Recombinant Factor Viii Having Enhanced Stability Following Mutation At The A1-c2 Domain Interface

Abstract

The invention relates to a recombinant factor VIII that includes one or more mutations at an interface of A1 and C2 domains of recombinant factor VIII. The one or more mutations include substitution of one or more amino acid residues with either a cysteine or an amino acid residue having a higher hydrophobicity. This results in enhanced stability of factor VIII. Methods for making the recombinant factor VIII, pharmaceutical compositions containing the recombinant factor VIII, and use of the recombinant factor VIII for treating hemophilia A are also disclosed.

Description

[0001] This application is a continuation of U.S. patent application Ser. No. 14/141,983, filed Dec. 27, 2013, now U.S. Pat. No. 8,865,154, which is a division of U.S. patent application Ser. No. 13/231,948, filed Sep. 13, 2011, now U.S. Pat. No. 8,637,448, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/382,919, filed Sep. 14, 2010, which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

[0003] Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. There is no cure for Hemophilia A and treatment consists of replacement therapy using preparations of (purified) plasma or the recombinant protein.

[0004] Factor VIII circulates as a non-covalent, metal ion-dependent heterodimer. This procofactor form of the protein contains a heavy chain (HC) comprised of A1(a1)A2(a2)B domains and a light chain (LC) comprised of (a3)A3C1C2 domains, with the lower case a representing short (.about.30-40 residue) segments rich in acidic residues (see Fay, "Activation of Factor VIII and Mechanisms of Cofactor Action," Blood Rev. 18:1-15 (2004)). Factor VIII is activated by proteolytic cleavages at the A1A2, A2B and A3A3 junctions catalyzed by thrombin or factor Xa. The product of this reaction, factor VIIIa, is a heterotrimer comprised of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (see Fay, "Activation of Factor VIII and Mechanisms of Cofactor Action," Blood Rev. 18:1-15 (2004)).

[0005] Reconstitution studies have shown that the factor VIII heterodimeric structure is supported by both electrostatic and hydrophobic interactions (Fay, "Reconstitution of Human Factor VIII from Isolated Subunits," Arch Biochem Biophys. 262:525-531 (1988); Ansong et al., "Factor VIII A1 Domain Residues 97-105 Represent a Light Chain-interactive Site," Biochemistry 45:13140-13149 (2006)), and the inter-chain affinity is further strengthened by factor VIII binding von Willebrand factor (Fay, "Reconstitution of Human Factor VIII from Isolated Subunits," Arch Biochem Biophys. 262:525-531 (1988); Kaufman et al., "Regulation of Factor VIII Expression and Activity by von Willebrand Factor," Thromb Haemost. 82:201-208 (1999)). Metal ions also contribute to the inter-chain affinity and activity parameters (Wakabayashi et al., "Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and Inter-subunit Affinity," Biochemistry 40:10293-10300 (2001)). Calcium is required to yield the active factor VIII conformation. Mutagenesis studies mapped a calcium-binding site to a segment rich in acidic residues within the A1 domain (residues 110-126) and identified specific residues within this region prominent in the coordination of the ion (Wakabayashi et al., "Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca.sup.2+ Binding Site Required for Cofactor Activity," J Biol Chem. 279:12677-12684 (2004)). A recent intermediate resolution X-ray structure (Shen et al., "The Tertiary Structure and Domain Organization of Coagulation Factor VIII," Blood 111:1240-1247 (2008)) confirmed this calcium-binding site as well as suggested a second potential site within the A2 domain. This structure also showed occupancy of the two type 1 copper ion sites within the A1 and A3 domains. Earlier functional studies have shown that copper ions facilitate the association of the heavy and light chains to form the heterodimer, increasing the inter-chain affinity by several-fold at physiologic pH (Fay et al., "Human Factor VIIIa Subunit Structure: Reconstruction of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit," J Biol Chem. 266:8957-8962 (1991); Wakabayashi et al., "pH-dependent Association of Factor VIII Chains: Enhancement of Affinity at Physiological pH by Cu.sup.2+," Biochim Biophys Acta. 1764:1094-1101 (2006); Ansong et al., "Factor VIII A3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site," Biochemistry 44:8850-8857 (2005)).

[0006] The instability of factor VIIIa results from weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer (Fay et al., "Human Factor VIIIa Subunit Structure: Reconstruction of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit," J Biol Chem. 266:8957-8962 (1991); Lollar et al., "pH-dependent Denaturation of Thrombin-activated Porcine Factor VIII," J Biol Chem. 265:1688-1692 (1990)) and leads to dampening of factor Xase activity (Lollar et al., "Coagulant Properties of Hybrid Human/Porcine Factor VIII Molecules," J Biol Chem. 267:23652-23657 (1992); Fay et al., "Model for the Factor VIIIa-dependent Decay of the Intrinsic Factor Xase: Role of Subunit Dissociation and Factor IXa-catalyzed Proteolysis," J Biol Chem. 271:6027-6032 (1996)). Limited information is available regarding the association of the A2 subunit in factor VIIIa, and residues in both the A1 and A3 domains appear to make contributions to the retention of this subunit. Several factor VIII point mutations have been shown to facilitate the dissociation of A2 relative to WT and these residues localize to either the A1-A2 domain interface (Pipe et al., "Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa in vivo," Blood 93:176-183 (1999); Pipe et al., "Hemophilia A Mutations Associated with 1-stage/2-stage Activity Discrepancy Disrupt Protein-protein Interactions within the Triplicated A Domains of Thrombin-activated Factor VIIIa," Blood 97:685-691 (2001)) or the A2-A3 domain interface (Hakeos et al., "Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/Two-stage Activity Discrepancy," Thromb Haemost. 88:781-787 (2002)). These factor VIII variants demonstrate a characteristic one-stage/two-stage assay discrepancy (Duncan et al., "Familial Discrepancy Between the One-stage and Two-stage Factor VIII Methods in a Subgroup of Patients with Haemophilia A," Br J Haematol. 87:846-848 (1994); Rudzki et al., "Mutations in a Subgroup of Patients with Mild Haemophilia A and a Familial Discrepancy Between the One-stage and Two-stage Factor VIII:C Methods," Br J Haematol. 94:400-406 (1996)), with significant reductions in activity values determined by the latter assay as a result of increased rates of A2 subunit dissociation.

[0007] Significant interest exists in stabilizing factor VIIIa, since a more stable form of the protein would represent a superior therapeutic for hemophilia A, potentially requiring less material to treat the patient (Fay et al., "Mutating Factor VIII: Lessons from Structure to Function," Blood Reviews 19:15-27 (2005)). To this end, preparations of factor VIII have been described where mutations have been made in the recombinant protein to prevent the dissociation of the A2 subunit by introducing novel covalent bonds between A2 and other factor VIII domains (Pipe et al., "Characterization of a Genetically Engineered Inactivation-resistant Coagulation Factor VIIIa," Proc Natl Acad Sci USA 94:11851-11856 (1997); Gale et al., "An Engineered Interdomain Disulfide Bond Stabilizes Human Blood Coagulation Factor VIIIa," J. Thromb. Haemostasis 1:1966-1971 (2003)). However, it has since been suggested that these types of mutation may not be desirable in a therapeutic factor VIII, because they substantially eliminate means for down-regulation. This situation could yield a prothrombotic condition, which may cause harm. Thus, it would be desirable to enhance the stability of both factor VIII and factor VIIIa, but in a manner that minimizes the likelihood of promoting prothrombotic conditions.

[0008] In U.S. Patent Application Publ. No. 20090118184, a number of recombinant factor VIII proteins are identified that possess one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa. These recombinant factor VIII proteins have one or more substitutions of a charged amino acid residue with a hydrophobic amino acid residue at either or both of the A1-A2 or A2-A3 domain interfaces. Despite the improvements made in the stability of recombinant factor VIII proteins (and their active forms, factor VIIIa), the need for further improvements continue to exist.

[0009] The intermediate resolution X-ray structures of factor VIII (Shen et al., "The Tertiary Structure and Domain Organization of Coagulation Factor VIII," Blood 111:1240-1247 (2008); Ngo et al., "Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa:Factor VIIIa Complex," Structure 16:597-606 (2008)) show close contact between A1 (heavy chain) and C2 (light chain) domains. It was recently reported that a factor VIII variant lacking the C2 domain retained the capacity to bind phospholipid membranes, albeit with a marked reduction in affinity (Wakabayashi et al., "Factor VIII Lacking the C2 Domain Retains Cofactor Activity in vitro," J. Biol. Chem 285:25176-25184 (2010)), supporting a direct role for the C1 domain in this interaction. Furthermore, deletion of the C2 domain did not grossly alter a number of functional properties including the rate of procofactor activation by thrombin, affinity of factor VIIIa for factor IXa, K.sub.m of factor Xase for substrate factor X, or k.sub.cat for factor Xa generation. However, this deletion did significantly destabilize the cofactor, as judged by increased rates of activity decay following exposure to elevated temperature or chemical denaturants. While contacts between the A1 and C2 domains of factor VIII appear to contribute to protein, and in particular heterodimer stability, little information is available on specific interactions and their functional significance. It would be desirable, therefore, to identify amino acid residues that can be modified to enhance stability between the A1 (heavy chain) and C2 (light chain) domains.

[0010] The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

[0011] A first aspect of the present invention relates to a recombinant factor VIII that includes one or more mutations at an interface of A1 and C2 domains of recombinant factor VIII. This results in enhanced stability of factor VIII. The one or more mutations include substitution of one or more amino acid residues with either a cysteine or an amino acid residue having a higher hydrophobicity.

[0012] According to one embodiment, the recombinant factor VIII includes one or more mutations at an interface of A1 and C2 domains of recombinant factor VIII that result in enhanced stability of factor VIII, wherein the one or more mutations comprise substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity.

[0013] According to another embodiment, the recombinant factor VIII includes (i) two or more mutations at an interface of A1 and C2 domains of recombinant factor VIII, wherein the two or more mutations comprise substitution of two or more amino acid residues with a Cysteine residue to afford a disulfide bond between the A1 and C2 domains; and (ii) one or more mutations at an interface of A1 and A2 domains or an interface of A2 and A3 domains of recombinant factor VIII, said one or more mutations comprising substitution of one or more charged amino acid residues with a hydrophobic amino acid residue. The recombinant factor VIII possessing these mutations exhibits enhanced stability of both factor VIII and factor VIIIa.

[0014] A second aspect of the present invention relates to the recombinant factor VIII according to the first aspect of the present invention, wherein the recombinant factor VIII further includes one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VIII for one or both of factor IXa and factor X; (ii) modified sites that enhance secretion in culture; (iii) modified serum protein binding sites that enhance the circulating half-life thereof; (iv) at least one glycosylation recognition sequence that is effective in decreasing antigenicity and/or immunogenicity thereof; (v) a modified A1 domain calcium-binding site that improves specific activity of the recombinant factor Villa; (vi) modified activated protein C-cleavage site; (vii) a modified A1 and A2 domain interface; and (viii) a modified A2 and A3 domain interface.

[0015] A third aspect of the present invention relates to a pharmaceutical composition that includes the recombinant factor VIII according to the present invention.

[0016] A fourth aspect of the present invention relates to an isolated nucleic acid molecule encoding a recombinant factor VIII of the present invention. Also included within this aspect of the present invention are recombinant DNA expression systems that contain a DNA molecule encoding the recombinant factor VIII of the present invention, and recombinant host cells that contain the DNA molecule and/or recombinant expression system.

[0017] A fifth aspect of the present invention relates to a method of making a recombinant factor VIII that includes: growing a host cell according to the fourth aspect of the present invention under conditions whereby the host cell expresses the recombinant factor VIII; and isolating the recombinant factor VIII.

[0018] A sixth aspect of the present invention relates to a method of treating an animal for hemophilia A. This method of treatment includes: administering to an animal exhibiting hemophilia A an effective amount of the recombinant factor VIII of the present invention, whereby the animal exhibits effective blood clotting following vascular injury.

[0019] The present invention demonstrates that a number of residues at the interface of A1 and C2 domains do not participate in non-covalent bonding, but instead may be destabilizing to factor VIII structure. Replacement of these residues with more hydrophobic residues--with the aim of increasing the buried hydrophobic area and reducing the buried hydrophilic area--was shown in the accompanying Examples to enhance inter-domain binding affinity. Stability parameters were assessed by following the activity of the factor VIII variants. Results from these studies demonstrated that a number of mutations yielded increased stability parameters. These stabilized variants of factor VIII and activated cofactor VIIIa should afford an improved therapeutic for treatment of hemophilia A.

[0020] To explore the role of this region in factor VIII and factor VIIIa stability, a variant containing a disulfide bond between A1 and C2 domains was generated by mutating Arg121 and Leu2302 to Cys (R121C-L2302C) and a second variant was generated by substituting a bulkier hydrophobic group (Ala108Ile) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C-L2302C variant was >90% efficient as judged by western blots. Binding affinity between the Ala108Ile A1 and A3C1C2 subunits was increased .about.3.7-fold in the variant compared with WT as judged by changes in fluorescence of acrylodan labeled-A1 subunits. Factor VIII thermal and chemical stability were monitored following rates of loss of factor VIII activity at 57.degree. C. or in guanidinium by factor Xa generation assays. The rate of decay of factor VIIIa activity was monitored at 23.degree. C. following activation by thrombin. Both R121C-L2302C and Ala108Ile variants showed up to .about.4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of Ala108Ile reconstituted with the A3C1C2 subunit showed a .about.4.6-fold increase in thermal stability while reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances factor VIII stability. Moreover, by combining these mutations with one or more mutations at the A1-A2 and/or A2-A3 domain interfaces, including Asp519Ala, Asp519Val, Glu665Ala, Glu665Val, Glu1984Ala, and Glu1984Val, double and triple mutants displayed .about.2-10 fold increases in the stability of factor VIIIa.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows alignment of partial A1 domain sequences of eight mammalian factor VIII molecules starting from amino acid residue A100 of human factor VIII. The partial amino acid sequences are from human factor VIII (residues 100-140 of SEQ ID NO: 2); pig factor VIII (SEQ ID NO: 3); canine factor VIII (SEQ ID NO: 4); mouse factor VIII (SEQ ID NO: 5); rabbit factor VIII (SEQ ID NO: 6); bat factor VIII (SEQ ID NO: 7); rat factor VIII (SEQ ID NO: 8); and sheep factor VIII (SEQ ID NO: 9). The figure also shows the consensus amino acid sequence (SEQ ID NO: 10) for the partial A1 domain. Underlined residues represent a C2 domain interface of the A1 domain.

[0022] FIG. 2 shows alignment of partial C2 domain sequences from eight mammalian factor VIII molecules starting from amino acid residue Q2231 of human factor VIII. The partial amino acid sequences are from human factor VIII (residues 2231-2334 of SEQ ID NO: 2); pig factor VIII (SEQ ID NO: 11); canine factor VIII (SEQ ID NO: 12); mouse factor VIII (SEQ ID NO: 13); rabbit factor VIII (SEQ ID NO: 14); bat factor VIII (SEQ ID NO: 15); rat factor VIII (SEQ ID NO: 16); and sheep factor VIII (SEQ ID NO: 17). The figure also shows the consensus amino acid sequence (SEQ ID NO: 18) for the partial C2 domain. Underlined residues represent an A1 domain interface of the C2 domain.

[0023] FIGS. 3A-C illustrate a model of selected residues at the A1-C2 domain interface. In FIG. 3A, the molecular surface of the factor VIII X-ray crystal structure, drawn by Swiss PDB viewer, shows the A1 domain (residues 1-336) in yellow, A2 domain (residues 373-711) in blue, A3 domain (residues 1690-2020) in red, C1 domain (residues 2021-2169) in green, and C2 domain (residues 2170-2332) in grey (FIG. 3A). Higher magnification of the A1-C2 contact region (yellow circle) is presented in FIGS. 3B-C. Side chains of Arg121 and Leu2302 (drawn as stick models) mutated to Cys residues are modeled by Swiss PDB viewer (FIG. 3B). In FIG. 3C, residues surrounding Ala108 (Leu2302, Ala2328, and Gln2329) are highlighted as stick models. C.beta. carbon of Ala108, C.delta. of Leu2302, C.beta. of Ala2328, and C.gamma. of Gln2329 are indicated by arrows (FIG. 3C, left panel). Ala108 mutated to Ile was modeled by Swiss PDB viewer (FIG. 3C, right panel). In the stick models hydrogen, carbon, oxygen, nitrogen, and sulfur are colored as cyan, white, red, blue, and yellow, respectively. Ribbon structures represent .alpha.-helix (red) and .beta.-strand (green).

[0024] FIG. 4 is a Western blot analysis of R121C-L2302C and WT factor VIII. Purified WT and mutant factor VIII proteins were electrophoresed under non-reducing (lanes 1, 2, 5, and 6) or reducing (lanes 3, 4, 7, and 8) conditions, transferred, and probed with 58.12 (anti-A1 domain antibody, lane 1-4) or 2D2 (anti-A3 domain antibody, lane 5-8). Protein bands were visualized by chemifluorescence as described in the accompanying Examples. Shown are WT (lane 1, 3, 5, and 7) and the R121C-L2302C factor VIII variant (lane 2, 4, 6, and 8) proteins.

[0025] FIG. 5 is a graph showing the binding affinity of A1 subunit from WT or Ala108Ile for the A3C1C2 subunit. Acrylodan-labeled A1 subunit from WT (circles) or Ala108Ile (triangles) was titrated with A3C1C2 and binding was detected by the change of fluorescence as described in the accompanying Examples. Data points averaged from 3 separate determinations were fitted to a quadratic equation curve by non-linear least squares regression.

[0026] FIGS. 6A-C are graphs that illustrate factor VIII R121C-L2302C and Ala108Ile variant stability. FIG. 6A shows the factor VIII activity decay at elevated temperature. Factor VIII (4 nM) was incubated at 57.degree. C. and at the indicated times aliquots were removed and activity was measured by factor Xa generation assays as described in the accompanying Examples. Data were fitted to a single exponential decay curve by non-linear least squares regression. FIG. 6B shows the inhibition of factor VIII by guanidinium. FVIII (50 nM) in 0-1.2 M guanidinium chloride was incubated for 2 hrs at 23.degree. C., diluted 1/50 and factor VIII activity was measured by factor Xa generation. Data were fitted to a linear equation by least squares regression. Bars represent standard error values of 3 measurements. FIG. 6C shows factor VIIIa decay. Thrombin-activated factor VIIIa (1.5 nM) was incubated at 23.degree. C., aliquots were taken at indicated time points and activity was measured by factor Xa generation assay as described in the accompanying Examples. Data were fitted to a single exponential decay curve by non-linear least squares regression. Symbols denote WT (circles), R121C-L2302C (triangles), and Ala108Ile (squares). Each point represents the value averaged from three separate determinations.

[0027] FIGS. 7A-B are graphs showing the thermal stability of A1 subunit reconstituted with A3C1C2 or A3C1 subunit. Thermal decay of reconstituted heterodimer of A1 subunit from WT (circles) or Ala108Ile (triangles) with WT A3C1C2 subunit at 55.degree. C. (FIG. 7A) or A3C1 subunit at 52.degree. C. (FIG. 7B) was detected by residual factor VIIIa activity following addition of A2 subunit as described in the accompanying Examples. Data were fitted to a single exponential decay curve by non-linear least squares regression. Data points averaged from 3 separate determinations were fitted to a single exponential decay curve to obtain rates.

[0028] FIGS. 8A-C are bar graphs comparing the thermal stability (FIG. 8A), chemical stability in the presence of guanidinium (FIG. 8B), and factor VIIIa decay (FIG. 8C) of WT and combination mutants. The combination mutants include one or more of the A1-C2 domain interface mutations (A108I or R121C-L2302C) in combination with one or more A1-A2 or A2-A3 domain interface mutations. Factor VIII activity decay, guanidinium inhibition, and factor VIIIa decay were measured as described in the accompanying Examples.

[0029] FIG. 9 is a graph comparing the effects of Ala108Ile, Asp519Val/Glu665Val, and the combined Ala108Ile/Asp519Val/Glu665Val variants, relative to wildtype, as measured using a thrombin generation assay.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to a recombinant factor VIII having one or more mutations that result in enhanced stability of factor VIII, in particular improved thermal and/or chemical stability of factor VIII.

[0031] The recombinant factor VIII of the present invention can be prepared by modifying the amino acid sequence of a wild-type factor VIII or a mutant factor VIII that has otherwise been modified to affect other properties of the factor VIII, such as antigenicity, factor VIIIa stability, circulating half-life, protein secretion, affinity for factor IXa and/or factor X, altered factor VIII-inactivation cleavage sites, immunogenicity, shelf-life, etc.

[0032] Suitable wild-type factor VIII that can be modified in accordance with the present invention can be from various animals including, without limitation, mammals such as humans (see, e.g., GenBank Accession Nos. AAA52484 (amino acid) and K01740 (nucleotide); and GenBank Accession Nos. CAD97566 (amino acid) and AX746360 (nucleotide), which are hereby incorporated by reference in their entirety), rats (see, e.g., GenBank Accession Nos. AAQ21580 (amino acid) and AY362193 (nucleotide), which are hereby incorporated by reference in their entirety), mice (see, e.g., GenBank Accession Nos. AAA37385 (amino acid) and L05573 (nucleotide), which are hereby incorporated by reference in their entirety), guinea pigs, dogs (see, e.g., GenBank Accession Nos. AAB87412 (amino acid) and AF016234 (nucleotide); and GenBank Accession Nos. AACO5384 (amino acid) and AF049489 (nucleotide), which are hereby incorporated by reference in their entirety), cats, monkeys, chimpanzees (see, e.g., GenBank Accession Nos. XP.sub.--529212 (amino acid) and XM.sub.--529212 (nucleotide), which are hereby incorporated by reference in their entirety), orangutans, cows, horses, sheep, pigs (see, e.g., GenBank Accession Nos. NP.sub.--999332 (amino acid) and NM.sub.--214167 (nucleotide), which are hereby incorporated by reference in their entirety), goats, rabbits, and chickens. These and other sequences are also available electronically via the Haemophilia A Mutation, Structure, Test and Resource Site (or HAMSTeRS), which further provides an alignment of human, porcine, murine, and canine factor VIII proteins. Thus, the conservation and homology among mammalian factor VIII proteins is well known.

[0033] By way of example, the human factor VIII cDNA nucleotide and predicted amino acid sequences are shown below in SEQ ID NOs: 1 and 2, respectively. Human factor VIII is synthesized as an approximately 300 kDa single chain protein with internal sequence homology that defines the "domain" sequence NH.sub.2-A1-A2-B-A3-C1-C2-COOH. In a factor VIII molecule, a "domain," as used herein, is a continuous sequence of amino acids that is defined by internal amino acid sequence identity and sites of proteolytic cleavage by thrombin. Unless otherwise specified, factor VIII domains include the following amino acid residues, when the sequences are aligned with the human amino acid sequence (SEQ ID NO: 2):

[0034] A1, residues Ala.sub.1-Arg.sub.372;

[0035] A2, residues Ser.sub.373-Arg.sub.740;

[0036] B, residues Ser.sub.741-Arg.sub.1648;

[0037] A3, residues Ser.sub.1690-Ile.sub.2032;

[0038] C1, residues Arg.sub.2033-Asn.sub.2172; and

[0039] C2, residues Ser.sub.2173-TYr.sub.2332.

[0040] The A3-C1-C2 sequence includes residues Ser.sub.1690-Tyr.sub.2332. The remaining sequence, residues Glu.sub.1649-Arg.sub.1689, is usually referred to as the factor VIII light chain activation peptide. Factor VIII is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor, forming factor VIIIa, which has procoagulant function. The biological function of factor VIIIa is to increase the catalytic efficiency of factor IXa toward factor X activation by several orders of magnitude. Thrombin-activated factor VIIIa is a 160 kDa A1/A2/A3-C1-C2 heterotrimer that forms a complex with factor IXa and factor X on the surface of platelets or monocytes. A "partial domain" as used herein is a continuous sequence of amino acids forming part of a domain.

[0041] As used herein, a residue corresponding to a particular position refers to the residue at that position in wild type human factor VIII; however the same position will retain its numbering even though the residue may be at a different location in a recombinant factor VIII of the present invention. For example, a B domainless factor VIII lacks the B domain yet it still retains the conventional residue numbering for domains A3, C1, and C2.

[0042] The cDNA encoding wild-type human factor VIII has a nucleotide sequence of SEQ ID NO: 1, as follows:

TABLE-US-00001 1 GCCACCAGAA GATACTACCT GGGTGCAGTG GAACTGTCAT GGGACTATAT 51 GCAAAGTGAT CTCGGTGAGC TGCCTGTGGA CGCAAGATTT CCTCCTAGAG 101 TGCCAAAATC TTTTCCATTC AACACCTCAG TCGTGTACAA AAAGACTCTG 151 TTTGTAGAAT TCACGGATCA CCTTTTCAAC ATCGCTAAGC CAAGGCCACC 201 CTGGATGGGT CTGCTAGGTC CTACCATCCA GGCTGAGGTT TATGATACAG 251 TGGTCATTAC ACTTAAGAAC ATGGCTTCCC ATCCTGTCAG TCTTCATGCT 301 GTTGGTGTAT CCTACTGGAA AGCTTCTGAG GGAGCTGAAT ATGATGATCA 351 GACCAGTCAA AGGGAGAAAG AAGATGATAA AGTCTTCCCT GGTGGAAGCC 401 ATACATATGT CTGGCAGGTC CTGAAAGAGA ATGGTCCAAT GGCCTCTGAC 451 CCACTGTGCC TTACCTACTC ATATCTTTCT CATGTGGACC TGGTAAAAGA 501 CTTGAATTCA GGCCTCATTG GAGCCCTACT AGTATGTAGA GAAGGGAGTC 551 TGGCCAAGGA AAAGACACAG ACCTTGCACA AATTTATACT ACTTTTTGCT 601 GTATTTGATG AAGGGAAAAG TTGGCACTCA GAAACAAAGA ACTCCTTGAT 651 GCAGGATAGG GATGCTGCAT CTGCTCGGGC CTGGCCTAAA ATGCACACAG 701 TCAATGGTTA TGTAAACAGG TCTCTGCCAG GTCTGATTGG ATGCCACAGG 751 AAATCAGTCT ATTGGCATGT GATTGGAATG GGCACCACTC CTGAAGTGCA 801 CTCAATATTC CTCGAAGGTC ACACATTTCT TGTGAGGAAC CATCGCCAGG 851 CGTCCTTGGA AATCTCGCCA ATAACTTTCC TTACTGCTCA AACACTCTTG 901 ATGGACCTTG GACAGTTTCT ACTGTTTTGT CATATCTCTT CCCACCAACA 951 TGATGGCATG GAAGCTTATG TCAAAGTAGA CAGCTGTCCA GAGGAACCCC 1001 AACTACGAAT GAAAAATAAT GAAGAAGCGG AAGACTATGA TGATGATCTT 1051 ACTGATTCTG AAATGGATGT GGTCAGGTTT GATGATGACA ACTCTCCTTC 1101 CTTTATCCAA ATTCGCTCAG TTGCCAAGAA GCATCCTAAA ACTTGGGTAC 1151 ATTACATTGC TGCTGAAGAG GAGGACTGGG ACTATGCTCC CTTAGTCCTC 1201 GCCCCCGATG ACAGAAGTTA TAAAAGTCAA TATTTGAACA ATGGCCCTCA 1251 GCGGATTGGT AGGAAGTACA AAAAAGTCCG ATTTATGGCA TACACAGATG 1301 AAACCTTTAA GACTCGTGAA GCTATTCAGC ATGAATCAGG AATCTTGGGA 1351 CCTTTACTTT ATGGGGAAGT TGGAGACACA CTGTTGATTA TATTTAAGAA 1401 TCAAGCAAGC AGACCATATA ACATCTACCC TCACGGAATC ACTGATGTCC 1451 GTCCTTTGTA TTCAAGGAGA TTACCAAAAG GTGTAAAACA TTTGAAGGAT 1501 TTTCCAATTC TGCCAGGAGA AATATTCAAA TATAAATGGA CAGTGACTGT 1551 AGAAGATGGG CCAACTAAAT CAGATCCTCG GTGCCTGACC CGCTATTACT 1601 CTAGTTTCGT TAATATGGAG AGAGATCTAG CTTCAGGACT CATTGGCCCT 1651 CTCCTCATCT GCTACAAAGA ATCTGTAGAT CAAAGAGGAA ACCAGATAAT 1701 GTCAGACAAG AGGAATGTCA TCCTGTTTTC TGTATTTGAT GAGAACCGAA 1751 GCTGGTACCT CACAGAGAAT ATACAACGCT TTCTCCCCAA TCCAGCTGGA 1801 GTGCAGCTTG AGGATCCAGA GTTCCAAGCC TCCAACATCA TGCACAGCAT 1851 CAATGGCTAT GTTTTTGATA GTTTGCAGTT GTCAGTTTGT TTGCATGAGG 1901 TGGCATACTG GTACATTCTA AGCATTGGAG CACAGACTGA CTTCCTTTCT 1951 GTCTTCTTCT CTGGATATAC CTTCAAACAC AAAATGGTCT ATGAAGACAC 2001 ACTCACCCTA TTCCCATTCT CAGGAGAAAC TGTCTTCATG TCGATGGAAA 2051 ACCCAGGTCT ATGGATTCTG GGGTGCCACA ACTCAGACTT TCGGAACAGA 2101 GGCATGACCG CCTTACTGAA GGTTTCTAGT TGTGACAAGA ACACTGGTGA 2151 TTATTACGAG GACAGTTATG AAGATATTTC AGCATACTTG CTGAGTAAAA 2201 ACAATGCCAT TGAACCAAGA AGCTTCTCCC AGAATTCAAG ACACCCTAGC 2251 ACTAGGCAAA AGCAATTTAA TGCCACCACA ATTCCAGAAA ATGACATAGA 2301 GAAGACTGAC CCTTGGTTTG CACACAGAAC ACCTATGCCT AAAATACAAA 2351 ATGTCTCCTC TAGTGATTTG TTGATGCTCT TGCGACAGAG TCCTACTCCA 2401 CATGGGCTAT CCTTATCTGA TCTCCAAGAA GCCAAATATG AGACTTTTTC 2451 TGATGATCCA TCACCTGGAG CAATAGACAG TAATAACAGC CTGTCTGAAA 2501 TGACACACTT CAGGCCACAG CTCCATCACA GTGGGGACAT GGTATTTACC 2551 CCTGAGTCAG GCCTCCAATT AAGATTAAAT GAGAAACTGG GGACAACTGC 2601 AGCAACAGAG TTGAAGAAAC TTGATTTCAA AGTTTCTAGT ACATCAAATA 2651 ATCTGATTTC AACAATTCCA TCAGACAATT TGGCAGCAGG TACTGATAAT 2701 ACAAGTTCCT TAGGACCCCC AAGTATGCCA GTTCATTATG ATAGTCAATT 2751 AGATACCACT CTATTTGGCA AAAAGTCATC TCCCCTTACT GAGTCTGGTG 2801 GACCTCTGAG CTTGAGTGAA GAAAATAATG ATTCAAAGTT GTTAGAATCA 2851 GGTTTAATGA ATAGCCAAGA AAGTTCATGG GGAAAAAATG TATCGTCAAC 2901 AGAGAGTGGT AGGTTATTTA AAGGGAAAAG AGCTCATGGA CCTGCTTTGT 2951 TGACTAAAGA TAATGCCTTA TTCAAAGTTA GCATCTCTTT GTTAAAGACA 3001 AACAAAACTT CCAATAATTC AGCAACTAAT AGAAAGACTC ACATTGATGG 3051 CCCATCATTA TTAATTGAGA ATAGTCCATC AGTCTGGCAA AATATATTAG 3101 AAAGTGACAC TGAGTTTAAA AAAGTGACAC CTTTGATTCA TGACAGAATG 3151 CTTATGGACA AAAATGCTAC AGCTTTGAGG CTAAATCATA TGTCAAATAA 3201 AACTACTTCA TCAAAAAACA TGGAAATGGT CCAACAGAAA AAAGAGGGCC 3251 CCATTCCACC AGATGCACAA AATCCAGATA TGTCGTTCTT TAAGATGCTA 3301 TTCTTGCCAG AATCAGCAAG GTGGATACAA AGGACTCATG GAAAGAACTC 3351 TCTGAACTCT GGGCAAGGCC CCAGTCCAAA GCAATTAGTA TCCTTAGGAC 3401 CAGAAAAATC TGTGGAAGGT CAGAATTTCT TGTCTGAGAA AAACAAAGTG 3451 GTAGTAGGAA AGGGTGAATT TACAAAGGAC GTAGGACTCA AAGAGATGGT 3501 TTTTCCAAGC AGCAGAAACC TATTTCTTAC TAACTTGGAT AATTTACATG 3551 AAAATAATAC ACACAATCAA GAAAAAAAAA TTCAGGAAGA AATAGAAAAG 3601 AAGGAAACAT TAATCCAAGA GAATGTAGTT TTGCCTCAGA TACATACAGT 3651 GACTGGCACT AAGAATTTCA TGAAGAACCT TTTCTTACTG AGCACTAGGC 3701 AAAATGTAGA AGGTTCATAT GACGGGGCAT ATGCTCCAGT ACTTCAAGAT 3751 TTTAGGTCAT TAAATGATTC AACAAATAGA ACAAAGAAAC ACACAGCTCA 3801 TTTCTCAAAA AAAGGGGAGG AAGAAAACTT GGAAGGCTTG GGAAATCAAA 3851 CCAAGCAAAT TGTAGAGAAA TATGCATGCA CCACAAGGAT ATCTCCTAAT 3901 ACAAGCCAGC AGAATTTTGT CACGCAACGT AGTAAGAGAG CTTTGAAACA 3951 ATTCAGACTC CCACTAGAAG AAACAGAACT TGAAAAAAGG ATAATTGTGG 4001 ATGACACCTC AACCCAGTGG TCCAAAAACA TGAAACATTT GACCCCGAGC 4051 ACCCTCACAC AGATAGACTA CAATGAGAAG GAGAAAGGGG CCATTACTCA 4101 GTCTCCCTTA TCAGATTGCC TTACGAGGAG TCATAGCATC CCTCAAGCAA 4151 ATAGATCTCC ATTACCCATT GCAAAGGTAT CATCATTTCC ATCTATTAGA 4201 CCTATATATC TGACCAGGGT CCTATTCCAA GACAACTCTT CTCATCTTCC 4251 AGCAGCATCT TATAGAAAGA AAGATTCTGG GGTCCAAGAA AGCAGTCATT 4301 TCTTACAAGG AGCCAAAAAA AATAACCTTT CTTTAGCCAT TCTAACCTTG 4351 GAGATGACTG GTGATCAAAG AGAGGTTGGC TCCCTGGGGA CAAGTGCCAC 4401 AAATTCAGTC ACATACAAGA AAGTTGAGAA CACTGTTCTC CCGAAACCAG 4451 ACTTGCCCAA AACATCTGGC AAAGTTGAAT TGCTTCCAAA AGTTCACATT 4501 TATCAGAAGG ACCTATTCCC TACGGAAACT AGCAATGGGT CTCCTGGCCA 4551 TCTGGATCTC GTGGAAGGGA GCCTTCTTCA GGGAACAGAG GGAGCGATTA 4601 AGTGGAATGA AGCAAACAGA CCTGGAAAAG TTCCCTTTCT GAGAGTAGCA 4651 ACAGAAAGCT CTGCAAAGAC TCCCTCCAAG CTATTGGATC CTCTTGCTTG 4701 GGATAACCAC TATGGTACTC AGATACCAAA AGAAGAGTGG AAATCCCAAG 4751 AGAAGTCACC AGAAAAAACA GCTTTTAAGA AAAAGGATAC CATTTTGTCC 4801 CTGAACGCTT GTGAAAGCAA TCATGCAATA GCAGCAATAA ATGAGGGACA 4851 AAATAAGCCC GAAATAGAAG TCACCTGGGC AAAGCAAGGT AGGACTGAAA 4901 GGCTGTGCTC TCAAAACCCA CCAGTCTTGA AACGCCATCA ACGGGAAATA 4951 ACTCGTACTA CTCTTCAGTC AGATCAAGAG GAAATTGACT ATGATGATAC 5001 CATATCAGTT GAAATGAAGA AGGAAGATTT TGACATTTAT GATGAGGATG 5051 AAAATCAGAG CCCCCGCAGC TTTCAAAAGA AAACACGACA CTATTTTATT 5101 GCTGCAGTGG AGAGGCTCTG GGATTATGGG ATGAGTAGCT CCCCACATGT 5151 TCTAAGAAAC AGGGCTCAGA GTGGCAGTGT CCCTCAGTTC AAGAAAGTTG 5201 TTTTCCAGGA ATTTACTGAT GGCTCCTTTA CTCAGCCCTT ATACCGTGGA 5251 GAACTAAATG AACATTTGGG ACTCCTGGGG CCATATATAA GAGCAGAAGT 5301 TGAAGATAAT ATCATGGTAA CTTTCAGAAA TCAGGCCTCT CGTCCCTATT 5351 CCTTCTATTC TAGCCTTATT TCTTATGAGG AAGATCAGAG GCAAGGAGCA 5401 GAACCTAGAA AAAACTTTGT CAAGCCTAAT GAAACCAAAA CTTACTTTTG 5451 GAAAGTGCAA CATCATATGG CACCCACTAA AGATGAGTTT GACTGCAAAG 5501 CCTGGGCTTA TTTCTCTGAT GTTGACCTGG AAAAAGATGT GCACTCAGGC 5551 CTGATTGGAC CCCTTCTGGT CTGCCACACT AACACACTGA ACCCTGCTCA 5601 TGGGAGACAA GTGACAGTAC AGGAATTTGC TCTGTTTTTC ACCATCTTTG 5651 ATGAGACCAA AAGCTGGTAC TTCACTGAAA ATATGGAAAG AAACTGCAGG 5701 GCTCCCTGCA ATATCCAGAT GGAAGATCCC ACTTTTAAAG AGAATTATCG 5751 CTTCCATGCA ATCAATGGCT ACATAATGGA TACACTACCT GGCTTAGTAA 5801 TGGCTCAGGA TCAAAGGATT CGATGGTATC TGCTCAGCAT GGGCAGCAAT 5851 GAAAACATCC ATTCTATTCA TTTCAGTGGA CATGTGTTCA CTGTACGAAA 5901 AAAAGAGGAG TATAAAATGG CACTGTACAA TCTCTATCCA GGTGTTTTTG 5951 AGACAGTGGA AATGTTACCA TCCAAAGCTG GAATTTGGCG GGTGGAATGC 6001 CTTATTGGCG AGCATCTACA TGCTGGGATG AGCACACTTT TTCTGGTGTA 6051 CAGCAATAAG TGTCAGACTC CCCTGGGAAT GGCTTCTGGA CACATTAGAG 6101 ATTTTCAGAT TACAGCTTCA GGACAATATG GACAGTGGGC CCCAAAGCTG 6151 GCCAGACTTC ATTATTCCGG ATCAATCAAT GCCTGGAGCA CCAAGGAGCC 6201 CTTTTCTTGG ATCAAGGTGG ATCTGTTGGC ACCAATGATT ATTCACGGCA

6251 TCAAGACCCA GGGTGCCCGT CAGAAGTTCT CCAGCCTCTA CATCTCTCAG 6301 TTTATCATCA TGTATAGTCT TGATGGGAAG AAGTGGCAGA CTTATCGAGG 6351 AAATTCCACT GGAACCTTAA TGGTCTTCTT TGGCAATGTG GATTCATCTG 6401 GGATAAAACA CAATATTTTT AACCCTCCAA TTATTGCTCG ATACATCCGT 6451 TTGCACCCAA CTCATTATAG CATTCGCAGC ACTCTTCGCA TGGAGTTGAT 6501 GGGCTGTGAT TTAAATAGTT GCAGCATGCC ATTGGGAATG GAGAGTAAAG 6551 CAATATCAGA TGCACAGATT ACTGCTTCAT CCTACTTTAC CAATATGTTT 6601 GCCACCTGGT CTCCTTCAAA AGCTCGACTT CACCTCCAAG GGAGGAGTAA 6651 TGCCTGGAGA CCTCAGGTGA ATAATCCAAA AGAGTGGCTG CAAGTGGACT 6701 TCCAGAAGAC AATGAAAGTC ACAGGAGTAA CTACTCAGGG AGTAAAATCT 6751 CTGCTTACCA GCATGTATGT GAAGGAGTTC CTCATCTCCA GCAGTCAAGA 6801 TGGCCATCAG TGGACTCTCT TTTTTCAGAA TGGCAAAGTA AAGGTTTTTC 6851 AGGGAAATCA AGACTCCTTC ACACCTGTGG TGAACTCTCT AGACCCACCG 6901 TTACTGACTC GCTACCTTCG AATTCACCCC CAGAGTTGGG TGCACCAGAT 6951 TGCCCTGAGG ATGGAGGTTC TGGGCTGCGA GGCACAGGAC CTCTACTGA

[0043] The wild-type human factor VIII encoded by SEQ ID NO: 1 has an amino acid sequence of SEQ ID NO: 2, as follows:

TABLE-US-00002 1 ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL 51 FVEFTVHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA 101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD 151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA 201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR 251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL 301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL 351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL 401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG 451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD 501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP 551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG 601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS 651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR 701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNSRHPS 751 TRQKQFNATT IPENDIEKTD PWFAHRTPMP KIQNVSSSDL LMLLRQSPTP 801 HGLSLSDLQE AKYETFSDDP SPGAIDSNNS LSEMTHFRPQ LHHSGDMVFT 851 PESGLQLRLN EKLGTTAATE LKKLDFKVSS TSNNLISTIP SDNLAAGTDN 901 TSSLGPPSMP VHYDSQLDTT LFGKKSSPLT ESGGPLSLSE ENNDSKLLES 951 GLMNSQESSW GKNVSSTESG RLFKGKRAHG PALLTKDNAL FKVSISLLKT 1001 NKTSNNSATN RKTHIDGPSL LIENSPSVWQ NILESDTEFK KVTPLIHDRM 1051 LMDKNATALR LNHMSNKTTS SKNMEMVQQK KEGPIPPDAQ NPDMSFFKML 1101 FLPESARWIQ RTHGKNSLNS GQGPSPKQLV SLGPEKSVEG QNFLSEKNKV 1151 VVGKGEFTKD VGLKEMVFPS SRNLFLTNLD NLHENNTHNQ EKKIQEEIEK 1201 KETLIQENVV LPQIHTVTGT KNFMKNLFLL STRQNVEGSY EGAYAPVLQD 1251 FRSLNDSTNR TKKHTAHFSK KGEEENLEGL GNQTKQIVEK YACTTRISPN 1301 TSQQNFVTQR SKRALKQFRL PLEETELEKR IIVDDTSTQW SKNMKHLTPS 1351 TLTQIDYNEK EKGAITQSPL SDCLTRSHSI PQANRSPLPI AKVSSFPSIR 1401 PIYLTRVLFQ DNSSHLPAAS YRKKDSGVQE SSHFLQGAKK NNLSLAILTL 1451 EMTGDQREVG SLGTSATNSV TYKKVENTVL PKPDLPKTSG KVELLPKVHI 1501 YQKDLFPTET SNGSPGHLDL VEGSLLQGTE GAIKWNEANR PGKVPFLRVA 1551 TESSAKTPSK LLDPLAWDNH YGTQIPKEEW KSQEKSPEKT AFKKKDTILS 1601 LNACESNHAI AAINEGQNKP EIEVTWAKQG RTERLCSQNP PVLKRHQREI 1651 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI 1701 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG 1751 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA 1801 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG 1851 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR 1901 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN 1951 ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC 2001 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL 2051 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ 2101 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR 2151 LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF 2201 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS 2251 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP 2301 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LY

[0044] A first aspect of the present invention relates to a recombinant factor VIII that includes one or more mutations at an interface of A1 and C2 domains of the recombinant factor VIII. This mutation results in enhanced stability, particularly enhanced thermal and/or chemical stability, of factor VIII. The one or more mutations include substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity, or substitution of two or more amino acid residues with Cysteine to afford a disulfide bond between the A1 and C2 domains.

[0045] As used herein, an amino acid having a higher hydrophobicity refers to a residue having a higher measurement or ranking of hydrophobicity relative to a particular wild type residue at the location of interest. The hydrophobic effect represents the tendency of water to exclude non-polar molecules. Hydropathy scale is a ranking list for the relative hydrophobicity of amino acid residues and proteins. The "hydropathy index" of a protein or amino acid is a number representing its hydrophilic or hydrophobic properties. Different methods have been used in the art to calculate the relative hydrophobicity of amino acid residues and proteins (Kyte et al., "A Simple Method for Displaying the Hydropathic Character of a Protein," J. Mol. Biol. 157: 105-32 (1982); Eisenberg D, "Three-dimensional Structure of Membrane and Surface Proteins," Ann. Rev. Biochem. 53: 595-623 (1984); Rose et al., "Hydrogen Bonding, Hydrophobicity, Packing, and Protein Folding," Annu. Rev. Biomol. Struct. 22: 381-415 (1993); Kauzmann, "Some Factors in the Interpretation of Protein Denaturation," Adv. Protein Chem. 14: 1-63 (1959), which are hereby incorporated by reference in their entirety). Any one of these hydrophobicity scales can be used for the purposes of the present invention; however, the Kyte-Doolittle hydrophobicity scale is perhaps the most often referenced scale. The hydropathy index is directly proportional to the hydrophobicity of the amino acid or the protein.

[0046] As used herein, the term "interface" is used to describe a protein surface where the atoms of the protein come in contact with the solvent (solvent-protein interface) or with another domain (domain interface). Domain interfaces can be either inter-domain (between domains) or intra-domain (within domains). Various methods are known in the art to identify interfaces. For example, the geometric distance between atoms that belong to same or different domains can be used to identify intra-domain or inter-domain interfaces (structural information, such as atomic coordinates, is available at the Protein Databank; Berman et al., "The Protein Data Bank," Nucleic Acid Res 28:235-242 (2000), which is hereby incorporated by reference in its entirety). Another approach is the Accessible Surface Area (ASA), which detects the buried region of a protein that is detached from a solvent (Jones et al., "Analysis of Protein-protein Interaction Sites Using Surface Patches," J Mol Biol 272:121-132 (1997), which is hereby incorporated by reference in its entirety). A further approach is the Voronoi diagram, a computational geometry method that uses a mathematical definition of interface regions (Ban et al., "Interface Surfaces for Protein-protein Complexes," Proceedings of the Research in Computational Molecular Biology, San Diego 27-31 (2004); Poupon A, "Voronoi and Voronoi-Related Tessellations in Studies of Protein Structure and Interaction," Curr Opin Struct Biol 14:233-241 (2004); Kim et al., "Euclidean Voronoi Diagrams of 3D Spheres and Applications to Protein Structure Analysis," Japan Journal of Industrial and Applied Mathematics 2005, 22:251-265 (2005), which are hereby incorporated by reference in their entirety). As described herein, the factor VIII crystal structure (Shen et al., "The Tertiary Structure and Domain Organization of Coagulation Factor VIII," Blood 111:1240-1247 (2008); Ngo et al., "Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa:Factor VIIIa Complex," Structure 16:597-606 (2008), which is hereby incorporated by reference in its entirety) can be modeled using Swiss PDB Viewer to identify residues that upon substitution with Cysteine will allow for disulfide bond formation (see FIG. 3B) or residues that are suitable for substitution with an amino acid having higher hydrophobicity (see FIG. 3C).

[0047] As used herein, a region of the A1 domain that interfaces with the C2 domain is referred to as a "C2 domain interface" and a region of the C2 domain that interfaces with the A1 domain is referred to as an "A1 domain interface."

[0048] The wild type (WT) factor VIII can be such that the A1 domain of the WT factor VIII includes a C2 domain interface having the amino acid sequence of (i) KXS (aa 8-10 of SEQ ID NO: 10) where "X" is A or S; (ii) SXXE (aa 20-23 of SEQ ID NO: 10) where "X" at the 2.sup.nd position can be Q, K, or P, and "X" at the 3.sup.rd position can be R, K, M, T, or A; and/or (iii) TYXW (aa 36-39 of SEQ ID NO: 10), where "X" at the 3.sup.rd position can be V or A. These are illustrated in FIG. 1.

[0049] The WT factor VIII can be such that the C2 domain of the WT factor VIII includes an A1 domain interface having the amino acid sequence of (i) XVT (aa 9-11 of SEQ ID NO: 18), where "X" is K or R; (ii) PPXX (aa 68-71 of SEQ ID NO: 18), where "X" at the 3.sup.rd position is L or R, and "X" at the 4.sup.th position is L, F, or V; and/or (iii) XXQX (aa 96-99 of SEQ ID NO: 18), where the "X" at the 1.sup.st position is E or D, "X" at the 2.sup.nd position is A or T, and "X" at the 4.sup.th position is D or Q. These are illustrated in FIG. 2.

[0050] According to one embodiment, one or both of the A1 domain interface and C2 domain interface include a substitution to introduce a Cysteine residue. In preferred embodiments, the substitution of amino acid residues at the interface of A1 and C2 domains is carried out such that at least a pair of amino acids are substituted with Cysteine. In this embodiment, the pair of Cysteine residues form an inter-domain (A1 to C2 domain) disulfide bond such that the stability of the recombinant factor VIII is enhanced.

[0051] In another embodiment of the recombinant factor VIII of the present invention, the one or more mutations at the interface of A1 and C2 domains of the recombinant factor VIII is the replacement of one or more amino acid residues with an amino acid incapable of forming disulfide bonds but having a higher hydrophobicity index.

[0052] In a further embodiment, multiple mutations are introduced at several interfaces of the A1 and C2 domains of factor VIII, including: (i) a pair of substitutions to introduce a pair of Cysteine residues that are capable of forming an inter-domain (A1 to C2 domain) disulfide bond; and (ii) the replacement of one or more amino acid residues with an amino acid incapable of forming disulfide bonds but having a higher hydrophobicity index.

[0053] The recombinant factor VIII according to several embodiments of the present invention are characterized by an A1 domain that includes a C2 domain interface having the amino acid sequence of (i) KXS (SEQ ID NO: 19), where "X" is T, G, A, M, C, F, L, V, or I; (ii) SXXX (SEQ ID NO: 20), where "X" at the 2.sup.nd position is wild type (Q, K, or P) or E, D, N, H, Y, W, S, T, G, A, M, C, F, L, V, or I, "X" at the 3.sup.rd position can be any amino acid other than R or preferably any amino acid other than R, K, M, T, or A; and "X" at the 4.sup.th position is wild type (E) or Q, D, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I; and/or (iii) TYXW (SEQ ID NO: 21), where "X" is M, C, F, L, V, or I. In at least one of the C2 domain interfaces, one of the X residues represents a substitution of a wild type residue.

[0054] The recombinant factor VIII according to several embodiments of the present invention are characterized by a C2 domain that includes an A1 domain interface having the amino acid sequence of (i) XVT (SEQ ID NO: 22), where "X" can be any amino acid other than K or R; (ii) PPXX (SEQ ID NO: 23), where "X" at the 3.sup.rd position can be any amino acid besides L or R and "X" at the 4.sup.th position is L, V, or I; and/or (iii) XXQX (SEQ ID NO: 24), where "X" at the 1.sup.st position is wild type (E or D) or Q, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I, "X" at the 2.sup.nd position is wild type (A or T) or G, M, C, F, L, V, or I, and "X" at the 4.sup.th position is wild type (D or Q) or N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I. In at least one of the A1 domain interfaces, one of the X residues represents a substitution of a wild type residue.

[0055] In certain embodiments, where a disulfide linkage is formed between A1 and C2 domains using a cysteine substitution, the cysteine substitution occurs at residue 121 of human factor VIII (i.e., the C2 domain interface is SEQ ID NO: 20, where X at the third position is cysteine) and residue 2302 of human factor VIII (i.e., the A1 domain interface is SEQ ID NO: 23, where X at the fourth position is cysteine). In other embodiments, the cysteine substitution occurs at a residue other than residues 121 and 2302 of human factor VIII.

[0056] One embodiment of the recombinant factor VIII of the present invention includes an A1 domain having a C2 domain interface that includes the amino acid sequence KXS (SEQ ID NO: 19), where the second residue (corresponding to position 108 of SEQ ID NO: 2) is Valine, Isoleucine, or Leucine.

[0057] A further embodiment of the recombinant factor VIII of the present invention includes a C2 domain having an A1 domain interface that includes the amino acid sequence of XVT (SEQ ID NO: 22), where X (corresponding to position 2328 of SEQ ID NO: 2) is other than Lysine or Arginine.

[0058] Another embodiment of the recombinant factor VIII includes an A1 domain having a C2 domain interface that includes the amino acid sequence of SXXE (SEQ ID NO: 25), where the second residue can be Q, K, P, E, D, N, H, Y, W, S, T, G, A, M, C, F, L, V, or I and the third residue (corresponding to position 121 of SEQ ID NO: 2) is cysteine; and a C2 domain having an A1 domain interface that includes the amino acid sequence of PPXX (SEQ ID NO: 23), where X at the 3.sup.rd position is any amino acid besides L or R, and X at the 4.sup.th position (corresponding to position 2302 of SEQ ID NO: 2) is cysteine.

[0059] Yet another embodiment of the recombinant factor includes a C2 domain having an A1 domain interface that includes the amino acid sequence of XXQX (SEQ ID NO: 24), where the 1.sup.st residue is E, D, Q, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I; the 2.sup.nd residue (corresponding to position 2328 of SEQ ID NO: 2) is Valine, Isoleucine, or Leucine; and the 4.sup.th residue is D, Q, N, H, P, Y, W, S, T, G, A, M, C, F, L, V, or I.

[0060] The recombinant factor VIII according to the present invention can also have more than one mutation as described supra. In one preferred embodiment the recombinant factor VIII has two or more amino acid substitutions.

[0061] According to one embodiment, the recombinant factor VIII includes (i) an A1 domain having a C2 domain interface that includes the amino acid sequence of KXS (SEQ ID NO: 19), where X (corresponding to position 108 of SEQ ID NO: 2) is Isoleucine, Leucine, or Valine; and (ii) a C2 domain having an A1 domain interface that includes the amino acid sequence XXQX (SEQ ID NO: 24), where the 1.sup.st and 4.sup.th residues are as described above and the 2.sup.nd residue (corresponding to position 2328 of SEQ ID NO: 2) is Isoleucine, Leucine, or Valine.

[0062] Suitable mutant factor VIII sequences that can be modified in accordance with the present invention can also include any previously known or subsequently identified mutant factor VIII sequences that have modified properties with regard to various attributes, including, without limitation, antigenicity, circulating half-life, factor VIIIa stability, protein secretion, affinity for factor IXa and/or factor X, altered factor VIII-inactivation cleavage sites, altered activated Protein C cleavage sites, enhanced specific activity of factor VIIIa, immunogenicity, and shelf-life.

[0063] In one embodiment the recombinant factor VIII of the present invention further comprises one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VIII for one or both of factor IXa and factor X; (ii) modified sites that enhance secretion in culture; (iii) modified serum protein binding sites that enhance the circulating half-life thereof; (iv) at least one glycosylation recognition sequence that is effective in decreasing antigenicity and/or immunogenicity thereof; (v) a modified A1 domain calcium-binding site that improves specific activity of the recombinant factor Villa; (vi) modified activated protein C-cleavage site; (vii) a modified A1 and A2 domain interface to enhance factor VIIIa stability; and (viii) a modified A2 and A3 domain interface to enhance factor VIIIa stability.

[0064] The recombinant factor VIII of the present invention can be one that has a combination of mutations, including one or more mutations at an interface of A1 and C2 domains of recombinant factor VIII as described supra and one or more mutations at the A1 and A2 domain interface and/or the A2 and A3 domain interface. Such a recombinant factor VIII in addition to the one or more mutations at the A1 and C2 domain interface also includes substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at either or both of the A1 and A2 or A2 and A3 domain interfaces.

[0065] Preferably, the charged residue to be replaced is either a Glu or Asp residue that does not participate in hydrogen bonding between the A1 and A2 or A2 and A3 domains. The hydrophobic amino acid residue that replaces the charged residue can be any of Ala, Val, Ile, Leu, Met, Phe, or Trp. Particularly preferred recombinant factor VIII of the present invention includes a substitution of the residue corresponding to Glu287 of wild type factor VIII, a substitution of the residue corresponding to Asp302 of wild type factor VIII, a substitution of the residue corresponding to Asp519 of wild type factor VIII, a substitution of the residue corresponding to Glu665 of wild type factor VIII, a substitution of the residue corresponding to Glu1984 of wild type factor VIII, or combinations thereof. The D302A, E287A, E665A, E665V, D519A, D519V, E1984A, and E1984V substitutions are preferred for achieving a recombinant factor VIII that has enhanced stability of both factor VIII and factor VIIIa. Preferred combinations of these substitutions include, without limitation, those corresponding to D519AE665V, D519VE665V, and D519VE1984A mutants, as well as D519AE665VE1984A and D519VE665VE1984A mutants. The enhanced stability of these mutants is believed to be achieved by stabilizing the inter-domain interface in factor VIII as well as reducing A2 subunit dissociation from A1/A3C1C2 as compared to wild type factor VIIIa. Exemplary mutants of this type are described in U.S. Patent Application Publ. No. US2009/0118184 to Fay et al., which is hereby incorporated by reference in its entirety.

[0066] Examples of mutant factor VIII possessing substitutions at the A1-C2 domain interface as well as one or both of the A1-A2 and A2-A3 domain interfaces include, without limitation, A108ID519AE665V, A108ID519VE665V, A108ID519VE1984A, A108ID519AE665VE1984A, A108ID519VE665VE1984A, R121C-L2302C/D519AE665V, R121C-L2302C/D519VE665V, R121C-L2302C/D519VE1984A, R121C-L2302C/D519AE665VE1984A, and R121C-L2302C/D519VE665VE1984A. Each of these mutant factor VIII can be expressed in a B-domainless form, as described below.

[0067] Another example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a factor VIII having a modified calcium binding site, preferably at an amino acid corresponding to residue 113 of SEQ ID NO: 2. This affords a factor VIIIa having enhanced specific activity. Exemplary mutants of this type are described in U.S. Patent Application Publ. No. US2007/0265199 to Fay et al., which is hereby incorporated by reference in its entirety. Preferably, the residue 113 mutant also is modified in accordance with one or more of the mutations described above (e.g., at positions 287, 302, 519, 665, and/or 1984) to afford a high stability/high specific activity factor VIII protein. Exemplary high stability/high specific activity factor VIII proteins include, without limitation: those possessing combined substitutions E113AD519A, E113AD519V, E113AE665A, E113AE665V, E113AE1984V, E113AD519AE665V, E113AD519VE665V, E113AD519VE1984A, E113AD519AE665VE1984A, and E113AD519VE665VE1984A.

[0068] Examples of mutant factor VIII possessing substitutions at the A1-C2 domain interface as well as the residue 113 substitution, and one or both of the A1-A2 and A2-A3 domain interface substitutions include, without limitation, A108IE113AD519AE665V, A108IE113AD519VE665V, A108IE113AD519VE1984A, A108IE113AD519AE665VE1984A, A108IE113AD519VE665VE1984A, R121C-L2302C/E113AD519AE665V, R121C-L2302C/E113AD519VE665V, R121C-L2302C/E113AD519VE1984A, R121C-L2302C/E113AD519AE665VE1984A, and R121C-L2302C/E113AD519VE665VE1984A. Each of these mutant factor VIII can be expressed in a B-domainless form, as described below.

[0069] A third example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a B-domainless factor VIII that contains amino acid residues 1-740 and 1690-2332 of SEQ ID NO: 2 (see, e.g., U.S. Pat. No. 6,458,563 to Lollar, which is hereby incorporated by reference in its entirety).

[0070] In one embodiment of the B-domainless recombinant factor VIII of the present invention, the B-domain is replaced by a DNA linker segment and at least one codon is replaced with a codon encoding an amino acid residue that has the same charge as a corresponding residue of porcine factor VIII (see, e.g., U.S. Patent Application Publication No. 2004/0197875 to Hauser et al., which is hereby incorporated by reference in its entirety).

[0071] In another embodiment of the B-domainless recombinant factor VIII of the present invention, the modified mutant factor VIII is encoded by a nucleotide sequence having a truncated factor IX intron 1 inserted in one or more locations (see, e.g., U.S. Pat. No. 6,800,461 to Negrier and U.S. Pat. No. 6,780,614 to Negrier, each of which is hereby incorporated by reference in its entirety). This recombinant factor VIII can be used for yielding higher production of the recombinant factor VIII in vitro as well as in a transfer vector for gene therapy (see, e.g., U.S. Pat. No. 6,800,461 to Negrier, which is hereby incorporated by reference in its entirety). In a particular example of this embodiment, the recombinant factor VIII can be encoded by a nucleotide sequence having a truncated factor IX intron 1 inserted in two locations, and having a promoter that is suitable for driving expression in hematopoietic cell lines, and specifically in platelets (see, e.g., U.S. Pat. No. 6,780,614 to Negrier, which is hereby incorporated by reference in its entirety).

[0072] Regardless of the embodiment, the B-domainless factor VIII preferably contains one or more of the mutations described above (e.g., modified A1 domain interface and/or C2 domain interface, as well as any other mutations to affect other properties of the resulting factor VIII). Recombinant factor VIII proteins prepared in accordance with the Examples herein are B-domainless.

[0073] A fourth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a chimeric human/animal factor VIII that contains one or more domains, or portions thereof, from human factor VIII and one or more domains, or portions thereof, from a non-human mammalian factor VIII. One or more animal amino acid residues can be substituted for human amino acid residues that are responsible for the antigenicity of human factor VIII. In particular, animal (e.g., porcine) residue substitutions can include, without limitation, one or more of the following: R484A, R488G, P485A, L486S, Y487L, Y487A, S488A, S488L, R489A, R4895, R490G, L491S, P492L, P492A, K493A, G494S, V495A, K496M, H497L, L498S, K499M, D500A, F501A, P502L, 1503M, L504M, P505A, G506A, E507G, 1508M, 1508A, M2199I, F2200L, L2252F, V2223A, K2227E, and/or L2251 (U.S. Pat. No. 5,859,204 to Lollar, U.S. Pat. No. 6,770,744 to Lollar, and U.S. Patent Application Publication No. 2003/0166536 to Lollar, each of which is hereby incorporated by reference in its entirety). Preferably, the recombinant chimeric factor VIII contains one or more of the mutations described above (e.g., modified A1 domain interface and/or C2 domain interface).

[0074] A fifth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a factor VIII that has enhanced affinity for factor IXa (see, e.g., Fay et al., "Factor VIIIa A2 Subunit Residues 558-565 Represent a Factor IXa Interactive Site," J. Biol. Chem. 269(32):20522-7 (1994); Bajaj et al., "Factor IXa: Factor VIIIa Interaction. Helix 330-338 of Factor IXa Interacts with Residues 558-565 and Spatially Adjacent Regions of the A2 Subunit of Factor VIIIa," J. Biol. Chem. 276(19):16302-9 (2001); and Lenting et al., "The Sequence Glu1811-Lys1818 of Human Blood Coagulation Factor VIII Comprises a Binding Site for Activated Factor IX," J. Biol. Chem. 271(4):1935-40 (1996), each of which is hereby incorporated by reference in its entirety) and/or factor X (see, e.g., Lapan et al., "Localization of a Factor X Interactive Site in the A1 Subunit of Factor VIIIa," J. Biol. Chem. 272:2082-88 (1997), which is hereby incorporated by reference in its entirety). Preferably, the enhanced-affinity factor VIII contains one or more of the mutations described above (e.g., modified A1 domain interface and/or C2 domain interface).

[0075] A sixth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a factor VIII that is modified to enhance secretion of the factor VIII (see, e.g., Swaroop et al., "Mutagenesis of a Potential Immunoglobulin-Binding Protein-Binding Site Enhances Secretion of Coagulation Factor VIII," J. Biol. Chem. 272(39):24121-4 (1997), which is hereby incorporated by reference in its entirety). Preferably, the secretion enhanced mutant factor VIII contains one or more of the mutations identified above (e.g., modified A1 domain interface and/or C2 domain interface).

[0076] A seventh example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a factor VIII with an increased circulating half-life. This modification can be made using various approaches, including, without limitation, by reducing interactions with heparan sulfate (Sarafanov et al., "Cell Surface Heparan Sulfate Proteoglycans Participate in Factor VIII Catabolism Mediated by Low Density Lipoprotein Receptor-Related Protein," J. Biol. Chem. 276(15):11970-9 (2001), which is hereby incorporated by reference in its entirety) and/or low-density lipoprotein receptor-related protein ("LRP") (Saenko et al., "Role of the Low Density Lipoprotein-Related Protein Receptor in Mediation of Factor VIII Catabolism," J. Biol. Chem. 274(53):37685-92 (1999); and Lenting et al., "The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-Related Protein," J. Biol. Chem. 274(34):23734-9 (1999), each of which is hereby incorporated by reference in its entirety). Preferably, the half-life enhanced mutant factor VIII contains one or more of the mutations described above (e.g., modified A1 domain interface and/or C2 domain interface).

[0077] An eighth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a modified factor VIII encoded by a nucleotide sequence modified to code for amino acids within known, existing epitopes to produce a recognition sequence for glycosylation at asparagines residues (see, e.g., U.S. Pat. No. 6,759,216 to Lollar, which is hereby incorporated by reference in its entirety). The mutant factor VIII of this example can be useful in providing a modified factor VIII that escapes detection by existing inhibitory antibodies (low antigenicity factor VIII) and which decreases the likelihood of developing inhibitory antibodies (low immunogenicity factor VIII). In one particular embodiment of this example, the modified factor VIII is mutated to have a consensus amino acid sequence for N-linked glycosylation. An example of such a consensus sequence is N--X-S/T, where N is asparagine, X is any amino acid, and S/T stands for serine or threonine (see U.S. Pat. No. 6,759,216 to Lollar, which is hereby incorporated by reference in its entirety). Preferably, the glycosylation site-modified factor VIII contains one or more of the mutations identified above (e.g., modified A1 domain interface and/or C2 domain interface).

[0078] A ninth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a modified factor VIII that is a procoagulant-active factor VIII having various mutations (see, e.g., U.S. Patent Application Publication No. 2004/0092442 to Kaufman et al., which is hereby incorporated by reference in its entirety). One example of this embodiment relates to a modified factor VIII that has been modified to (i) delete the von Willebrand factor binding site, (ii) add a mutation at Arg 740, and (iii) add an amino acid sequence spacer between the A2- and A3-domains, where the amino acid spacer is of a sufficient length so that upon activation, the procoagulant-active factor VIII protein becomes a heterodimer (see U.S. Patent Application Publication No. 2004/0092442 to Kaufman et al., which is hereby incorporated by reference in its entirety). Preferably, procoagulant active factor VIII is also modified to contain one or more of the mutations described above (e.g., at positions modified A1 domain interface and/or C2 domain interface).

[0079] A tenth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a modified factor VIII that includes a substitution of one or more amino acid residues within a region surrounding an activated protein C cleavage site, except that the cleavable Arg scissile bond (at Arg336 and/or Arg562) is not substituted (see, e.g., U.S. Patent Application Publication No. 2009/0118185 to Fay et al., which is hereby incorporated by reference in its entirety). In the most preferred embodiments, the one or more substitutions appears within the P4-P3' activated protein C cleavage site, which can be the site corresponding to wild type residues 333-339 of the A1 domain or the site corresponding to residues 559-565 of the A2 domain. Exemplary mutant P4-P3' regions, which include the substitution of one or more amino acids include, without limitation, VDQRGNQ (SEQ ID NO: 26) neighboring Arg562, VDQRMKN (SEQ ID NO: 27) neighboring Arg562, and PQLRGNQ (SEQ ID NO: 28) neighboring Arg336, PDLRMKN (SEQ ID NO: 29) neighboring Arg336, PQQRMKN (SEQ ID NO: 30) neighboring Arg336, PQRRMKN (SEQ ID NO: 31) neighboring Arg336, PQLRGKN (SEQ ID NO: 32) neighboring Arg336, PQLRMIN (SEQ ID NO: 33) neighboring Arg336, and PQLRMNN (SEQ ID NO: 34) neighboring Arg336. These substitutions are preferred for achieving a mutant factor VIIIa having a reduced rate of inactivation by activated protein C, but unlike mutants having single mutation replacements of the P1 Arg residue the resulting factor VIIIa is capable of being inactivated by activated protein C. Preferably, factor VIII having a modified activated protein C cleavage site is also modified to contain one or more of the mutations described above (e.g., at positions modified A1 domain interface and/or C2 domain interface).

[0080] Further, the mutant factor VIII can be modified to take advantage of various advancements regarding recombinant coagulation factors generally (see, e.g., Saenko et al., "The Future of Recombinant Coagulation Factors," J. Thrombosis and Haemostasis 1:922-930 (2003), which is hereby incorporated by reference in its entirety).

[0081] The recombinant factor VIII of the present invention can be modified at any residue to stabilize the A1/C2 domain interfaces (includes positions corresponding to 108, 121, 2302, 2328 of the WT factor VIII), as well as be modified at any charged residue that destabilizes the A1A2 or A2A3 domain interfaces (including positions 287, 302, 519, 665, or 1984), be modified to be B-domainless, to be chimeric, to have modified calcium binding sites that enhance factor VIIIa activity (e.g., at position 113), to have altered inactivation cleavage sites, to have enhanced factor IXa and/or factor X affinity, to have enhanced secretion, to have an increased circulating half-life, or to have mutant glycosylation sites; or to possess any one or more of such modifications in addition to the one or more modifications to charged residues, including a modified calcium-binding site that improves activity of the recombinant factor VIII. A number of exemplary B-domainless high stability recombinant factor VIII proteins are described in the Examples.

[0082] The recombinant factor VIII is preferably produced in a substantially pure form. In a particular embodiment, the substantially pure recombinant factor VIII is at least about 80% pure, more preferably at least 90% pure, most preferably at least 95% pure. A substantially pure recombinant factor VIII can be obtained by conventional techniques well known in the art. Typically, the substantially pure recombinant factor VIII is secreted into the growth medium of recombinant host cells. Alternatively, the substantially pure recombinant factor VIII is produced but not secreted into growth medium. In such cases, to isolate the substantially pure recombinant factor VIII, the host cell carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove cell debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the substantially pure recombinant factor VIII is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the recombinant factor VIII. If necessary, a protein fraction (containing the substantially pure recombinant factor VIII) may be further purified by high performance liquid chromatography ("HPLC").

[0083] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes the recombinant factor VIII of the present invention. The isolated nucleic acid molecule encoding the recombinant factor VIII can be either RNA or DNA.

[0084] In another embodiment the isolated nucleic acid molecule can have a nucleotide sequence encoding a recombinant factor VIII according to the present invention which further comprises one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VIII for one or both of factor IXa and factor X; (ii) modified sites that enhance secretion in culture; (iii) modified serum protein binding sites that enhance the circulating half-life thereof; (iv) at least one glycosylation recognition sequence that is effective in decreasing antigenicity and/or immunogenicity thereof; (v) a modified calcium-binding site that improves specific activity of the recombinant factor Villa; (vi) modified activated protein C-cleavage site; (vii) a modified A1 and A2 domain interface; and (viii) a modified A2 and A3 domain interface.

[0085] In one embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a modified A1/A2 domain interface or A2/A3 domain interface (e.g., at positions corresponding to positions 287, 302, 519, 665, 1984 and/or 332-340 of SEQ ID NO: 2), as modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0086] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a mutation at position 113 that enhances factor VIII specific activity, as modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0087] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a B-domainless factor VIII of the type described above, as modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0088] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a chimeric human/porcine of the type described above, as modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0089] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII whose inactivation sites have been modified as described above, as further modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0090] In yet another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII whose affinity for factor IXa and/or factor X has been enhanced, as further modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0091] In a still further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII whose affinity for various serum-binding proteins has been altered to increase its circulating half-life, as further modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0092] In a further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII that has increased secretion in culture, as further modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0093] In a further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII that possesses one or more non-naturally occurring glycosylation site, as further modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0094] In a still further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII that has a modified activated protein C cleavage site, as further modified with one or more of the substitutions affecting the A1/C2 domain interfaces (e.g., SEQ ID NOS: 19-25).

[0095] In yet another embodiment, the isolated nucleic acid molecule encodes a recombinant factor VIII that is modified at any one or more charged residues as described above and is also modified to possess any two or more of the following: modified to be B-domainless, modified to be chimeric, modified to have altered inactivation cleavage sites, modified to have enhanced factor IXa and/or factor X affinity, modified to have enhanced secretion, modified to have an increased circulating half-life, modified to possess one or more non-naturally occurring glycosylation site, modified within a calcium-binding site (e.g., at position 113) such that the specific activity of the recombinant factor VIII is improved, modified activated protein C-cleavage site, a modified A1 and A2 domain interface, and a modified A2 and A3 domain interface.

[0096] Another aspect of the present invention relates to a recombinant DNA expression system that includes an isolated DNA molecule of the present invention, which expression system encodes a recombinant factor VIII. In one embodiment, the DNA molecule is in sense orientation relative to a promoter.

[0097] A further aspect of the present invention relates to a host cell including an isolated nucleic acid molecule encoding the recombinant factor VIII of the present invention. In a particular embodiment, the host cell can contain the isolated nucleic acid molecule in DNA molecule form, either as a stable plasmid or as a stable insertion or integration into the host cell genome. In another embodiment, the host cell can contain a DNA molecule in an expression system. Suitable host cells can be, without limitation, animal cells (e.g., baby hamster kidney ("BHK") cells), bacterial cells (e.g., E. coli), insect cells (e.g., Sf9 cells), fungal cells, yeast cells (e.g., Saccharomyces or Schizosaccharomyces), plant cells (e.g., Arabidopsis or tobacco cells), or algal cells.

[0098] The recombinant DNA expression system and host cells can be produced using various recombinant techniques well-known in the art, as further discussed below.

[0099] The DNA molecule encoding the recombinant factor VIII of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. Thus, one embodiment of the present invention provides a DNA construct containing the isolated nucleic acid of the present invention, which is operably linked to both a 5' promoter and a 3' regulatory region (i.e., transcription terminator) capable of affording transcription and expression of the encoded recombinant factor VIII of the present invention in host cells or host organisms.

[0100] With respect to the recombinant expression system of the present invention, an expression vector containing a DNA molecule encoding the recombinant factor VIII of the present invention can be made using common techniques in the art. The nucleic acid molecules of the present invention can be inserted into any of the many available expression vectors using reagents that are well known in the art. In preparing a DNA vector for expression, the various DNA sequences may normally be inserted or substituted into a bacterial plasmid. Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker which allows for selection in a bacterium, and generally one or more unique, conveniently located restriction sites. The selection of a vector will depend on the preferred transformation technique and target host for transformation.

[0101] A variety of host-vector systems may be utilized to express the recombinant factor VIII-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria (e.g., Agrobacterium). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

[0102] When recombinantly produced, the factor VIII protein or polypeptide (or fragment or variant thereof) is expressed in a recombinant host cell, typically, although not exclusively, a eukaryote.

[0103] Suitable vectors for practicing the present invention include, but are not limited to, the following viral vectors such as lambda vector system gt11, gtWES.tB, Charon 4, and plasmid vectors such as pCMV, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993)), pQE, pIH821, pGEX, pET series (Studier et al, "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Methods in Enzymology 185:60-89 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. Any appropriate vectors now known or later described for genetic transformation are suitable for use with the present invention.

[0104] Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor, N.Y.: Cold Springs Laboratory, (1982), which is hereby incorporated by reference in its entirety.

[0105] U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.

[0106] Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).

[0107] Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.

[0108] Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology 68:473 (1979), which is hereby incorporated by reference in its entirety.

[0109] Promoters vary in their "strength" (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is generally desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in Escherichia coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P.sub.R and P.sub.L promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

[0110] Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

[0111] Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5' to the initiation codon ("ATG") to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

[0112] In one embodiment, the nucleic acid molecule of the present invention is incorporated into an appropriate vector in the sense direction, such that the open reading frame is properly oriented for the expression of the encoded protein under control of a promoter of choice. This involves the inclusion of the appropriate regulatory elements into the DNA-vector construct. These include non-translated regions of the vector, useful promoters, and 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.

[0113] A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism.

[0114] An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed.

[0115] The DNA construct of the present invention can also include an operable 3' regulatory region, selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a DNA molecule which encodes for a protein of choice.

[0116] The vector of choice, promoter, and an appropriate 3' regulatory region can be ligated together to produce the DNA construct of the present invention using well known molecular cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. Current Protocols in Molecular Biology, New York, N.Y.: John Wiley & Sons (1989), each of which is hereby incorporated by reference in its entirety.

[0117] As noted, one alternative to the use of prokaryotic host cells is the use of eukaryotic host cells, such as mammalian cells, which can also be used to recombinantly produce the recombinant factor VIII of the present invention. Mammalian cells suitable for carrying out the present invention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (e.g., ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, and NS-1 cells.

[0118] Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcription and translation control sequences known in the art. Common promoters include SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.

[0119] Once the DNA construct of the present invention has been prepared, it is ready to be incorporated into a host cell. Accordingly, another aspect of the present invention relates to a method of making a recombinant cell. Basically, this method is carried out by transforming a host cell with a DNA construct of the present invention under conditions effective to yield transcription of the DNA molecule in the host cell. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.

[0120] In view of the recombinant technology discussed herein, another aspect of the present invention relates to a method of making a recombinant factor VIII of the present invention. This method involves growing a host cell of the present invention under conditions whereby the host cell expresses the recombinant factor VIII of the present invention. The recombinant factor VIII is then isolated. In one embodiment, the host cell is grown in vitro in a growth medium. In a particular embodiment, suitable growth media can include, without limitation, a growth medium containing a von Willebrand Factor (referred to herein as "VWF"). In this embodiment, the host cell can contain a transgene encoding a VWF or the VWF can be introduced to the growth medium as a supplement. VWF in the growth medium will allow for greater expression levels of the recombinant factor VIII. Once the recombinant factor VIII is secreted into the growth medium, it can then be isolated from the growth medium using techniques well-known by those of ordinary skill in the relevant recombinant DNA and protein arts (including those described herein). In another embodiment, the method of making the recombinant factor VIII of the present invention further involves disrupting the host cell prior to isolation of the recombinant factor VIII. In this embodiment, the recombinant factor VIII is isolated from cellular debris.

[0121] The modifications at positions corresponding to 108, 121/2302, 2328 of the WT factor VIII are particularly preferred, because they result in enhanced stability of factor VIII and, when used in combination with modifications at residues 519, 665, and/or 1984, result in significantly enhanced stability of both factor VIII and factor VIIIa. This increased stability is important with regard to circulating half-life of factor VIII and the activity of factor VIIIa during blood clotting. Furthermore, this property is significant in terms of enhancing the recovery of usable factor VIII during the purification and preparation of the protein for therapeutic use, particularly given the improved thermal and chemical stability of factor VIII.

[0122] When an expression vector is used for purposes of in vivo transformation to induce factor VIII expression in a target cell, promoters of varying strength can be employed depending on the degree of enhancement desired. One of skill in the art can readily select appropriate mammalian promoters based on their strength as a promoter. Alternatively, an inducible promoter can be employed for purposes of controlling when expression or suppression of factor VIII is desired. One of skill in the art can readily select appropriate inducible mammalian promoters from those known in the art. Finally, tissue specific mammalian promoters can be selected to restrict the efficacy of any gene transformation system to a particular tissue. Tissue specific promoters are known in the art and can be selected based upon the tissue or cell type to be treated.

[0123] Another aspect of the present invention relates to a method of treating an animal for a blood disorder such as hemophilia, particularly hemophilia A. This method involves administering to an animal exhibiting hemophilia A an effective amount of the recombinant factor VIII of the present invention, whereby the animal exhibits effective blood clotting following vascular injury. A suitable effective amount of the recombinant factor VIII can include, without limitation, between about 10 to about 50 units/kg body weight of the animal. The animal can be any mammal, but preferably a human, a rat, a mouse, a guinea pig, a dog, a cat, a monkey, a chimpanzee, an orangutan, a cow, a horse, a sheep, a pig, a goat, or a rabbit.

[0124] The recombinant factor VIII of the present invention can be used to treat uncontrolled bleeding due to factor VIII deficiency (e.g., intraarticular, intracranial, or gastrointestinal hemorrhage) in hemophiliacs with and without inhibitory antibodies and in patients with acquired factor VIII deficiency due to the development of inhibitory antibodies. In a particular embodiment, the recombinant factor VIII, alone, or in the form of a pharmaceutical composition (i.e., in combination with stabilizers, delivery vehicles, and/or carriers) is infused into patients intravenously according to the same procedure that is used for infusion of human or animal factor VIII.

[0125] Alternatively, or in addition thereto, the recombinant factor VIII can be administered by administering a viral vector such as an adeno-associated virus (Gnatenko et al., "Human Factor VIII Can Be Packaged and Functionally Expressed in an Adeno-associated Virus Background: Applicability to Hemophilia A Gene Therapy," Br. J. Haematol. 104:27-36 (1999), which is hereby incorporated by reference in its entirety), or by transplanting cells genetically engineered to produce the recombinant factor VIII, typically via implantation of a device containing such cells. Such transplantation typically involves using recombinant dermal fibroblasts, a non-viral approach (Roth et al., "Nonviral Transfer of the Gene Encoding Coagulation Factor VIII in Patients with Sever Hemophilia," New Engl. J. Med. 344:1735-1742 (2001), which is hereby incorporated by reference in its entirety).

[0126] The treatment dosages of recombinant factor VIII that should be administered to a patient in need of such treatment will vary depending on the severity of the factor VIII deficiency. Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and duration of each patient's bleeding episode. Accordingly, the recombinant factor VIII is included in a pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the protein to stop bleeding, as measured by standard clotting assays.

[0127] Factor VIII is classically defined as that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A. The coagulant activity in vitro of purified and partially-purified forms of factor VIII is used to calculate the dose of recombinant factor VIII for infusions in human patients and is a reliable indicator of activity recovered from patient plasma and of correction of the in vivo bleeding defect. There are no reported discrepancies between standard assay of novel factor VIII molecules in vitro and their behavior in the dog infusion model or in human patients, according to Lusher et al., "Recombinant Factor VIII for the Treatment of Previously Untreated Patients with Hemophilia A--Safety, Efficacy, and Development of Inhibitors," New Engl. J. Med. 328:453-459 (1993); Pittman et al., "A2 Domain of Human Recombinant-derived Factor VIII is Required for Procoagulant Activity but not for Thrombin Cleavage," Blood 79:389-397 (1992); and Brinkhous et al., "Purified Human Factor VIII Procoagulant Protein: Comparative Hemostatic Response After Infusions into Hemophilic and von Willebrand Disease Dogs," Proc. Natl. Acad. Sci. 82:8752-8755 (1985), which are hereby incorporated by reference in their entirety.

[0128] Usually, the desired plasma factor VIII activity level to be achieved in the patient through administration of the recombinant factor VIII is in the range of 30-100% of normal. In one embodiment, administration of the therapeutic recombinant factor VIII is given intravenously at a preferred dosage in the range from about 5 to 50 units/kg body weight, and particularly in a range of 10-50 units/kg body weight, and further particularly at a dosage of 20-40 units/kg body weight; the interval frequency is in the range from about 8 to 24 hours (in severely affected hemophiliacs); and the duration of treatment in days is in the range from 1 to 10 days or until the bleeding episode is resolved. See, e.g., Roberts and Jones, "Hemophilia and Related Conditions--Congenital Deficiencies of Prothrombin (Factor II, Factor V, and Factors VII to XII)," Ch. 153, 1453-1474, 1460, in Hematology, Williams, W. J., et al., ed. (1990), which is hereby incorporated by reference in its entirety. Patients with inhibitors may require a different amount of recombinant factor VIII than their previous form of factor VIII. For example, patients may require less recombinant factor VIII because of its higher specific activity than the wild-type VIII and its decreased antibody reactivity. As in treatment with human or plasma-derived factor VIII, the amount of therapeutic recombinant factor VIII infused is defined by the one-stage factor VIII coagulation assay and, in selected instances, in vivo recovery is determined by measuring the factor VIII in the patient's plasma after infusion. It is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed recombinant factor VIII.

[0129] Treatment can take the form of a single intravenous administration of the recombinant factor VIII or periodic or continuous administration over an extended period of time, as required. Alternatively, therapeutic recombinant factor VIII can be administered subcutaneously or orally with liposomes in one or several doses at varying intervals of time.

[0130] The recombinant factor VIII can also be used to treat uncontrolled bleeding due to factor VIII deficiency in hemophiliacs who have developed antibodies to human factor VIII.

[0131] It has been demonstrated herein that the recombinant factor VIII of the present invention can differ in specific activity from the wild-type factor VIII and retain a higher specific activity for a longer duration following activation. Factor VIII proteins having greater procoagulant activity from wild-type factor VIII are useful in treatment of hemophilia because lower dosages will be required to correct a patient's factor VIII deficiency. This will not only reduce medical expense for both the patient and the insurer, but also reduce the likelihood of developing an immune response to the factor VIII (because less antigen is administered).

EXAMPLES

[0132] The following examples are provided to illustrate embodiments of the present invention, but they are by no means intended to limit its scope.

Materials & Methods

[0133] Reagents:

[0134] Recombinant factor VIII (KOGENATE.TM.) and the monoclonal antibodies 58.12 and 2D2 were generous gifts from Dr. Lisa Regan of Bayer Corporation (Berkeley, Calif.). Phospholipid vesicles containing 20% phosphatidylcholine (PC), 40% phosphatidylethanolamine (PE), and 40% phosphatidylserine (PS) were prepared using octylglucoside as described previously (Mimms et al., "Phospholipid Vesicle Formation and Transmembrane Protein Incorporation Using Octyl Glucoside," Biochemistry 20:833-840 (1981), which is hereby incorporated by reference in its entirety). The reagents .alpha.-thrombin, factor VIIa, factor IXa.beta., factor X, and factor Xa (Enzyme Research Laboratories, South Bend, Ind.), hirudin (DiaPharma, West Chester, Ohio), phospholipids (Avanti Polar Lipids, Alabaster, Ala.), the chromogenic Xa substrate, Pefachrome Xa (Pefa-5523, CH.sub.3OCO-D-Cha-Gly-Arg-pNAAcOH; Centerchem Inc. Norwalk Conn.), recombinant human tissue factor (rTF), Innovin (Dade Behring, Newark, Del.), fluorogenic substrate, Z-Gly-Gly-Arg-AMC (Calbiochem, San Diego, Calif.), thrombin calibrator (Diagnostica Stago, Parsippany, N.J.), and acrylodan (Molecular Probes, Eugene, Oreg.) were purchased from the indicated vendors.

[0135] Expression and Purification of WT and Variant Factor VIII:

[0136] Recombinant WT and variant factor VIII forms were stably expressed in BHK cells and purified as described previously (Wakabayashi et al., "Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca.sup.2+ Binding Site Required for Cofactor Activity," J Biol Chem. 279:12677-12684 (2004), which is hereby incorporated by reference in its entirety). After transfection there were no significant differences in the amounts of factor VIII secretion among the variants. Protein yields for the variants ranged from >10 to .about.100 .mu.g from two 750 cm.sup.2 culture flasks, with purity from .about.85% to >95% as judged by SDS-PAGE. The primary contaminant in the factor VIII preparations was albumin and at the concentrations present in the factor VIII showed no effect on stability or activity parameters. Factor VIII concentration was measured by ELISA and factor VIII activity was determined by one-stage clotting assay and two-stage chromogenic factor Xa generation assay, both of which are described below.

[0137] Western Blotting:

[0138] Factor VIII proteins (0.34 .mu.g) were activated by thrombin (20 nM) for 30 min at 23.degree. C. and subjected to electrophoresis under either non-reducing or reducing (0.1 M dithiothreitol) conditions using 10% polyacrylamide gels run at constant voltage (150V). Gels were transferred to a polyvinylidene fluoride membrane, probed with an anti-A1 domain (58.12) or anti-A3 domain (2D2) monoclonal antibody and protein bands were visualized using chemifluorescence. The chemifluorescence substrate (ECF substrate, GE Healthcare, Piscataway, N.J.) was reacted and the fluorescence signal scanned using a phosphoimager (Storm 860, GE Healthcare).

[0139] ELISA:

[0140] A sandwich ELISA was performed to measure the concentration of factor VIII proteins as previously described (Wakabayashi et al., "A Glu113Ala Mutation within a Factor VIII Ca.sup.2+-Binding Site Enhances Cofactor Interactions in Factor Xase," Biochemistry 44:10298-10304 (2005), which is hereby incorporated by reference in its entirety) using purified commercial recombinant factor VIII (KOGENATE.TM., Bayer Corporation) as a standard. Factor VIII capture used the anti-C2 monoclonal antibody (GMA8003, Green Mountain Antibodies) and the anti-A2 monoclonal antibody, R8B12 (GMA8012, Green Mountain Antibodies) was employed for factor VIII detection following biotinylation.

[0141] One-Stage Clotting Assay:

[0142] One-stage clotting assays were performed using substrate plasma chemically depleted of factor VIII (Over, "Methodology of the One-stage Assay of Factor VIII (VIII:C)," Scand J Haematol Suppl. 41:13-24 (1984), which is hereby incorporated by reference in its entirety) and assayed using a Diagnostica Stago clotting instrument. Plasma was incubated with APTT reagent (General Diagnostics) for 6 min at 37.degree. C. after which a dilution of factor VIII was added to the cuvette. After 1 min the mixture was recalcified, and clotting time was determined and compared to a pooled normal plasma standard.

[0143] Two-Stage Chromogenic Factor Xa Generation Assay:

[0144] The rate of conversion of factor X to factor Xa was monitored in a purified system (Lollar et al., "Factor VIII and Factor VIIIa," Methods Enzymol. 222:128-143 (1993), which is hereby incorporated by reference in its entirety) according to methods previously described (Wakabayashi et al., "Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and Inter-subunit Affinity," Biochemistry 40:10293-10300 (2001); Wakabayashi et al., "Ca.sup.2+ Binding to Both the Heavy and Light Chains of Factor VIII Is Required for Cofactor Activity," Biochemistry 41:8485-8492 (2002), each of which is hereby incorporated by reference in its entirety). Factor VIII (1 nM) in buffer containing 20 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH 7.2, 0.1 M NaCl, 0.01% Tween 20, 0.01% BSA, 5 mM CaCl.sub.2 (Buffer B), and 10 .mu.M PSPCPE vesicles was activated with 20 nM .alpha.-thrombin for 1 min. The reaction was stopped by adding hirudin (10 U/ml) and the resulting factor VIIIa was reacted with factor IXa (40 nM) for 1 min. Factor X (300 nM) was added to initiate reactions which were quenched after 1 min by the addition of 50 mM EDTA. Factor Xa generated was determined following reaction with the chromogenic substrate Pefachrome Xa (0.46 mM final concentration). All reactions were run at 23.degree. C.

[0145] Factor VIII Activity at Elevated Temperature:

[0146] WT factor VIII or factor VIII variants (4 nM) in buffer B were incubated at 57.degree. C. (pH at this temperature=6.94). Aliquots were removed at the indicated times, cooled to room temperature, and residual factor VIII activity was determined using a two-stage factor Xa generation assay.

[0147] Factor Villa Activity Decay:

[0148] WT and mutant factor VIII (1.5 nM) in buffer B containing 20 .mu.M PSPCPE vesicles were activated using 20 nM thrombin for 1 mM at 23.degree. C. Reactions were immediately quenched by hirudin (10 U/ml) to inactivate thrombin, aliquots removed at the indicated times, and activity was determined using the factor Xa generation assay following addition of factor IXa (40 nM) and factor X (300 nM).

[0149] Factor VIII Activity Inhibition by Guanidinium Chloride:

[0150] WT and factor VIII variants (50 nM) in buffer B plus 0-1.8 M guanidinium chloride were incubated for 2 hrs at 23.degree. C. Aliquots were diluted (1/50) in buffer A containing 20 .mu.M PSPCPE vesicles and activated by 5 nM thrombin for 1 min. Reactions were immediately quenched with hirudin (10 U/ml) and activity was determined by factor Xa generation assay following addition of factor IXa (40 nM) and FX (300 nM). Residual guanidinium chloride (<36 mM) did not inhibit the proteolytic activation of factor VIII or its cofactor activity.

[0151] Thermal Denaturation of Reconstituted A1 and A3C1C2 or A3C1 Subunit as Detected by FXa Generation Assay--

[0152] A1 subunit (50 nM) from WT or Ala108Ile factor VIII was reconstituted with A3C1C2 (200 nM) or A3C1 (500 nM) at 37.degree. C. for 2 hr in 10 mM MES, pH 6.5, 0.15 M NaCl, 0.01% Tween 20, 0.01% BSA, 5 mM CaCl.sub.2. Samples were incubated at 55.degree. C. (A3C1C2) or 52.degree. C. (A3C1) (pH at this temperature=6.94), aliquots were taken at indicated times, and further incubated with 200 nM A2 subunit at 23.degree. C. for 30 min Samples were then diluted 1:20 with buffer B containing 20 .mu.M PSPCPE vesicles and reconstituted factor VIIIa activity was measured directly by factor Xa generation assay in the absence of the thrombin activation step. Data were fitted to the single exponential decay equation by non-linear least squares regression and parameter values were obtained.

[0153] Thrombin Generation Assay--

[0154] The amount of thrombin generated in plasma was measured by Calibrated Automated Thrombography using methods previously described (Wakabayashi et al., "Combining Mutations of Charged Residues at the A2 Domain Interface Enhances Factor VIII Stability over Single Point Mutations," J. Thromb. Haemost. 7:438-444 (2009), which is hereby incorporated by reference in its entirety). Briefly, factor VIII deficient plasma (<1% residual activity, platelet-poor) from severe hemophilia A patients lacking factor VIII inhibitor (George King Bio-Medical, Overland Park, Kans.) was mixed at 37.degree. C. with a final concentration of 0.3 nM factor VIII, 0.5 pM rTF, 4 .mu.M PSPCPE vesicles, 433 .mu.M fluorogenic substrate, 13.3 mM CaCl.sub.2, and 105 nM thrombin calibrator. The development of a fluorescent signal was monitored at 8 second intervals using a Microplate Spectrofluorometer (Spectramax Gemini, Molecular Devices, Sunnyvale, Calif.) with a 355 nm (excitation)/460 nm (emission) filter set. Fluorescent signals were corrected by the reference signal from the thrombin calibrator samples and actual thrombin generation in nM was calculated.

[0155] Data Analysis:

[0156] For activity decay analysis of factor VIII/VIIIa, activity values as a function of time were fitted to a single exponential decay curve by non-linear least squares regression using the equation,

A=A.sub.0e.sup.-kt

where A is residual factor VIIIa activity (nM/min/nM factor VIII), A.sub.0 is the initial activity, k is the apparent rate constant, and t is the time (minutes) of reaction of factor VIII (for factor VIII thermal decay experiments) or of factor VIIIa after thrombin was quenched (for factor VIIIa decay measurements). Factor VIII activity inhibition by guanidinium was fitted to a linear equation by least squares regression using the equation,

A=50k(X-IC.sub.50)

where A is the normalized activity [=100(%)], IC.sub.50 is the inhibitor (guanidinium chloride) concentration (M) at 50% activity, X is the guanidinium chloride concentration (M), and k is the slope. Determinations for A1-A3C1C2 binding affinity used the quadratic equation:

F = F max B ( B + K d + X ) 2 - ( B + K d + X ) 2 - 4 B X 2 ##EQU00001##

where F.sub.max is the maximum increase in fluorescence at saturation, B is the A1 concentration (=15 nM), K.sub.d is the dissociation constant, and X is the concentration of A3C1C2 in nM, Nonlinear least-squares regression analysis was performed using Kaleidagraph (Synergy, Reading, Pa.). A Student's t-test was performed for statistical analysis.

Example 1

Recombinant Expression and Purification of Factor VIII Mutants Possessing Modified A1 and C2 Domain Interactions

[0157] The A1 and C2 domains show close proximity to one another in the factor VIII crystal structure (FIG. 3A). These regions were investigated with the aim towards enhancing inter-domain interactions to positively alter affinity and stability parameters. Examination of the A1-C2 interface provided two possible approaches for increasing the inter-domain affinity. Although the orientation of side chains cannot be discerned due to the resolution (.about.4 .ANG.) of the structure (4), the putative spatial separation of several paired residues indicated that mutagenesis of these residues to Cys could result in formation of a nascent inter-domain disulfide bridge. Paired A1/C2 domain residues were identified that appeared to meet this distance requirement and included Ser119/Pro2300, Gln120/Pro2299, Arg121/Leu2302, Ala108/Ala2328, and Trp106/Ala2328.

[0158] Double mutations where each residue of the respective pair was replaced with Cys were prepared as B-domainless factor VIII--lacking residues Gln744-Ser1637 in the B-domain (Doering et al., "Expression and Characterization of Recombinant Murine Factor VIII," Thromb. Haemost 88:450-458 (2002), which is hereby incorporated by reference in its entirety)--using previously described methods (Wakabayashi et al., "Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca.sup.2+-Binding Site Required for Cofactor Activity," J. Biol. Chem 279:12677-12684 (2004), which is hereby incorporated by reference in its entirety). Briefly, B-domainless factor VIII cDNA was restricted from the factor VIII expression construct HSQ-MSAB-NotI-RENeo using the endonucleases XhoI and NotI, and then cloned into the Bluescript II K/S-vector. Factor VIII molecules bearing one or more point mutations were constructed by introducing mutations into shuttle constructs using the Stratagene QuikChange site-directed mutagenesis kit. Upon confirmation of the presence of only the desired mutations by sequencing, the appropriate fragment was restricted and cloned back into the factor VIII expression construct. Presence of only the desired mutations was again confirmed by a second round of sequencing. Of the five variants examined, only the Arg121Cys/Leu2302Cys variant (FIG. 3B) retained a wild-type like specific activity (.about.86% the WT value, Table 1) and was further evaluated.

[0159] Recombinant WT and variant factor VIII forms were stably expressed in BHK cells and purified as previously described (Lollar and Parker, "pH-dependent Denaturation of Thrombin-activated Porcine Factor VIII," J. Biol. Chem 265:1688-1692 (1990), which is hereby incorporated by reference in its entirety). Protein yields for the variants ranged from >10 to .about.100 .mu.g from two 750 cm.sup.2 culture flasks, with purity >90% as judged by SDS-PAGE. The primary contaminant in the factor VIII preparations was albumin. Factor VIII concentrations were measured using an Enzyme-Linked Immunoadsorbant Assay (ELISA) and factor VIII activity was determined by one-stage clotting and two-stage chromogenic factor Xa generation assays described supra.

[0160] In addition, a small cavity in the A1-C2 interface that potentially exists was noted, this putative cavity is mainly surrounded by alkyl groups from Ala108, Ala2328, Leu2302, and Gln2329 (FIG. 3C), with the side chain of Ala108 proposed to be directed towards the C2 domain. In an effort to increase hydrophobic interactions within this area, several variants were prepared to introduce bulky hydrophobic groups in these domains. This was carried out by mutating Ala residues to Ile, Leu, and Val (Ala108Ile, Ala108Leu, Ala108Val, Ala232811e, Ala2328Leu, and Ala2328Val) in a B-domainless factor VIII cDNA using the procedures described in the preceding paragraph. Of the variants prepared, the Ala108Ile factor VIII variant showed minimal effects on factor VIII specific activity (.about.74% the WT value, Table 1); the Ala108Leu and Ala108Val variants showed greater diminution of factor VIII specific activity.

[0161] The A1 subunit was purified from WT or Ala108Ile factor VIII. Factor VIII (1-3 .mu.M) was reacted with thrombin (50 nM) in 20 mM HEPES, pH 7.2, 0.1 M NaCl, 0.01% Tween 20 (buffer A) for 30 min and treated with 50 mM EDTA overnight at 4.degree. C. After a 1:4 dilution with buffer A, the samples were subjected to chromatography using a heparin Sepharose column (1.5 cm.times.0.7 cm in diameter, GE Healthcare, Piscataway, N.J.). The flow through fraction was collected and applied to a Q-Sepharose column (1.5 cm.times.0.7 cm in diameter, GE Healthcare). After the column was washed with buffer A, bound A1 subunit was eluted with 20 mM HEPES, pH 7.2, 0.8 M NaCl, 0.01% Tween20, and purified A1 subunit was kept frozen at 80.degree. C. until use. A2 and A3C1C2 subunits were completely absorbed by the heparin Sepharose column step and the final A1 product was >95% pure as judged by SDS-PAGE. A2 and A3C1C2 subunits were purified from recombinant factor VIII (Kogenate.TM.) as described previously (Fay and Smudzin, "Characterization of the Interaction Between the A2 Subunit and A1/A3-C1-C2 Dimer in Human Factor VIIIa," J. Biol. Chem 267:13246-13250 (1992), which is hereby incorporated by reference in its entirety). A3C1 subunit was purified from C2 domain-deleted factor VIII (Wakabayashi et al., "Factor VIII Lacking the C2 Domain Retains Cofactor Activity in vitro," J. Biol. Chem 285:25176-25184 (2010), which is hereby incorporated by reference in its entirety) using the same method for A3C1C2 purification.

[0162] Purified A1 subunit from WT and Ala108Ile factor VIII was labeled with acrylodan by sulfhydryl specific protein modification as previously described (Wakabayashi et al., "Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and Inter-subunit Affinity," Biochemistry 40:10293-10300 (2001), which is hereby incorporated by reference in its entirety). A1 (15 nM) from WT or Ala108Ile factor VIII was reconstituted with A3C1C2 subunit (0-300 nM) at 37.degree. C. for 2 h in buffer B at pH 7.4. Fluorescence measurements were performed using an Aminco-Bowman Series 2 Luminescence Spectrometer (Thermo Spectronic, Rochester, N.Y.) at 23.degree. C. at an excitation wavelength of 395 nm (2 nm bandwidth). Fluorescence emission was monitored at 480-490 nm (8 nm bandwidth) and all spectra were corrected for background. Data were fitted to a quadratic equation by non-linear least squares regression and parameter values were obtained.

Example 2

Confirmation of Disulfide Bridge in Arg121Cys/Leu2302Cys Factor VIII Variant

[0163] Evidence for high efficiency disulfide bridging between factor VIII A1 and A3C1C2 domains in this double mutant, as judged by Western Blotting is shown in FIG. 4. For this analysis, WT factor VIII and the Arg121Cys/Leu2302Cys factor VIII variant were cleaved with thrombin to generate the factor VIIIa heterotrimer prior to SDS-PAGE, which was then run in the absence and presence of disulfide bond reduction using DTT. Blots were probed with an anti-A1 antibody (58.12, lanes 1-4) and an anti-A3 antibody (2D2, lanes 5-8). Both A1 and A3C1C2 subunits derived from the factor VIII Arg121Cys/Leu2302Cys variant were detected at the .about.120 kDa band, consistent with the sum of their mol masses under non-reducing conditions (lanes 2 and 6), while reduction by 0.1 M DTT yielded the separated subunits (lanes 4 and 8). Based upon the band densities of bridged and free subunits in the non-reduced lanes, it appeared that >90% of the variant molecules were disulfide-linked.

Example 3

Affinity of the WT and Ala108Ile Factor VIII Variant A1 Subunits for A3C1C2

[0164] To assess the affinity of the Ala108Ile A1 domain for C2 domain, the purified factor VIII variant and WT were treated with thrombin and the A1 subunits were separately purified as described supra. A1 subunits were then reacted with the environment-sensitive fluorescent probe, acrylodan, and these reagents were used to probe binding with the A3C1C2 subunit. The site for acrylodan attachment is likely the lone free thiol in A1 at Cys310, which is in close proximity (<15 .ANG.) to residues in the A3 domain of light chain (Ngo et al., "Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa:Factor VIIIa Complex," Structure 16:597-606 (2008), which is hereby incorporated by reference in its entirety). Indeed, increases in the emission fluorescence from acrylodan-labeled A1 (AcA1) subunit have been previously observed when A3C1C2 was bound to the molecule (Wakabayashi et al, "Metal-ion Independent Factor VIII Subunit Association and the Role of Calcium and Copper for Its Affinity and Activity," Biochemistry 40:10293-10300 (2001); Ansong et al., "Factor VIII A3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site," Biochemistry 44:8850-8857 (2005), each of which is hereby incorporated by reference in its entirety). Titration of AcA1 with A3C1C2 was performed as described supra and the results are shown in FIG. 5. AcA1 (15 nM) fluorescence from both the WT and Ala108Ile subunits saturably increased as the A3C1C2 concentration increased. The estimated K.sub.d of this interaction for WT and Ala108Ile A1 subunits were 88.7.+-.9.8 nM and 24.1.+-.4.1 nM, respectively. The K.sub.d value for WT was somewhat higher than a previously reported value (.about.50 nM) (Ansong et al., "Factor VIII A3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site," Biochemistry 44:8850-8857 (2005), which is hereby incorporated by reference in its entirety) likely due to slightly higher pH (7.4) employed for the binding conditions (Wakabayashi et al., "pH-dependent Association of Factor VIII Chains: Enhancement of Affinity at Physiological pH by Cu.sup.2+," Biochim. Biophys. Acta 1764:1094-1101 (2006), which is hereby incorporated by reference in its entirety). This result indicated a .about.4-fold increase in affinity of Ala108Ile A1 for A3C1C2 as compared with the WT A1 subunit for A3C1C2. The estimated maximal values in fluorescence for WT and Ala108Ile were 0.221.+-.0.009 and 0.245.+-.0.011 respectively, and were not significantly different (p>0.1).

Example 4

Stability of Factor VIII Arg121Cys/Leu2302Cys and Ala108Ile Variants

[0165] The above results demonstrate that introduction of the disulfide bridge or increasing the hydrophobic character at the A1-C2 interface stabilizes this inter-domain interaction. To test the functional consequences of these mutations, stability parameters of factor VIII (factor VIIIa) were evaluated by several methods. Thermal denaturation experiments were performed at 57.degree. C. as described supra. Data shown in FIG. 6A were fitted to a single exponential decay curve using non-linear least squares regression. WT factor VIII (circles) decayed to .about.40% the initial activity level in 6-7 min at 57.degree. C. On the other hand, the Arg121Cys/Leu2302Cys variant (triangles) retained >40% activity up to 20 min, whereas the Ala108Ile variant (squares) retained this level for >30 min. Overall, decay rates for the Arg121Cys/Leu2302Cys and Ala108Ile variants obtained by curve-fit were reduced by 3.1- and 4.2-fold, respectively, compared to the WT factor VIII value (see Table 1 below).

TABLE-US-00003 TABLE 1 Properties of Wild type Factor VIII and Variants Specific Factor VIII Factor VIIIa Activity Decay Rate IC.sub.50 Decay Rate (%) (min.sup.-1) (M) (min.sup.-1) WT 100.0 .+-. 8.2 0.143 .+-. 0.003 (1.0) 0.814 .+-. 0.010 (1.00) 0.154 .+-. 0.006 (1.00) R121C/L2302C 86.4 .+-. 3.8 0.047 .+-. 0.001 (0.32) 0.826 .+-. 0.005 (1.01) 0.132 .+-. 0.004 (0.86) A108I 73.7 .+-. 3.9 0.034 .+-. 0.002 (0.24) 0.892 .+-. 0.008 (1.10) 0.119 .+-. 0.008 (0.77) Specific activity was determined by factor Xa generation assay as described above and expressed as a relative activity compared to WT value. Factor VIII decay data at 57.degree. C. as shown in FIG. 6A, and factor Villa spontaneous decay data as shown in FIG. 6C were fitted to a single exponential decay curve. Guanidinium denaturation data as shown in FIG. 6B were fitted to a linear response curve by non-linear least squares regression and IC.sub.50 values with standard deviations were obtained. Values in parentheses are relative to the WT value.

[0166] In a complementary series of experiments, factor VIII stability was examined following a 2 h exposure to 0.6-1.2 M guanidinium (FIG. 6B). As the concentration of guanidinium increased, factor VIII activity was reduced to near zero as an indication of denaturation. Using the range of linear response (.about.0.6-1 M), data points were fitted by a linear equation and the IC.sub.50 values were obtained (see Table 1). Factor VIII activity of the Ala108Ile variant was significantly more stable than WT showing a .about.10% higher IC.sub.50 values compared with WT (p<0.001), while the IC.sub.50 determined for the Arg121Cys/Leu2302Cys variant was only slightly increased (.about.2% greater than WT, Table 1). Overall, the stability data for the disulfide bridged variant suggested that the covalent bond between A1 and C2 subunits significantly increased factor VIII thermal stability while showing little stabilizing effect in the presence of guanidinium. This result indicated that dissociation of factor VIII heavy and light chains may be a prominent cause for activity loss at elevated temperature, but that chain dissociation may not represent a primary mode for activity loss due to chemical denaturation. Alternatively, the Ala108Ile mutation demonstrated a more global protective effect in increasing factor VIII stability towards either thermal or chemical denaturation. Control experiments were performed to determine whether there was any time-dependent change in activity following the thermal or chemical denaturation step and return of factor VIII to either ambient temperature or dilution of denaturant, respectively. Factor VIII was assayed from 30 seconds to 1 hour, and no significant change in activity was observed.

[0167] Factor VIIIa activity is labile due to A2 subunit dissociation following proteolytic activation (Fay et al., "Human Factor VIIIa Subunit Structure: Reconstitution of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit," J. Biol. Chem 266:8957-8962 (1991); Lollar et al., "pH-dependent Denaturation of Thrombin-activated Porcine Factor VIII," J. Biol. Chem 265:1688-1692 (1990); Lollar et al., "Coagulant Properties of Hybrid Human/Porcine Factor VIII Molecules," J. Biol. Chem 267:23652-23657 (1992), each of which is hereby incorporated by reference in its entirety) To determine whether these mutations affected factor VIIIa decay, experiments were performed to assess rates of loss of factor VIIIa activity over time. As shown in FIG. 6C, reaction conditions employed resulted in the loss of .about.50% of WT factor VIIIa activity at .about.6 min after thrombin activation, while .about.10% activity remained after 16 min. The observed factor VIIIa activity decay was slightly reduced for both Arg121Cys/Leu2302Cys and Ala108Ile variants which showed .about.40% activity in 7-8 min and demonstrated decay rates that were 1.2- and 1.3-fold greater than WT factor VIII, respectively (see Table 1). These results demonstrated only minor effects on the inter-subunit interactions involving A2 subunit following modification of the A1 and C2 domain interface.

Example 5

Stability of Reconstituted Ala108Ile or WT A1 Subunit with A3C1C2 or A3C1 Subunits

[0168] The thermal stability for the A1/A3C1C2 dimer was assessed following its reconstitution from isolated subunits. Purified WT A1 or Ala108Ile A1 subunits was reconstituted with A3C1C2 subunit at 37.degree. C. for 2 hrs and the stability of the A1/A3C1C2 dimer at elevated temperature (55.degree. C.) was measured by factor Xa generation assay following addition of A2 subunit as described in Methods. As shown in FIG. 7A, factor VIIIa activity reconstituted from WT-A1 decayed to .about.25% its original value at 20 min (circles) while .about.80% of the original activity level remained for the A1 subunit containing the Ala108Ile mutation (triangles). The estimated decay rates for WT and the mutant factor VIIIa forms were 0.066.+-.0.005 and 0.014.+-.0.001 min.sup.-1, respectively, showing a 4.6-fold rate reduction for the variant compared to WT factor VIII.

[0169] Similar reconstitution experiments were performed using the A3C1 subunit derived from the C2 domain-deleted factor VIII variant. The rationale for this experiment was that if the enhanced stability of the Ala108Ile A1 were due to interaction with the C2 domain following reassociation with A3C1C2, then use of the truncated A3C1 for reconstitution would abrogate the enhanced stability of the variant. Experiments performed with a C2-domain deleted factor VIII, described in an earlier report (Wakabayashi et al., "Factor VIII Lacking the C2 Domain Retains Cofactor Activity in vitro," J. Biol. Chem 285:25176-25184 (2010), which is hereby incorporated by reference in its entirety), showed that this variant was marked less stable than WT factor VIII at elevated temperatures and rates of decay needed to be monitored at a relatively lower temperature (52.degree. C.). Under these conditions, the C2-domain deleted factor VIII variant decayed .about.40-fold faster than WT factor VIII. For this reason, stability studies following factor VIII reconstitutions with the A3C1 light chain were performed at 52.degree. C. Furthermore, in an earlier study (Wakabayashi et al., "Generation of Enhanced Stability Factor VIII Variants by Replacement of Charged Residues at the A2 Domain Interface," Blood 112:2761-2769 (2008), which is hereby incorporated by reference in its entirety), it was demonstrated that factor VIII stability measured over a range of temperatures from 52-60.degree. C. yielded similar relative rates of decay when comparing a given factor VIII variant to WT. Consistent with observations using the C2 domain-deleted factor VIII, reconstitutions using either A1 form with A3C1 were observed to yield an overall faster decay (FIG. 7B), with .about.50% activity reduction at 4 min at 52.degree. C., than results observed following reconstitutions with the intact A3C1C2. The estimated decay rates for WT and the variant factor VIIIa forms were similar (0.166.+-.0.001 and 0.154.+-.0.013 min.sup.-1, respectively). That the observed increase in thermal stability of the Ala108Ile variant was also observed following reconstitutions using purified components supports the conclusion that the enhanced stability of the variant as compared with WT derived from improved interaction(s) between A1 and A3C1C2 subunits and required the presence of C2 subunit.

Discussion of Examples 1-5

[0170] The preceding Examples illustrate interactions at the interface between factor VIII A1 and C2 domains following preparation of two factor VIII variants, Arg121Cys/Leu2302Cys factor VIII, which possesses a nascent disulfide bond spanning these domains, and Ala108Ile factor VIII, which has a larger hydrophobic side chain to better fill the inter-domain space. Several other mutations were prepared at this region in an attempt to create a disulfide bond or to increase hydrophobicity. These variants yielded low specific activity values, possibly resulting from unfavorable changes in conformation, and their characterization was not pursued further. However, both variants studied exhibited enhanced inter-A1-C2 domain affinity resulting in increases in the observed stability of the factor VIII variants, especially related to thermal denaturation.

[0171] The intermediate resolution (.about.4 .ANG.) X-ray structure of factor VIII (Ngo et al., "Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa:Factor VIIIa Complex," Structure 16:597-606 (2008), which is hereby incorporated by reference in its entirety) predicts the close proximity of Arg121 and Leu2302 with 7.7 .ANG. separating C.alpha. atoms (PDB#3CDZ). This spatial separation suggested the potential to bridge this distance by a disulfide bond (.about.4-6 .ANG.) following replacement of these residues with Cys, and provided that the side chains were in an acceptable orientation. Results evaluating the Arg121Cys/Leu2302Cys factor VIII protein by western blotting in the absence and presence of disulfide bond reduction showed high efficiency bridging (>90%) constituting experimental proof for the opposing orientation of side chains of these two residues in factor VIII. In addition, the X-ray structure also showed that the A1-C2 junction adjacent to Ala108 is rich in hydrophobic groups represented by the C.beta. carbon of Ala108 from the C.delta. of Leu2302, the C.beta. of Ala2328, or the C.gamma. of Gln2329 (see FIG. 3C). Thus, it was believed that extended alkyl groups of side chains larger than the methyl group of Ala might contribute to enhanced binding energy. Of several variants prepared to this region, replacement of Ala108 with Ile yielded a variant possessing near WT-like specific activity.

[0172] Both Arg121Cys/Leu2302Cys and Ala108Ile variants exhibited superior stability parameters as compared with the WT protein. For example, the thermal decay rates for the Arg121Cys/Leu2302Cys and Ala108Ile factor VIII variants were reduced by 3.1- and 4.2-fold, respectively, as compared with WT. Further dissection of the interaction with the Ala108Ile variant was obtained following reconstitution of the A1/A3C1C2 dimer using WT and variant A1 subunits as well as the truncated A3C1 subunit derived from the C2 domain-deleted factor VIII. Enhanced stability of the variant compared with WT was observed using native A3C1C2, but similar stability parameter values for variant and WT were observed even in the absence of the C2 domain. Taken together, these observations support the belief that modulating the A1-C2 interface, either through covalent bridging or increased hydrophobic interaction, appeared to make an important contribution to overall protein stability.

[0173] The primary cause for thermal decay of FVIII is attributed to dissociation of the heavy and light chains (Ansong et al., "Factor VIII A3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site," Biochemistry 44:8850-8857 (2005), which is hereby incorporated by reference in its entirety). This result is supported by the present study showing that bridging the factor VIII heavy chain and light chain via a disulfide bond between A1 and C2 domains preferentially reduced thermal decay as compared with chemical denaturation. Thus, chemical denaturation appears to represent a more global effect on factor VIII structure and less specific for chain dissociation. It was reported earlier that several residues at the A2-A3 interface (Tyr1792, Tyr1786 and Asp666) possibly contributed to the binding energy only in the active factor VIIIa form (Wakabayashi et al., "Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa," J. Biol. Chem 283:11645-11651 (2008), which is hereby incorporated by reference in its entirety). Thus, interactions between the A2 domain of the heavy chain and A3C1C2 domains of the light chain may be minimal in the pro-cofactor. Based upon that earlier report and the present study, it is believed that in the factor VIII heterodimer, the predominant sources for binding energy likely derive from A1 interactions with both the A3 and C2 domains.

[0174] On the other hand, the instability of factor VIIIa results from weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer (Fay et al., "Human Factor VIIIa Subunit Structure: Reconstitution of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit," J. Biol. Chem 266:8957-8962 (1991); Lollar et al., "pH-dependent Denaturation of Thrombin-activated Porcine Factor VIII," J. Biol. Chem 265:1688-1692 (1990), each of which is hereby incorporated by reference in its entirety) and its dissociation leads to dampening of factor Xase activity (Lollar et al., "Coagulant Properties of Hybrid Human/Porcine Factor VIII Molecules," J. Biol. Chem 267:23652-23657 (1992); Fay et al., "Model for the Factor VIIIa-dependent Decay of the Intrinsic Factor Xase: Role of Subunit Dissociation and Factor IXa-catalyzed Proteolysis," J. Biol. Chem 271:6027-6032 (1996), each of which is hereby incorporated by reference in its entirety). Several factor VIII point mutations have been shown to facilitate the rate of dissociation of A2 relative to wild type (WT) and these residues localize to either the A1-A2 domain interface (Pipe et al., "Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa in vivo," Blood 93:176-183 (1999); Pipe et al., "Hemophilia A Mutations Associated with 1-stage/2-stage Activity Discrepancy Disrupt Protein-protein Interactions within the Triplicated A Domains of Thrombin-activated Factor VIIIa," Blood 97:685-691 (2001), each of which is hereby incorporated by reference in its entirety) or the A1 and C2 domains (Hakeos et al., "Hemophilia A Mutations Within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-Stage/Two-Stage Activity Discrepancy," Thromb. Haemost 88: 781-787 (2002), which is hereby incorporated by reference in its entirety). In U.S. Patent Application Publ. No. 20090118184 to Fay et al., which is hereby incorporated by reference in its entirety, it was demonstrated that replacing the charged residues Asp519, Glu665, and Glu1984 with Ala or Val yielded increased factor VIII stability and in particular enhanced retention of the A2 subunit in factor VIIIa. Interestingly, neither the single mutants nor combinations of these mutations yielded factor VIII variants that showed reductions in the rate of thermal decay of greater than 2.3-fold, whereas the variants examined in the present study showed thermal decay rate reductions of 3- to 4-fold. Thus, the magnitude of stability enhancement observed for the A1-C2 interface variants appears somewhat larger than for the A2 domain-mediated interactions. However, while these variants clearly showed superior factor VIII stability, results from this study indicated essentially little if any effect of the interactions involving the A1 and C2 domains in stabilizing the factor VIIIa cofactor, suggesting no linkage of these sites with sites involved in A2 subunit retention. For this reason, it is the combinations of these A1-C2 domain stabilizing mutations with A1-A2 or A3-A2 domain stabilizing mutations that appear to be most desirable.

[0175] A1 domain residues 110-126 are in close contact to the C2 domain. These residues contain a Ca.sup.2+ binding site predicted by Ala-scanning mutagenesis (Wakabayashi et al., "Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca.sup.2+ Binding Site Required for Cofactor Activity," J. Biol. Chem 279:12677-12684 (2004), which is hereby incorporated by reference in its entirety) and subsequently identified in the X-ray crystal structure (Shen et al., "The Tertiary Structure and Domain Organization of Coagulation Factor VIII," Blood 111:1240-1247 (2008); Ngo et al., "Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa:Factor VIIIa Complex," Structure 16:597-606 (2008), each of which is hereby incorporated by reference in its entirety). Interestingly, preliminary experiments assessing chelation of Ca.sup.2+ (and/or Cu.sup.2+) in factor VIII by EGTA yielded dramatic losses in activity of WT factor VIII while showing more minimal effects on the activity of Arg121Cys/Leu2302Cys and Ala108Ile variants. Without being bound by belief, it is believed the functional effects of Ca.sup.2+ occupancy at 110-126 in factor VIII were replaced by enhanced stabilizing interactions between the A1 and C2 domains in the variants.

[0176] In conclusion, results from Examples 1-5 demonstrate that interactions between the A1 and C2 domains of factor VIII contribute to the integrity of the protein, providing significant energy for stabilizing the multi-domain structure of factor VIII. Furthermore, observations for enhancing factor VIII stability, in particular by increasing non-covalent, hydrophobic interactions at the A1-C2 domain interface suggests that these variants could potentially represent superior therapeutics in the treatment of hemophilia.

Example 6

Combination of R121C/L2302C or Ala108Ile Substitution with One or More Substitutions at the A1-A2 or A2-A3 Domain Interfaces

[0177] Based on the improved thermal and/or chemical stability afforded by the A1-C2 variants, it was also determined whether the mutations at the A1-C2 interface can be combined with the mutation at one of the A1-A2 or A2-A3 interfaces to generate a factor VIII with even greater stability. Previously tested stable factor VIII mutants, as described in U.S. Patent Application Publ. No. 2009/0118184 to Fay et al., which is hereby incorporated by reference in its entirety, showed up to 2-fold increases in thermal stability compared with WT factor VIII in any combination of the mutation with Asp519Ala, Asp519Val, Glu665Ala, Glu665Val, Glu1984Ala, and Glu1984Val (A1-A2 or A2-A3 domain mutants).

[0178] These single point mutations or the double mutation Asp519Val/Glu665Val were combined with either R121C/L2302C or Ala108Ile in a B-domainless factor VIII cDNA using the procedures described in Example 1. These factor VIII mutants were expressed and purified using the procedures described in Example 1. Specific activity values showed that most of the mutants (10 out of 13) showed normal factor VIII activity values (>60% WT) as measured by factor Xa generation assay (see Table 2 below). Thermal stability values were determined as described above in Example 4. Many of the resulting mutants exhibited >5 fold increase in thermal stability (10/13 mutants, FIG. 8A), with Ala108Ile/Glu665Val and Ala108Ile/Asp519Val/Glu665Val being the most stable mutants (.about.10 fold increase relative to WT). Most of the mutants also showed 15-30% increases in IC.sub.50 value (guanidinium experiment) compared with WT (FIG. 8B). In addition, the high factor VIIIa stability of the A domain mutants was mostly preserved (mutants (11/13) showed 2-10 fold increase in factor VIIIa stability relative to WT, FIG. 8C). Collectively, modification at the A1-C2 contacting region by covalent attachment or increasing hydrophobic interaction improved factor VIII stability and these modifications can be combined with A2 domain interface mutations to provide essentially additive effects compared with either type of mutation alone.

TABLE-US-00004 TABLE 2 Specific Activity of Factor VIII Variants FVIII variants Activity (%) FVIII variants Activity (%) WT 100 D519V/E665V 90.0 Ala108Ile 73.7 121CL 86.4 A108I/D519A 77.7 121CL/D519A 60.7 A108I/D519V 69.5 121CL/D519V 79.5 A108I/E665A 98.8 121CL/E665A 0 A108I/E665V 83.8 121CL/E665V 22.2 A108I/E1984A 43.4 121CL/E1984A 63.4 A108I/E1984V 94.1 121CL/E1984V 90.3 A108I/D519V/E665V 76.5 Activity was measured by FXa generation assay and expressed as relative values compared to WT activity. The single letter code is used to designate amino acid residues: I (Ile), E (Glu), D (Asp), A (Ala) and V (Val). The variant 121CL represents R121C/L2302C.

[0179] Thrombin generation assays were performed as previously described (Wakabayashi et al., "Combining Mutations of Charged Residues at the A2 Domain Interface Enhances Factor VIII Stability over Single Point Mutations," J. Thromb. Haemost. 7:438-444 (2009), which is hereby incorporated by reference in its entirety) to determine the effects of combining the Ala108Ile mutation with an A2 domain interface mutation. For this analysis, the Asp519Val/Glu665Val double mutation was employed. Results in FIG. 9 compare WT factor VIII with Ala108Ile, Asp519Val/Glu665Val and the combined Ala108Ile/Asp519Val/Glu665Val variants. Both the Ala108Ile and the Asp519Val/Glu665Val variants showed improved thrombin generation parameter values compared with WT (see Table 3 below). In addition, the combined Ala108Ile/Asp519Val/Glu665Val mutation yielded somewhat greater thrombin peak values and endogenous thrombin potential ("ETP", which is the area under the curve and represents total thrombin generated) than either individual variant. This indicates a positive effect in combining these mutations.

TABLE-US-00005 TABLE 3 Thrombin Generation Assay Parameter Values Latent Time Peak Time Peak Value ETP FVIII variants (min) (min) (nM) (nM min) WT 7.59 .+-. 0.09 (1.00) 16.1 .+-. 0.22 (1.00) 88.2 .+-. 6.60 (1.00) 1217 .+-. 34 (1.00) A108I 7.09 .+-. 0.41 (0.93) 12.7 .+-. 0.56 (0.79) 175.3 .+-. 17.0 (1.98) 1831 .+-. 183 (1.50) D519V/E665V 8.19 .+-. 0.18 (1.07) 13.4 .+-. 0.16 (0.83) 203.4 .+-. 18.2 (2.30) 1939 .+-. 251 (1.59) A108I/D519V/E665V 7.05 .+-. 0.27 (0.92) 11.9 .+-. 0.31 (0.73) 222.8 .+-. 10.7 (2.52) 1990 .+-. 122 (1.64) Thrombin generation assays in the presence of 0.5 nM factor VIII proteins, 0.5 pM rTF, and 4 .mu.M PSPCPE vesicles were performed and parameter values were calculated. Data represents the average values of triplicate samples. Values in parentheses are relative to the WT value. The single letter code is used to designate amino acid residues: I (Ile), E (Glu), D (Asp), A (Ala) and V (Val).

[0180] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Sequence CWU 1

1

3416999DNAHuman 1gccaccagaa gatactacct gggtgcagtg gaactgtcat gggactatat gcaaagtgat 60ctcggtgagc tgcctgtgga cgcaagattt cctcctagag tgccaaaatc ttttccattc 120aacacctcag tcgtgtacaa aaagactctg tttgtagaat tcacggatca ccttttcaac 180atcgctaagc caaggccacc ctggatgggt ctgctaggtc ctaccatcca ggctgaggtt 240tatgatacag tggtcattac acttaagaac atggcttccc atcctgtcag tcttcatgct 300gttggtgtat cctactggaa agcttctgag ggagctgaat atgatgatca gaccagtcaa 360agggagaaag aagatgataa agtcttccct ggtggaagcc atacatatgt ctggcaggtc 420ctgaaagaga atggtccaat ggcctctgac ccactgtgcc ttacctactc atatctttct 480catgtggacc tggtaaaaga cttgaattca ggcctcattg gagccctact agtatgtaga 540gaagggagtc tggccaagga aaagacacag accttgcaca aatttatact actttttgct 600gtatttgatg aagggaaaag ttggcactca gaaacaaaga actccttgat gcaggatagg 660gatgctgcat ctgctcgggc ctggcctaaa atgcacacag tcaatggtta tgtaaacagg 720tctctgccag gtctgattgg atgccacagg aaatcagtct attggcatgt gattggaatg 780ggcaccactc ctgaagtgca ctcaatattc ctcgaaggtc acacatttct tgtgaggaac 840catcgccagg cgtccttgga aatctcgcca ataactttcc ttactgctca aacactcttg 900atggaccttg gacagtttct actgttttgt catatctctt cccaccaaca tgatggcatg 960gaagcttatg tcaaagtaga cagctgtcca gaggaacccc aactacgaat gaaaaataat 1020gaagaagcgg aagactatga tgatgatctt actgattctg aaatggatgt ggtcaggttt 1080gatgatgaca actctccttc ctttatccaa attcgctcag ttgccaagaa gcatcctaaa 1140acttgggtac attacattgc tgctgaagag gaggactggg actatgctcc cttagtcctc 1200gcccccgatg acagaagtta taaaagtcaa tatttgaaca atggccctca gcggattggt 1260aggaagtaca aaaaagtccg atttatggca tacacagatg aaacctttaa gactcgtgaa 1320gctattcagc atgaatcagg aatcttggga cctttacttt atggggaagt tggagacaca 1380ctgttgatta tatttaagaa tcaagcaagc agaccatata acatctaccc tcacggaatc 1440actgatgtcc gtcctttgta ttcaaggaga ttaccaaaag gtgtaaaaca tttgaaggat 1500tttccaattc tgccaggaga aatattcaaa tataaatgga cagtgactgt agaagatggg 1560ccaactaaat cagatcctcg gtgcctgacc cgctattact ctagtttcgt taatatggag 1620agagatctag cttcaggact cattggccct ctcctcatct gctacaaaga atctgtagat 1680caaagaggaa accagataat gtcagacaag 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gcctccaatt aagattaaat 2580gagaaactgg ggacaactgc agcaacagag ttgaagaaac ttgatttcaa agtttctagt 2640acatcaaata atctgatttc aacaattcca tcagacaatt tggcagcagg tactgataat 2700acaagttcct taggaccccc aagtatgcca gttcattatg atagtcaatt agataccact 2760ctatttggca aaaagtcatc tccccttact gagtctggtg gacctctgag cttgagtgaa 2820gaaaataatg attcaaagtt gttagaatca ggtttaatga atagccaaga aagttcatgg 2880ggaaaaaatg tatcgtcaac agagagtggt aggttattta aagggaaaag agctcatgga 2940cctgctttgt tgactaaaga taatgcctta ttcaaagtta gcatctcttt gttaaagaca 3000aacaaaactt ccaataattc agcaactaat agaaagactc acattgatgg cccatcatta 3060ttaattgaga atagtccatc agtctggcaa aatatattag aaagtgacac tgagtttaaa 3120aaagtgacac ctttgattca tgacagaatg cttatggaca aaaatgctac agctttgagg 3180ctaaatcata tgtcaaataa aactacttca tcaaaaaaca tggaaatggt ccaacagaaa 3240aaagagggcc ccattccacc agatgcacaa aatccagata tgtcgttctt taagatgcta 3300ttcttgccag aatcagcaag gtggatacaa aggactcatg gaaagaactc tctgaactct 3360gggcaaggcc ccagtccaaa gcaattagta tccttaggac cagaaaaatc tgtggaaggt 3420cagaatttct tgtctgagaa aaacaaagtg gtagtaggaa agggtgaatt tacaaaggac 3480gtaggactca aagagatggt ttttccaagc agcagaaacc tatttcttac taacttggat 3540aatttacatg aaaataatac acacaatcaa gaaaaaaaaa ttcaggaaga aatagaaaag 3600aaggaaacat taatccaaga gaatgtagtt ttgcctcaga tacatacagt gactggcact 3660aagaatttca tgaagaacct tttcttactg agcactaggc aaaatgtaga aggttcatat 3720gacggggcat atgctccagt acttcaagat tttaggtcat taaatgattc aacaaataga 3780acaaagaaac acacagctca tttctcaaaa aaaggggagg aagaaaactt ggaaggcttg 3840ggaaatcaaa ccaagcaaat tgtagagaaa tatgcatgca ccacaaggat atctcctaat 3900acaagccagc agaattttgt cacgcaacgt agtaagagag ctttgaaaca attcagactc 3960ccactagaag aaacagaact tgaaaaaagg ataattgtgg atgacacctc aacccagtgg 4020tccaaaaaca tgaaacattt gaccccgagc accctcacac agatagacta caatgagaag 4080gagaaagggg ccattactca gtctccctta tcagattgcc ttacgaggag tcatagcatc 4140cctcaagcaa atagatctcc attacccatt gcaaaggtat catcatttcc atctattaga 4200cctatatatc tgaccagggt cctattccaa gacaactctt ctcatcttcc agcagcatct 4260tatagaaaga aagattctgg ggtccaagaa agcagtcatt tcttacaagg agccaaaaaa 4320aataaccttt ctttagccat tctaaccttg gagatgactg gtgatcaaag agaggttggc 4380tccctgggga caagtgccac aaattcagtc acatacaaga aagttgagaa cactgttctc 4440ccgaaaccag acttgcccaa aacatctggc aaagttgaat tgcttccaaa agttcacatt 4500tatcagaagg acctattccc tacggaaact agcaatgggt ctcctggcca tctggatctc 4560gtggaaggga gccttcttca gggaacagag ggagcgatta agtggaatga agcaaacaga 4620cctggaaaag ttccctttct gagagtagca acagaaagct ctgcaaagac tccctccaag 4680ctattggatc ctcttgcttg ggataaccac tatggtactc agataccaaa agaagagtgg 4740aaatcccaag agaagtcacc agaaaaaaca gcttttaaga aaaaggatac cattttgtcc 4800ctgaacgctt gtgaaagcaa tcatgcaata gcagcaataa atgagggaca aaataagccc 4860gaaatagaag tcacctgggc aaagcaaggt aggactgaaa ggctgtgctc tcaaaaccca 4920ccagtcttga aacgccatca acgggaaata actcgtacta ctcttcagtc agatcaagag 4980gaaattgact atgatgatac catatcagtt gaaatgaaga aggaagattt tgacatttat 5040gatgaggatg aaaatcagag cccccgcagc tttcaaaaga aaacacgaca ctattttatt 5100gctgcagtgg agaggctctg ggattatggg atgagtagct ccccacatgt tctaagaaac 5160agggctcaga gtggcagtgt ccctcagttc aagaaagttg ttttccagga atttactgat 5220ggctccttta ctcagccctt ataccgtgga gaactaaatg aacatttggg actcctgggg 5280ccatatataa gagcagaagt tgaagataat atcatggtaa ctttcagaaa tcaggcctct 5340cgtccctatt ccttctattc tagccttatt tcttatgagg aagatcagag gcaaggagca 5400gaacctagaa aaaactttgt caagcctaat gaaaccaaaa cttacttttg gaaagtgcaa 5460catcatatgg cacccactaa agatgagttt gactgcaaag cctgggctta tttctctgat 5520gttgacctgg aaaaagatgt gcactcaggc ctgattggac cccttctggt ctgccacact 5580aacacactga accctgctca tgggagacaa gtgacagtac aggaatttgc tctgtttttc 5640accatctttg atgagaccaa aagctggtac ttcactgaaa atatggaaag aaactgcagg 5700gctccctgca atatccagat ggaagatccc acttttaaag agaattatcg cttccatgca 5760atcaatggct acataatgga tacactacct ggcttagtaa tggctcagga tcaaaggatt 5820cgatggtatc tgctcagcat gggcagcaat gaaaacatcc attctattca tttcagtgga 5880catgtgttca ctgtacgaaa aaaagaggag tataaaatgg cactgtacaa tctctatcca 5940ggtgtttttg agacagtgga aatgttacca tccaaagctg gaatttggcg ggtggaatgc 6000cttattggcg agcatctaca tgctgggatg agcacacttt ttctggtgta cagcaataag 6060tgtcagactc ccctgggaat ggcttctgga cacattagag attttcagat tacagcttca 6120ggacaatatg gacagtgggc cccaaagctg gccagacttc attattccgg atcaatcaat 6180gcctggagca ccaaggagcc cttttcttgg atcaaggtgg atctgttggc accaatgatt 6240attcacggca tcaagaccca gggtgcccgt cagaagttct ccagcctcta catctctcag 6300tttatcatca tgtatagtct tgatgggaag aagtggcaga cttatcgagg aaattccact 6360ggaaccttaa tggtcttctt tggcaatgtg gattcatctg ggataaaaca caatattttt 6420aaccctccaa ttattgctcg atacatccgt ttgcacccaa ctcattatag cattcgcagc 6480actcttcgca tggagttgat gggctgtgat ttaaatagtt gcagcatgcc attgggaatg 6540gagagtaaag caatatcaga tgcacagatt actgcttcat cctactttac caatatgttt 6600gccacctggt ctccttcaaa agctcgactt cacctccaag ggaggagtaa tgcctggaga 6660cctcaggtga ataatccaaa agagtggctg caagtggact tccagaagac aatgaaagtc 6720acaggagtaa ctactcaggg agtaaaatct ctgcttacca gcatgtatgt gaaggagttc 6780ctcatctcca gcagtcaaga tggccatcag tggactctct tttttcagaa tggcaaagta 6840aaggtttttc agggaaatca agactccttc acacctgtgg tgaactctct agacccaccg 6900ttactgactc gctaccttcg aattcacccc cagagttggg tgcaccagat tgccctgagg 6960atggaggttc tgggctgcga ggcacaggac ctctactga 699922332PRTHuman 2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830 Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950 955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065 Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080 Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095 Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125 Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140 Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155 Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170 Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185 Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200 Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215

Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230 Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260 His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320 Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335 Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350 Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365 Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380 Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395 Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410 Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425 Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440 Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455 Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470 Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515 Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545 Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560 Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575 Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590 Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605 Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620 Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635 Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650 Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665 Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680 Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695 Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710 Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770 Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815 Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830 Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1835 1840 1845 Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1850 1855 1860 Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875 Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890 Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905 Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915 1920 Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925 1930 1935 Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950 His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1970 1975 1980 Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995 Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010 Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025 Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035 2040 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 2045 2050 2055 Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070 Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 2075 2080 2085 Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090 2095 2100 Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115 Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130 Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145 Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160 Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 2170 2175 Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2180 2185 2190 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205 Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 2210 2215 2220 Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235 Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250 Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265 His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290 2295 Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310 Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315 2320 2325 Gln Asp Leu Tyr 2330 341PRTPorcine 3Ala Val Gly Val Ser Phe Trp Lys Ser Ser Glu Gly Ala Glu Tyr Glu 1 5 10 15 Asp His Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val Leu Pro Gly 20 25 30 Lys Ser Gln Thr Tyr Val Trp Gln Val 35 40 441PRTCanine 4Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Glu 1 5 10 15 Asp Gln Thr Ser Gln Lys Glu Lys Glu Asp Asp Asn Val Ile Pro Gly 20 25 30 Glu Ser His Thr Tyr Val Trp Gln Val 35 40 541PRTMouse 5Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Asp Glu Tyr Glu 1 5 10 15 Asp Gln Thr Ser Gln Met Glu Lys Glu Asp Asp Lys Val Phe Pro Gly 20 25 30 Glu Ser His Thr Tyr Val Trp Gln Val 35 40 641PRTRabbit 6Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp 1 5 10 15 Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Ile Phe Pro Gly 20 25 30 Glu Ser His Thr Tyr Val Trp Gln Val 35 40 741PRTBat 7Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Glu 1 5 10 15 Asp Glu Thr Ser Lys Thr Glu Lys Glu Asp Asp Lys Val Ile Pro Gly 20 25 30 Glu Ser His Thr Tyr Val Trp His Val 35 40 841PRTRat 8Ala Val Gly Met Ser Phe Trp Lys Ala Ser Glu Gly Ala Ala Tyr Asp 1 5 10 15 Asp His Ser Ser Pro Ala Glu Lys Asp Asp Asp Lys Val Leu Pro Gly 20 25 30 Glu Ser His Thr Tyr Ala Trp Gln Val 35 40 941PRTSheep 9Ala Ile Gly Val Ser Tyr Trp Lys Ser Ser Glu Gly Ala Ala Tyr Lys 1 5 10 15 Asp Glu Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val Ile Pro Gly 20 25 30 Lys Ser His Thr Tyr Val Trp His Ile 35 40 1041PRTArtificialConsensus 10Ala Xaa Gly Xaa Ser Xaa Trp Lys Xaa Ser Glu Gly Xaa Xaa Tyr Xaa 1 5 10 15 Asp Xaa Xaa Ser Xaa Xaa Glu Lys Xaa Asp Asp Xaa Xaa Xaa Pro Gly 20 25 30 Xaa Ser Xaa Thr Tyr Xaa Trp Xaa Xaa 35 40 11102PRTPorcine 11Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln 1 5 10 15 Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val 20 25 30 Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly 35 40 45 His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val 50 55 60 Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro 65 70 75 80 Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys 85 90 95 Glu Ala Gln Asp Leu Tyr 100 12102PRTCanine 12Gln Val Asp Phe Arg Lys Thr Met Lys Val Thr Gly Ile Thr Thr Gln 1 5 10 15 Gly Val Lys Ser Leu Leu Ile Ser Met Tyr Val Lys Glu Phe Leu Ile 20 25 30 Ser Ser Ser Gln Asp Gly His Asn Trp Thr Leu Phe Leu Gln Asn Gly 35 40 45 Lys Val Lys Val Phe Gln Gly Asn Arg Asp Ser Ser Thr Pro Val Arg 50 55 60 Asn Arg Leu Glu Pro Pro Leu Val Ala Arg Tyr Val Arg Leu His Pro 65 70 75 80 Gln Ser Trp Ala His His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys 85 90 95 Asp Thr Gln Gln Pro Ala 100 13102PRTMouse 13Gln Val Asp Leu Gln Lys Thr Met Lys Val Thr Gly Ile Ile Thr Gln 1 5 10 15 Gly Val Lys Ser Leu Phe Thr Ser Met Phe Val Lys Glu Phe Leu Ile 20 25 30 Ser Ser Ser Gln Asp Gly His His Trp Thr Gln Ile Leu Tyr Asn Gly 35 40 45 Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Met Met 50 55 60 Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro 65 70 75 80 Gln Ile Trp Glu His Gln Ile Ala Leu Arg Leu Glu Ile Leu Gly Cys 85 90 95 Glu Ala Gln Gln Gln Tyr 100 14102PRTRabbit 14Gln Val Asp Leu Arg Lys Thr Met Lys Val Thr Gly Ile Thr Thr Gln 1 5 10 15 Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Thr Glu Phe Leu Ile 20 25 30 Ser Ser Ser Gln Asp Gly His His Trp Thr Leu Val Leu Gln Lys Gly 35 40 45 Lys Leu Lys Val Phe Lys Gly Asn Gln Asp Ser Phe Thr Pro Val Leu 50 55 60 Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro 65 70 75 80 Lys Ser Trp Val His Gln Ile Ala Leu Arg Leu Glu Val Leu Gly Cys 85 90 95 Glu Ala Gln Gln Leu Tyr 100 15102PRTBat 15Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Ile Thr Thr Gln 1 5 10 15 Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile 20 25 30 Ser Ser Ser Gln Asp Gly His Asn Trp Thr Pro Phe Leu Gln Asn Gly 35 40 45 Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Leu 50 55 60 Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro 65 70 75 80 Gln Ser Trp Val His Gln Ile Ala Leu Arg Leu Glu Val Leu Gly Cys 85 90 95 Glu Ala Gln Gln Leu Tyr 100 16102PRTRat 16Gln Val Asp Leu Gln Arg Thr Val Lys Val Thr Gly Val Val Thr Gln 1 5 10 15 Gly Ala Arg Ser Leu Leu Thr Ala Met Phe Val Lys Lys Phe Leu Val 20 25 30 Ser Thr Ser Gln Asp Gly Arg His Trp Thr His Val Leu Gln Asp Gly 35 40 45 Lys Val Lys Val Phe Gln Gly Asn Arg Asp Ala Ser Thr Pro Met Val 50 55 60 Asn Ser Leu His Pro Pro Arg Phe Thr Arg Tyr Leu Arg Ile His Pro 65 70 75 80 Gln Val Trp Glu Arg Gln Ile Ala Leu Arg Leu Glu Ile Leu Gly Cys 85 90 95 Glu Ala Gln Gln Leu Asp 100 17102PRTSheep 17Gln Val Asp Phe Gln Lys Thr Met Arg Val Thr Gly Ile Thr Thr Gln 1 5 10 15 Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile 20 25 30 Ser Ser Ser Gln Glu Gly His Asn Trp Thr Pro Phe Leu Gln Asn Gly 35 40 45 Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 50 55 60 Asn Thr Leu Asp Pro Pro Leu Phe Thr Arg Phe Leu Arg Ile His Pro 65 70 75 80 Gln Ser Trp Val His His Ile Ala Leu Arg Leu Glu Phe Trp Gly Cys 85 90 95 Glu Ala Gln Gln Gln Tyr 100 18102PRTArtificialConsensus 18Gln Val Asp Xaa Xaa Lys Thr Xaa Xaa Val Thr Gly Xaa Xaa Thr Gln 1 5 10 15 Gly Xaa Xaa Ser Leu Xaa Xaa Xaa Met Xaa Val Xaa Xaa Phe Leu Xaa 20 25 30 Ser Xaa Ser Gln Xaa Gly Xaa Xaa Trp Thr Xaa Xaa Xaa Xaa Xaa Gly 35 40 45 Xaa Xaa Lys Val Phe Xaa Gly Asn Xaa Asp Xaa Xaa Thr Pro Xaa Xaa 50

55 60 Asn Xaa Leu Xaa Pro Pro Xaa Xaa Xaa Arg Xaa Xaa Arg Xaa His Pro 65 70 75 80 Xaa Xaa Trp Xaa Xaa Xaa Ile Ala Leu Arg Xaa Glu Xaa Xaa Gly Cys 85 90 95 Xaa Xaa Gln Xaa Xaa Xaa 100 193PRTArtificialMutant 19Lys Xaa Ser 1 204PRTArtificialMutant 20Ser Xaa Xaa Xaa 1 214PRTArtificialMutant 21Thr Tyr Xaa Trp 1 223PRTArtificialMutant 22Xaa Val Thr 1 234PRTArtificialMutant 23Pro Pro Xaa Xaa 1 244PRTArtificialMutant 24Xaa Xaa Gln Xaa 1 254PRTArtificialMutant 25Ser Xaa Xaa Glu 1 267PRTArtificialMutant 26Val Asp Gln Arg Gly Asn Gln 1 5 277PRTArtificialMutant 27Val Asp Gln Arg Met Lys Asn 1 5 287PRTArtificialMutant 28Pro Gln Leu Arg Gly Asn Gln 1 5 297PRTArtificialMutant 29Pro Asp Leu Arg Met Lys Asn 1 5 307PRTArtificialMutant 30Pro Gln Gln Arg Met Lys Asn 1 5 317PRTArtificialMutant 31Pro Gln Arg Arg Met Lys Asn 1 5 327PRTArtificialMutant 32Pro Gln Leu Arg Gly Lys Asn 1 5 337PRTArtificialMutant 33Pro Gln Leu Arg Met Ile Asn 1 5 347PRTArtificialMutant 34Pro Gln Leu Arg Met Asn Asn 1 5

Claims

1. A recombinant factor VIII comprising substitution of two or more amino acid residues with a Cysteine residue at an interface of A1 and C2 domains of the recombinant factor VIII to afford a disulfide bond between the A1 and C2 domains, and one or more of the following: (i) a substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at a C2 domain interface of the A1 domain; (ii) a substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at an A1 domain interface of the C2 domain; and (iii) a substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at an interface of A1 and A2 domains or an interface of A2 and A3 domains of the recombinant factor VIII.

2. The recombinant factor VIII according to claim 1, wherein the A1 domain having the Cysteine substitution comprises the sequence SXXX (SEQ ID NO: 20) wherein X at the third position is Cysteine, and the C2 domain having the Cysteine substitution comprises the sequence PPXX (SEQ ID NO: 23) wherein X at the fourth position is Cysteine.

3. The recombinant factor VIII according to claim 1, wherein the two or more amino acid residues substituted with a Cysteine residue comprise an Arg.fwdarw.Cys substitution at a position corresponding to residue 121 of SEQ ID NO: 2 and an Leu.fwdarw.Cys substitution at a position corresponding to residue 2302 of SEQ ID NO: 2.

4. The recombinant factor VIII according to claim 1, wherein the substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at a C2 domain interface of the A1 domain is present.

5. The recombinant factor VIII according to claim 4, wherein the C2 domain interface of the A1 domain comprises the amino acid sequence of: KXS (SEQ ID NO: 19), where the substitution is at the second position and X is Valine, Isoleucine, or Leucine; or TYXW (SEQ ID NO: 21), where the substitution is at the third position and X is Leucine, Isoleucine, or Valine.

6. The recombinant factor VIII according to claim 4, wherein the substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at the C2 domain interface of the A1 domain comprises an Ala.fwdarw.Ile substitution at a position corresponding to residue 108 of SEQ ID NO: 2.

7. The recombinant factor VIII according to claim 1, wherein the substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at an A1 domain interface of the C2 domain is present.

8. The recombinant factor VIII according to claim 1, wherein the substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at an interface of A1 and A2 domains or an interface of A2 and A3 domains of the recombinant factor VIII is present.

9. The recombinant factor VIII according to claim 8, wherein the charged amino acid residue is either Glu or Asp, and the hydrophobic amino acid substitution is one of Ala, Val, Ile, Leu, Met, Phe, or Trp.

10. The recombinant factor VIII according to claim 8 wherein the substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at the interface of A1 and A2 domains or the interface of A2 and A3 domains of the recombinant factor VIII comprises substitution of a Glu287 residue of wild type factor VIII, substitution of an Asp302 residue of wild type factor VIII, substitution of an Asp519 residue of wild type factor VIII, substitution of a Glu665 residue of wild type factor VIII, substitution of a Glu1984 residue of wildtype factor VIII, or a combination thereof.

11. The recombinant factor VIII according to claim 8, wherein the substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at an interface of A1 and A2 domains or an interface of A2 and A3 domains of the recombinant factor VIII comprises: (i) an Asp.fwdarw.Ala substitution at a position corresponding to residue 302 of SEQ ID NO: 2; (ii) a Glu.fwdarw.Ala substitution at a position corresponding to residue 287 of SEQ ID NO: 2; (iii) a Glu.fwdarw.Ala or Glu.fwdarw.Val substitution at a position corresponding to residue 665 of SEQ ID NO: 2; (iv) an Asp.fwdarw.Ala or Asp.fwdarw.Val substitution at a position corresponding to residue 519 of SEQ ID NO: 2; (v) a Glu.fwdarw.Ala or Glu.fwdarw.Val substitution at a position corresponding to residue 1984 of SEQ ID NO: 2; or (vi) combinations of any two or more of the substitutions (i)-(v).

12. The recombinant factor VIII according to claim 1, wherein the recombinant factor VIII further comprises one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VIII for one or both of factor IXa and factor X; (ii) modified sites that enhance secretion in culture; (iii) modified serum protein binding sites that enhance the circulating half-life thereof; (iv) at least one glycosylation recognition sequence that is effective in decreasing antigenicity and/or immunogenicity thereof; (v) a modified A1 domain calcium-binding site that improves specific activity of the recombinant factor VIIIa; and (vi) a modified activated protein C-cleavage site.

13. A pharmaceutical composition comprising the recombinant factor VIII according to claim 1 and a pharmaceutically acceptable carrier.

14. A method of treating an animal for hemophilia A, said method comprising: administering to an animal exhibiting hemophilia A an effective amount of the recombinant factor VIII of claim 1, whereby the animal exhibits effective blood clotting following vascular injury.

15. An isolated nucleic acid molecule encoding the recombinant factor VIII according to claim 1.

16. The isolated nucleic acid molecule according to claim 15, wherein the nucleic acid is DNA.

17. A recombinant expression system comprising a DNA molecule of claim 16.

18. A recombinant host cell comprising the nucleic acid molecule according to claim 15.

19. An implantable device comprising a plurality of the recombinant host cells according to claim 18 contained within the device.

20. A method of treating an animal for hemophilia A, said method comprising: administering to an animal exhibiting hemophilia A a recombinant host cell of claim 18, whereby the recombinant host cell expresses the recombinant factor VIII and the animal exhibits effective blood clotting following vascular injury.