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Publication numberWO2009058446 A1
Publication typeApplication
Application numberPCT/US2008/071170
Publication dateMay 7, 2009
Filing dateJul 25, 2008
Priority dateNov 1, 2007
Also published asCA2703948A1, CN101965409A, EP2209908A1, EP2209908A4, EP2711436A1, US8183345, US8338571, US20090118184, US20090118185, US20130085110, US20140056861
Publication numberPCT/2008/71170, PCT/US/2008/071170, PCT/US/2008/71170, PCT/US/8/071170, PCT/US/8/71170, PCT/US2008/071170, PCT/US2008/71170, PCT/US2008071170, PCT/US200871170, PCT/US8/071170, PCT/US8/71170, PCT/US8071170, PCT/US871170, WO 2009/058446 A1, WO 2009058446 A1, WO 2009058446A1, WO-A1-2009058446, WO2009/058446A1, WO2009058446 A1, WO2009058446A1
InventorsPhilip J Fay, Hironao Wakabayashi
ApplicantUniv Rochester, Philip J Fay, Hironao Wakabayashi
Export CitationBiBTeX, EndNote, RefMan
External Links: Patentscope, Espacenet
Recombinant factor viii having increased stability
WO 2009058446 A1
Abstract
The present invention relates to a recombinant factor VIII that includes one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa. Methods of making and using the recombinant factor VlIl, and pharmaceutical compositions containing the same are also disclosed. The present invention further relates to an isolated nucleic acid molecule that encodes the recombinant factor VIII, as well as DNA expression systems and host cells containing the isolated nucleic acid molecule.
Claims  (OCR text may contain errors)
WHAT IS CLAIMED:
1. A recombinant factor VIlI comprising one or more mutations that result in enhanced stability of both factor VlIl and factor VIIIa, wherein the one or more mutations comprise substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at either or both of the A1A2 or A2A3 domain interfaces.
2. The recombinant factor VIII according to claim 1 , wherein the charged amino acid residue is either Glu or Asp. and the hydrophobic amino acid substitution is one of Ala, VaI, Ile, Leu, Met. Phe, or Trp.
3. The recombinant factor VIIl according to claim 1 , wherein the one or more mutations comprises a substitution of a Glu287 residue of wildtype factor VIII, a substitution of an Asp302 residue of wildtype factor VIII, a substitution of an Asp519 residue of wildtype factor VIII, a substitution of a Glu665 residue of wildlype factor VHl. a substitution of a Glu1984 residue of wildlype factor VlII. or combinations thereof.
4. The recombinant factor VIlI according to claim 3. wherein the substitution of the Asp302 residue is D302A.
5. The recombinant factor VIII according to claim 3, wherein the substitution of the Glu287 residue is H287A.
6. The recombinant factor VIlI according to claim 3, wherein the substitution of the Glυ665 residue is E665A or E665V.
7. The recombinant factor VIIl according to claim 3, wherein the substitution of the Asp519 residue is D519A or D519V.
8. The recombinant factor VIlI according to claim 3, wherein the substitution of the Glu1984 residue is E1984A or E1984V.
9. The recombinant factor VIII according to claim 3, wherein the one or more mutations comprise two or more substitutions selected from the G)u665 residue, the Asp519 residue, and the Glu 1984 residue.
10. The recombinant factor VlII according to claim 9, wherein the two or more substitutions include D519VE665V, D519AE665V, D519VE1984A, E665VE1984A, E665AE1984V, D519AE665VE1984A, D519VE665VE1984A, or D519VE665VE1984V.
11. The recombinant factor VIII according to claim 1 , wherein the recombinant factor VIII consists of domains A1, A2, A3, C1, and C2, or portions thereof.
12. The recombinant factor VIlI according to claim 1 1 wherein domains A1 and A2 are present on a heavy chain and domains A3, Cl . and C2 are present on a light chain.
13. The recombinant factor VIII according to claim 1 , wherein the recombinant factor VIII comprises one or more domains, or portions thereof, from human factor VIlI and one or more domains, or portions thereof, from a non-human mammalian factor VIlI.
14. The recombinant factor VIIl according to claim 1, wherein the recombinant factor VIII is substantially pure.
15. The recombinant factor VIIl according to claim 1 wherein the recombinant factor VIlI further comprises one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VlII 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; and (v) a modified calcium- binding site that improves specific activity of the recombinant factor VIIIa.
16. The recombinant factor VIlI according to claim 1 further comprising a substitution of a GluI 13 residue of wildtype factor VlU, which substitution of the GluI 13 residue enhances activity of activated factor VIIIa.
17. The recombinant factor VIlI according to claim 16, wherein the one or more mutations comprises a substitution of a Glu287 residue of wildtype factor VIII, a substitution of an Asp302 residue of wildtype factor Viii, a substitution of an Asp519 residue of wildtype factor VIII, a substitution of a Glu665 residue of wildtype factor VIlI, a substitution of a Glu 1984 residue of wildtype factor VIII, or combinations thereof.
18. The recombinant factor VIlI according to claim 17, wherein the substitutions comprise E113AD519A. E1 13AD519V, E113AE665A, E113AE665V, or E113AE 1984V.
19. A pharmaceutical composition comprising the recombinant factor VIII according to claim 1.
20. The pharmaceutical composition according to claim 19 further comprising a stabilizer.
21. The pharmaceutical composition according to claim 19 further comprising a delivery vehicle.
22. The pharmaceutical composition according to claim 19 further comprising a pharmaceutically acceptable carrier.
23. An isolated nucleic acid molecule encoding a recombinant factor VHl according to claim 1.
24. The isolated nucleic acid molecule according to claim 23, wherein the one or more mutations comprises a substitution of a G)u287 residue of wildtype factor VIlI, a substitution of an Asp302 residue of wildtype factor VIII, a substitution of an Asp519 residue of wildtype factor VIlI, a substitution of a Glu665 residue of wildtype factor VIII, a substitution of a Glu 1984 residue of wildtype factor VIlI, or combinations thereof.
25. The isolated nucleic acid molecule according to claim 24, wherein the substitution of the Asp302 residue is D302A.
26. The isolated nucleic acid molecule according to claim 24, wherein the substitution of the Glu287 residue is E287A.
27. The isolated nucleic acid molecule according to claim 24, wherein the substitution of the Glu665 residue is E665A or H665V.
28. The isolated nucleic acid molecule according to claim 24, wherein the substitution of the Asp519 residue is D519A or D519V.
29. The isolated nucleic acid molecule according to claim 24, wherein the substitution of the Glu 1984 residue is E 1984A or El 984V.
30. The isolated nucleic acid molecule according to claim 24, wherein the one or more mutations comprise two or more substitutions selected from the Glu665 residue, the Asp519 residue, and the Glu1984 residue.
31. The isolated nucleic acid molecule according to claim 30, wherein the two or more substitutions include D519VE665V, D519AE665W D519VE1984A, E665VE1984A, E665AE1984V, D519AE665VE 1984A, D5I 9VE665VE1984A. or D519VE665 VEl 984V.
32. The isolated nucleic acid molecule according to claim 23, wherein the recombinant factor VlIl 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 scrum 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; and (v) a modified calcium-binding site that improves activity of the recombinant factor VIIIa.
33. The isolated nucleic acid molecule according to claim 23, wherein the nucleic acid is RNA. 34. The isolated nucleic acid molecule according to claim 23. wherein the nucleic acid is DNA.
35. A recombinant DNA expression system comprising a DNA molecule according to claim 34.
36. The recombinant DNA expression system according to claim 35, wherein the DNA molecule is in sense orientation relative to a promoter.
37. A host cell comprising a nucleic acid molecule according to claim 23.
38. A host cell comprising the DNA molecule according to claim 34.
39. The host cell according to claim 38. wherein the DNA molecule is in an expression system.
40. The host cell according to claim 38. wherein the host cell is an animal cell, a bacterial cell, an insect cell, a fungal cell, a yeast cell, a plant cell, or an algal cell.
41. A method of making a recombinant factor VIIl comprising: growing a host cell according to claim 38 under conditions whereby the host cell expresses the recombinant factor VllI; and isolating the recombinant factor VIII.
42. The method according to claim 41 , wherein said growing is carried out in vitro in a growth medium.
43. The method according to claim 42, wherein the growth medium comprises von Willebrand Factor.
44. The method according to claim 43, wherein the host cell comprises a transgene encoding von Willebrand Factor. 45. The method according to claim 44, wherein the recombinant factor VIII is secreted into the growth medium, said isolating comprises isolating the recombinant factor VIII from the growth medium.
46. The method according to claim 41 further comprising: disrupting the host cell prior to said isolating, wherein said isolating comprises isolating the recombinant factor VIII from cellular debris.
47. A method of treating an animal for hemophilia A, the method comprising: administering to an animal exhibiting hemophilia A an effective amount of the recombinant factor VIII according to claim 1 , whereby the animal exhibits effective blood clotting following vascular injury.
48. The method according to claim 47. wherein the effective amount comprises between about 10 to about 50 units/kg body weight of the animal.
49. The method according to claim 47 wherein the animal is a mammal.
50. The method according to claim 47 wherein the animal is selected from the group consisting of human, rat, mouse, guinea pig, dog, cat, monkey, chimpanzee, orangutan, cow, horse, sheep, pig. goat, rabbit, and chicken.
51. The method according to claim 47 further comprising periodically repeating said administering.
Description  (OCR text may contain errors)

RECOMBINANT FACTOR VIII HAVING INCREASED STABILITY

[0001] This application claims the priority benefit of U.S. Provisional Patent

Application Serial No. 60/984,518, filed November 1. 2007, and U.S. Provisional Patent Application Serial No. 60/991,304, filed November 30, 2007, each of which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under grant numbers HL 76213 and HL 38199 awarded by the National Institutes of Health. The government has certain rights in the invention.

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 VlII. 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 (-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 VIlI is activated by proteolytic cleavages at the A1 A2, A2B and A3 A3 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] Rcconstitution studies have shown that the factor VIlI 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 VIlI 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 Sυbunits," Arch Biochem Biophys. 262:525-531 (1988); Kaufman et al. "Regulation of Factor VIlI 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 VIlI Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and lnter- 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 1 10-126 in the A1 Domain of Factor VlIl Contain a Ca2+ 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 1 1 1 : 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. Karlicr 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 VUIa 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 Cu2+," Biochim Biophys Acta. 1764: 1094-1 101 (2006); Among 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/A3CIC2 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 VIlI." 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 VIlI Molecules." J Biol Chem. 267:23652-23657 (1992); Fay et al., "Model for the Factor Vllla-dcpcndcnt Decay of the Intrinsic Factor Xase: Role of Subunit Dissociation and Factor IXa- catalyzed Proteolysis/1./ 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 subunil. Several factor VlII 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 Λ Domains of Thrombin-activated Factor Villa." Blood 97:685-691 (2001)) or the A2-A3 domain interface (Hakcos et al., "Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/Two-stagc 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 VIIl 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] Examination of the ceruloplasmin-based homology model for the A domains of factor VIII (Pemberton et al., "A Molecular Model for the Triplicated A Domains of Human Factor VlIl Based on the Crystal Structure of Human

Ceruloplasmin." Blood 89:2413-2421 (1997)) suggests an extended interface between the A2 domain and each of the A1 and A3 domains, with multiple potential contacts contributing to binding interactions. [0008] 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 subυnit by introducing novel covalent bonds between A2 and other factor VHl domains (Pipe et al., "Characterization of a Genetically Engineered Inactivation-resistant Coagulation Factor VIIIa," Proc Natl Acad Sci USA 94: 1 1851 - I l 856 ( 1997); Gale et al., "An Engineered lnterdomain Disulfide Bond Stabilizes Human Blood Coagulation Factor Villa." 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 VIH, 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 VlHa, but in a manner that minimizes the likelihood of promoting prothrombotic conditions. [0009] The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION [0010] A first aspect of the present invention relates to a recombinant factor

VIII that includes one or more mutations that result in enhanced stability of both factor VlI 1 and factor VIIIa.

[0011] Preferably, the one or more mutations constitute a replacement of one or more charged amino acid residues with a hydrophobic amino acid residue at either or both of the A1 A2 or A2A3 domain interfaces. Particularly preferred recombinant factor VlII of the present invention includes a substitution of a Glu287 residue of wildtype factor VIII, a substitution of an Asp302 residue of wildtype factor VIII, a substitution of an Asp519 residue of wildtype factor VIII, a substitution of a Glu665 residue of wildtype factor VIH, a substitution of a Glu1984 residue of wildtype factor VIIl, or combinations thereof.

[0012] Λ second aspect of the present invention relates to a pharmaceutical composition that includes the recombinant factor WlH according to the first aspect of the present invention. [0013] A third aspect of the present invention relates to an isolated nucleic acid molecule encoding a recombinant factor VHl according to the first aspect 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. [0014] A fourth aspect of the present invention relates to a method of making a recombinant factor VIII that includes: growing a host cell according to the third aspect of the present invention under conditions whereby the host celt expresses the recombinant factor VIII; and isolating the recombinant factor VIII. [0015] A fifth 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 VIIl according to the first aspect of the present invention, whereby the animal exhibits effective blood clotting following vascular injury.

[0016] The present invention demonstrates that a number of charged residues at the A1 A2 and A2Λ3 domain interfaces do not participate in hydrogen bonding, but instead may be destabilizing to factor VlII structure and/or may facilitate the dissociation of the A2 subunit following activation of the factor VIII procofactor. Replacement of these charged residues with 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 following the activity of the factor VlI) variants at elevated temperature and time courses for the decay of factor VIIIa activity resulting from A2 subunit dissociation. Results from these studies demonstrated that a number of mutations yielded increased stability parameters consistent with the elimination of destabilizing forces likely due to buried charge at the A2 domain interface. These stabilized variants of factor VIII and activated cofactor VIIIa should afford an improved therapeutic for treatment of hemophilia A.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a graph illustrating the activity of factor VIII mutants relative to WT factor VIlI, as measured by one-stage clotting assay (black bar) and two-stage chromogcnic factor Xa generation assay (grey bar). Activity for WT and mutant factor VIII forms were measured as described in the Examples. Error bars show the values for standard deviation averaged from three separate determinations. [0018] Figures 2A-B illustrate the activity decay of WT and mutant factor

VIII and factor VIIIa, respectively. In Figure 2A, factor VIlI (4 nM) was incubated at 55"C and at the indicated times aliquots were removed and assayed for activity by factor Xa generation assays as described in the Examples. Results are shown for WT (dashed line, open circles), R282A (open triangles), S524A (open squares), N684Λ (open diamonds), R531 A (closed circles), S650A (closed triangles), E287A (closed squares), and D302A (closed diamonds). In Figure 2B, thrombin-activated factor VIIIa (4 nM) in the presence of 40 nM factor IXa was incubated at 23C, aliquots were taken at indicated time points and activity was measured by factor Xa generation assay as described in the Examples. Results are shown for WT (dashed line, open circles), R282A (open triangles), S524A (open squares), Y1792F (open diamonds). N684A (closed circles), Y1786F (closed Iriangles), R531A (closed squares), E287A (closed diamonds), and D302A (grey circles). Results for selected fast decay variants are shown in an expanded scale in the inset to Figure 2B. [0019] Figures 3A-B illustrate SDS-PAGE and Western blot analysis of factor

VIII mutants and WT factor VIII. Figure 3A shows purified WT and mutant factor VtII proteins (0.77 μg) following SDS-PAGE on 8% polyacrylamidc gels that were visualized by GelCode. Figure 3B shows purified WT and mutant factor VIlI proteins (0.34 μg) that were electrophoresed on 8% polyacryl amide gels, transferred to PVDF membranes, and probed by biotinylaled R8B12 antibody. Bands were visualized by chemifluorescence as described in accompanying Examples. WT (lane 1), Glu272Ala (lane 2), Glu272Val (lane 3). Asp519Ala (lane 4). AspS IWal (lane 5), Glu665Ala (lane 6), Glu665Val (lane 7). Glu1984Ala (lane 8), and Glu 1984VaI (lane 9). MW, molecular weight marker: sFVlII, single chain form factor VIII: HC, heavy chain: LC, light chain. An apparent stoichiometry ratios oϊ single chain form to heterodimer of WT and mutant factor VIII forms were 0.96 (WT), 0.64 (Glu272Ala), 0.92 (Glu272Val), 0.74 (Asp519Ala). 0.8 (Asp519Val), 0.64 (Glu665Ala). 0.63 (Glu665Val), 0.91 (Glu1984Ala), and 0.5 (Glu 1984VaI). [0020] Figures 4A-D illustrate specific activity of factor VIII mutants relative to WT factor VlII and thrombin generation assays. Figure 4A shows activity values that were determined using a one-stage clotting assay (grey bar) and two-stage chromogenic factor Xa generation assay (solid bar) as described in the accompanying Examples. Figure 4B -C illustrate thrombogram of factor VIIl proteins. WT (dashed line). Glu272Ala (open squares), Glu272Val (closed squares), Asp519Ala (open circles), Asp519Val (closed circles), Glu665Ala (open triangles), Glu665Val (closed triangles), Glu 1984Ala (open diamonds), and GluI 984 Vai (closed diamonds). Figure 4D illustrates parameter values obtained from thrombin generation assays. Thrombin generation assays were performed as described in the accompanying Examples.

Thrombograms show the average values of triplicated samples. The parameter values were expressed as values (%) relative to WT. The actual values for WT were 7.5 0.5 min (lag time), 13.7 0.3 min (peak time), 157.3 14.7 nM (peak value), 979.8 37.9 nM/min (ETP). Lag time (open bar). Peak time (grey bar). Peak Value (closed bar), and ETP (lined bar) are shown. Error bars show the standard deviation values averaged from three separate determinations.

[0021] Figures 5 A-B illustrate activity decay of WT and mutant factor VlH.

Factor VIIl (4 nM) was incubated at various temperatures (52-60C) and at the indicated times aliquots were removed and assayed for activity by factor Xa generation assays as described in the accompanying Examples. Data were fitted by non-linear least squares regression, and decay rates were obtained. Each point represents the value averaged from three separate determinations. Results are shown for WT (dashed line, cross symbols), Ghi272Aia (open squares), Glu272Val (closed squares). Asp519Ala (open circles), Asp519Val (closed circles), Glu665Ala (open triangles), Glu665Val (closed triangles), Glu 1984Ala (open diamonds). Glu 1984VaI (closed diamonds), and full-length Kogenate factor VIII (grey circles). Figure 5Λ illustrates representative factor VIII decay curves after 55C incubation. Figure 5B illustrates plots of factor VIII decay rate at various temperatures. The inset graph in Figure 5B is an enlargement of the decay results over the temperature range of 52- 55C.

[0022] Figure 6 is a graph illustrating activity decay of factor VlH in plasma at 37C. Factor VIII ( 1 nM) was incubated at 37C in factor VIII deficient plasma and at the indicated times aliquots were removed and assayed for one-stage clotting assays as described in the accompanying Examples. Results are shown for WT (dashed line. cross symbols). Asp519Ala (open circles), Asp519Val (closed circles), Glu665Ala (open triangles). Glu665Val (closed triangles). Glu 1984Ala (open diamonds), and Glu 1984VaI (closed diamonds). Data were fitted by non-linear least squares regression and each point represents the value averaged from three separate determinations.

[0023] Figures 7A--B are graphs illustrating the activity decay of WT and mutant factor VIlIa in the absence or presence of factor IXa, Figure 7 A shows thrombin-activated factor VIIIa (4 nM) that was incubated at 23C. Aliquots were taken at indicated time points and activity was measured by factor Xa generation assay as described in the accompanying Examples. Figure 7B shows activity decay of WT and mutant factor VIIIa in the presence of factor IXa. Factor VIII (4 nM) was activated with thrombin in the presence of 40 nM factor IXa. Aliquots were taken at indicated time points and activity was measured by factor Xa generation assay as described in the accompanying Examples. Results are shown for WT (dashed line, cross symbols), Glu272Ala (open squares), Glu272Val (closed squares), Asp519Ala {open circles), Asp519Val (closed circles), Glu665Ala (open triangles). Glu665Val (closed triangles), Glu 1984Ala (open diamonds), and Glu 1984VaI (closed diamonds). Data were fitted by non-linear least squares regression and each point represents the value averaged from three separate determinations.

[0024] Figure 8 is a graph illustrating the specific activity of factor VIIl double or triple combination mutants having Asp519, Glu665, and/or GluI 984 residues changed to Ala or VaI. Activity values were determined using a one-stage clotting assay (grey bar) and two-stage chromogenic factor Xa generation assay (black bar) as described in the Examples. Error bars show the standard deviation values averaged from three separate determinations.

[0025] Figure 9 is a graph illustrating factor VIII activity decay rates for WT and factor VIII double or triple combination mutants having Asp519, Glu665, and/or Glu 1984 residues changed to Ala or VaI. Factor VIlI activity decay experiments were performed and decay rates were estimated by non-linear least squares regression as described in the Examples. Grey bars show the rates relative to the best single mutants (see Example 5, Figure 5A) and were calculated after division by the rate of best (lowest) value. For example, the rate relative values to the best single mutant of the D519 AE665 A pairing equals the decay rate for D519AE665A divided by the decay rate of D519A. Black bars show the actual decay rate parameter values represented * 10. [0026] Figure 10 is a graph illustrating factor VIIIa activity decay rate of WT and factor VlIl double or triple combination mutants having Asp519, Olu665. and Glu1984 residues changed to Ala or VaI. Factor VIIIa activity decay measurements after incubation of 1.5 nM factor VIIIa in the absence of factor IXa were performed and decay rates were estimated by non-linear least squares regression as described in the Examples. Grey bars show the rates relative to best single mutants (see Example 7. Figure 7A), and were calculated as described in the legend to Figure 9. Black bars show the actual decay rate parameter values represented * 10. [0027] Figures 1 1A-B illustrate the results of thrombin generation assay using the combination mutants. Figure 1 1 A shows the thrombogram of factor VIIl proteins. Thrombin generation assays were performed as described in the Examples. Final concentrations of reagents were 0.2 nM (factor VIlI), 0.5 pM (rTF), 4 μM (PSPCPE vesicles), 433 μM (flυorogenic substrate), 13.3 mM CaICb, and 105 nM (thrombin calibrator). The results are shown for WT (dashed line), D519AE665V (open circles), D5 I9VE665V (closed circles), D519VE1984A (open triangles), and

D519VE665VE1984A (closed triangles). Figure 1 1 B shows parameter values obtained from thrombin generation assay. Thrombograms show the average values of triplicated samples. The parameter values were expressed as values (%) relative to WT. The actual values for WT were 8.5 0.4 mtn (lag time), 21.3 0.6 min (peak time), 58.5 15.6 nM (peak value). 883.6 199.8 nM/min (ETP). Lag time (open bar). Peak time (grey bar), Peak Value (closed bar), and ETP (lined bar). Error bars show the standard deviation values averaged from three separate determinations. [0028] Figures 12A-C illustrate the specific activity and activity decay rates for factor VlII and factor VIIIa relative to WT for Ala or VaI mutants at residues Asp519, Glu665, andOr Glu 1984 in combination with Glu 1 13Ala mutation. Figure 12A shows specific activity of the combination mutants versus WT, as determined using a one-stage clotting assay (grey bar) and two-stage chromogenic factor Xa generation assay (black bar) as described in the Examples. Error bars show the standard deviation values averaged from three separate determinations. Figure 12B shows the results of factor VIII activity decay assays at 55C; decay rates were estimated by non-linear least squares regression as described in the Examples. Figure 12C shows the results of factor VIIIa activity decay measurements after incubation of 1.5 nM factor VIIIa in the absence of factor IXa; decay rates were estimated by nonlinear least squares regression as described in the Examples.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention relates to a recombinant factor VIII having one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa.

[0030] The recombinant factor VlIl of the present invention can be prepared by modifying the amino acid sequence of a wild-type factor VIII or a mutant factor VIlI that has otherwise been modified to affect other properties of the factor VIII, such as antigenicity, circulating half-life, protein secretion, affinity for factor IXa and/or factor X, altered factor Vlll-inactivation cleavage sites, immunogcnicity, shelf- life, etc.

[0031] 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 K0I 740 (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. AAC05384 (amino acid) and AF049489 (nucleotide), which are hereby incorporated by reference in their entirety), cats, monkeys, chimpanzees (see, e.g., GenBank Accession Nos. XP 529212 (amino acid) and XM 529212 (nucleotide), which are hereby incorporated by reference in their entirety), orangutans, cows, horses, sheep, pigs (see, e.g., GenBank Accession Nos. NP 999332 (amino acid) and NM_214167 (nucleotide), which are hereby incorporated by reference in their entirety), goats, rabbirs, 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 VlII proteins. Thus, the conservation and homology among mammalian factor VlH proteins is well known. [0032] 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 VllI is synthesized as an approximately 300 kDa single chain protein with internal sequence homology that defines the "domain" sequence NH2-A1-A2-B-A3-C 1 -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 VlIl domains include the following amino acid residues, when the sequences are aligned with the human amino acid sequence (SEQ ID NO: 2): A1, residues Ala|-Arg372

A2. residues Ser3-jj-Arg74o;

B, residues Ser74i-Axgi64; A3, residues Serι<i<>o-Ile2032;

Cl , residues Arg203.w\sn2i7_; and

C2. residues Se^m-Tyrjjm-

[0033] The A3-C 1 -C2 sequence includes residues Seriao-Tyr-vMz- The remaining sequence, residues Glui6--Argi6g(>, 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.

[0034] The gene encoding the wild-type human factor VIII has a nucleotide sequence of SEQ ID NO: 1 , as follows: gccaccagaagatactacctgggtgcagtggaactgtcatgggactatatgcaaagt gatctcggtgagctgcctgtggacgcaagatttcctcctagagεgccaaaatctttt ccattcaacacctcagtcgtgtacaaaaagactctgtttgtagaattcacggatcac ctcttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatc caggctgaggcttatgacacagtggtcattacacccaagaacatggcttcccatcct gccagtcttcatgctgttggtgtatcctactggaaagcttccgagggagctgaatat gatgatcagaccagtcaaagggagaaagaagatgataaagtcctccctggtggaage catacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactg cgccctacctactcaεatcttεctcatgtggacctggtaaaagacttgaattcaggc cccactggagccctactagtatgtagagaagggagtctggccaaggaaaagacacag accttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcac tcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctgg cctaaaacgcacacagtcaatggttatgtaaacaggtctctgccaggtccgattgga tgccacaggaaatcagtctattggcatgtgatcggaacgggcaccacccctgaagtg cactcaaeactcctcgaaggtcacacatctcttgtgaggaaccatcgccaggcgccc ttggaaatctcgccaacaactteccctactgctcaaacactcttgatggacctcgga cagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcetaε gtcaaagtagacagcegtccagaggaaccccaactacgaatgaaaaataatgaagaa gcggaagactatgatgatgac.ee tactgatεcegaaatggatgtggεcaggtttgat gatgacaactctcctccctttatccaaattcgctcagttgccaagaagcatcctaaa acttgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtc ctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcgg attggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaag actcgtgaagctattcagcatgaatcaggaatcttgggacctttacttcatggggaa gttggagacacactgttgattacatctaagaatcaagcaagcagaccatataacatc taccctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggt gtaaaacatttgaaggattttccaactctgccaggagaaatattcaaatataaatgg acagtgaccgtagaagacgggccaactaaatcagatcctcggtgσctgacccgctat tactctagtttcgtcaatatggagagagacctagcttcaggactcattggccctctc ctcatctgctacaaagaatctgcagatcaaagaggaaaccagataatgccagacaag aggaacgtcatcctgttttctgtatttgacgagaaccgaagctggtacctcacagag aatatacaacgctttctccccaatccagctggagtgeagcccgaggatccagagttc caagcctccaacatcatgcacagcatcaatggctatgtttttgatagtctgcagctg tcagtttgtttgcatgaggtggcatactggtacattctaagcaccggagcacagact gacttcctctctgtcttcttctctggatataccttcaaacacaaaacggtctatgaa gacacactcaccccattcccattctcaggagaaactgtcttcatgtcgatggaaaac ccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgacc gccttactgaaggtccccagctgtgacaagaacactggtgattattacgaggacagt tatgaagatattccagcatacttgctgagcaaaaacaatgccactgaaccaagaagc ttctcccagaattcaagacaccctagcactaggcaaaagcaatttaatgccaccaca attccagaaaatgacatagagaagactgacccttggtttgcacacagaacacctatg cccaaaatacaaaatgtctcctctagtgatttgttgatgctcttgcgacagagtcct actccacacgggctatccttatctgatctccaagaagccaaatatgagactttttct gatgatccatcacctggagcaacagacagtaataacagcctgcccgaaatgacacac ttcaggccacagctccatcacagtggggacatggtatttacccctgagtcaggcctc caattaagattaaatgagaaactggggacaactgcagcaacagagttgaagaaacct gatttcaaagtttccagtacatcaaataacctgatttcaacaattccatcagacaat ttggcagcaggtactgataacacaagctccttaggacccccaagtatgccagctcat tatgatagtcaattagataccactccacttggcaaaaagtcatctccccttactgag tctggtggacctctgagcttgagtgaagaaaataatgactcaaagttgttagaatca ggtttaatgaatagccaagaaagttcatggggaaaaaatgtatcgtcaacagagagt ggeaggttatttaaagggaaaagagetcatggacctgctttgttgactaaagataat gccttattcaaagttagcatctctttgttaaagacaaacaaaactcccaataattca gcaactaatagaaagactcacactgatggcccatcattattaattgagaatagtcca tcagcctggcaaaatatattagaaagtgacactgagtttaaaaaagtgacacctttg attcatgacagaatgcttatggacaaaaatgctacagctttgaggctaaatcatatg tcaaataaaactacttcatcaaaaaacatggaaacggtccaacagaaaaaagagggc cccattccaccagatgcacaaaatccagatatgccgtcctctaagatgctattcttg ccagaatcagcaaggtggatacaaaggactcatggaaagaactctctgaactctggg caaggccccagtccaaageaattagtatccttaggaccagaaaaatctgtggaaggt cagaaεttcttgtctgagaaaaacaaagtggtagtaggaaagggtgaatttacaaag gacgtaggactcaaagagatggttttcccaagcagcagaaacccattccttactaac tcggataatttacatgaaaataatacacacaatcaagaaaaaaaaactcaggaagaa atagaaaagaaggaaacaccaatccaagagaatgtagctttgcctcagatacataca gtgactggcaccaagaacttcatgaagaacctcttcttactgagcaetaggcaaaat gtagaaggttcatatgacggggcatatgctccagtactccaagattttaggtcatta aatgatccaacaaatagaacaaagaaacacacagcccatttctcaaaaaaaggggag gaagaaaacttggaaggcttgggaaatcaaaccaagcaaattgtagagaaatatgca tgcaccacaaggatatctcctaatacaagccagcagaattttgtcacgcaacgtagt aagagagctttgaaacaattcagactcccactagaagaaacagaacttgaaaaaagg ataattgtggatgacacctcaacccagtggtccaaaaacatgaaacatttgaccccg agcaccctcacacagatagactacaatgagaaggagaaaggggccattactcagtct cccttaεcagattgccttacgaggagtcatagcatccctcaagcaaatagatctcca ttacccattgcaaaggtatcatcatttccatctattagacctatatatctgaccagg gtcctattccaagacaactcttctcatcttccagcagcatcttatagaaagaaagat tctggggtccaagaaagcagtcatttcttacaaggagccaaaaaaaataacctttct ttagccattctaaccttggagatgactggtgatcaaagagaggttggctccctgggg acaagtgccacaaattcagtcacatacaagaaagttgagaacactgttctcccgaaa ccagacttgcccaaaacatctggcaaagttgaattgcttccaaaagttcacatttat cagaaggacctattccctacggaaactagcaatgggtctcctggccatctggatctc gtggaagggagccttcttcagggaacagagggagcgattaagtggaacgaagcaaac agacccggaaaagttccctttctgagagtagcaacagaaagctctgcaaagactccc tccaagctattggatccccttgcttgggataaccactatggtactcagataccaaaa gaagagtggaaaccccaagagaagtcaccagaaaaaacagcttttaagaaaaaggat accattttgtccctgaacgcttgtgaaagcaatcatgcaatagcagcaataaatgag ggacaaaataagcccgaaatagaagtcacctgggcaaagcaaggtaggactgaaagg ctgtgctctcaaaacccaccagtcttgaaacgccatcaacgggaaataactcgtact actcttcagtcagatcaagaggaaattgactatgatgataccatatcagttgaaatg aagaaggaagattctgacacttatgatgaggatgaaaatcagagcccccgcagcttt caaaagaaaacacgacactattttattgctgcagtggagaggctctgggattacggg acgagtagctccccacatgttctaagaaacagggctcagagtggcagCgtccctcag ttcaagaaagttgttttccaggaatttactgatggctcctttactcagcccttatac cgtggagaaccaaacgaacatttgggactcctggggccatatataagagcagaagtt gaagataatatcatggtaactctcagaaatcaggcctctcgtccctattccttctat tctagcctcatttcttatgaggaagatcagaggcaaggagcagaacctagaaaaaac tttgtcaagcctaatgaaaccaaaacttacttttggaaagcgcaacatcatatggca cccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctg gaaaaagatgtgcactcaggcctgattggaccccttctggtctgccacactaacaca ctgaaccctgctcatgggagacaagtgacagtacaggaatttgctctgtttttcacc atctttgatgagaccaaaagctggtacttcactgaaaatatggaaagaaactgcagg gctccctgcaatatccagatggaagatcccacttttaaagagaattatcgcttccat gcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatcaa aggattcgatggtatctgctcagcatgggcagcaatgaaaacatccattctattcat tccagtggacacgtgttcactgtacgaaaaaaagaggagtataaaatggcactgtac aatctctatccaggtgtttttgagacagtggaaatgttaccatccaaagctggaatt tggcgggtggaatgccttattggcgagcatctacatgctgggatgagcacactttct ctggtgtacagcaataagtgtcagactcccctgggaatggcctccggacacattaga gattttcagattacagcttcaggacaatatggacagtgggccccaaagctggccaga ctccactactccggatcaatcaatgcctggagcaccaaggagcccctttcttggacc aaggtggatctgttggcaccaatgattattcacggcatcaagacccagggtgcccgt cagaagttctccagcccctacatctcccagtttatcatcacgtatagtcttgatggg aagaagtggcagacttatcgaggaaattccactggaaccLtaatggtcttctttggc aatgtggattcatctgggataaaacacaatatctttaaccctccaattattgctcga tacatccgtttgcacccaactcattatagcattcgcagcactctccgcatggagttg atgggctgtgatttaaatagttgcagcatgccactgggaatggagagcaaagcaata tcagatgcacagattactgcttcatcctactttaccaatatgtctgccacctggtct ccctcaaaagctcgacttcacctccaagggaggagtaacgcctggagacctcaggcg aacaacccaaaagagcggccgcaagtggacttccagaagacaatgaaagtcacagga gtaactacccagggagtaaaatctctgcttaccagcatgtatgcgaaggagt ccctc atctccagcagtcaagatggccatcagtggaccctcttttttcagaatggcaaagca aaggttcttcagggaaatcaagactccttcacacctgtggtgaactctctagaccca ccgttactgactcgccaccttcgaatrcacccccagagttgggtgcaccagattgcc ctgaggatggaggtcccgggctgcgaggcacaggacctctactga [0035] The wild-type human factor VIII encoded by SEQ ID NO: 1 has an amino acid sequence of SHQ ID NO:2, as follows:

ATRRY'ifLGAVELSWDyMOSDLGEIL.PVDARFPPRVPKSFPFHTSWYKKTLFVEFTVH LFNIAKPRPPWWGLIΛPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW KASEGAEY DDQTSQREKSDDKVF-5GGSHTYWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSG

LIGALLVCREGSLAXEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHS I FLEGHTFLVRNHRQAS LE I S PI TFLTAQTLI-MDIIGQFLLFCH I SSHQHDGMEAr5ZKVDSCPBEPQLRMKNNEE

AEDYDDDLTDSEMDWRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDMDYAPLV LAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGE VGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGBTFKYKM TVTVEDGPTKSDPRCLTR YYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK RNVILFSVFDENRSWYLTENIQRFLPNPAC-VQLUDPEFQASNIMKSINGYVFDSLQL SVCLHEVAYWYΣLSIGAQTDFLSVFFSGYTFKHKMVΥEDTLTLFPFSGETVFMSMEN PGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDIYEDISAYLLSKNNAIEPRS FSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSP TPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGL QLRLNEKLGTTAATELKKLDKKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVM YDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSVIGKNVSSTES GRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSP SVWQNILESDTEFKLON1PLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEG PIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEG QNFLSEKNKVWGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEE IEKKETLIQENWLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYEGAYAPVLQDFRSL NDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRS KRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQS PLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKD SG VQESSHFLQGAKKNNLS LAI LTLEMTGDQR E VGS LGTS ATNSVTYKKVENTVLPK PDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEAN RPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKD

TILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPP1TLKRHQREITRT TLQSDQEE I DYDDT I S VEMKKEDFDI YDEDENQS PRS FQKKTRHYFI AAVERLWDYG MSSSPHVLRNRAQSGSVPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEV F,DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAE PRKN FVKPNETKTYFWKVQHHMA PTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTSNMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQ RIRWYLLSMGSNEN1HS1HFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGI WR\ΈCLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLAR LHYSGSINAWSTKEPFSWIKVDLLAPMI1HGIKTQGARQKFSSLYISQFIIMYSLDG KKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMEL MGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATIISPSKARLHLQGRSNAWRPQV NNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV

KVFQGNQDS FTP WNSLDP PLLTR YLR 1H PQS WVHQ I ALRME VLGCE AQDLY In the above sequence, several charged residues are identified by bold typeface and underlining, including Glu287, Asp302, Asp519, Glu665. and Glu 1984 [0036] The recombinant factor VIlI of the present invention is characterized by the replacement of one or more charged amino acid residues with a hydrophobic amino acid residue at either or both of the A1 A2 or A2A3 domain interfaces. Preferably, the charged residue to be replaced is either a Glu or Asp residue that docs not participate in hydrogen bonding between the A1 A2 or A2A3 domains. The hydrophobic amino acid residue that replaces the charged residue can be any of Ala, VaI, Ile. Leu. Met, Phe, or Tip. Particularly preferred recombinant factor VIIl of the present invention includes a substitution of the Glu287 residue of wildtype factor VIII, a substitution of the Asp302 residue of wildtype factor VIIl, a substitution of the Asp519 residue of wildtype factor VIII, a substitution of the Glu665 residue of wildtype factor VUI, a substitution of the Glu 1984 residue of wildtype factor VIII, or combinations thereof. The D302A. E287A, E665A. 1-665 V, D519A. D519V, El 984A, and I- 1984V substitutions are preferred for achieving a recombinant factor VIII that has enhanced stability of both factor VIIl and factor Villa. Preferred combinations of these substitutions include, without limitation, D519AE665V, D519VE665V, and D519VE1984A double mutants, as well as D519AE665VE1984A and D519VE665VE1984A triple mutants. The enhanced stability of these mutants is believed to be achieved by stabilizing the inter-domain interface in factor VIlI as well as reducing A2 subunit dissociation from A1/A3C1C2 as compared to wildtype factor VIIIa. [0037] 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 VIlI sequences that have modified properties with regard to various attributes, including, without limitation, antigenicity, circulating half-life, protein secretion, affinity for factor IXa and/or factor X, altered factor Vlll-inactivation cleavage sites, enhanced specific activity of factor VIIIa, immunogenicity. and shelf-life.

[0038] One example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a factor VIH having a modified calcium binding site, preferably at 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. 10/581,471 to Fay et al., which is hereby incorporated by reference in its entirety. Preferably, the residue 1 13 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 EI 13AD519A, Kl 13AD519V, E113ΛE665A, El 13AH665V. or E l 13AE 1984V substitutions. [0039] A second example of a suitable mutant factor VIIl that can be modified in accordance with the present invention is a B-domainless factor VIlI that contains amino acid residues 1-740 and 1690-2332 of SCQ ID NO: 2 (see. e.g., U.S. Patent No. 6,458,563 to Lollar, which is hereby incorporated by reference in its entirety). [0040] 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 VIIl (see. e.g., U.S. Patent Application Publication No. 2004/0197875 to Hauser el al., which is hereby incorporated by reference in its entirety).

[0041] In another embodiment of the B-domainless recombinant factor VIII of the present invention, the modified mutant factor VIlI is encoded by a nucleotide sequence having a truncated factor lX inlron 1 inserted in one or more locations (see. e.g., U.S. Patent No. 6,800,461 to Negrier and U.S. Patent No. 6.780,614 to Negricr, each of which is hereby incorporated by reference in its entirety). This recombinant factor VMl 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. Patent 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 VIlI 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. Patent No. 6,780,614 to Negrier, which is hereby incorporated by reference in its entirely). [0042] Regardless of the embodiment, the B-domainless factor VIII preferably contains one or more of the mutations described above (e.g.. at positions 287, 302, 519. 665, and/or 1984). Recombinant factor VlIl proteins prepared in accordance with the Examples herein are B-domainless.

[0043] A third example of a suitable mutant factor VHl that can be modified in accordance with the present invention is a chimeric human/animal factor VIIl that contains one or more animal amino acid residues as substifution(s) for human amino acid residues that are responsible for the antigenicity of human factor VlII. In particular, animal (e.g.. porcine) residue substitutions can include, without limitation, one or more of the following: R484A, R488CΪ, P485A, L486S, Y487L, Y487A, S488A, S488L, R489A, R489S, R490G, L491S, P492L, P492A, K493A, G494S, V495A. K496M. H497L, L498S, K499M, D500A, F501 A. P502L, 1503M, I.504M, P505A, G506A, F.507G. I508M, I508A. M2199I, F2200L, L2252F, V2223A. K2227E, and/or L2251 (U.S. Patent No. 5.859,204 to Lollar. U.S. Patent No. 6,770.744 to Lollar. and U.S. Patent Application Publication No. 2003/0166536 Io 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., at positions 287, 302, 519. 665. and/or 1984). [0044] A fourth 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 Sυbunit Residues 558-565 Represent a Factor IXa Interactive Site." J. Biol. Chem. 269(32):20522-7 (1994); Bajaj ct al., "Factor IXa: Factor VIlIa 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 Glul 81 1 - Lysl818 of Human Blood Coagulation Factor VIII Comprises a Binding Site for Activated Factor IX/1 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., I^apan 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 VIlI contains one or more of the mutations described above (e.g.. at positions 287. 302, 519, 665, and/or 1984). [0045] A fifth example of a suitable mutant factor VIH 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 Immunoglobυlin-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., at positions 287. 302. 519, 665. and/or 1984).

[0046] A sixth 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 VIIl Catabolism Mediated by Low Density Lipoprotein Receptor-Related Protein." J. Biol. Chem. 276( 15): 1 1970-9 (2001 ). which is hereby incorporated by reference in its entirety) and/or low-density lipoprotein receplor-related protein ("LRP") (Saenko et al., "Role of the Low Density Lipoprotein-Related Protein Receptor in Mediation of f-actor VIIl Catabolism/' J. Biol, Cham. 274(53):37685-92 (1999); and LenUng et al., "The Light Chain of Factor VIIl 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 VlIl contains one or more of the mutations described above (e.g.. at positions 287, 302, 519, 665, and/or 1984).

[0047] A seventh 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. Patent No. 6,759,216 to Lollar, which is hereby incorporated by reference in its entirety). The mutant factor VIlI of this example can be useful in providing a modified factor VIlI 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-Ii nked 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. Patent 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.. at positions 287, 302, 519, 665, and/or 1984). [0048] An eighth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a modified factor VlIl that is a procoagulant-activc factor VIIl 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 VIlI 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 procoagulanl-active factor VUI 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 VIIt is also modified to contain one or more of the mutations described above (e.g., at positions 287, 302, 519, 665, and/or 1984). [0049] Further, the mutant factor VIII can be modified to take advantage of various advancements regarding recombinant coagulation factors generally (see. e.g ., Sacnko 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). [0050] The recombinant factor VI(I of the present invention can be modified at any charged residue that destabilizes the A1 A2 or A2A3 domain interfaces (including positions 287, 302, 519. 665, or 1984). as well as be modified to be B- domainless, to be chimeric, to have modified calcium binding sites that enhance factor VIIIa activity (e.g., at position 1 13), to have altered inaclivation 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, enhanced specific activity, high stability recombinant factor VlIl proteins are described in the Examples.

[0051] The recombinant factor VIIl is preferably produced in a substantially pure form. In a particular embodiment, the substantially pure recombinant factor VIlI 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 V1H 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 sonicaiion, 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 VlIl is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the recombinant factor VlIl. If necessary, a protein fraction (containing the substantially pure recombinant factor VlU) may be further purified by high performance liquid chromatography ("1-IPLC'1). [0052] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes the recombinant factor VIlI of the present invention. The isolated nucleic acid molecule encoding the recombinant factor VIII can be either RNA or DNA.

[0053] In one embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a mutation at position 1 13 that enhances factor VlII specific activity, as modified with one or more of the substitutions of charged residues (e.g., at positions 287, 302, 519, 665, 1984, and/or 332-340 of SEQ ID NO: 2). [0054] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a B-domainless factor VIIl of the type described above, as modified with one or more of the substitutions of charged residues (e.g., at positions 287, 302, 519, 665, and/or 1984 of SEQ ID NO:2).

[0055] 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 of charged residues (e.g., at positions 287, 302, 519. 665, and/or 1984 of SEQ ID NO:2). [0056] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIIl whose inactivation sites have been modified as described above, as further modified with one or more of the substitutions of charged residues (e.g.. at positions 287. 302, 519, 665. and/or 1984 of SEQ ID NO:2). [0057] In yet another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIlI whose affinity for factor IXa and/or factor X has been enhanced, as further modified with one or more of the substitutions of charged residues (e.g., at positions 287, 302, 519, 665, and/or 1984 of SEQ ID NO:2).

[0058] 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 of charged residues (e.g., at positions 287, 302. 519, 665, and/or 1984 of SEQ ID NO:2).

[0059] 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 modi Med with one or more of the substitutions of charged residues (e.g. , at positions 287, 302, 519, 665, and/or 1984 of SRQ ID NO:2).

[0060] In a further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a lacloT VIII that possesses one or more non-naturally occurring glycosylation site, as further modified with one or more of the substitutions of charged residues (e.g., at positions 287, 302. 519. 665, and/or 1984 of SKQ ID NO:2).

[0061] 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, and modified within a calcium-binding site (e.g., at position 113) such that the specific activity of the recombinant factor VIII is improved.

[0062] 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. [0063] 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. , Λrabidopsϊs or tobacco cells), or algal cells.

[0064] The recombinant DNA expression system and host cells can be produced using various recombinant techniques well-known in the art, as further discussed below. [0065] The DNA molecule encoding the recombinant factor VIIl 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 VUl of the present invention in host cells or host organisms.

[0066] With respect to the recombinant expression system of the present invention, an expression vector containing a DNA molecule encoding the recombinant factor VIIl 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. [0067 j A variety of host-vector systems may be utilized to express the recombinant factor VUl-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.

[0068] When recombinantly produced, the factor VIlI protein or polypeptide

(or fragment or variant thereof) is expressed in a recombinant host cell, typically, although not exclusively, a eυkaryote. [0069] Suitable vectors for practicing the present invention include, but are not limited to, the following viral vectors such as lambda vector system gtl I , gtWES.tB, Charon 4, and plasmid vectors such as pCMV, pBR322, pBR.325. PACYC177, pACYC184, pUC8, pUC9, pUC18, pUC 19, pLG339, pR290, pKC37, pKC10l, SV 40, pBluescript 11 SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog ( 1993 )), pQE . p1H821 , 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. [0070] 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 ct al.. Molecular Cloning: A Laboratory Manual Cold Springs Harbor, N. Y.: Cold Springs Laboratory, ( 1982), which is hereby incorporated by reference in its entirety.

[0071] U.S. Patent 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.

[0072] Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation). [0073] 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.

[0074] 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-Dalgamo ("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 ami no-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. [0075] Promoters vary in their ''strength" (i.e., their ability to promote transcription). Tor 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 to//, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, irp promoter, recA promoter, ribosomal RNA promoter, the PR and Pt promoters of coliphage lambda and others, including but not limited, to /tϊcUV5, ompP, bla, Ipp. and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacW5 (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. [0076] 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 IPTU (isopropylthio-beta-D-galactoside). A variety of other operons, such as irp,pro, etc., are under different controls. j0077| 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 ("ATCi") 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 ofcoliphage lambda, or from the 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.

[0078] 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. [0079] A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism. [0080] 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. [0081] 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 tnRNA for expression in the host cell of choice, operably linked to a DNA molecule which encodes for a protein of choice. [0082] 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 ct al., Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor Press, NY (1989), and Λusυbel. 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.

[0083] 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 recombinanily 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., AlCC No. CRL 6281), CHO (e.g.. ATCC No. CCL 61), HeLa (e.g.. ATCC No. CCL 2). 293 (ATCC No. 1573), CHOP, and NS-I cells. [0084] 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 EIa, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR. [0085] 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. Recombinanl molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.

10ϋ861 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. 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 Wtllebrand Factor (referred to herein as "VWF"). In this embodiment, the host cell can contain a transgene encoding a VWT 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 VIlI. 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 VlIl 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. [0087] The modifications at positions 287, 302, 519. 665. and/or 1984 are particularly preferred, because they result in enhanced stability of both factor VIIl 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.

[0088] When an expression vector is used for purposes of in vivo transformation to induce factor VIlI 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.

[0U89J 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. [0090] The recombinant factor VIIl of the present invention can be used to treat uncontrolled bleeding due to factor VIfI 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.

[0091] Alternatively, or in addition thereto, the recombinant factor VlH can be administered by administering a viral vector such as an adeno-associated virus (Gnatenko et al., 'Ηuman 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 ct 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). [0092] 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 VIlI 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. [0093] 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 trie dog infusion model or in human patients, according to Lusher et al., "Recombinant Factor VIlI 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 ct at., "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 VIlI Procoagulant Protein: Comparative Hemostatic Response After Infusions into Hemophilic and von Willcbrand Disease Dogs." Proc. Natl. Acad Sci. 82:8752-8755 (1985), which are hereby incorporated by reference in their entirety. [0094] 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 IL Factor V, and Factors VIl to XIl)," 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 VIlI than their previous form of factor VIIl. 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 VIlI 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.

[0095] 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 VlII can be administered subcutaneousiy or orally with liposomes in one or several doses at varying intervals of time.

[0096] The recombinant factor VlII can also be used to treat uncontrolled bleeding due to factor VlII deficiency in hemophiliacs who have developed antibodies to human factor VIII. [0097] H has been demonstrated herein that the recombinant factor VIII of the present invention can differ in specific activity from the wild-type factor VIN. Factor VUI proteins having greater procoagulant activity from wild-type factor VlH 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 VlIl (because less antigen is administered). EXAMPLES

[0098] 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

Reagents

[0099] Recombinant factor VIII (Kogenate) was a generous gift from Dr.

Lisa Regan of Bayer Corporation (Berkeley. CA). 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 Glucosidc," Biochemistry 20:833-840 ( 1981 ), which is hereby incorporated by reference in its entirety). The reagents α-thrombin. factor Vila, factor IXaβ, factor X, and factor Xa (Enzyme Research Laboratories, South Bend, IN), hirudin and phospholipids (DiaPharma. West Chester, OH), the chromogenic Xa substrate, Pefachrome Xa (Pefa-5523, CH-jOCO-D-CHA-Gly-Arg- pNA AcOH; Centerchem Inc. Norwalk CT), recombinant human tissue factor (rTF), Innovin (Dade Behring, Newark, DE), fluorogenic substrate, Z- G Iy-G Iy- Arg- AMC (Calbiochem, San Diego, CA), and thrombin calibrator (Diagnosttca Stago, Parsippany. NJ) were purchased from the indicated vendors.

Construction, Expression and Purification of WT and Variant Factor VUI: [0100] Ala mutants (at D27. H281, R282, E287, D302, S313, 11317, T522,

S524, R53 I, N538, E540, S650, S654, D666, E683, N684, S695, D696, Sl 791, D 1795, Q 1820, E 1829, S 1949, N 1950, and R 1966); Phe mutants (at Y476, Y664, Y 1786, and Y 1792); Ala and VaI mutants (at charged residues E272, D519. E665. and E1984): and WT factor VIII forms were individually constructed as a B- domainless factor VIlI, lacking residues Gln744-Serl637 in the B-domain (Doering et al. "Expression and Characterization of Recombinant Murine Factor VIH." Thromb Haemost. 88:450-458 (2002), which is hereby incorporated by reference in its entirely). The cloning and expression constructs were generous gifts from Dr. Pete Lollar and John Healey. Recombinant WT and variant factor VIII forms were stably expressed in BHK cells and purified as described previously (Wakabayashi et al. "Residues 1 10-126 in the A1 Domain of Factor VlIl Contain a Ca2^ 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 ~100 μg from two 750 cm2 culture flasks, with purity from -85% to >95% as judged by SUS-PACili. The primary contaminant in the factor VIII preparations was albumin and at the concentrations present in the factor VlII showed no effect on stability of activity parameters. Factor VNI concentration was measured using an Enzyme-Linked Immunoadsorbant Assay (ELISA) and factor VIlI activity was determined by an one-stage clotting assay and a two-stage chromogenic factor Xa generation assay as described below.

SDS-PAGE and Western Blotting

[0101] Factor VIII proteins (0.77 μg for gel staining and 0.34 μg for Western blot) were electrophoresed on 8% polyacrylamide gel at constant voltage (100V). Gels were stained with Gelcode Blue (Thermo Scientific, Rockford, II.) or transferred to a polyvinylidene fluoride membrane and probed with biotinylated anti-A2 antibody (R8I312. Green Mountain Antibodies, Burlington, VT) followed by the incubation with peroxidase-conjugated streptoavidin (Calbiochem, San Diego, CA). The chemifluorescence substrate (ECF substrate. GE Healthcare, Piscataway, NJ) was reacted and the fluorescence signal scanned using a phosphoimager (Storm 860, GE Healthcare). The density of single chain factor VIlI form ( 170 kDa) and heavy chain (HC, 90 kDa) were quantified using ImageQuant software (GE Healthcare) and the amount ratios were calculated.

EUSA [0102] A sandwich ELISA was performed to measure the concentration of factor VIII proteins as previously described ( Wakabayashi et al., "A Glul 13Ala Mutation within a Factor VIII Ca2*-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 VlII (Kogenate, Bayer Corporation) as a standard. Factor VIII capture used the anti-C2 antibody (ESH-8. American Diagnostica Inc., Stamford, CT) and a biotinylated R8B 12 antibody, was employed for factor VIII detection. One-stage Clotting Assay

[0103] One-stage dotting assays were performed using substrate plasma chemically depleted of factor VIII (Over, "Methodology of the One-stage Assay of Factor VIII (VIIIrC)," ScandJ 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 37C after which a dilution of factor VIII was added to the cuvette. After I min the mixture was recalcified, and cloning time was determined and compared to a pooled normal plasma standard. Two-Stage Chromogenic Factor Xa Generation Assay

[0104] 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 VIIl Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and lnter- subunit Affinity." Biochemistry 4O:\Q293-\0y0O (2001); Wakabayashi et al.. "Ca2' 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]pipcrazine-N'-(2-ethanesulfonic acid) (HHPBS), pH 7.2, 0.1 M NaCl,

0.01% Tween 20, 0.01% BSA, 5 mM CaCb, and 10 μM PSPCPE vesicles (Buffer A) was activated with 20 nM α-thrombin for I min. The reaction was stopped by adding hirudin (10 U/ml) and the resulting factor VIIIa was reacted with factor IXa (40 nM) for I min. Factor X (300 nM) was added to initiate reactions which were quenched after 1 min by the addition of 50 mM ODTA. Factor Xa generated was determined following reaction with the chromogenic substrate Pefachrome Xa (0.46 mM final concentration). All reactions were run at 23C.

Thrombin Generation Assay

[0105] The amount of thrombin generated in plasma was measured by Calibrated Automated Thrombography (Hemker et al., "Calibrated Automated Thrombin Generation Measurement in Clotting Plasma," Pathophysiol Haemost Thromb. 33:4-15 (2003); Hemker et al.. "Ilirombin Generation in Plasma: Its Assessmenl via the Endogenous Thrombin Potential," Thromb Haemυst. 74:134-138 (1995), each of which is hereby incorporated by reference in its entirety). In a 96- well plate, 80 μl of factor VIII deficient plasma (<1% residual activity, platelet-poor) from severe hemophilia A patient tacking factor VIIl inhibitor (George King Bio- Medical. Overland Park, KS) was mixed with factor VIII samples (20 μl: 6 n.M) in HEPES-BSA buffer (20 mM HEPES, pH 7.35. 0.15 M NaCl, 6% BSA) containing 3 pM rTF (the concentration of rTK stock was determined by factor Xa generation assay using known concentrations of factor Vila), PSPCPE vesicles (24 μM) or 20 μl thrombin calibrator (630 nM) and reactions were immediately started by mixing with 20 μl fluorogenic substrate (2.5 mM. Z-Gly-Gly- Arg-AMC) in HRPES-BSA buffer including 0.1 M CaCl2. All reagents were pre-warmed at 37C. Final concentrations of reagents were 1 nM factor VIΪΪ (except as otherwise noted), 0.5 pM rTF, 4 μM PSPCPIi vesicles, 433 μM fluorogenic substrate, 13.3 mM CaICl2, and 105 nM thrombin calibrator. The development of a fluorescent signal at 37C was monitored at 8 second intervals using a Microplate Spectra fluorometer (Spetramax Gemini, Molecular Devices, Sunnyvale, CA) with a 355 nm ( excitation )/460 nm (emission) filter set. Fluorescent signals were corrected by the reference signal from the thrombin calibrator samples (Hemker et al., "Calibrated Automated Thrombin Generation Measurement in Clotting Plasma," Pathophysiol Haemost Thromb. 33:4- 15 (2003), each of which is hereby incorporated by reference in its entirety) and actual thrombin generation in nM was calculated as previously described (1 leinker et al.. "Thrombin Generation in Plasma: Its Assessment via the Endogenous Thrombin Potential," Thromb Haemost. 74:134-138 (1995), which is hereby incorporated by reference in its entirety). Factor VlII Activity at Elevated Temperature

[0106] WT factor VIII or factor VIII variants (4 nM) in buffer A were incubated at 52-60 C. Aliquots were removed at the indicated times and residual factor VIII activity was determined using a two-stage chromogenic factor Xa generation assay. Factor VJIh Activity Time Course

[0107] WT and mutant factor VIlI (4 nM) in buffer A containing 10 μM

PSPCPE vesicles were activated by 20 nM thrombin for I min at 23C. Reactions were immediately quenched by hirudin ( 10 U/ml), 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). For decay measurements run in the presence of factor IXa. factor IXa (40 nM) was added to reactions prior to thrombin addition.

Factor VIII Stability in Plasma

[0108] WT or variant factor VlIl ( 1 nM) was added to factor VIII deficient plasma (<I% residual activity) from severe hemophilia A patient lacking factor VHl inhibitor (George King Bio-Medical). Plasma was supplemented with 0.02% NaN3 to prevent the growth of microorganisms and samples were incubated at 37C. Aliquots were removed at the indicated times and residual activity was determined by a one- stage clotting assay.

Data analysis

[0109] Factor 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,

where A is residual factor VlIIa activity (nM/min/nM factor VIII), Ao is the initial activity, k is the apparent rate constant, and / is the time (minutes) of reaction of either factor VIIl at elevated temperature (for factor VIII decay experiments) or after thrombin activation was quenched (for factor VIIIa decay measurements). Nonlinear least-squares regression analysis was performed by Kaleidagraph (Synergy, Reading, PA). Comparison of average values was performed by the Student's /-test. The factor VIII A domain modeled structure (Pemberton et al., UA Molecular Model for the Triplicated A Domains of Human Factor VIII Based on the Crystal Structure of Human Ceruloplasmin," Blood 89:2413-2421 (1997), which is hereby incorporated by reference in its entirety) was analyzed using Swiss PDB Viewer to identify charged residues that were located at the A2 domain interface and that showed little potential for hydrogen bonding interactions based on a threshold of >2.8 A separating the polar atoms of the complementary domains (Weiner ct al.. "A New Force Field for Molecular Mechanical Simulation of Nucleic Acids Proteins," J Am Chem Soc. 106:765-784 ( 1984), which is hereby incorporated by reference in its entirety). Example 1: Activity Values for Factor VIU Mutants Targeting Hydrogen Bonding Interactions

[0110] Bonding interactions involving the A2 domain in factor VIlI remain poorly understood yet represent a primary mechanism for the regulation of cofactor activity. The factor VIIl homology model (Pcmberton et al.. "A Molecular Model for the Triplicated A Domains of Human Factor VIII Based on the Crystal Structure of Human Ceruloplasmin," Blood 89:2413-2421 (1997), which is hereby incorporated by reference in its entirety) identifies the potential for many hydrogen bonds linking residues in the A2 domain with those in the A1 or A3 domains. Using a criterion for a spatial separation of <2.8 A between hydrogen donor and acceptor atoms (Weiner et al., "A New Force Field for Molecular Mechanical Simulation of Nucleic Acids Proteins," J Am Cheni Soc. 106:765-784 (1984), which is hereby incorporated by reference in its entirety) thirty residues were identified as having a side chain atom that may be involved in hydrogen bonding with an atom from a complementary A domain (see Table 1 below). In approximately half of the residues identified, side chain atoms were juxtaposed with either backbone carbonyl oxygen or amide hydrogen atoms, while the remainder represented possible interactions between neighboring side chains. Target residues in the factor VIlI A domains were individually mutated to Ala. with the exception that Tyr residues were replaced with Phe. and the point mutations were stably expressed as B-domainlcss factor VIIl. [0111] Factor VIII activity was measured for the purified proteins using a one- stage clotting assay and a (two-stage) factor Xa generation assay. Results from the one-stage assay (Figure 1) indicated that 9 of the 30 point mutants showed <50% activity relative to WT factor VIII. Five of these variants demonstrated a one- stage/two stage assay discrepancy (>1.5 fold difference), with three mutants (S524A, H281 A, and E287A) showing reduction in only the two-stage assay. The reduced activity values for mutation in several targeted residues were consistent with a contribution of those side chains to the structural stability of factor VIlI and/or factor VIIIa. Table 1 : Amino Acid Residues Capable of Hydrogen Bonding

Example 2: Thermostability of Factor VlII Variants

[0112] To assess the heat-stability of the WT procofactor and variants, a temperature at 55C was employed based upon factor VTII inactivation results described in an earlier study (Ansong ct 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). For these reactions, factor VIII was incubated for indicated times at the elevated temperature, after which the reaction mixture was immediately cooled to room temperature, and factor VlII reacted with thrombin and assayed fυr cefaclor activity using a factor Xa generation assay. Rates of loss for factor VIlI activity to the heat treatment, as judged by residual cofactor function, was determined as described in Methods. Figure 2A shows results for variants showing the greatest and the least sensitivities to the heat treatment compared with WT. (0U3J Table 2 (below) summarizes the results obtained from factor VIlI thermostability assays for the 30 variants. Overall, these activity data fit well to a single exponential decay function with correlation coefficients in most cases >0.98. While a number of mutations were benign with respect to the amino acid replacement (21 showing <2-fold differences in rates of decay), several residues including Axg282 (A 1 domain), and A2 domain residues Ser524. Asn684 and Ser650 showed -5- to ~20-fold increased rates in factor VIII decay suggesting an important role for these residues in maintaining factor VIIl stability. Furthermore, the R282A and N684A variants showed significantly reduced specific activity values suggesting both activity and stability parameters were affected by the single point mutations. On the other hand, replacement of E287 and D302 with Ala yielded reduced rates for factor VIII decay at the elevated temperature. This apparent increase in protein stability following mutation is consistent with these acidic side chains destabilizing inter- domain interactions in the WT cofactor. Table 2: Factor ViII and Villa Decay Rates and Activity Values

Example 3: Factor Villa Decay Rates

[0114] Factor VIIIa activity is labile due to A2 subunit dissociation (Fay ct 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-depcndent Denaturation of Thrombin-activated Porcine Factor Will" J Biol Chem. 265: 1688-1692 (1990), each of which is hereby incorporated by reference in its entirety). Results from earlier studies showed that inclusion of factor IXa and phospholipid vesicles with factor VIIIa to form the Xase complex reduced the lability of the cofactor (Lollar et al., "Stabilization of Thrombin-activated Porcine Factor VI1I:C by Factor IXa Phospholipid." Blood 63: 1303- 1308 (1984); Lamphear et al., "Factor IXa Enhances Reconstitution of Factor VIIIa from Isolated A2 Subunit and AI/A3-C1-C2 Dimer/V. Biol. Chem. 267:3725-3730 (1992), each of which is hereby incorporated by reference in its entirety) by partially stabilizing the A2 subunit within factor Xasc (Fay ct 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), which is hereby incorporated by reference in its entirety). This approach was recently used to examine the decay rate for an E1829A factor VIIIa mutant (Wakabayashi ct al., "A3 Domain Residue Glu 1829 Contributes to A2 Subunit Retention in Factor VIIIa." J Thromb. Haemost. 5:996-1001(2007), which is hereby incorporated by reference in its entirety) since the activity decay of this variant factor VIIIa, in the absence of factor IXa and membrane, was too rapid to measure accurately. This approach was similarly employed to assess rates for factor VIIIa decay for the panel of variants described in this Example. Factor VIII (4 nM) was incubated with a molar excess of factor IXa (40 nM) and phospholipid vesicles, rapidly activated with thrombin and subsequent factor Xase activity was measured over a time course at 23"C. Rates of decay of factor Xasc activity was attributed to A2 subunit dissociation and data were fitted using a single exponential decay. Civen the high Kt value for the affinity of A2 subunit within factor VIIIa (144 nM) and the low factor VIIIa concentration (4 nM) used in the reactions, the effect of re-association of dissociated A2 subunit is negligible, supporting use of a simple single exponential applied for this regression analysis. [0115] Results are presented in Figure 2B, which shows data for the most severely affected variants as well as those variants showing a positive response to the mutation. Seven variants possessing significant (>5-fold) increases in rates of factor VIIIa decay compared with WT (Table 2). These mutations included R282A, S524A, N684A, E1829A. Y1786F, D666A. and Y1792F. Factor VlIl activity values for these variants as measured by a two-stage assay were significantly lower than those determined by one-stage assay (Figure 1), consistent with the mutations leading to appreciable rates of A2 subunit dissociation. Furthermore, several of these mutations (including R282A. N684A and Yl 792F) showed overall low specific activity in the onc-stagc assay. As is the case for factor VIIl mutants possessing this assay discrepancy, activity determined from the one-stage assay was also reduced (Pipe ct al., "Mild Hemophilia A Caused by Increased Rate of Fador VIIl 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-stagc/2- stage Activity Discrepancy Disrupt Protein-protein Interactions within the Triplicated A Domains o.'Thrombin-activated Factor VIIIa," Blood 97:685-691 (2001 ): Hakcos et al.. "Hemophilia A Mutations within the Factor VlII A2-A3 Subunit Interface Destabilize Factor VUIa and Cause One-stage/Two-stage Activity Discrepancy," Thromb Haemυst. 88:781-787 (2002), each of which is hereby incorporated by- reference in its entirety), possibly reflecting direct effects of A2 dissociation rates on determining factor VIII activity. [0116] Conversely, the variants E287A and D302A that possessed greater thermostabilities than WT factor VIII also yielded enhanced stability of factor VIIIa as judged by reductions in the rates of cofactor decay following activation by thrombin. Results wilh the D302A variant were more pronounced and showed an -2- fold reduced rate of cofactor decay relative to WT factor VIIIa, retaining -90% of its original activity after 40 min. This observation was consistent with the mutations primarily altering conformation at the inter-domain interface in the procofactor. [0117] Taken together, these results in Examples I -3 identify contributions of multiple residues to inter-A2 (domain) subunit interactions in the procofactor and cofactor forms of factor VIII. with selected residues making disparate contributions to protein stability. While the observed effects of mutation at the target residues were for the most part either benign or detrimental, the mutations at two A1 domain acidic residues, D302 and E287, yielded modest enhancement in stability in both pro- and active cofactor forms. The relative activity of E287 was somewhat reduced compared with WT, whereas the activity values for the D302 variant were indistinguishable from the WT protein, and suggest the latter represents a gain-of-function mutation. These results indicate that some destabiϋzation may result from burying the (negative) charge at the interface and/or an increase in stability when these residue side chains are hydrophobic.

Example 4: Identification of Additional Target Residues and Generation of Point Mutants at GIu272, Asp519, GIu665, and Glu1984

[0118] Based on the results of the preceding Examples, the substitution of other charged residues was explored. Using the ceruloplasmin-based homology model (Pemberton el al., "A Molecular Model for the Triplicated A Domains of

Human Factor VIIl Based on the Crystal Structure of Human Ccruloplasmin," Blood 89:2413-2421 ( 1997), which is hereby incorporated by reference in its entirety) for the A domains of factor VIII, four charged residues were identified (Glu272, Asp519, Glu665, and G Iu 1984). These four residues appear to be buried at the interface of the A2 domain with either the A1 domain (Glu272 and Asp519) or the A3 domain

(Olu665. and Glul 984), but did not appear to contribute to H-bonding interactions based upon spatial separations of >2,8 A with potential bonding neighbors. These residues were mutated to either Ala or VaI to eliminate charge as well as provide for potential hydrophobic interactions with similar side chains from other buried residues. Factor VIII variants were prepared as B-domainless factor VIIl in stable-expressing BHK cell lines.

[0119] Factor VIIl was expressed as a mixture of single chain and heterodimer forms. The purified proteins ranged from -85% to >95% as judged by SDS-PAGE (Figure 3A). Western blotting using an anti-A2 domain antibody was used to quantitate the stoichiometry of the single chain and heterodimer forms (Figure 3B). This value was near unity for WT and was somewhat lower and variable for the factor VlH variants.

[0120] Purified proteins were assessed for specific activity using both one- stage and two-stage assays (Figure 4A) and thrombin generation parameters (Figures 4B-D). All but the Glu272Ala variant yielded specific activity values that were at least 80% that of WT, suggesting the remaining mutations had little if any effect on factor VIIl cofactor function. Thrombin generation performed at low rTF concentration (0.5 pM) and a physiologic concentration (1 nM) factor VIIl yielded results that paralleled the specific activity values. Parameter values shown in Figure 4D indicated the peak value and ETP for the Glu272Ala were reduced compared to WT, whereas all other parameter values for the remaining variants ranged from >80 - 1 10% the WT value.

Example 5: Thermostability of Clu272, Asp519, Clu665 and C Iu 1984 Factor VIII Variants

[0121] The purified factor VlIl mutant proteins were assessed for stability at elevated temperatures as judged by rates of activity loss. Factor VIlI (4 nM) was incubated at 52-60C and at the indicated times an aliquot was removed, cooled to room temperature, reacted with thrombin, and residual cofactor activity was measured using a factor Xa generation assay as described in the Materials and Methods. Results shown in Figure 5 A illustrate the time course for activity decay of the factor VIII WT and variants at 55C. This temperature was chosen based upon an earlier study (Ansong et al., "Factor VIII A1 Domain Residues 97-105 Represent a Light Chain- interactive Site," Biochemistry 45:13140-13149 (2006). which is hereby incorporated by reference in its entirety) showing near-complete activity loss within 1h for WT factor VIII. The WT protein lost 50% activity in -15 min. It was observed that the Glu272Ala and Glu272Val variants displayed reduced stability as judged by somewhat faster activity decay, and this property may be related to the reduced specific activities observed for mutations at this site. On the other hand, Ala and VaI replacements for Asp519, Glu665, and Glu 1984 all showed improved stability at the elevated temperature with variants possessing mutations at the two former sites retaining 50% activity through -20-25 min while mutations at the latter site yielded variants that maintained this activity level through >30 min. Comparison of the decay rate values from the fitted curves (Table 1, below) indicated that factor VIII thermal stability was improved ^2-fold for the GluI 984 variants relative to WT with mutation to VaI appearing somewhat preferred to Ala. [0122] Results assessing a range of temperatures (Figure 5B) indicated that both Ala and VaI variants of Asp519, Glu665. and Glu 1984 consistently showed reductions of decay rate up to 2-fold compared to WT at all temperatures tested. However, the presence of both single chain and heterodimer forms in somewhat varying ratios may impact these decay rate results should one form show greater stability. A control experiment using Kogenate factor VIII which is essentially all in the heterodimer form (Wakabayashi et ah, ''Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Colactor Activity and lnter-subunit Affinity," Biochemistry 40:10293-10300 (2001), which is hereby incorporated by reference in its entirety) yielded decay rates that were ~2-fold that of WT (Figure 5B), consistent with the heterodimer form showing less stability to elevated temperature than single chain factor VIIl. Thus, the decay rates measured are apparently due to heterogeneity of single chain and two chain content in the various factor VIII forms. However, given that all the variants possessed less relative single chain factor VIM compared with the WT (see Figure 5B), these data indicated that decay rate values for these variants underestimate the increase in stability between the mutants and WT.

Table 3: Factor VIII and Villa Decay Rates

Example 6: Factor VIII Stability in Plasma at 37*C

[0123] To lest the effects of the mutations on factor VIlI stability under more native conditions, a near physiological concentration of the proteins ( I nM) was incubated in (anti-coagulated) factor VIII deficient plasma from a hemophilia A patient free from factor VlII inhibitor activity at 37C for up to 4 days. Residual factor VIII was assayed daily using a one-stage dotting assay. Activity of the WT factor VIIl was reduced to -50% after 2 days as was that of the Asp519Val variant, while the Glu665Ala variant showed a modest (-15%) reduction in the rate of activity decay (Figure 6 and Table 3). However, the activity values for the Asp519Ala, Glu665VaI and both Glu 1984 variants were >50% the initial value at day 4. The results obtained from the plasma incubation in large part parallel those from the incubations performed at elevated temperature with the Glu665Val variant and the two Glu 1984 variants demonstrating significant increases in stability under the two reaction conditions as judged by retention of function. While both Asp519 variants showed improved stability at elevated temperature, only the Ala variant showed improvement in the plasma assay.

Example 7: Factor VIIIa Decay Rates of Glu272, Asp519, Glu665 and Glu 1984 Variants

[0124] The above results indicate that mutations consistent with replacing buried charged residues with hydrophobic residues in general yielded factor VlIl protein showing enhanced stability. Inasmuch as these mutations are at or near the interface of the A2 domain with A1 or A3, it was predicted that they could positively impact the lability of factor VIIIa by reducing rates for dissociation of the A2 subunit. Rates of loss of factor VIIIa activity resulting from this mechanism were assessed under two conditions. In the first, the WHI and factor VlII variants were activated with thrombin and at indicated times the remaining cofactor activity was determined following addition of factor IXa and factor X and monitoring rates of factor Xa generation. In the second method, the above assay was modified to include addition of factor IXa prior to factor VIII activation to allow for immediate formation of factor Xase. Incorporation of factor VIIIa in the factor Xase complex has been shown to partially stabilize cofactor activity by reducing its decay rate as much as 10-fυld by a mechanism consistent with factor IXa tethering the A2 and A3C1C2 subunits with Xase (Fay et al., "Model for the Factor VIIIa-dependent Decay of the Intrinsic Factor Xasc: Role of Sυbυnit Dissociation and Factor IXa-catalyzed Proteolysis," J Biol Chem. 271 :6027-6032 (1996), which is hereby incorporated by reference in its entirety). [0125] Results obtained in the absence or presence of added factor IXa are shown in Figures 7A and 7B, respectively. In the absence of factor IXa. WT factor VIIIa lost 50% of its activity in -8 min (Figure 7A), whereas this level of activity persisted for -40 min when factor IXa was included during factor VIII activation (Figure 7B). Decay rate values are shown in Table 3 and indicate a >5-fold stabilization of cofactor activity by formation of factor Xase. Evaluation of the variants revealed that both Olu272Ala and -VaI forms possessed 2- and 3-fold increased rates of decay, respectively, in the absence of factor IXa as compared to the WT control. These results indicate a weakened inter-sυbunit affinity with either mutation, possibly the result of loss of a relatively weak affinity bonding interaction involving the acidic side chain. In the presence of factor IXa, decay rates for the two variants were essentially indistinguishable from that of WT, indicating that inclusion of factor IXa eliminated any detrimental interaction generated by the mutations at this residue. [0126] Mutations at the other three sites (Asp519, Glu665 and GluI 984) all resulted in reductions in factor VIIIa decay rates with the degree of reduction variable depending upon the specific residue changed and in one case, the replacement residue. Mutations at Asp519 yielded -30% reductions in decay rales that were similar for both the Ala and VaI variants when factor IXa was absent. Rates for activity decay of these variants were decreased >2-fold in the presence of factor IXa, suggesting a synergy of the mutations with the stabilizing effects of binding the enzyme. While the Glu665Ala variant showed similar values to the two Asp519 variants, the Glu665Val variant showed 5-fold and 3-fold reductions in decay rates in the absence and presence of factor IXa, respectively, suggesting replacement with the larger hydrophobic residue yielded a more favorable interaction with neighboring residues for A2 subunit retention. Finally, both Glu 1984 variants showed -4-fold reductions in factor VIIIa decay compared with WT in the absence of factor IXa, and 5-8-fold reductions when factor IXa was present. The significance of this enhanced stability is observed in Figure 7B which shows >90% factor VUIa activity remaining after 40 min in factor Xase comprised of either G)u1984 variant. The similarity in responses with cither Ala or VaI at Glu 1984 suggested that both residues were well tolerated with perhaps a slightly stronger inter-subunit affinity achieved in the presence of VaI. Overall, these results demonstrate significant enhancement in factor VIIIa stability resulting from improved A2 subunit retention following selective replacement of charged residues with hydrophobic residues.

Discussion of Examples 1-7

[0127] The above Examples demonstrate that substitution of selected charged residues with hydrophobic ones at sites predicted to interface the A2 domain resulted in a general, though variable, increase in the stability- of factor VlIl. This stability was assessed following activity retention at elevated temperature as well as by reduction in the rate of A2 subunit dissociation in the cefaclor.

[0128] In the initial analysis of Examples 1 -3, 30 residues localized to the factor VIII A2 domain interface were selected for mutational analysis based upon spatial separations of <2.8 A. which could potentially form hydrogen-bonding partners. The 30 charged/polar residues were mutated to Ala (or Phc for Tyr residues), recombinant proteins stably expressed, and rates of loss of activity were measured. Fourteen of the 30 residues examined showed >2-fold increases in rates of factor VlII decay at 55C and/or rates for factor VIIIa decay relative to WT, suggesting that multiple residues at the A1 A2 and A2 A3 domain interfaces contribute to the stabilization of factor VlII. Interestingly, two acidic residues that were examined, Asp302 and Glu287, yielded modest (<2-fold) enhancement in stability in both the procofactor and active cofactor forms when mutated to Ala. Both of these acidic residues are conserved in human, canine, porcine, mouse, rabbit, rat, and bat factor VIlI. These initial results suggested that these acidic side chains did not contribute to stabilizing hydrogen-bonding interactions but rather were somewhat detrimental to factor VIII structure as assessed by functional stability. [0129] Based on these initial studies, the creation of additional hydrophobic interaction.; were assessed for gain of function. The four acidic residues examined in Examples 4-7 are conserved in human, canine, porcine, mouse, rabbit, and bat factor VIIl, while Glu665 is Ala and ϋlu1984 is Thr in rat factor (see Swiss Institute of Bioinformatics, online analysis UniProtKB/Swiss-Prot Release 55.5 and UniProtK-B/Trl-MBL Release 38.5 (2008), which is hereby incorporated by reference in its entirety). The results of Examples 4-7 demonstrate that three of these residues, Asp519, Glu665 and Glu 1984, when replaced with Ala and/or VaI, resulted in enhancements in protein stability. Only one acidic residue evaluated in Examples 4-7 yielded results that were detrimental to activity when mutated. Mutation at Glu272 to Ala yielded a low specific activity factor VIII form with reduced thrombin generation parameters values; and both Ala and Vai replacements possessed moderately decreased thermostability and 2-3-fold higher rates of A2 subunit dissociation in the cofactor form as compared with WT, From these observations, it is believed that Glu272 may indeed participate in bonding interaction^) with neighboring residues, and subsequent mutations at this site disrupt these interactions. This conclusion is consistent with examination of the Hemophilia A database (Kcmball-Cook et al., " The Factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4," Nucleic Acids Res. 26:216-219 (1998); Kemball-Cook (MRC Clinical Sciences Centre), Haemophilia A Mutation Database (accessed July 2. 2008), each of which is hereby incorporated by reference in its entirety), which lists Lys (charge reversal) or Gly (small side chain) at position 272 as a moderate/mild phenotype with reduced factor VlII antigen. The latter observation is consistent with the mutations yielding increased plasma instability. However, there was no significant effect of these mutations on levels of expression in cell culture following mutations at this site to Ala or VaI. Conversely, no mutations at Asp519, Glu665 and Glu1984 are listed in the database.

[0130] Proteins tend to fold so that the charged or polar moieties remain solvent exposed while hydrophobic groups are buried (Pace et al., ' Forces Contributing to the Conformational Stability of Proteins/' FASEBJ. 10:75-83 ( 1996), which is hereby incorporated by reference in its entirety). Therefore, based upon the observed gain-of-ftmction mutations at Glu287. Asp302, Asp519, Glu665 and Glu 1984 when these residues are replaced with hydrophobic ones, it is believed that these charged residues are buried at the A2 domain interfaces. Furthermore, these results suggest that these acidic residues do not contribute to electrostatic bonding interactions and are likely destabilizing to WT protein structure and/or subunit interactions. [0131] Because mutagenesis using either Λla or VaI resulted in a hydrophobic residue (to replace the charged acidic residues), the Ala or VaI substitution would tend to stabilize other hydrophobic contacts at the interface. Furthermore, the VaI side chain is larger than Ala, so comparison of effects on activity following replacement at a given site may offer some insights into residue packing and volume at that site. For example, that replacement of Glu 1984 with either residue yielded similar results, suggesting both were well-tolerated at that site; whereas Glu665Val showed a 3-fold reduced factor VFIIa decay rate compared with Glu665Ala. suggesting the larger volume side chain of VaI was better tolerated in the putative hydrophobic binding pocket.

[0132] Overall, the results of Example 1 -7 contribute significantly to the understanding of factor VlIl A domain structure, which has previously been limited to models derived from homology with a high resolution structure for ceruloplasmin (Pemberton et al., "A Molecular Model for the Triplicated A Domains of Human Factor VIII Based on the Crystal Structure of Human Ceruloplasmin," Blood 89:2413- 2421 (1997), which is hereby incorporated by reference in its entirety) and a recent, intermediate resolution structure (3.75 Λ) of human factor VIII (Shen et al., "The Tertiary Structure and Domain Organization of Coagulation Factor VIII," Blood 1 11 : 1240-1247 (2008), which is hereby incorporated by reference in its entirety). While the latter structure does not allow for assignments of hydrogen bonding interactions (<2.8 A), the authors of that study indicate that the A domains of factor VIlI can be superimposed onto those of ceruloplasmin with a high degree of accuracy. [0133] While the ceruloplasmin model suggests Asp302 and Glu287 could contribute hydrogen bonding interactions, the stability studies of Example 1-3 demonstrate that this in unlikely. Instead, these acidic side chains are believed to be buried in a hydrophobic environment. Conversely, results from Examples 4-7 support the belief that Glu272 likely contributes a hydrogen bonding interaction at the A2 domain interface, because loss of this charge reduces factor VIIl (VIIIa) stability. The remaining three acidic residues evaluated in Examples 4-7 appear to be buried at the interface as predicted by the model, in that no polar atom from a neighboring residue on a complementary domain appears to localize near the carboxylic groups of these residues. Rather, it is noted that these moieties appear to be proximal with hydrophobic groups. For example, the model predicts that the carboxyl oxygen of Asp519 and methyl carbon of Thr275 are separated by ~4.2 A, the carboxyl oxygen of Glu665 and methyl carbon of VaI 1982 are separated by -8.1 A, and the carboxyl oxygen of Glu 1984 and methyl carbon of Va1663 are separated by -6.2 A. [0134] Factor VlU variants demonstrating enhanced stability and reduced rates of co factor activity decay represent positive attributes for a therapeutic preparation. The former property should allow for increased yields of active protein during its purification and formulation, resulting in overall higher specific activity values. These reagents may also possess a longer circulating half-life relative to WT (see Figure 6), exclusive of various cellular clearance mechanisms. Two groups have previously reported on factor VIlI variants where cofactor activity has been stabilized by reducing/eliminating the rate of A2 subunit dissociation. In both cases, mutations were employed to covalently link the A2 domain to other regions of the molecule. In one case, an inactivation-resistant factor VIII was prepared by linking the A2 domain to a segment of B-domain contiguous with the A3C1 C2 domains and lacking thrombin cleavage sites that would release either the A2 domain or B-domain fragment following procofactor activation (Pipe et al., "Characterization of a Genetically Fngineered Inactivation-resistant Coagulation Factor VIIIa," Proc Nail Acad Set USA 94:1 1851-1 1856 (1997), which is hereby incorporated by reference in its entirety). In a second case, selected residues in the A3 and A2 domains that were in close proximity were replaced with Cys residues so as to form disulfide bridges between the two domains such that A2 would remain covalent with A3 following thrombin activation (Gale et al., "An Engineered Interdomain Disulfide Bond Stabilizes Human Blood Coagulation Factor VIIIa," J Thromb Huemost. 1 : 1966- 1971 (2003); Radtke et al. "Disulfide Bond-stabilized Factor VIlI has Prolonged Factor VIIIa Activity and Improved Potency in Whole Blood Clotting Assays," J Thromb Haemost. 5: 102-108 (2007), each of which is hereby incorporated by reference in its entirety). The latter mutants also demonstrated augmented activity in thrombin generation assays, although the reaction conditions employed in these studies used a sub-physiologic (<0.5 nM) concentration of factor VIII. [0135] The results of Examples 1 -7 using a physiologic factor VIII level ( 1 nM) showed little difference between WT and the variants demonstrating higher stability, although the Glu272Ala yielded reduced thrombin generation parameters consistent with its lower specific activity. The failure to observe a significant difference with the high stability variants may reflect differences in reaction conditions and/or that these mutations do not covalently bridge the A2 domain and the rales for factor VfIIa decay are not sufficiently reduced.

[0136] Results presented in Examples 1 -7 demonstrate several-fold decreases in rates for cofacior inactivation can be achieved following single point mutations to convert acidic residues to hydrophobic ones. In each of these case, these mutations occur at interfaces where the altered residues are likely buried and not surface exposed, and do not alter covalent interactions within the protein. Based on preliminary results, the cofactor forms of the Glu 1984VaI and -Ala variants and the WT cofactor show similar rates of inactivation as measured by activated protein C- catalyzed cleavages at Arg336 and Arg562 (Varfaj Ct al., "Residues Surrounding Arg336 and Arg562 Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C," J. Biol. Chem. 282(28):20264-72 (2007), which is hereby incorporated by reference in its entirety). This supports the belief that down- regulation of these higher stability variants should also proceed via the protein C pathway in much the same way as WT cofactor. Thus, the stabilized variants of the present invention should be free from the problems associated with the inactivation- resistant mutants described above.

Example 8: Stability Analysis of Di- and Tri-substituted Factor V1H Variants

[0137] To determine whether additive or synergistic effects will result in further enhancements in factor VIlI (VIIIa) stability, combinations of the point mutations described in the preceding Examples have been prepared using the same procedures described in the Materials and Methods. In particular, double or triple combination mutants were prepared with the Ala or VaI substitutions of residues Asp519, Glu665. and Glu 1984. These combination mutants (amino acids are identified using the single letter code) include: D519AE665A, D519AE665V, D519AE 1984A, D519AE1984V, D5 I 9VE665V. D519VE1984A, D519VEI 984V, E665AH1984A, E665AE1984V, E665VE1984A, E665VE1984V,

D519AE665VE1984A, D519VE665VE1984A, D519VE665VE1984V.

D519VE665 A factor VlII was excluded from this analysis, because this mutant showed atypical characteristics in EUSA and Western blot results. [0138] To produce triple mutants, D519A or D519V was combined with either E665VE1984A or E665VE1984V. The other combinations were eliminated because the E665AE1984A and E665AE1984V double mutants did not enhance boih factor VIII and factor VIIIa stability as compared with each single mutant. Results using D519AE665VE1984V were excluded for the same reasons as observed for D519VE665A.

[0139] The first group of new mutants (Group A), which combined mutation

(Ala or VaI) at Asp519 with mutation at either Glu(>65 or (Jlu1984, retained normal values for specific activity (>80% the wild type (WT) value, Figure 8). Interestingly D519AH665A, D519VE665V, D519VE1984A, and D519VE1984V showed significantly increased specific activity values up to - 1.8 fold compared to WT factor VIlI as measured by a one stage clotting assay (Figure 8). The specific activity of the second group of mutants (Group B), which were the combination of mutations at Glu665 and Glu 1984 unexpectedly showed reduction in specific activity of up to ~2 fold compared lo WT factor VIII. with the exception of E665VE1984A which was somewhat greater than the WT value (Figure 8). The third group (Group C) represents the triple mutations and showed normal to modestly increased activity by the one-stage assay (D519VE665VE1984V). However, the activity of D519VE665VE1984V as measured by two-stage assay was significantly reduced. Since Asp519 is located at the A1 and A2 interface while Glu665 and Glu 1984 arc located at the A2 and A3 interface, it is believed that the tendency for elevated specific activity for the Group A mutations compared with Group B may result from more favorable interactions at the A1 -A2 junction that afϊcct conformation and preserve the active cofactor form. [0140] Figure 9 shows the summary of the results from factor VIIi thermal stability experiments conducted at 55C. The rate values obtained for the combination mutants were compared with rate values from the best single mutant in that particular combination using data for the single mutations obtained from Example 5 (Figure 5A). Figure 9 also shows the actual value for the rate of factor VIII decay (see also Table 4). The degree of reduction of the relative decay rates appear to relate to the enhancement observed for the combination of mutations. In Group A, mutants D519AE665Λ, D519AD665V. and D519VE665V showed significant enhancement in stability (reductions in decay rates) and most of the mutants also maintained an absolute decay rate that was ~ 50% the WT value. On the other hand, the relative rates for two of the Group B mutants were somewhat increased (E665AR1984A and E665AE1984V) as compared with the better single mutation. In Group C, mutants D519AE665VE1984A and D519VE665VE1984A showed no significant change in the rate while the rate value for D519VE665 VE 1984V was slightly increased. [0141] Interestingly, the enhancement of stability observed for the combination of mutations was more easily observed for the factor VIIIa forms (Figure 10). To increase the sensitivity of the factor VIIIa decay assay for highly stable mutants, a lower concentration of factor VIIIa ( 1.5 nM) for the incubation than was employed in the preceding Examples. I .arge stability enhancements of up to -4 fold compared to the single mutants were observed for all Group A mutants. Actual values for the factor VIIIa decay rates of D519VE665V and D519VE1984A were 14 and 12% of that of WT factor VfII. respectively (Figure 10 and Table 4). Group B mutants in general yielded poorer results when compared with the better individual mutation in the pairing, with E665 AE 1984A and E665 AE 1984V, showing ~2.2 and -2.7 told increases, respectively, in relative decay rate values. The triple mutations (Group C) showed the largest factor VlUa stability enhancements with maximal stability observed for D519VE665VE1984A, which showed a decay rate that was -10% of WT (Figure 10 and Table 4).

Table 4: Factor VIII and Villa Decay Rates and Activity Values for Combination Mutants

[0142] A thrombin generation assay was performed on selected mutants and the results are shown in Figures 11A-B. There was no significant improvement in thrombin generation profiles when the single mutants were tested using a final concentration of 1 nM factor VIIl (see Materials & Methods, and Example 4 above). To bcπcr compare the more stable factor VIIl mutants, a lower factor VlII concentration (0.2 nM) was used. Results from this analysis showed that -2.3 fold increase in the peak height and -1.5 fold increase in endogenous thrombin potential (ETP) compared to WT factor VIIl (Figures 1 1A-B). Although the lag time and peak time values for D519AE665V, D519VH1984A, and D519VE665VE1984A were not changed significantly relative to WT, the peak height and ETP values were significantly greater than WT (-20% to 70%). Overall, these results indicate that the selected four combination mutants ali possessing enhanced factor VIIIa stability showed improved thrombin generation profiles. This observation indicates that these mutants may have greater capacity for increased thrombin generation per unit concentration factor VIIl in a physiologic situation.

Example 9: Ala or VaI Mutants at Asp519, Glu665, and CIu 1984 in

Combination with High Specific Activity GluI 13Ala (E113A) Mutation

[0143] The El 13A mutation is known to enhance factor VlII specific activity as judged by a one-stage clotting assay (U.S. Patent Application Publ. No. 10/581.471 to Fay et al.; Wakabayashi et al.. ' A Glul 13Ala Mutation within a Factor VIII Ca(2+)-Binding Site Enhances Cofactor Interactions in Factor Xase," Biochemistry 44:10298-10304 (2005), each of which is hereby incorporated by reference in its entirety). Since the generation of factor VIII with both high stability and high specific activity represents a unique class of reagents for potential therapeutic application in the treatment of hemophilia, the effect of combined mutation of El 13A with the high stability1 mutants described in the preceding Examples was analyzed. [0144] Ala or VaI mutants at Asp519, Glu665, and Glu1984 were prepared in combination with the E113A mutation using the same procedures described in the Materials and Methods. These double mutants (amino acids are identified using the single letter code) include: E113AD519A, El 13AD519V, El 13AE665A, E113AE665V, and F.I 13AE1984V.

[0145] Specific activity values determined using the one-stage assay for the combined mutants were -2 to -3.3 fold higher than WT factor VIII while keeping the normal level of activity by two-stage assay. These results indicate that mutations at Asp519, Glu665, or Glul 984 did not adversely alϊect the activity enhancement observed for the El 13A mutation (Figure 12A). In addition, the factor VIlI and VIIIa decay rates of EI 13 A in combination with the high stability mutants were not significantly different from the value of each original single high stability mutant (see Figures 5B-C; Table 5), suggesting that the EH 3A mutation did not affect the enhanced stability parameters for these mutants.

Table 5: Factor VIlI and Villa Decay Rates and Activity Values

[0146] From the foregoing results, the mutation of El 13 A can be combined with any of the currently described increased stability mutations for the purpose of generating a recombinant factor VIlI characterized by both increased specific activity and enhanced factor Vlll/factor VlIIa stability. This includes the combination of il 13A (or other suitable El 13 substitutions as described in U.S. Patent Application Serial No. 10/581 ,471 to Fay el al., which is hereby incorporated by reference in its entirety) with single or multiple stability-enhanced mutants of the type described herein. [0147] 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.

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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
WO2011101277A1 *Feb 9, 2011Aug 25, 2011Novo Nordisk A/SConjugated proteins
Classifications
International ClassificationC12Q1/68, C12P21/06, C07K14/755
Cooperative ClassificationA61K35/33, C07K14/755, C12N15/86
European ClassificationC07K14/755
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