1-POLY, where --S--S-- indicates the central disulfide bond and the other variable s are as defined above. Due to the symmetry of the resulting disulfide polymer, cleavage with a reducing agent such as dithiothreitol results in formation of two moles of the corresponding thiol-reactive polymer derivative.

[0121] Based on the structure of the particular reactive groups E and NU, other reagents used and the reaction conditions chosen, one of ordinary skill in the art can readily determine the structure of the resulting X group. Similarly, based on a linker's thiol-reactive group, reagents used, and reaction conditions chosen, one of ordinary skill can readily determine the structure of the resulting Z group.

[0122] In some non-limiting embodiments, X can be an amide, e.g., --C(.dbd.O)--NH--, a urethane, e.g., --O--C(O)--NH--, --(CH.sub.3)--N--, --(CH.sub.3--CH.sub.2)--N--, --(CH.sub.3--CH.sub.2--CH.sub.2)--N--, --((CH.sub.3).sub.2--CH)--N--, --HN--, --HN--O--, --HN--NH--, --HN--NH--(C.dbd.O)--, --HN--NH--(C.dbd.O)--NH--, --HN--NH--(C.dbd.O)--O-- and --HN--NH--SO.sub.2--. In yet other embodiments, X can be represented as --C(.dbd.O)G-, where G is heteroatom or group such as --O, --NH, --S, or --NR, where R is lower alkyl.

[0123] In some embodiments where the polymer is HAS or HES, the terminal glucose moiety at the reducing end of the polymer is coupled to a linker. In these embodiments, the C.sub.1 carbon in the terminal glucose can serve as an electrophile for reaction with the nucleophile of the linker. However, in other embodiments, the reducing end terminal glucose residue may be derivatized so that a different electrophilic group is available for reaction, or in yet other embodiments, the reducing end terminal glucose residue may be derivatized so that a nucleophilic group is present that can then be reacted with a corresponding electrophilic group of a linker.

[0124] In some embodiments the reducing end terminal glucose residue is used in its unoxidized state (for example at carbon C.sub.1) for coupling with a linker, while in other embodiments, the reducing end terminal glucose is used in its oxidized state (for example at carbon C.sub.1) for coupling with a linker. In some embodiments, oxidation of the reducing end terminal glucose residue results in a lactone or carboxylic acid whereby carbon C.sub.1 of the glucose residue is part of a carbonyl group. Oxidation can be carried out using reagents and reaction conditions familiar to those of ordinary skill in the art, for example using an alkaline iodine solution as described in WO 2005/014050, which is incorporated by reference.

[0125] In some embodiments, a bifunctional linker comprising at least one amine group can be reacted with the oxidized reducing end of a HAS or HES polymer to form an amide. Non-limiting exemplary embodiments of such linkers include 1,8-diamino octane, 1,7-diamino heptane, 1,6-diamino hexane, 1,5-diamino pentane, 1,4-diamino butane, 1,3-diamino propane, and 1,2-diamino ethane. Other embodiments include diaminopolyethyleneglycol according to the formula H.sub.2N--(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2--CH.sub.2--NH.sub.2, where n can be 1, 2, 3, 4, 5, or a higher integer. Where, as in these examples, the second functional group is also an amine, the polymer-linker combination can be reacted with a monohalogen-substituted acetic acid, for example, bromoacetic acid, chloroacetic acid or iodoacetic acid, and an activating agent, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to generate a thiol reactive haloacetamide derivative that can then be coupled to a thiol of a substituted cysteine to form a thioether linkage. This reaction scheme is explained in additional detail in WO 2005/014050.

[0126] In another embodiment described further in WO 2005/014050, the polymer-linker combination formed from HAS or HES and a diamino linker can be reacted with a second bifunctional linker comprising a reactive ester group and a maleimide group. A non-limiting example of a reactive ester is succinimide ester and non-limiting example of a bifunctional linker comprising a reactive ester and maleimide is N-(.alpha.-maleimidoacetoxy)succinimide ester. The resulting derivative of HAS or HES can then be reacted with the thiol of a substituted cysteine forming a thioether linkage.

[0127] In some embodiments, a thiol reactive polymer, such as those described above, can be reacted with a second compound that is also thiol reactive. Examples of such second thiol-reactive compounds include CH.sub.2.dbd.CH--S(O).sub.2--CH.dbd.CH.sub.2, as described in WO 2014/147173 which is incorporated by reference, and the genus of compounds represented by formula (III) as described in WO 2014/147175, which is incorporated by reference. After coupling the polymer derivative with the second compound, the thiol reactive group of said second compound can be reacted with the thiol of the substituted cysteine, thereby forming a conjugate of polymer and FVIII mutein.

[0128] Following is a non-exhaustive and non-limiting list of linkers containing reactive groups capable of reacting with the thiol group of a substituted cysteine of the muteins of the disclosure: 1,11-Bis-Maleimidotriethyleneglycol; 1,4-Bis-Maleimidobutane; 1,4-Bis-Maleimmidyl-2,3-dihydroxy-butane; 1,8-Bis-Maleimidodiethylene-glycol; 2-{2-[2-(2-[pyrid-2-yl]-disulfanyl-ethoxy)-ethoxy]-ethyl-disulfanyl}-pyri- dine; 2-[N2-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)- -L-lysinyl]ethylmethanethiosulfate; 2-{N-[4N6-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-- L-lysinyl]}ethylmethanethiosulfate; 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide; 2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide; 3-(2-Pyridyldithio)propionylhydrazide; 4-(4-N-Maleimidophenyl)-butyric acid hydrazide.HCl; 4-(N-Maleimidomethyl)-cyclohexane-1-carboxyl-hydrazide HCl 1/2 dioxane; 4-Succinimidyloxycarbonyl-methyl-.alpha.-(2-pyridyldithio)toluene; Bis-Maleimidoethane; Bis-Maleimidohexane; Cystamine; Cysteamine; Dithiobis-maleimidoethane; Divinyl sulfone; m-Maleimidobenzoyl-N-hydroxysuccinimide ester; m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester; N-(2-amino-ethyl)-3-maleimido-propionamide; N-(.beta.-Maleimidopropionic acid)hydrazide.TFA; N-(.beta.-Maleimidopropyloxy)succinimide ester; N-(p-Maleimidophenyl)isocyanate; N-(.alpha.-Maleimidoacetoxy)-succinimide ester; N-(.gamma.-Maleimidobutryloxy)sulfosuccinimide ester; N-(.gamma.-Maleimidobutyryloxy)succinimide ester; N-(.epsilon.-Maleimidocaproic acid)hydrazide; N-(.epsilon.-Maleimidocaproyloxy)succinimide ester; N-(.epsilon.-Maleimidocaproyloxy)sulfosuccinimide ester; N-(K-Maleimidoundecanoic acid)hydrazide; N-(K-Maleimidoundecanoyloxy)sulfosuccinimide ester; N,N'-Bis(vinylsulfonylacetyl)ethylenediamine; NHS-PEG12-Maleimide; NHS-PEG24-Maleimide; NHS-PEG2-Maleimide; NHS-PEG4-Maleimide; NHS-PEG6-Maleimide; NHS-PEG8-Maleimide; N-Succinimidyl 3-(2-pyridyldithio)propionate; N-Succinimidyl iodoacetate; N-Succinimidyl S-acetylthio-acetate; N-Succinimidyl S-acetylthio-propionate; N-Succinimidyl(4-iodoacetyl)aminobenzoate; N-Succinimidyl-(4-vinylsulfonyl) benzoate; N-.gamma.-Maleimidobutryloxy-sulfosuccinimide ester; N-.gamma.-Maleimidobutyryloxy-succinimide ester; N-.epsilon.-Maleimidocaproic acid; Succinimdyl 3-(bromoacetamido)propionate; Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate); Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; Succinimidyl 4-(p-maleimidophenyl)butyrate; Succinimidyl 6-(3'-[2-pyridyldithio] propionamido)hexanoate; Succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate; Sulfo-NHS-(2-6-[Biotinamido]-2-(p-azidobezamido); Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate; Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate; Sulfosuccinimidyl 6-(.alpha.-methyl-.alpha.-[2-pyridyldithio]-toluamido) hexanoate; Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate; Tris-(2-Maleimidoethyl)amine; H.sub.2N--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH(COOH)--CH.sub.2--SH; H.sub.2N--CH(COOH)--C(CH.sub.3).sub.2--SH; H.sub.2N--CH(CH.sub.2OH)--CH.sub.2--SH; H.sub.2N--CH(CH.sub.2OH)--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH(CONH.sub.2)--C(CH.sub.3).sub.2--SH; H.sub.2N--CH(CONH.sub.2)--CH.sub.2--SH; H.sub.2N--CH(COOH)--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.- 2--SH; H.sub.2N--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--C- H.sub.2--O--CH.sub.2--CH.sub.2--SH; H.sub.2N--CH(COOH)--CH.sub.2--CH.sub.2--C(.dbd.O)NH--CH--C(.dbd.O)(--NH--- CH.sub.2--COOH)--CH.sub.2--SH; H.sub.2N--O--CH.sub.2--CH.sub.2--SH; H.sub.2N--O--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--O-CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--O--CH(COOH)--CH.sub.2--SH; H.sub.2N--O--CH(COOH)--C(CH.sub.3).sub.2--SH; H.sub.2N--O--CH(CH.sub.2OH)--CH.sub.2--SH; H.sub.2N--O--CH(CH.sub.2OH)--CH.sub.2--CH.sub.2--SH; H.sub.2N--O--CH(CONH.sub.2)--C(CH.sub.3).sub.2--SH; H.sub.2N--NH--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--CH(COOH)--CH.sub.2--SH; H.sub.2N--NH--CH(COOH)--C(CH.sub.3).sub.2--SH; H.sub.2N--NH--CH(CH.sub.2OH)--CH.sub.2--SH; H.sub.2N--NH--CH(CH.sub.2OH)--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--CH(CONH.sub.2)--C(CH.sub.3).sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH.sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2- --SH; H.sub.2N--NH--C(.dbd.O)--CH(COOH)--CH.sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH(COOH)--C(CH.sub.3).sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH(CH.sub.2OH)--CH.sub.2--SH; H.sub.2N--NH--C(.dbd.O)--CH(CH.sub.2OH)--CH.sub.2--CH.sub.2--SH; and H.sub.2N--NH--C(.dbd.O)--CH(CONH.sub.2)--C(CH.sub.3).sub.2--SH.

[0129] Additional information about certain of linkers listed above may be found in the Thermo Scientific Crosslinking Technical Handbook (2012), which is incorporated by reference.

[0130] The linkers listed above (and others not expressly disclosed) may be used to attach a biocompatible polymer to a cysteine substitution mutein of the disclosure. As will be appreciated by one of ordinary skill in the art, certain of these linkers are homobifunctional, comprising reactive groups that react exclusively or predominantly with thiol. Where a biocompatible polymer includes a thiol, these linkers may be used directly without modifying the polymer to link the polymer and mutein using methods and reagents familiar to those of ordinary skill in the art. However, where a polymer naturally lacks a thiol, or lacks a thiol at a desired location in the polymer, one may be created chemically, again using methods and reagents familiar to those of ordinary skill in the art. Certain of the foregoing linkers are heterobifunctional, comprising a reactive group specific for thiol, and at least one other reactive group that is non-reactive with thiol, or that substantially favors reaction with a different functional group than thiol, for example an amine group, or a carboxyl group. Where a biocompatible polymer includes one of these other groups (e.g., --NH.sub.2 or --COOH), these linkers may be used directly without modifying the polymer to link the polymer and mutein using methods and reagents familiar to those of ordinary skill in the art. However, where a polymer naturally lacks one of these groups, or lacks one of these groups at a desired location in the polymer, such groups may be created chemically using methods and reagents familiar to those of ordinary skill in the art.

[0131] Additional linkers and methods, conditions and reagents for carrying out reactions to join linkers with bioactive polymers, such as PEG, HAS and HES, and cysteine thiol groups are disclosed in U.S. Pat. Nos. 7,910,661 and 7,863,421, which are incorporated by reference, and international patent application publication nos. WO 2002/080979, WO 2003/070772, WO 2004/024761, WO 2005/014050, WO 2014/147173 and WO 2014/147175, which are incorporated by reference. Further information and details regarding conjugating linkers to thiol groups may be found in G. T., Hermanson, Bioconjugate Techniques, 3rd Ed., Academic Press (ISBN 978-O-12-382239-0), and Chalker, J. M., et al., Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology, Chem. Asian J., 4:630-640 (2009), each of which is incorporated by reference.

Compositions

[0132] The present disclosure also provides compositions comprising FVIII muteins conjugated with a biocompatible polymer for administration to subjects suffering from a deficiency of FVIII activity. Such compositions may further comprise a pharmaceutically acceptable carrier, vehicle, excipients, or stabilizers. These may include, without limitation, solvents, dispersion media, coatings, antibacterial and antifungal agents, bulking agents, wetting agents, emulsifiers, antioxidants, chelating agents, metal ions, proteins, preservatives, pH buffers, zwitterions (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine), absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers include without limitation water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.

[0133] According to other embodiments, compositions may further include agents for maintaining isotonic balance between the composition and bodily fluids, including without limitation sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride.

[0134] A composition for use according to the invention may be in any suitable form for administration to a subject, including without limitation as liquid solutions (e.g., injectable and infusible solutions). The form depends on the intended mode of administration and therapeutic application. In certain embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).

[0135] Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating conjugates of FVIII muteins and biocompatible polymers in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization.

[0136] Dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other desired ingredients, for example, from among those enumerated above.

[0137] In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0138] In some non-limiting embodiments of the FVIII conjugates of the disclosure, a pharmaceutical composition comprising such conjugates can additionally comprise sucrose, calcium chloride dihydrate, L-histidine, polysorbate 80 and sodium chloride. In some embodiments, these compositions can be supplied in a premixed aqueous solution, or as a lyophilized cake ready for reconstitution with sterile water or with a saline solution, for example, one containing 0.9% (w/v) of sodium chloride.

Methods of Treatment or Prevention

[0139] FVIII muteins, conjugates of such muteins with biocompatible polymers, and compositions comprising such muteins or conjugates are useful for treating or preventing bleeding in subjects. Thus, in some embodiments, a FVIII mutein of the disclosure or conjugate of a mutein and a biocompatible polymer are administered in a therapeutically effective amount to a subject in need of treatment for bleeding. In other embodiments, a FVIII mutein of the disclosure or conjugate of a mutein and a biocompatible polymer are administered in a prophylactically effective amount to a subject in need of prophylaxis for bleeding. What constitutes a therapeutically or prophylactically effective amount can be determined according to the knowledge of those ordinarily skilled in the art.

[0140] In some embodiments treatment is intended to reduce the amount of bleeding and/or time that bleeding continues before stopping where bleeding has already commenced, for example due to trauma or spontaneous bleeding, for example into joints, muscles or the brain, or other tissues. In some embodiments, prevention is intended to reduce or stop bleeding before it begins. In some embodiments, administration of a mutein or conjugate of the disclosure is provided before a procedure, such as a dental procedure, or surgery (elective or non-elective) where there is a substantial risk that bleeding will occur. In other embodiments, prophylactic administration is provided to reduce or stop bleeding that would occur unexpectedly due, for example, to an accident or spontaneous bleeding.

[0141] In some embodiments, subjects in need of treatment or prophylaxis have a deficiency of FVIII activity. In some embodiments, subjects in need of treatment or prophylaxis have hemophilia, for example Hemophilia A (HA). In some subjects, the hemophilia is caused by a genetic mutation (hereditary or spontaneous), while in other subjects it is acquired, for example, due to production of autoantibodies against FVIII. In some embodiments, subjects in need of treatment or prophylaxis have mild, moderate or severe Hemophilia A. In some embodiments, subjects in need of treatment or prophylaxis are pediatric or adult human patients with Hemophilia A.

[0142] In some embodiments, treatment or prophylaxis of a subject with a mutein or conjugate of the disclosure is provided in combination with a different drug intended to treat or prevent bleeding. Non-limiting examples of such drugs include desmopressin (such as DDAVP or desmopressin acetate) and aminocaproic acid. Administration of a composition comprising a mutein or conjugate of the disclosure to a subject can occur before, contemporaneously with, or after administration of the other drug or drugs intended to help treat or prevent bleeding. In some embodiments, the mutein or conjugate of the disclosure can be combined with such other drug in the same composition for more convenient dosing, or in different compositions.

Determination of Plasma Half-Life

[0143] Plasma half-life and other pharmacokinetic properties of any mutein or conjugate of the disclosure can be determined after administration to a subject using methods familiar to those of ordinary skill in the art. For example, a composition comprising a predetermined amount of a mutein or conjugate thereof with a biocompatible polymer of the disclosure can be administered to a subject with Hemophilia A. Often, the administration is made after a washout period of some time, e.g., 72 hours, to permit any previous dose of Factor VIII replacement therapy to be eliminated. Then, at predetermined times, a blood sample is taken, plasma isolated, and the plasma samples tested for FVIII activity. Non-limiting exemplary times for taking blood samples after administration are 0.25 hours, 0.5 hr, 1 hr, 3 hr, 6 hr, 9 hr, 24 hr, 28 hr, 32 hr and 48 hr, although other time points are possible as long as they, according to the judgment of those ordinarily skilled in the art, permit sampling of plasma FVIII activity over a sufficient span of time and at close enough intervals. Non-limiting examples of assays for testing FVIII activity include the aPTT assay and chromogenic assay, but use of other assays is also possible as explained elsewhere herein, or as would be familiar to those of ordinary skill in the art.

[0144] After FVIII activity as a function of time is determined, plasma half-life (t.sub.1/2) of the FVIII mutein or conjugate thereof administered at the beginning of the experiment can readily be determined using statistical methods such as regression analysis or other statistical methods familiar to those of ordinary skill in the art. In addition, other pharmacokinetic values, such as area under the curve (AUC), can be estimated using other mathematical techniques applied to the data. Publications explaining in greater detail how t.sub.1/2 and other PK values can be calculated include Bjorkman, et al., J. Thomb. Haemo., 8:730-6 (2010); Bjorkman, et al., Blood, 119(2):612-8 (2012); Morfini, et al., Thromb. Haemost., 66(3):384-6 (1991); Lee, et al., Scientific and Standardization Committee Communication, posted on ISTH website Mar. 21, 2001, downloaded from www.isth.org/resource/group/d4a6f49a-f4ec-450f-9e0f-7be9f0c2ab2e/official- _communications/fiviiipharmaco.pdf on Feb. 23, 2015; and Vlot, et al., Thromb. Haemost., 83:65-9 (2000), each of which is incorporated by reference.

[0145] In some embodiments, plasma half-life in non-human animals, such as monkeys, mice or rats, can be determined using similar techniques as those described above, and the results scaled to predict corresponding plasma half-life in human subjects. The scaling factors to be employed are familiar to those of ordinary skill in the art.

Dosing and Frequency of Administration

[0146] Identifying suitable dosages to treat or prevent uncontrolled bleeding in subjects with Hemophilia A (or other deficiency of FVIII activity) with FVIII muteins and conjugates thereof with biocompatible polymers will depend on a variety of factors. Some of these factors are drug-dependent, including without limitation mutein or conjugate half-life, specific activity and side effects, if any. Other factors are subject-dependent, including subject age, body mass, site and severity of bleeding, if any, severity of hemophilia, and general health. Other factors may be relevant as well.

[0147] By convention in the art, one International Unit (IU) of FVIII activity is equivalent to the FVIII activity in one milliliter of normal human plasma. Thus, once the activity of a purified preparation of a FVIII mutein or conjugate thereof is determined using an art-recognized method, one of ordinary skill in the art can readily calculate the amount of the FVIII mutein required to formulate a pharmaceutical composition containing some predefined level of FVIII activity expressed in IUs, or concentration expressed in IUs per volume (if liquid) or mass (if solid, e.g., powder). Once so formulated, healthcare providers or patients can then administer an amount of the composition calculated to deliver a desired amount of FVIII activity for treatment or prophylaxis.

[0148] Exemplary non-limiting dosages include 0.001 IU/kg, 0.01 IU/kg, 0.1 IU/kg, 0.5 IU/kg, 1 IU/kg, 2 IU/kg, 3 IU/kg, 4 IU/kg, 5 IU/kg, 6 IU/kg, 7 IU/kg, 8 IU/kg, 9 IU/kg, 10 IU/kg, 15 IU/kg, 20 IU/kg, 25 IU/kg, 30 IU/kg, 35 IU/kg, 40 IU/kg, 45 IU/kg, 50 IU/kg, 100 IU/kg, 150 IU/kg, 200 IU/kg, 250 IU/kg, 300 IU/kg, 350 IU/kg, 400 IU/kg, 450 IU/kg, 500 IU/kg, or 1000 IU/kg, where IU/kg is International Units/kilogram of subject body mass. Other dosages are also possible, as are ranges between and among the foregoing values.

[0149] FVIII muteins of the disclosure and conjugates thereof with biocompatible polymers having extended half-lives may be administered to subjects at intervals sufficient to maintain any desired level of therapeutic or prophylactic effect. The necessary frequency of administration will depend on a variety of drug and subject specific factors, including those influencing the choice of dosage. Methods for determining the necessary frequency of administration to achieve a desired level of therapeutic or prophylactic efficacy are within the knowledge of those of ordinary skill in the art.

[0150] In certain embodiments, conjugates of FVIII muteins and biocompatible polymers are administered to a subject suffering from a deficiency of FVIII activity 5 times per day, 4 times per day, 3 times per day, 2 times per day, once per day, once per 2 days, once per 3 days, once per 4 days, once per 5 days, once per 6 days, once per 7 days, once per 8 days, once per 9 days, once per 10 days, once per 11 days, once per 12 days, once per 13 days, once per 14 days, once per 15 days, once per 16 days, once per 17 days, once per 18 days, once per 19 days, once per 20 days, once per 21 days, once per 22 days, once per 23 days, once per 24 days, once per 25 days, once per 26 days, once per 27 days, once per 28 days, once weekly, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, or even longer.

[0151] The optimal dose and frequency of administration of a pharmaceutical composition comprising a FVIII mutein or conjugate thereof of the disclosure for treating or preventing uncontrolled bleeding in a subject can be determined according to the knowledge of those ordinarily skill in the art. For example, a starting dose can be administered based on upon information such as the severity of the subject's hemophilia, and the subject's age and body mass, or other factors. Blood samples can then be taken at various times thereafter to test FVIII activity in the plasma. If the care provider determines that too low or too high FVIII activity is present, the dose and/or frequency of administration can be changed and the subject's plasma then retested until the amount of FVIII activity is deemed optimal to control the subject's condition.

[0152] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document was individually indicated to be incorporated by reference for all purposes.

EXAMPLES

Example 1: Construction of FVIII Mutein Expression Vectors and Transient Transfection

[0153] Using standard molecular biology techniques, cysteine substitution mutations were introduced into cDNAs encoding human B domain deleted Factor VIII (BDD FVIII) having the amino acid sequence of SEQ ID NO:2 (FIG. 2). The substitution positions are identified in Table 1 in which the numbers correspond to the positions in the amino acid sequence of mature human FVIII shown in FIG. 1 (SEQ ID NO:1). The same positions are also highlighted in the amino acid sequences shown in FIG. 1 and FIG. 2 using bold and underline font. For transient expression, single cysteine substitutions were introduced into a cDNA encoding BDD FVIII with a spacer and FLAG tag epitope at the carboxy-terminus (SEQ ID NO:7). Later a subset of single cysteine substitutions and combinations with substitutions at positions 336 and/or 1680 were stably transfected using a cDNA without the spacer and FLAG tag (SEQ ID NO:8).

[0154] After verifying the sequence of the substitutions, cDNAs encoding the various cysteine substitution muteins were cloned into a mammalian expression vector using an Infusion Dry-Down Kit (Clontech Laboratories, Mountain View, Calif.). Expression vector plasmids were transfected into COS-1 cells as follows. TransiT (Mirus Bio) was diluted (40 .mu.l) in 2 ml Opti-Mem (Life Technologies), vortexed and incubated at room temperature for 15 minutes. Plasmid DNA (16 .mu.g) was added to the mixture and incubated at room temperature for 15 minutes. The 2 ml TransiT/DNA/Opti-MEM mixture was added to a COS-1 cells in a P100 tissue culture plate containing 8 ml of growth medium in the presence of 10% fetal calf serum and incubated overnight at 37.degree. C. in 5% CO.sub.2. Transfection medium was removed and the cells were carefully rinsed with 10 ml of cell growth medium. To each plate 10 ml of medium containing penicillin/streptomycin and glutamine was added and incubated at 34.degree. C. or 37.degree. C. in 5% CO.sub.2. Seventy-two hours later conditioned medium was harvested, centrifuged to remove cellular debris and reserved for further characterization of transiently expressed FVIII muteins.

Example 2: Quantitation of Transiently Expressed FVIII Muteins

[0155] Expression of FVIII muteins from the transiently transfected cell culture supernatants was analyzed by Western blot. First, proteins in samples of supernatants were separated by SDS-PAGE and then blotted to membranes. FVIII was detected using a specific anti-FVIII antibody. Supernatants from COS-1 cells transiently transfected with an expression vector encoding unmutated B domain deleted (BDD) FVIII were included as controls. Apparent expression levels of FVIII muteins compared to unmutated BDD FVIII were estimated by eye and scored as follows: (-) much lower or undetectable compared to control; (+) lower compared to control; (++) same as control; (+++) greater compared to control. The results of expression analysis are set forth in Table 2, column 2. Because substitution with certain cysteines could have altered the epitope of the antibody used to detect expressed protein, it is possible that the actual protein expression level is greater than or less than the apparent expression level.

Example 3: Activity of Transiently Expressed FVIII Muteins

[0156] Activity of control unmutated BDD FVIII and the FVIII muteins identified in Table 1 was tested using an activated partial thromboplastin time (aPTT) assay. Cell culture supernatants containing the transiently expressed FVIII muteins were diluted and 50 .mu.l added to 50 .mu.l of aPTT reagent (Actin.RTM. FSL, Siemens) and 50 .mu.l of FVIII deficient plasma (George King Bio-Medical, Inc., Overland Park Kans.) and incubated for 3 minutes at 37.degree. C. The reaction was initiated immediately with the addition of 50 .mu.l of 25 mM calcium chloride. The time to clot was measured using a StarT4 coagulation instrument (Diagnostica Stago, Parsippany, N.J.). Standard curves were prepared by dilution of pooled normal plasma (FACT, George King Bio-Medical, Inc.). One international unit (IU) of FVIII activity was defined as that amount measured in 1 ml of normal human pooled plasma. Activity of the FVIII muteins was compared to that of unmutated BDD FVIII and expressed as a percentage of the activity of the positive control. The activity of the muteins is set forth in Table 2, column 3. Presence of a FLAG tag at the carboxy-terminus of the unmutated BDD FVIII light chain did not affect its activity.

TABLE-US-00002 TABLE 2 Cysteine substitution Transient Percent FVIII mutation expression level activity F59C - 13% N239C - 4% P333C + 22% R336C ++ 137% P379C ++ 28% T481C ++ 70% R484C ++ 75% L486C ++ 20% S488C + 25% R489C + 28% R490C - 17% P492C + 21% K493C ++ 50% V495C +++ 100% K496C ++ 65% H497C ++ 76% K499C + 18% D500C - 18% F501C ++ 94% E507C ++ 66% Y555C ++ 60% R562C ++ 39% S568C + 49% R571C + 46% N582C - 7% Y1680C + 27% Q1778C ++ 105% E1793C ++ 48% E1794C ++ 75% R1797C ++ 42% Q1798C ++ 66% G1799C ++ 77% A1800C + 53% E1801C ++ 31% F1806C +++ 61% N1810C + 21% E1811C ++ 67% T1814C + 27% F1816C ++ 26% K1818C - 8% F1891C + 48% F2035C ++ 55% F2068C ++ 74% K2092C + 33% F2093C ++ 137% S2094C ++ 95% S2095C ++ 71% N2118C + 38% V2125C - 21% K2183C ++ 63% S2186C ++ 64% T2191C ++ 20% F2196C ++ 59% S2204C - 52% S2206C ++ 57% L2212C ++ 15%

Example 4: Production of Stably Transfected CHO Pools Expressing Active FVIII Muteins

[0157] To produce cells stably expressing FVIII muteins, expression vectors for certain of the muteins described in Example 1 were transfected into CHO host cells by electroporation or using polycationic lipid transfection techniques. Two days later, media conditions were changed to select transfected cells and eliminate untransfected cells by growth in media lacking nucleotides. Growth of selected cells was appreciable by about 10-14 days after selection began. CHO pools stably expressing FVIII muteins were then used to produce conditioned media. Media containing expressed FVIII muteins was harvested 7 or 8 days post seeding so that cell viability did not fall below 50%. Cells were removed from media by centrifugation or filtration. Conditioned media from stably transfected CHO cells was then tested for FVIII activity as in Example 3. Results are shown in Table 3.

TABLE-US-00003 TABLE 3 Cysteine substitution FVIII activity (mIU/ml) mutation in CHO pool media 481 322 484 864 493 1561 495 748 496 780 497 457 507 370 555 1081 562 1534 568 254 1778 15899 1794 241 1798 490 1799 1703 1800 1185 1806 1206 1811 1285 2035 698 2068 502 2093 20190 2094 8024 2095 2576 2183 5924 2186 8947 2196 700 2204 1853 2206 1974

Example 5: Purification of FVIII Muteins

[0158] Conditioned media from stably transfected CHO cells expressing FVIII muteins was harvested and polysorbate 80 added to a final concentration of 0.05%. Media was then passed through an anion exchange column, washed at pH 7.0 in the presence of CaCl.sub.2, and bound protein step eluted with 1 M NaCl. Fractions containing FVIII were pooled and passed through an immunoaffinity column using an antibody to FVIII (Eriksson et al., Semin Hematol. 38(2 Suppl 4):24-31 (2001), which is incorporated by reference). After elution of bound protein, FVIII muteins were further purified on MonoQ resin (GE) column with a linear NaCl gradient at pH 6.25 in the presence of CaCl.sub.2) and 0.1% polysorbate 80. Purified proteins were then dialyzed into buffer at pH 7.0 and protein concentration determined.

Example 6: Thrombin Cleavage of Selected FVIII Muteins

[0159] Purified B domain deleted FVIII (BDD FVIII) and selected FVIII muteins (1.25 .mu.g each) were incubated with or without 1 U/mL of a thrombin (Haematologic Technologies, Essex Junction, Vt.) in 150 mM NaCl, 2 mM CaCl.sub.2), 20 mM Tris-HCL, and 5% glycerol, at pH 7.5. Reactions were incubated at 37.degree. C. for 30 minutes after which the reactions were stopped by adding D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) to a final concentration of 351 nM. Enyzmatic reaction products were then analyzed by denaturing SDS-polyacrylamide gel electrophoresis on a 4-20% bis-tris gel (Invitrogen) and visualized by silver staining. The results are shown in FIG. 4. Thrombin digestion of the muteins and control produced the expected cleavage fragments.

Example 7: PEGylation of Selected FVIII Muteins and Effect on Activity

[0160] Selected purified FVIII muteins were treated with a reducing agent to remove the cap from the substituted cysteine and then reacted with a thiol-reactive PEG derivative to create the conjugates. In the reduction step, 100 .mu.g/ml of the FVIII mutein was treated with 50 molar equivalents of tris (2-carboxyethyl)phosphine (TCEP) for 1 hour at 4.degree. C. after which TCEP and released cap were removed using a desalting column (Zeba Spin 7K MWCO from Thermo Scientific). Uncapped muteins were then incubated at 4.degree. C. for at least 30 minutes to allow any native disulfide bonds reduced in the previous step to reoxidize. Uncapped muteins were then treated with 10 molar equivalents of maleimide-PEG 60K (SUNBRIGHT.RTM. GL2-600MA, NOF Corp.) at 4.degree. C. overnight in buffer containing 19 mM histidine, 310 mM NaCl, 3.4 mM CaCl.sub.2), and 0.02% polysorbate 80.

[0161] The extent of PEGylation was determined using a LabChip GXII (PerkinElmer). In a typical experiment, 500 ng of PEGylated FVIII reaction mixture was denatured, reduced and run on the LabChip. Protein was then detected with a fluorescent dye and data output as an electropherogram allowing quantification of the extent of PEGylation. The extent of PEGylation was expressed as a percentage of the starting amount of protein for each FVIII mutein and is shown in Table 4, column 2.

[0162] FVIII activity of the PEGylated muteins was measured using the aPTT assay as described in Example 3. Activity after PEGylation was calculated as the percent activity relative to the activity of each mutein before PEGylation and is shown in Table 4, column 3. The specific activity for three PEGylated muteins comprising the S2094C mutation was also determined. Activity was measured by two methods, including the aPTT assay described in Example 3 and a chromogenic FVIII activity assay according to the manufacturer's instructions (DiaPharma). Results are shown in Table 5.

TABLE-US-00004 TABLE 4 Percent FVIII Cysteine substitution activity after mutation Percent PEGylation PEGylation R484C 0 106 K493C 73 119 K496C 33 104 Y555C 6 109 R562C 8 113 Q1778C 1 103 G1799C 13 113 F1806C 12 114 F2035C 27 102 F2093C 70 49 S2094C 41 81 S2095C 20 92 K2183C 4 112 S2186C 21 88 F2196C 14 92 S2204C 38 92 S2206C 9 78 S2094C + R336A 45 85 S2094C + R336A + 47 82 Y1680F S2094C + Y1680F 38 88 BDD FVIII 2 111

TABLE-US-00005 TABLE 5 Specific activity PEGylated (PEG 60K) Specific activity chromogenic assay FVIII mutein aPTT assay (IU/mg) (IU/mg) S2094C 1240 3900 S2094C + R336A 1380 4300 S2094C + Y1680F 1180 4000 S2094C + R336A 1050 4175 Y1680F

Example 9: In Vivo Clotting Activity of PEGylated FVIII Muteins

[0163] PEGylated FVIII muteins were tested for their ability to control severe bleeding in male hemophilic mice genetically null for FVIII (strain B6; 129S-F8tm1Kaz/J) at different times after administration. Bi, et al., Nat Genet 10:119-21 (1995), which is incorporated by reference. The muteins tested were S2094C, S2094C+R336A, and S2094C+R336A+Y1680F, in each case conjugated with PEG 60K on the cysteine substituted at position 2094.

[0164] Mice were anesthetized with isoflurane and placed on a heated platform. Tails were immersed for 5 minutes in 50 mL phosphate buffered saline (PBS) warmed to 37.degree. C. and then removed from the PBS. Mice were then intravenously administered saline, a single dose of unmutated BDD FVIII or one of three PEGylated FVIII muteins (200 IU/kg based on activity measured by the aPTT assay). Ten animals were used for saline, and three to five animals were used in each treatment group. At 5 minutes, 24 hours and 48 hours after dosing, the animal's tails were transected 3 mm from the end and placed for 10 minutes into a tube containing PBS warmed to 37.degree. C. For animals given saline, tail transection was performed 5 min after administration. The collection tube was then centrifuged and the pelleted erythrocytes resuspended in 5 ml lysis buffer (8.3 g/L ammonium chloride, 1.0 g/L potassium bicarbonate, and 0.037 g/L EDTA). Light absorbance of the samples was measured spectroscopically at 575 nm and total blood loss calculated from a standard curve. The experiment using BDD FVIII was performed separately from the experiment using the PEGylated FVIII muteins. Results from saline control experiments conducted at different times were combined.

[0165] Results from the experiments are shown in FIG. 5. Data is the average .+-.SEM. After dosing hemophilic mice with BDD FVIII, blood loss was substantially reduced compared to negative control at the 5 min time point, but the procoagulant effect disappeared by 24 hours. In contrast, for each of the three PEGylated muteins tested, procoagulant effect in the hemophilic mice was evident 24 hours after dosing, and in one case (2094+336), the effect was evident 48 hours after dosing. Thus, the duration of action of each of the PEGylated muteins was substantially longer compared to unmodified BDD FVIII. The data demonstrates that compared to unmodified FVIII, it is possible to substantially extend the half-life of FVIII by conjugating a biocompatible polymer to a cysteine substitution mutation of the disclosure.

Example 10: Pharmacokinetics of FVIII Muteins in Hemophilic Mice

[0166] Purified FVIII muteins are administered to Hemophilia A mice via the tail vein. Unmutated BDD FVIII is also tested as a control. At different times after administering FVIII, mice are anesthetized and blood drawn into sodium citrate. Plasma is prepared by centrifuging the blood at 2500 g for 10 minutes. Plasma is then used in one or more assays, e.g., the aPTT assay or chromogenic assay, to measure plasma FVIII clotting activity. Plasma FVIII protein levels are determined by a FVIII specific ELISA using a FVIII specific monoclonal antibody. By comparing plasma FVIII activity and protein concentrations as a function of time after FVIII administration, it is possible to calculate the circulatory half-life of the FVIII muteins in comparison to unmutated BDD FVIII.

[0167] Additional embodiments of the disclosure include and encompass:

1. A modified FVIII protein comprising at least one cysteine substitution mutation at an amino acid corresponding to a position in the amino acid sequence of SEQ ID NO:1 selected from the group of amino acid positions consisting of: 59, 239, 333, 336, 379, 481, 484, 486, 488, 489, 490, 492, 493, 495, 496, 497, 499, 500, 501, 507, 555, 562, 568, 571, 582, 1680, 1778, 1793, 1794, 1797, 1798, 1799, 1800, 1801, 1806, 1810, 1811, 1814, 1816, 1818, 1891, 2035, 2068, 2092, 2093, 2094, 2095, 2118, 2125, 2183, 2186, 2191, 2196, 2204, 2206, and 2212. 2. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 59. 3. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 239. 4. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 333. 5. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 336. 6. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 379. 7. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 481. 8. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 484. 9. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 486. 10. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 488. 11. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 489. 12. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 490. 13. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 492. 14. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 493. 15. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 495. 16. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 496. 17. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 497. 18. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 499. 19. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 500. 20. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 501. 21. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 507. 22. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 555. 23. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 562. 24. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 568. 25. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 571. 26. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 582. 27. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1680. 28. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1778. 29. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1793. 30. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1794. 31. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1797. 32. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1798. 33. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1799. 34. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1800. 35. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1801. 36. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1806. 37. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1810. 38. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1811. 39. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1814. 40. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1816. 41. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1818. 42. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 1891. 43. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2035. 44. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2068. 45. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2092. 46. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2093. 47. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2094. 48. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2095. 49. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2118. 50. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2125. 51. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2183. 52. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2186. 53. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2191. 54. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2196. 55. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2204. 56. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2206. 57. The modified FVIII protein of embodiment 1, wherein the cysteine substitution mutation occurs at position 2212. 58. The modified FVIII protein of embodiment 1, wherein said FVIII protein further comprises at least one additional substitution mutation at an amino acid corresponding to position 336, 562, 1680 or 1968 in the amino acid sequence of SEQ ID NO:1 selected from the group of substitutions consisting of: R336A, R562A, Y1680F and K1968A. 59. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 493 and is combined with the substitution mutation R336A. 60. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 496 and is combined with the substitution mutation R336A. 61. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 1799 and is combined with the substitution mutation R336A. 62. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2094 and is combined with the substitution mutation R336A. 63. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2186 and is combined with the substitution mutation R336A. 64. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2204 and is combined with the substitution mutation R336A. 65. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2206 and is combined with the substitution mutation R336A. 66. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 493 and is combined with the substitution mutation Y1680F. 67. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 496 and is combined with the substitution mutation Y1680F. 68. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 1799 and is combined with the substitution mutation Y1680F. 69. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2094 and is combined with the substitution mutation Y1680F. 70. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2186 and is combined with the substitution mutation Y1680F. 71. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2204 and is combined with the substitution mutation Y1680F. 72. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2206 and is combined with the substitution mutation Y1680F. 73. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 493 and is combined with the substitution mutations R336A and Y1680F. 74. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 496 and is combined with the substitution mutations R336A and Y1680F. 75. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 1799 and is combined with the substitution mutations R336A and Y1680F. 76. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2094 and is combined with the substitution mutations R336A and Y1680F. 77. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2186 and is combined with the substitution mutations R336A and Y1680F. 78. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2204 and is combined with the substitution mutations R336A and Y1680F. 79. The modified FVIII protein of embodiment 58, wherein the cysteine substitution mutation occurs at position 2206 and is combined with the substitution mutations R336A and Y1680F. 80. The modified FVIII protein of any one of embodiments 1-79, wherein compared to unmodified FVIII said modified FVIII has a functional attribute selected from the group consisting of: higher expression, greater procoagulant activity, reduced immunogenicity, greater stability, reduced susceptibility to degradation, greater resistance to proteases, greater resistance to oxidation, improved ability to be activated by thrombin or other coagulation factors, improved ability to combine with other coagulation factors and components into the tenase complex, increased association or binding to vWF, improved shelf-life, reduced binding by inhibitory antibodies, reduced interaction with the low density lipoprotein receptor related protein, reduced interaction with low density lipoprotein receptor, reduced interaction with cell surface heparin sulphate proteoglycans, increased circulatory half-life, and improved pharmacokinetics. 81. The modified FVIII protein of any one of embodiments 1-79 further comprising a moiety. 82. The modified FVIII protein of embodiment 81, wherein said moiety is selected from the group consisting of: small organic molecule, macromolecule, antibody, antibody fragment, antigen binding domain, antibody Fc region, protein of immunologic origin, protein of immunologic function, intact clotting factor, functional fragment of a clotting factor, enzyme, nucleic acid, DNA, RNA, organometallic compound, lipid, fatty acyl chain, phospholipid, glycolipid, protein, peptide, amino acid, carbohydrate, monosaccharide, disaccharide, hydrophobic compound, hydrophilic compound, organic acid, and organic base. 83. The modified FVIII protein of embodiment 81, wherein said moiety is attached to said cysteine. 84. The modified FVIII protein of embodiment 83, wherein said moiety is attached to said cysteine covalently. 85. The modified FVIII protein of embodiment 84, wherein said moiety is covalently attached via the thiol of said cysteine. 86. The modified FVIII protein of embodiment 84, wherein said moiety is covalently attached via a reactive group. 87. The modified FVIII protein of embodiment 84, wherein said moiety further comprises a linker. 88. The modified FVIII protein of any one of embodiments 1-79, further comprising a biocompatible polymer attached directly or indirectly to said cysteine. 89. The modified FVIII protein of embodiment 88, further comprising a linker between said cysteine and said biocompatible polymer. 90. The modified FVIII protein of embodiment 88, wherein said biocompatible polymer is selected from the group consisting of: polyethylene glycol (PEG), hydroxyalkyl starch, hydroxyethyl starch (HES), polysialic acid (PSA), a zwitterionic brush polymer, and a poly-phosphorylcholine branched polymer. 91. The modified FVIII protein of embodiment 88, wherein said modified FVIII protein has an increased circulatory half-life compared to unmodified FVIII protein. 92. The modified FVIII protein of embodiment 91, wherein the circulatory half-life of said modified FVIII protein is increased at least about 2 times compared to unmodified FVIII protein. 93. The modified FVIII protein of embodiment 91, wherein the circulatory half-life of said modified FVIII protein is increased at least about 5 times compared to unmodified FVIII protein. 94. The modified FVIII protein of embodiment 91, wherein the circulatory half-life of said modified FVIII protein is increased at least about 10 times compared to unmodified FVIII protein. 95. A composition comprising the modified FVIII protein of embodiment 88 and a pharmaceutically acceptable excipient. 96. A nucleic acid encoding the modified FVIII protein of any one of embodiments 1-79. 97. A host cell comprising the nucleic acid of embodiment 96 operatively linked to a genetic regulatory sequence. 98. A method of treating bleeding in a subject having a deficiency of FVIII activity comprising the step of administering a therapeutically effective dose of a composition comprising the modified FVIII protein of embodiment 88.

99. The method of embodiment 98, wherein said subject has hemophilia A. 100. A method of preventing bleeding in a subject having a deficiency of FVIII activity comprising the step of administering a prophylactically effective dose of a composition comprising the modified FVIII protein of embodiment 88. 101. The method of embodiment 100, wherein said subject has hemophilia A. 102. The method of embodiment 100, wherein said step of administering said modified FVIII protein occurs not more often than a period selected from the group consisting of: 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, and 3 weeks. 103. A method of preparing a conjugate comprising a cysteine-substituted FVIII protein and a biocompatible polymer comprising the steps of:

[0168] reacting under suitable conditions a thiol-reactive polymer having the formula POLY-W.sub.0,1--X--Y-T with a cysteine substituted FVIII protein to form a conjugate having the formula POLY-W.sub.0,1-X--Y--Z-FVIII, where POLY is a biocompatible polymer, W is a spacer, X is a group formed by the reaction of an electrophile and a nucleophile, Y is a moiety, T is a thiol-reactive group or a protected thiol, Z is a group formed by the reaction of T and a thiol in a substituted cysteine, and FVIII is the remainder of FVIII protein.

[0169] 104. The method of embodiment 103, where the thiol-reactive polymer, POLY-W.sub.0,1--X--Y-T, is formed by reacting under suitable conditions an electrophilically active polymer having the formula POLY-W.sub.0,1-E and a nucleophilically active molecule having the formula NU-Y-T, where POLY is a biocompatible polymer, W is a spacer, E is an electrophilic group, NU is a nucleophilic group, Y is a moiety and T is a thiol-reactive group or a protected thiol.

[0170] 105. The method of embodiment 103, where the thiol-reactive polymer, POLY-W.sub.0,1--X--Y-T, is formed by reacting under suitable conditions a nucleophilically active polymer having the formula POLY-W.sub.0,1-NU and an electrophilically active molecule having the formula E-Y-T, where POLY is a biocompatible polymer, W is a spacer, E is an electrophilic group, NU is a nucleophilic group, Y is a moiety and T is a thiol-reactive group or a protected thiol.

[0171] 106. The method of embodiment 104 or 105, where said cysteine-substituted FVIII protein is the cysteine substituted FVIII protein of any one of embodiments 1 to 79.

[0172] 107. The method of embodiment 103, where spacer W or moiety Y includes a physiologically cleavable linkage.

[0173] 108. A conjugate comprising a cysteine-substituted FVIII protein and a biocompatible polymer prepared by the method of embodiment 103.

[0174] 109. The conjugate of embodiment 108, where said cysteine-substituted FVIII protein is the cysteine substituted FVIII protein of any one of embodiments 1 to 79.

Sequence CWU 1

1

812332PRTHomo sapiens 1Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490 1495 1500Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735 1740Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1835 1840 1845Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1850 1855 1860Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915 1920Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925 1930 1935Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1970 1975 1980Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035 2040Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 2045 2050 2055Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 2075 2080 2085Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090 2095 2100Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 2170 2175Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2180 2185 2190Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 2210 2215 2220Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290 2295Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315 2320 2325Gln Asp Leu Tyr 233021438PRTArtificial SequenceBDD FVIII 2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr

Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His 740 745 750Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile 755 760 765Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp 770 775 780Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys785 790 795 800Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly 805 810 815Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser 820 825 830Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser 835 840 845Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 850 855 860Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr865 870 875 880Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile 885 890 895Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe 900 905 910Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His 915 920 925Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 930 935 940Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro945 950 955 960Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln 965 970 975Val Thr Val Gln Glu Phe Ala Leu Phe Leu Thr Ile Phe Asp Glu Thr 980 985 990Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro 995 1000 1005Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg 1010 1015 1020Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu 1025 1030 1035Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 1040 1045 1050Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val 1055 1060 1065Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn 1070 1075 1080Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys 1085 1090 1095Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His 1100 1105 1110Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln 1115 1120 1125Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile 1130 1135 1140Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg 1145 1150 1155Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro 1160 1165 1170Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His 1175 1180 1185Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1190 1195 1200Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 1205 1210 1215Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe 1220 1225 1230Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro 1235 1240 1245Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser 1250 1255 1260Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn 1265 1270 1275Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 1280 1285 1290Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr 1295 1300 1305Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn 1310 1315 1320Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val 1325 1330 1335Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly 1340 1345 1350Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile 1355 1360 1365Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn 1370 1375 1380Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro 1385 1390 1395Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg 1400 1405 1410Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu 1415 1420 1425Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1430 14353754PRTArtificial SequenceBDD FVIII 3Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His 740 745 750Gln Arg4684PRTArtificial SequenceBDD FVIII 4Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr1 5 10 15Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 20 25 30Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg 35 40 45His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 50 55 60Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro65 70 75 80Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr 85 90 95Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 100 105 110Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 115 120 125Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr 130 135 140Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys145 150 155 160Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala 165 170 175Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp 180 185 190Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 195 200 205Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr 210 215 220Val Gln Glu Phe Ala Leu Phe Leu Thr Ile Phe Asp Glu Thr Lys Ser225 230 235 240Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 245 250 255Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 260 265 270Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln 275 280 285Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 290 295 300Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys305 310 315 320Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 325 330 335Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 340 345 350Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 355 360 365Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile 370

375 380Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro385 390 395 400Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 405 410 415Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile 420 425 430Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu 435 440 445Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 450 455 460Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly465 470 475 480Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 485 490 495Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser 500 505 510Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 515 520 525Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala 530 535 540Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala545 550 555 560Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn 565 570 575Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val 580 585 590Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 595 600 605Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr 610 615 620Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp625 630 635 640Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 645 650 655Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg 660 665 670Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 675 680514PRTArtificial SequencePartial B domain 5Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg1 5 1069048DNAHomo sapiens 6gcttagtgct gagcacatcc agtgggtaaa gttccttaaa atgctctgca aagaaattgg 60gacttttcat taaatcagaa attttacttt tttcccctcc tgggagctaa agatatttta 120gagaagaatt aaccttttgc ttctccagtt gaacatttgt agcaataagt catgcaaata 180gagctctcca cctgcttctt tctgtgcctt ttgcgattct gctttagtgc caccagaaga 240tactacctgg gtgcagtgga actgtcatgg gactatatgc aaagtgatct cggtgagctg 300cctgtggacg caagatttcc tcctagagtg ccaaaatctt ttccattcaa cacctcagtc 360gtgtacaaaa agactctgtt tgtagaattc acggatcacc ttttcaacat cgctaagcca 420aggccaccct ggatgggtct gctaggtcct accatccagg ctgaggttta tgatacagtg 480gtcattacac ttaagaacat ggcttcccat cctgtcagtc ttcatgctgt tggtgtatcc 540tactggaaag cttctgaggg agctgaatat gatgatcaga ccagtcaaag ggagaaagaa 600gatgataaag tcttccctgg tggaagccat acatatgtct ggcaggtcct gaaagagaat 660ggtccaatgg cctctgaccc actgtgcctt acctactcat atctttctca tgtggacctg 720gtaaaagact tgaattcagg cctcattgga gccctactag tatgtagaga agggagtctg 780gccaaggaaa agacacagac cttgcacaaa tttatactac tttttgctgt atttgatgaa 840gggaaaagtt ggcactcaga aacaaagaac tccttgatgc aggataggga tgctgcatct 900gctcgggcct ggcctaaaat gcacacagtc aatggttatg taaacaggtc tctgccaggt 960ctgattggat gccacaggaa atcagtctat tggcatgtga ttggaatggg caccactcct 1020gaagtgcact caatattcct cgaaggtcac acatttcttg tgaggaacca tcgccaggcg 1080tccttggaaa tctcgccaat aactttcctt actgctcaaa cactcttgat ggaccttgga 1140cagtttctac tgttttgtca tatctcttcc caccaacatg atggcatgga agcttatgtc 1200aaagtagaca gctgtccaga ggaaccccaa ctacgaatga aaaataatga agaagcggaa 1260gactatgatg atgatcttac tgattctgaa atggatgtgg tcaggtttga tgatgacaac 1320tctccttcct ttatccaaat tcgctcagtt gccaagaagc atcctaaaac ttgggtacat 1380tacattgctg ctgaagagga ggactgggac tatgctccct tagtcctcgc ccccgatgac 1440agaagttata aaagtcaata tttgaacaat ggccctcagc ggattggtag gaagtacaaa 1500aaagtccgat ttatggcata cacagatgaa acctttaaga ctcgtgaagc tattcagcat 1560gaatcaggaa tcttgggacc tttactttat ggggaagttg gagacacact gttgattata 1620tttaagaatc aagcaagcag accatataac atctaccctc acggaatcac tgatgtccgt 1680cctttgtatt caaggagatt accaaaaggt gtaaaacatt tgaaggattt tccaattctg 1740ccaggagaaa tattcaaata taaatggaca gtgactgtag aagatgggcc aactaaatca 1800gatcctcggt gcctgacccg ctattactct agtttcgtta atatggagag agatctagct 1860tcaggactca ttggccctct cctcatctgc tacaaagaat ctgtagatca aagaggaaac 1920cagataatgt cagacaagag gaatgtcatc ctgttttctg tatttgatga gaaccgaagc 1980tggtacctca cagagaatat acaacgcttt ctccccaatc cagctggagt gcagcttgag 2040gatccagagt tccaagcctc caacatcatg cacagcatca atggctatgt ttttgatagt 2100ttgcagttgt cagtttgttt gcatgaggtg gcatactggt acattctaag cattggagca 2160cagactgact tcctttctgt cttcttctct ggatatacct tcaaacacaa aatggtctat 2220gaagacacac tcaccctatt cccattctca ggagaaactg tcttcatgtc gatggaaaac 2280ccaggtctat ggattctggg gtgccacaac tcagactttc ggaacagagg catgaccgcc 2340ttactgaagg tttctagttg tgacaagaac actggtgatt attacgagga cagttatgaa 2400gatatttcag catacttgct gagtaaaaac aatgccattg aaccaagaag cttctcccag 2460aattcaagac accctagcac taggcaaaag caatttaatg ccaccacaat tccagaaaat 2520gacatagaga agactgaccc ttggtttgca cacagaacac ctatgcctaa aatacaaaat 2580gtctcctcta gtgatttgtt gatgctcttg cgacagagtc ctactccaca tgggctatcc 2640ttatctgatc tccaagaagc caaatatgag actttttctg atgatccatc acctggagca 2700atagacagta ataacagcct gtctgaaatg acacacttca ggccacagct ccatcacagt 2760ggggacatgg tatttacccc tgagtcaggc ctccaattaa gattaaatga gaaactgggg 2820acaactgcag caacagagtt gaagaaactt gatttcaaag tttctagtac atcaaataat 2880ctgatttcaa caattccatc agacaatttg gcagcaggta ctgataatac aagttcctta 2940ggacccccaa gtatgccagt tcattatgat agtcaattag ataccactct atttggcaaa 3000aagtcatctc cccttactga gtctggtgga cctctgagct tgagtgaaga aaataatgat 3060tcaaagttgt tagaatcagg tttaatgaat agccaagaaa gttcatgggg aaaaaatgta 3120tcgtcaacag agagtggtag gttatttaaa gggaaaagag ctcatggacc tgctttgttg 3180actaaagata atgccttatt caaagttagc atctctttgt taaagacaaa caaaacttcc 3240aataattcag caactaatag aaagactcac attgatggcc catcattatt aattgagaat 3300agtccatcag tctggcaaaa tatattagaa agtgacactg agtttaaaaa agtgacacct 3360ttgattcatg acagaatgct tatggacaaa aatgctacag ctttgaggct aaatcatatg 3420tcaaataaaa ctacttcatc aaaaaacatg gaaatggtcc aacagaaaaa agagggcccc 3480attccaccag atgcacaaaa tccagatatg tcgttcttta agatgctatt cttgccagaa 3540tcagcaaggt ggatacaaag gactcatgga aagaactctc tgaactctgg gcaaggcccc 3600agtccaaagc aattagtatc cttaggacca gaaaaatctg tggaaggtca gaatttcttg 3660tctgagaaaa acaaagtggt agtaggaaag ggtgaattta caaaggacgt aggactcaaa 3720gagatggttt ttccaagcag cagaaaccta tttcttacta acttggataa tttacatgaa 3780aataatacac acaatcaaga aaaaaaaatt caggaagaaa tagaaaagaa ggaaacatta 3840atccaagaga atgtagtttt gcctcagata catacagtga ctggcactaa gaatttcatg 3900aagaaccttt tcttactgag cactaggcaa aatgtagaag gttcatatga cggggcatat 3960gctccagtac ttcaagattt taggtcatta aatgattcaa caaatagaac aaagaaacac 4020acagctcatt tctcaaaaaa aggggaggaa gaaaacttgg aaggcttggg aaatcaaacc 4080aagcaaattg tagagaaata tgcatgcacc acaaggatat ctcctaatac aagccagcag 4140aattttgtca cgcaacgtag taagagagct ttgaaacaat tcagactccc actagaagaa 4200acagaacttg aaaaaaggat aattgtggat gacacctcaa cccagtggtc caaaaacatg 4260aaacatttga ccccgagcac cctcacacag atagactaca atgagaagga gaaaggggcc 4320attactcagt ctcccttatc agattgcctt acgaggagtc atagcatccc tcaagcaaat 4380agatctccat tacccattgc aaaggtatca tcatttccat ctattagacc tatatatctg 4440accagggtcc tattccaaga caactcttct catcttccag cagcatctta tagaaagaaa 4500gattctgggg tccaagaaag cagtcatttc ttacaaggag ccaaaaaaaa taacctttct 4560ttagccattc taaccttgga gatgactggt gatcaaagag aggttggctc cctggggaca 4620agtgccacaa attcagtcac atacaagaaa gttgagaaca ctgttctccc gaaaccagac 4680ttgcccaaaa catctggcaa agttgaattg cttccaaaag ttcacattta tcagaaggac 4740ctattcccta cggaaactag caatgggtct cctggccatc tggatctcgt ggaagggagc 4800cttcttcagg gaacagaggg agcgattaag tggaatgaag caaacagacc tggaaaagtt 4860ccctttctga gagtagcaac agaaagctct gcaaagactc cctccaagct attggatcct 4920cttgcttggg ataaccacta tggtactcag ataccaaaag aagagtggaa atcccaagag 4980aagtcaccag aaaaaacagc ttttaagaaa aaggatacca ttttgtccct gaacgcttgt 5040gaaagcaatc atgcaatagc agcaataaat gagggacaaa ataagcccga aatagaagtc 5100acctgggcaa agcaaggtag gactgaaagg ctgtgctctc aaaacccacc agtcttgaaa 5160cgccatcaac gggaaataac tcgtactact cttcagtcag atcaagagga aattgactat 5220gatgatacca tatcagttga aatgaagaag gaagattttg acatttatga tgaggatgaa 5280aatcagagcc cccgcagctt tcaaaagaaa acacgacact attttattgc tgcagtggag 5340aggctctggg attatgggat gagtagctcc ccacatgttc taagaaacag ggctcagagt 5400ggcagtgtcc ctcagttcaa gaaagttgtt ttccaggaat ttactgatgg ctcctttact 5460cagcccttat accgtggaga actaaatgaa catttgggac tcctggggcc atatataaga 5520gcagaagttg aagataatat catggtaact ttcagaaatc aggcctctcg tccctattcc 5580ttctattcta gccttatttc ttatgaggaa gatcagaggc aaggagcaga acctagaaaa 5640aactttgtca agcctaatga aaccaaaact tacttttgga aagtgcaaca tcatatggca 5700cccactaaag atgagtttga ctgcaaagcc tgggcttatt tctctgatgt tgacctggaa 5760aaagatgtgc actcaggcct gattggaccc cttctggtct gccacactaa cacactgaac 5820cctgctcatg ggagacaagt gacagtacag gaatttgctc tgtttttcac catctttgat 5880gagaccaaaa gctggtactt cactgaaaat atggaaagaa actgcagggc tccctgcaat 5940atccagatgg aagatcccac ttttaaagag aattatcgct tccatgcaat caatggctac 6000ataatggata cactacctgg cttagtaatg gctcaggatc aaaggattcg atggtatctg 6060ctcagcatgg gcagcaatga aaacatccat tctattcatt tcagtggaca tgtgttcact 6120gtacgaaaaa aagaggagta taaaatggca ctgtacaatc tctatccagg tgtttttgag 6180acagtggaaa tgttaccatc caaagctgga atttggcggg tggaatgcct tattggcgag 6240catctacatg ctgggatgag cacacttttt ctggtgtaca gcaataagtg tcagactccc 6300ctgggaatgg cttctggaca cattagagat tttcagatta cagcttcagg acaatatgga 6360cagtgggccc caaagctggc cagacttcat tattccggat caatcaatgc ctggagcacc 6420aaggagccct tttcttggat caaggtggat ctgttggcac caatgattat tcacggcatc 6480aagacccagg gtgcccgtca gaagttctcc agcctctaca tctctcagtt tatcatcatg 6540tatagtcttg atgggaagaa gtggcagact tatcgaggaa attccactgg aaccttaatg 6600gtcttctttg gcaatgtgga ttcatctggg ataaaacaca atatttttaa ccctccaatt 6660attgctcgat acatccgttt gcacccaact cattatagca ttcgcagcac tcttcgcatg 6720gagttgatgg gctgtgattt aaatagttgc agcatgccat tgggaatgga gagtaaagca 6780atatcagatg cacagattac tgcttcatcc tactttacca atatgtttgc cacctggtct 6840ccttcaaaag ctcgacttca cctccaaggg aggagtaatg cctggagacc tcaggtgaat 6900aatccaaaag agtggctgca agtggacttc cagaagacaa tgaaagtcac aggagtaact 6960actcagggag taaaatctct gcttaccagc atgtatgtga aggagttcct catctccagc 7020agtcaagatg gccatcagtg gactctcttt tttcagaatg gcaaagtaaa ggtttttcag 7080ggaaatcaag actccttcac acctgtggtg aactctctag acccaccgtt actgactcgc 7140taccttcgaa ttcaccccca gagttgggtg caccagattg ccctgaggat ggaggttctg 7200ggctgcgagg cacaggacct ctactgaggg tggccactgc agcacctgcc actgccgtca 7260cctctccctc ctcagctcca gggcagtgtc cctccctggc ttgccttcta cctttgtgct 7320aaatcctagc agacactgcc ttgaagcctc ctgaattaac tatcatcagt cctgcatttc 7380tttggtgggg ggccaggagg gtgcatccaa tttaacttaa ctcttaccta ttttctgcag 7440ctgctcccag attactcctt ccttccaata taactaggca aaaagaagtg aggagaaacc 7500tgcatgaaag cattcttccc tgaaaagtta ggcctctcag agtcaccact tcctctgttg 7560tagaaaaact atgtgatgaa actttgaaaa agatatttat gatgttaaca tttcaggtta 7620agcctcatac gtttaaaata aaactctcag ttgtttatta tcctgatcaa gcatggaaca 7680aagcatgttt caggatcaga tcaatacaat cttggagtca aaaggcaaat catttggaca 7740atctgcaaaa tggagagaat acaataacta ctacagtaaa gtctgtttct gcttccttac 7800acatagatat aattatgtta tttagtcatt atgaggggca cattcttatc tccaaaacta 7860gcattcttaa actgagaatt atagatgggg ttcaagaatc cctaagtccc ctgaaattat 7920ataaggcatt ctgtataaat gcaaatgtgc atttttctga cgagtgtcca tagatataaa 7980gccatttggt cttaattctg accaataaaa aaataagtca ggaggatgca attgttgaaa 8040gctttgaaat aaaataacaa tgtcttcttg aaatttgtga tggccaagaa agaaaatgat 8100gatgacatta ggcttctaaa ggacatacat ttaatatttc tgtggaaata tgaggaaaat 8160ccatggttat ctgagatagg agatacaaac tttgtaattc taataatgca ctcagtttac 8220tctctccctc tactaatttc ctgctgaaaa taacacaaca aaaatgtaac aggggaaatt 8280atataccgtg actgaaaact agagtcctac ttacatagtt gaaatatcaa ggaggtcaga 8340agaaaattgg actggtgaaa acagaaaaaa cactccagtc tgccatatca ccacacaata 8400ggatccccct tcttgccctc cacccccata agattgtgaa gggtttactg ctccttccat 8460ctgcctgacc ccttcactat gactacacag aatctcctga tagtaaaggg ggctggaggc 8520aaggataagt tatagagcag ttggaggaag catccaaaga ttgcaaccca gggcaaatgg 8580aaaacaggag atcctaatat gaaagaaaaa tggatcccaa tctgagaaaa ggcaaaagaa 8640tggctacttt tttctatgct ggagtatttt ctaataatcc tgcttgaccc ttatctgacc 8700tctttggaaa ctataacata gctgtcacag tatagtcaca atccacaaat gatgcaggtg 8760caaatggttt atagccctgt gaagttctta aagtttagag gctaacttac agaaatgaat 8820aagttgtttt gttttatagc ccggtagagg agttaacccc aaaggtgata tggttttatt 8880tcctgttatg tttaacttga taatcttatt ttggcattct tttcccattg actatataca 8940tctctatttc tcaaatgttc atggaactag ctcttttatt ttcctgctgg tttcttcagt 9000aatgagttaa ataaaacatt gacacataca aacaaaaaaa aaaaaaaa 904874410DNAArtificial SequenceBDD FVIII cDNA 7atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctaccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280ttctctcaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttctcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac

ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta cggaggcggt 4380ggggactaca aggacgatga cgacaagtaa 441084374DNAArtificial SequenceBDD FVIII cDNA 8atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctaccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280ttctctcaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttctcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctaa 4374

Claims

1. A modified FVIII protein comprising a cysteine substitution mutation at an amino acid corresponding to a position in the amino acid sequence of SEQ ID NO:1 selected from the group of amino acid positions consisting of: 2094, 2186, 2204, 2206, 59, 239, 333, 336, 379, 481, 484, 486, 488, 489, 490, 492, 493, 495, 496, 497, 499, 500, 501, 507, 555, 562, 568, 571, 582, 1680, 1778, 1793, 1794, 1797, 1798, 1799, 1800, 1801, 1806, 1810, 1811, 1814, 1816, 1818, 1891, 2035, 2068, 2092, 2093, 2095, 2118, 2125, 2183, 2191, 2196, and 2212.

2. The modified FVIII protein of claim 1, further comprising at least one additional substitution other than cysteine at a position corresponding to 336, 562, 1680 or 1968 in the amino acid sequence of SEQ ID NO:1 selected from the group consisting of: R336A, R562A, Y1680F and K1968A.

3. The modified FVIII protein of claim 1, further comprising two additional substitutions other than cysteine at positions corresponding to 336 and 1680 in the amino acid sequence of SEQ ID NO:1, wherein said substitutions are R336A and Y1680F.

4. (canceled)

5. The modified FVIII protein of claim 1, wherein said FVIII protein is a single chain.

6. The modified FVIII protein of claim 1, wherein said FVIII protein is an inactive two-chain form capable of being activated by thrombin.

7. The modified FVIII protein of claim 1, wherein said FVIII protein lacks all or part of the B domain.

8. The modified FVIII protein of claim 7, wherein in its single chain form said FVIII protein has the amino acid sequence of SEQ ID NO:2.

9. A conjugate comprising the modified FVIII protein of claim 1 and a biocompatible polymer covalently attached directly, or indirectly via a linker, to said cysteine substitution mutation.

10. The conjugate of claim 9, wherein the conjugate further comprises a spacer.

11. The conjugate of claim 9, wherein said covalent attachment is to the sulfur atom of the thiol group of said cysteine substitution mutation.

12. The conjugate of claim 9, wherein said biocompatible polymer is selected from the group consisting of: polyethylene glycol (PEG), hydroxyalkyl starch (HAS), hydroxyethyl starch (HES), polysialic acid (PSA), a zwitterionic brush polymer, and a poly-phosphorylcholine branched polymer.

13. The conjugate of claim 12, wherein said biocompatible polymer is hydroxyethyl starch (HES).

14. (canceled)

15. A nucleic acid encoding the modified FVIII protein of claim 1.

16. A host cell comprising the nucleic acid of claim 15.

17. A vector comprising the nucleic acid of claim 15.

18. A host cell comprising the vector of claim 17.

19. A method of producing the modified FVIII protein of claim 1, comprising culturing the host cell of claim 18 under conditions where the protein is expressed and isolating said protein.

20. (canceled)

21. A composition comprising the conjugate of claim 13 and a pharmaceutically acceptable excipient.

22. A method of preventing or treating bleeding in a subject having a deficiency of FVIII activity comprising the step of administering to a subject having a deficiency of FVIII activity a prophylactically or therapeutically effective dose of the composition of claim 21.

23. The method of claim 22, wherein said subject has Hemophilia A.

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)