Factor Viii Complex With Xten And Von Willebrand Factor Protein, And Uses Thereof

Abstract

The present invention provides a chimeric protein comprising a VWF protein comprising the D' domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain comprising a VWF fragment of the invention binds to or is associated with a polypeptide chain comprising a FVIII protein linked to an XTEN sequence and the polypeptide chain comprising the VWF fragment can prevent or inhibit binding of endogenous VWF to the FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to the FVIII protein, which is a half-life limiting factor for FVIII, the VWF fragment can induce extension of half-life of the chimeric protein comprising a FVIII protein. The invention also includes nucleotides, vectors, host cells, methods of using the VWF fragment, or the chimeric proteins.

Description

RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser. No. 16/154,310, filed Oct. 8, 2018, which is a division of U.S. patent application Ser. No. 14/413,765, filed Jan. 9, 2015, now U.S. Pat. No. 10,138,291, which is a 35 U.S.C. .sctn. 371 filing of International Patent Application No. PCT/US2013/049989, filed Jul. 10, 2013, which claims priority to U.S. Provisional Patent Application Nos. 61/840,811, filed Jun. 28, 2013; 61/827,158, filed May 24, 2013; 61/801,504, filed Mar. 15, 2013; 61/801,544, filed Mar. 15, 2013; 61/759,819, filed Feb. 1, 2013; and 61/670,401, filed Jul. 11, 2012, the entire disclosures of which are hereby incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The instant application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 23, 2021, is named "719865_SA9-428USDIV2_ST25.txt" and is 577,268 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Haemophilia A is a bleeding disorder caused by defects in the gene encoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients affected with hemophilia A can be treated with infusion of purified or recombinantly produced FVIII. All commercially available FVIII products, however, are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to the patients. See Weiner M. A. and Cairo, M. S., Pediatric Hematology Secrets, Lee, M. T., 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried in order to extend the FVIII half-life. For example, the approaches in development to extend the half-life of clotting factors include pegylation, glycopegylation, and conjugation with albumin. See Dumont et al., Blood. 119(13): 3024-3030 (Published online Jan. 13, 2012). Regardless of the protein engineering used, however, the long acting FVIII products currently under development are reported to have limited half-lives--only to about 1.5 to 2 hours in preclinical animal models. See id. Consistent results have been demonstrated in humans, for example, rFVIIIFc was reported to improve half-life up to .about.1.7 fold compared with ADVATE.RTM. in hemophilia A patients. See Id. Therefore, the half-life increases, despite minor improvements, may indicate the presence of other T1/2 limiting factors. See Liu, T. et al., 2007 ISTH meeting, abstract #P-M-035; Henrik, A. et al., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131.

[0004] Plasma von Willebrand Factor (VWF) has a half-life of approximately 12 hours (ranging from 9 to 15 hours). http://www.nhlbi.nih.gov/guidelines/vwd/2_scientificoverview.htm (last visited Oct. 22, 2011). The VWF half-life may be affected by a number of factors: glycosylation pattern, ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motif-13), and various mutations in VWF.

[0005] In plasma, 95-98% of FVIII circulates in a tight non-covalent complex with full-length VWF. The formation of this complex is important for the maintenance of appropriate plasma levels of FVIII in vivo. Lenting et al., Blood 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost. 5(7): 1353-60 (2007). The full-length wild-type FVIII is mostly present as a heterodimer having a heavy chain (MW 200 kD) and a light chain (MW 73 kD). When FVIII is activated due to proteolysis at positions 372 and 740 in the heavy chain and at position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII. The activated FVIII, together with activated factor IX, calcium, and phospholipid ("tenase complex"), induces the activation of factor X, generating large amounts of thrombin. Thrombin, in turn, then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form the soluble fibrin polymer. Thrombin also activates factor XIII, which, together with calcium, serves to crosslink and stabilize the soluble fibrin polymer, forming crosslinked (insoluble) fibrin. The activated FVIII is cleared fast from the circulation by proteolysis.

[0006] Due to the frequent dosing and inconvenience caused by the dosing schedule, there is still a need to develop FVIII products requiring less frequent administration, i.e., a FVIII product that has a half-life longer than the 1.5 to 2 fold half-life limitation.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention is directed to a chimeric protein comprising (i) a von Willebrand Factor (VWF) fragment comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker and wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein. The chimeric protein can comprise a single polypeptide chain comprising the VWF fragment, the XTEN sequence, and the FVIII protein, or two polypeptide chains, a first chain comprising the VWF fragment and the second chain comprising the FVIII protein, wherein the XTEN polypeptide is linked either to the VWF fragment or the FVIII protein.

[0008] In one embodiment, the chimeric protein of the invention comprises a formula comprising:

V-X-FVIII, (a)

FVIII-X-V, (b)

V-X:FVIII, (c)

X-V:FVIII, (d)

FVIII:V-X, or (e)

FVIII:X-V, (f)

wherein V comprises a VWF fragment, X comprises one or more XTEN sequences, and FVIII comprises a FVIII protein. The hyphen (-) can be a peptide bond or a linker, e.g., a cleavable linker, while the colon (:) represents a chemical association or a physical association between the polypeptides, for example a covalent or non-covalent bond.

[0009] In another embodiment, the chimeric protein further comprises (iv) an immunoglobulin (Ig) constant region or a portion thereof (also indicated as F1 or a first Ig constant region or a portion thereof) linked to the VWF fragment, the XTEN sequence, the FVIII protein, or any combinations thereof. In other embodiments, the chimeric protein further comprises an additional Ig constant region or a portion thereof (also indicated as F2 or a second Ig constant region or a portion thereof). The first Ig constant region or a portion thereof can be linked to the VWF fragment or the XTEN sequence, and the second Ig constant region can be linked to the FVIII protein. The first Ig constant region, the second Ig constant region or a portion thereof, or both can extend the half-life of the FVIII protein.

[0010] In some embodiments, the second Ig constant region or a portion thereof (F2) is linked to the VWF fragment by a linker, e.g., a processable linker. In other embodiments, the second Ig constant region or a portion thereof (F2) is associated with the (first) Ig constant region or a portion thereof (F1). The second Ig constant region or a portion thereof (F2) and the first Ig constant region or a portion thereof (F1) can be identical or different. The second Ig constant region or a portion thereof can be associated with the Ig constant region or a portion thereof by a covalent bond, e.g., a disulfide bond. The VWF fragment linked to the first Ig constant region or a portion thereof may also be associated with the FVIII protein linked to the second Fc region by a non-covalent bond. In certain embodiments, the FVIII protein can further comprise one or more additional XTEN sequences which are linked to the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites). In some embodiments, the half-life of the FVIII protein is extended, compared to wild type FVIII or a FVIII protein without the VWF fragment.

[0011] In some embodiments, the chimeric protein comprises a formula comprising:

V-L2-X-L1-F1:FVIII-L3-F2; (g)

V-L2-X-L1-F1:F2-L3-FVIII; (h)

F1-L1-X-L2-V:FVIII-L3-F2; (i)

F1-L1-X-L2-V:F2-L3-FVIII; (j)

V-L2-X-L1-F1-L4-FVIII-L3-F2; (k)

F2-L3-FVIII-L4-F1-L1-X-L2-V; (l)

FVIII-L3-F2-L4-V-L2-X-L1-F1; or (m)

F1-L1-X-L2-V-L4-F2-L3-FVIII, (n)

wherein V comprises a VWF fragment, each of L1, L2, and L3 comprises an optional linker, e.g., a cleavable linker, L4 is an optional linker, e.g., a processable linker, FVIII comprises a FVIII protein, X comprises one or more XTEN sequences, F1 comprises an optional first Ig constant region or a portion thereof, F2 comprises an optional second Ig constant region or a portion thereof, and (:) is a covalent bond or non-covalent bond.

[0012] The present invention is also directed to a chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more insertion sites) and wherein the Ig constant region or a portion thereof is linked to or associated with the FVIII protein or the XTEN sequence. In one embodiment, the Ig constant region or a portion thereof useful for the chimeric protein comprises a first Fc region. In another embodiment, the chimeric protein further comprises an additional Ig constant region or a portion thereof. The additional Ig constant region or a portion thereof useful for the invention can comprise a second Fc region, which is linked to or associated with the first Fc region, e.g., by a covalent bond. In other embodiments, the first Fc region is linked to the second Fc region by a linker, e.g., a processable linker.

[0013] In other aspects, a chimeric protein comprises (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, and (iv) an Ig constant region or a portion thereof, which comprises the D' domain and the D3 domain of VWF, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more insertion sites), the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof. Non-limiting examples of the chimeric proteins may comprise a formula, which comprises:

FVIII(X1)-L1-F1:V-L2-X2-L3-F2; (1)

FVIII(X1)-L1-F1:F2-L3-X2-L2-V; (2)

F1-L1-FVIII(X1):V-L2-X2-L3-F2; (3)

F1-L1-FVIII(X1):F2-L3-X2-L2-V; (4)

FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2; (5)

FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V; (6)

F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (7)

F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V, (8)

wherein FVIII(X1) comprises a FVIII protein and one or more XTEN sequences, wherein one or more of the XTEN sequences are linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites); each of L1, L2, or L3 comprises an optional linker, e.g., a cleavable linker; L4 is a linker, a processable linker; X2 comprises one or more XTEN sequences; F1 comprises an Ig constant region or a portion thereof; F2 comprises an optional additional Ig constant region or a portion thereof, and V comprises a VWF fragment; (-) is a peptide bond or one or more amino acids; and (:) comprises a covalent bond or a non-covalent bond.

[0014] One aspect of the invention is that the VWF fragment useful for the chimeric protein does not bind to a VWF clearance receptor, which prevents or inhibits interaction of the FVIII protein with endogenous VWF. The chimeric protein comprising the VWF fragment thus has reduced clearance or is not cleared through a VWF clearance pathway. Another aspect of the invention is that the VWF fragment is capable of protecting the FVIII protein from one or more protease cleavages, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors.

[0015] Because of the VWF fragment's ability to prevent or inhibit interaction between the FVIII protein and endogenous VWF, the half-life of the FVIII protein, is extended compared to a FVIII protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0016] The Ig constant region or a portion thereof useful for the chimeric protein comprises a first Fc region, which is linked to the VWF fragment by an optional linker, e.g., a cleavable linker. The chimeric protein can further comprise an additional Ig constant region or a portion thereof, which is linked to the FVIII protein or the XTEN sequence, the Ig constant region or a portion thereof, the VWF fragment, or any combinations thereof by an optional linker. In one embodiment, the additional Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker. The additional Ig constant region or a portion thereof can comprise a second Fc region.

[0017] The Ig constant region or a portion thereof useful in the present invention and the additional Ig constant region or a portion thereof useful in the present invention are identical or different.

[0018] In some aspects, the FVIII protein is linked to an XTEN sequence at the C-terminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in mature native human FVIII (e.g., one or more insertion sites) or any combinations thereof. One or more insertion sites in the FVIII protein can be located within one or more domains of the FVIII protein selected from the group consisting of the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the C1 domain, the C2 domain, and any combinations thereof or between one or more domains of the FVIII protein selected from the group consisting of the A1 domain and a1 acidic region, the a1 acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and C1 domain, the C1 domain and C2 domain, and any combinations thereof or between two domains of the FVIII protein selected from the group consisting of the A1 domain and a1 acidic region, the a1 acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and C1 domain, the C1 domain and C2 domain, and any combinations thereof.

[0019] In one embodiment, the one or more insertion sites are located immediately downstream of one or more amino acids in mature native human FVIII (e.g., SEQ ID NO: 4 [mature FVIII sequence-full length]) selected from the group consisting of the amino acid residues in Table 7, 8, 9, 10, 11, or any combinations thereof.

[0020] In another embodiment, the one or more insertion sites are located in one or more permissive loops of mature native human FVIII. In other embodiments, the one or more insertion sites are located in the a3 region of mature native human FVIII. For example, an XTEN sequence can be inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 (full length mature FVIII). In other embodiments, a FVIII protein is linked to at least two XTEN sequences, a first XTEN sequence inserted within the a3 region, and a second XTEN sequence inserted within a permissive loop in the FVIII protein (e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2). In still other embodiments, a FVIII protein is linked to at least three XTEN sequences, a first XTEN sequence inserted within the a3 region and a second XTEN sequence and a third XTEN sequence inserted within one or two permissive loop in the FVIII protein (e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2).

[0021] In certain embodiments, the one or more insertion sites for one or more XTEN insertions are immediately downstream of one or more amino acids (numbered relative to mature FVIII sequence) selected from the group consisting of:

TABLE-US-00001 (1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) amino acid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) amino acid 1937, (52) amino acid 2019, (53) amino acid 2068, (54) amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188, (58) amino acid 2227, (59) amino acid 2277, and (60) two or more combinations thereof.

[0022] In some embodiments, one XTEN is inserted in the FVIII protein. In some embodiments, two XTENs are inserted in the FVIII protein. In some embodiments, 3 XTENs are inserted in the FVIII protein.

[0023] In a particular example, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4 (full-length mature FVIII). In another example, a first XTEN is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In some examples, a first XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In other examples, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In yet other embodiments, a first XTEN is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In still other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In certain embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4 (full-length mature FVIII), a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 2, a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a fourth XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In another example, an XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4. In an additional example, a first XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a second XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In another example, a first XTEN is immediately inserted downstream of amino acid 403 corresponding to SEQ ID NO: 4 and a second XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 745 of corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4.

[0024] In some embodiments, the FVIII protein is a dual chain FVIII isoform. In some embodiments, the FVIII protein is a single chain FVIII isoform.

[0025] In some embodiments, the XTEN that is inserted is SEQ ID NO: 39 (AE288). In some examples, the XTENs that are inserted are SEQ ID NOs: 38 and 37 (AG144 and AE144). In some examples, the XTENs that are inserted are SEQ ID NOs: 37, 38 and 37 (AE144, AG144, and AE144). In some embodiments. the XTENs that are inserted are SEQ ID NOs: 37 and 40 (AE144 and AE288). In some embodiments, the XTENs that are inserted are AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combinations thereof.

[0026] The FVIII protein useful in the invention can comprise B domain or a portion thereof, e.g., SQ B domain deleted FVIII. In one embodiment, the FVIII protein comprises single chain FVIII. In another embodiment, the single chain FVIII contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). In other embodiments, the amino acid substitution is an amino acid other than arginine. In some embodiments, the FVIII protein comprises a heavy chain of FVIII and a light chain of FVIII, wherein the heavy chain and the light chain are associated with each other by a metal bond.

[0027] The FVIII protein can have a low affinity to or does not bind to a low-density lipoprotein receptor-related protein (LRP), e.g., by containing at least one amino acid substitution that lowers the affinity to or eliminates the binding to the LRP. Such at least one amino acid substitution can be at a residue corresponding to residue 471, residue 484, residue 487, residue 490, residue 497, residue 2092, residue 2093 or two or more combinations thereof of full-length mature FVIII. In a particular embodiment, the amino acid substitution at residue 471, 484, or 497 is an amino acid other than arginine, the amino acid substitution at residue 487 is an amino acid other than tyrosine, the amino acid substitution at residue 2092 is an amino acid other than lysine, or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine.

[0028] In some embodiments, the FVIII protein contains at least one amino acid substitution, which induces the FVIII protein to be more stable than a FVIII protein without the substitution. Such substitutions can be located in the A2 domain and the A3 domain of the FVIII protein, e.g., at a residue corresponding to residue 664, residue 1826, residue 662, residue 1828, or two or more combinations thereof of full-length mature FVIII.

[0029] The VWF fragment useful for the present invention comprises a D' domain and D3 domain, which together are capable of binding to FVIII. The VWF fragment can comprise the amino acid sequence of the D' domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2 and/or the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2. In one embodiment, the VWF fragment is a monomer. In another embodiment, the VWF fragment comprises at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments. In one embodiment, the two or more VWF fragments may be identical or they may be different. The VWF fragment can comprise an amino acid at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2. The VWF fragment may consist essentially of or consist of amino acids 764 to 1240 of SEQ ID NO: 2. In certain embodiments, the VWF fragment can contain at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. In other embodiments, the VWF fragment further comprises the D1 domain, the D2 domain, or the D1 and D2 domains of VWF.

[0030] The VWF fragment may further comprise a VWF domain selected from the group consisting of the A1 domain, the A2 domain, the A3 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, and any combinations thereof. For example, the VWF fragment can consist essentially of or consist of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the D1, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof, (4) the D1, D2, D', and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3, and A1 domains of VWF or fragments thereof. In some embodiments, the VWF fragment further comprises a signal peptide of VWF or FVIII which is operably linked to the VWF fragment.

[0031] One or more of the linkers useful in the invention have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. In some embodiments, one or more of the linkers have a length of about 1 to about 2000 amino acids. In one embodiment, one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids. In another embodiment, one or more of the linkers comprise a gly/ser peptide, an XTEN sequence, or both. Examples of the gly/ser peptide include, but are not limited to, a formula of (Gly.sub.4Ser).sub.n (SEQ ID NO: 139) or S(Gly.sub.4Ser).sub.n (SEQ ID NO: 140), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. For example, the (Gly.sub.4Ser).sub.n linker can be (Gly.sub.4Ser).sub.3 (SEQ ID NO: 63) or (Gly.sub.4Ser).sub.4 (SEQ ID NO: 138). In one embodiment, the linker comprises at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both. In another embodiment, the linker comprises 20 amino acids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acids thrombin cleavable linker. The cleavable linkers can comprise one or more of the cleavage sites by a protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, Granzyme-B, TEV, Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP-20, e.g., TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8). Non-limiting examples of one or more of the cleavage sites comprise an amino acid sequence selected from the group consisting of RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO:23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28), GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34), and LPKTGSES (SEQ ID NO: 35). In some embodiments, the first cleavage site and the second cleavage site are identical or different.

[0032] The XTEN sequence useful for the invention can be selected from the group consisting of AE42 (SEQ ID NO: 36), AE144 (SEQ ID NO: 37), AG144 (SEQ ID NO: 38), AE288 (SEQ ID NO: 39), AG288 (SEQ ID NO: 40), AE576 (SEQ ID NO: 41). AG576 (SEQ ID NO: 42), AE864 (SEQ ID NO: 43), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO:135), AG144_F (SEQ ID NO: 136), AE288_2 (SEQ ID NO: 137), or AG864 (SEQ ID NO: 44). In a particular embodiment, the XTEN sequence comprises AE288 or AG288.

[0033] The chimeric protein of the invention can be polysialylated, pegylated, or hesylated.

[0034] The present invention is also directed to a polynucleotide or a set of polynucleotides encoding the chimeric protein. The polynucleotide can further comprise a polynucleotide chain, which encodes PC5 or PC7. The invention is also directed to a vector comprising the polynucleotide or the set of polynucleotides and one or more promoter operably linked to the polynucleotide or the set of polynucleotides. The vector can further comprise an additional vector, which comprises a polynucleotide chain encoding PC5 or PC7. The invention is also drawn to a host cell comprising the polynucleotide or the vector. The host cell can be a mammalian cell, e.g., HEK293 cell, CHO cell, or BHK cell. In some embodiments, the PC5 or PC7 of the host cell cleaves the D1D2 domains of VWF.

[0035] The invention is also directed to a pharmaceutical composition comprising the chimeric protein, the polynucleotide, the vector, or the host cell, and a pharmaceutically acceptable carrier. The composition of the invention thus has an extended half-life compared to wild type FVIII protein. The half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. The half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0036] The composition of the present invention can be administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration. In one embodiment, the composition is administered via parenteral administration, e.g., intravenous or subcutaneous administration. The composition of the invention is useful to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof. In one embodiment, the subject treated with the chimeric protein is scheduled to undergo a surgery. In another embodiment, the treatment is prophylactic or on-demand.

[0037] The invention is also directed to a method of preventing or inhibiting binding of a FVIII protein with endogenous VWF comprising adding an effective amount of the chimeric protein, the polynucleotide vector, the host cell, or the composition to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus prevents or inhibits binding of endogenous VWF. The present invention is further directed to a method of extending or increasing the half-life of the FVIII protein, wherein the method comprises administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases the half-life of the FVIII protein. Also provided is a method of preventing or inhibiting clearance of a FVIII protein from a cell, wherein the method comprises administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition to a cell comprising a FVIII protein or a polynucleotide encoding the FVIII protein, wherein the protein having VWF activity binds to the FVIII protein. The subject useful for the present methods is an animal, e.g., a human, e.g., a patient suffering from hemophilia A.

[0038] The present invention also provides a method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition, wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath. The treatment can be prophylactic or on-demand. In one embodiment, the effective amount is 0.1 .mu.g/kg to 500 mg/kg.

[0039] The invention also includes a method of making a chimeric protein, comprising transfecting one or more host cell with the polynucleotide or the vector and expressing the chimeric protein in the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0040] FIG. 1A-D. Schematic diagrams of VWF fragments. FIG. 1A shows three exemplary VWF fragments useful for the invention, e.g., VWF-002, VWF-010, and VWF-013. VWF-002 contains amino acids 1 to 477 of SEQ ID NO: 124 (amino acids 764 to 1240 of SEQ ID NO: 2) and is synthesized without the pre/propeptide sequences. VWF-010 contains the D1D2 domains in addition to the D'D3 domains. VWF-013 contains the D1D2D'D3 domains in addition to alanine residues substituting cysteines at residues 336 and 379 of SEQ ID NO: 123 FIG. 1B shows VWF-031, which contains the D1D2D'D3 domains fused to an Ig constant region or a portion thereof, e.g., an Fc region, by a cleavable linker, e.g., a 48 amino acids thrombin cleavable linker. FIG. 1C shows VWF-025, which is a nucleotide sequence encoding D1D2D'D3 domains contained in pLIVE vector, and VWF-029, which is a nucleotide sequence encoding D1D2D'D3 domains with two amino acid substitutions, C336A and C379A, in pLIVE vector. FIG. 1D shows full-length VWF fragment comprising propeptide (the D1 and D2 domains) and mature subunits (the D', D3, A1, A2, A3, D4, B1-3, C1-2 domains). The VWF fragment is about 250 kDa protein and forms multimers (>20 MDa) by disulfide bonding. The VWF fragment associates with FVIII (95-98%) in non-covalent complex and then extends half-life of FVIII by protecting FVIII from protease cleavage/activation, stabilizing heavy & light chain, and preventing clearance of FVIII by scavenger receptors. The VWF fragment also can limit half-life of FVIII by clearance of FVIII-VWF complex through VWF receptors and preventing pinocytosis and recycling of rFVIIIFc.

[0041] FIG. 2. Pharmacokinetic profile of rFVIII-XTEN (rFVIII-AE288 or rFVIII-288AE) in VWF D'D3 expression mice or in FVIII and VWF double knockout (DKO) mice. FIG. 2A shows the timeline of hydrodynamic injection (HDI) of the D'D3 domain encoding plasmid DNA (VWF-025) (day -5), intravenous dosing of rFVIII-XTEN AE288 (day 0), and PK sample collection (day 5). FIG. 2B shows FVIII activity measured by a FVIII chromogenic assay after IV dosing of rFVIII-XTEN288 in D1D2D'D3 mice (inverted triangle) and rFVIII-XTEN288 in DKO mice (diamond). FIG. 2C shows the D'D3 plasma level (ng/mL) after administration of VWF-025. The X axis represents time in hours.

[0042] FIG. 3. Schematic diagram of exemplary VWF:FVIII heterodimer constructs. The constructs have the common structure represented as formula FVIII-F1-L1-V-X-L2-F2, but contain examples of different variable linkers. The construct (FVIII-161) shown contains a heterodimeric FVIII (the heavy chain and the light chain are associated by a metal bond) linked to a first Fc region and a VWF fragment, which is the D' and D3 domains of VWF (i.e., amino acids 1 to 477 of SEQ ID NO: 2 with amino acid substitutions C336A and C379A) linked to an XTEN sequence, which is further linked to a cleavable linker and a second Fc region. The XTEN sequence contained in FVIII-161 is an XTEN AE288 sequence, and the linker is a thrombin cleavable linker, which has 35 amino acids. In FVIII-161, the FVIII protein linked to the first Fc region is linked to the VWF fragment by a processable linker. Upon expression, the processable linker can be cleaved by an intracellular processing enzyme, thus making the construct three polypeptide chains associated with each other.

[0043] FIG. 4 is schematic diagrams of FVIII-VWF heterodimer or monomer examples. FVIII-168, FVIII-175, FVIII-172, FVIII-174, and FVIII170. Construct FVIII-168 comprises a single chain FVIII sequence (having an alanine residue substitute the arginine residues at residues 1645 and 1648) linked to a first Fc region, which is then fused to a VWF fragment linked to a second Fc region by a thrombin cleavable linker, which has 48 amino acids. AE288 XTEN is inserted in the B domain of the single chain FVIII sequence. The linkage between the first Fc region and the VWF fragment comprises a linker that is capable of being cleaved by an intracellular processing enzyme, i.e., processable linker. Construct FVIII-175 comprises a single chain FVIII (having an alanine residue substitute the arginine residues at residues 1645 and 1648) linked to AE288 XTEN and a first Fc region, which is linked to a second Fc region by a linker, e.g., a processable linker. AE288 XTEN is inserted in the B domain of the single chain FVIII sequence. Construct FVIII-172 comprises two polypeptide chains, a first chain comprising a heavy chain FVIII sequence fused to AE288 XTEN, a second chain comprising a light chain FVIII sequence, a first Fc region, a linker (e.g., a processable linker), a VWF fragment, a thrombin cleavable linker (e.g., 48 amino acids), and a second Fc region. Construct FVIII-174 comprises two polypeptide chains, a first chain comprising a heavy chain FVIII sequence fused to AE288 XTEN and a second chain comprises a light chain FVIII, a first Fc region, a linker (e.g., a processable linker), and a second Fc region. Construct FVIII-170 comprises a VWF fragment, AE288 XTEN, a linker (e.g., a thrombin cleavable linker, which is 35 amino acids in length), and a single chain FVIII sequence.

[0044] FIG. 5. Pharmacokinetic profile of FVIII/VWF heterodimers containing an XTEN sequence in combination with an Fc region. Constructs FVIII-161, FVIII-168, and FVIII-172 were administered to FVIII:VWF double knockout (DKO) mice by Hydrodynamic injection (HDI) at 100 ug/mouse dose. Construct FVIII-170 was administered to FVIII:VWF DKO mice by HDI at 50 .mu.g/mouse dose. The post-HDI plasma FVIII activity was analyzed by FVIII chromogenic assay for 24 hr post-HDI. The FVIII activity of the FVIII:VWF heterodimers containing an XTEN sequence and Fc domains was compared with the FVIII activity of BDD-FVIII without the VWF fragment, XTEN sequence, and Fc domains.

[0045] FIG. 6. Schematic diagrams of FVIII-VWF heterodimer examples co-transfection system. FIG. 6A. Construct FVIII-169 contains the full-length FVIII sequence (with an alanine residue substituting the arginine residues at 1645 and 1648 and with an XTEN sequence inserted in the single chain FVIII sequence), which is linked to an Fc region. VWF-031 contains the D1D2D'D3 fragment (with an alanine residue substituting the cysteine residues at 336 and 379) which is linked to another Fc region with a 48 thrombin cleavable linker. After intracellular processing, construct FVIII-169 produces a full length single chain FVIII (SCFVIII) fused to one Fc fragment and an XTEN sequence, and construct VWF-031 produces a 477 amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds can be formed between the Fc fragments that are linked to the SC FVIII or the D'D3 fragment, this in turn allows a non-covalent association of FVIII and D'D3. FIG. 6B. Construct FVIII-173 contains a heterodimeric FVIII sequence, a heavy chain FVIII sequence linked to an XTEN sequence and a light chain FVIII sequence linked to an Fc region. VWF-031 is described above. After intracellular processing, construct FVIII-173 produces a heterodimeric protein, a heavy chain FVIII fused to an XTEN sequence, a light chain FVIII fused to one Fc fragment, and construct VWF-031 produces a 477 amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds can be formed between the Fc fragments that are linked to the light chain FVIII or the D'D3 fragment, this in turn allows a non-covalent association of FVIII and D'D3.

[0046] FIG. 7. Binding Affinity of Exemplary FVIII:VWF containing an XTEN sequence and Fc domains to immobilized hVWF in Octet assay. The binding affinity for FVIII-169/VWF-031 and FVIII-057 (rFVIIIFc) fused to immobilized hVWF was tested using biolayer interferometry based measurements (Octet assay). FIG. 7A shows binding response in nanomoles of FVIII169 and FVIIIFc drug substance (a positive control) to immobilized hVWF. FIG. 7B shows binding response of human IgG1 (a negative control) to immobilized human VWF.

[0047] FIG. 8. Pharmacokinetic (PK) profile of FVIII-169 in HemA and FVIII:VWF double knockout (DKO) mice. FIG. 8A shows the PK profile of FVIII-169/VWF-031 and FVIIIFc in HemA mice. HemA mice were treated with a single intravenous dose of FVIII-169/VWF-031 at 200 IU/kg. Plasma samples collected from the mice were tested by FVIII chromogenic assay. Half-life of FVIII-169/VWF-031 was calculated using WinNonlin program. FIG. 8B shows the PK profile of FVIII-169/VWF-031, FVIII-169/Fc, and FVIIIFc in FVIII/VWF DKO mice.

[0048] FIG. 9. PK profile of FVIII-XTEN variants in D'D3 expressing FVIII/VWF DKO mice. FIG. 9A shows comparison of the PK profile of the FVIII-XTEN variants, FVIII with one XTEN, FVIII with two XTENs, and FVIII with three XTENs. One, two, or three XTENs were inserted in various portions of FVIII including C-terminus and B-domain. CT indicates that an XTEN is linked to the C-terminus of FVIII. Insertion site B/CT indicates that one XTEN is inserted between amino acid residue 745 and amino acid residue 746 of the FVIII protein and another XTEN is linked to the C-terminus of the FVIII protein. The amino acid residue numbering corresponds to the SQ BDD FVIII protein sequence. Insertion site 1900/B/CT indicates that a first XTEN is inserted between amino acid residue 1900 and amino acid residue 1901 of FVIII, a second XTEN is inserted between amino acid residue 745 and amino acid residue 746 of FVIII, and a third XTEN is linked to the C-terminus of FVIII. The mouse strain used to administer the FVIII-XTEN variants is a DKO mouse strain expressing D'D3 domains. FIG. 9B shows the PK profile of FVIII-XTEN with three XTEN insertions. The FVIII-XTEN (1900/B/CT) variant was administered to either the FVIII/VWF DKO mice or HemA mice. The half-life of FVIII-XTEN (1900/B/CT) is compared.

[0049] FIG. 10. FVIII activity of FVIIIFc (hollow triangle), FVIII169:Fc (filled circle), and FVIII169:VWF31 (hollow triangle) in mouse DKO plasma measured by chromogenic assay. FVIII:Fc contains a dual-chain FVIII (Heavy chain and Light chain) fused to an Fc dimer (i.e., monomer-dimer hybrid). FVIII169 is described above (containing AE288 in the B domain, immediately downstream of amino acid 745 corresponding to mature FVIII sequence). FVIII169:Fc contains FVIII169 fused to an Fc dimer. FVIII169:VWF31 contains VWF31 in addition to the Fc dimer, FVIII169 fused to the first Fc region and VWF31 fused to the second Fc region, wherein the first Fc region and the second Fc region form a covalent bond, e.g., one or more disulfide bonds.

[0050] FIG. 11. Effects of Fc, XTEN, and VWF-D'D3 fragments on FVIII half-life extension. BDD-FVIII (REFACTO.RTM.) (square), FVIIFc (circle), FVIII169/Fc (triangle), and FVIII169NWF031 (inverted triangle) were administered to FVIII and VWF double knockout (DKO) mice. The FVIII activity was measured by chromogenic assay, and the half-life was calculated using the WinNonlin-Phoenix program. X-axis shows time, and the Y-axis shows the FVIII plasma activity in mU/mL.

[0051] FIG. 12A-C. Effects of different XTENs in rFVIII-XTEN/VWF heterodimer in HemA mice. FIG. 12A shows the FVIII plasma activity normalized to 5 min value (%) of two XTENs inserted immediately downstream of residues 1900 and 1656 corresponding to mature FVIII sequence (i.e., FVIII-195 (dual chain FVIII isoform) and FVIII-199 (single chain FVIII isoform)), compared to FVIII-169 containing an XTEN immediately downstream of residue 745 corresponding to mature FVIII sequence. FVIII-169NWF-031 (filled circle), FVIII-199/VWF-031 (filled square), and FVIII-195NWF031 (hollow square) were administered in HemA mice to measure the FVIII plasma activity. FIG. 12B shows the half-life extension effect of the second XTEN insertion immediately downstream of residues 403 (A2 domain) and 745 (B domain) (i.e., FVIII-203) and residues 745 (B domain) and 1900 (A3 domain) (FVIII-204) corresponding to mature FVIII sequence compared to FVIII-169 (an XTEN insertion in B domain only). FVIII-204NWF031 (filled triangle), FVIII-169NWF-031 (filled circle), FVIII-203NWF-031 (filled square), and scBDD-FVIII (hollow diamond) were administered to HemA mice. The X-axis shows FVIII plasma activity normalized to 5 min value (%), and the y-axis shows time in hours. FIG. 12C shows the half-life extension effect of the two XTEN insertions immediately downstream of residues 18 (A1 domain) and 745 (B domain) (i.e., FVIII-205) compared to FVIII-169 (a single XTEN insertion in the B domain) and single chain FVIII without any Fc regions or any XTENs (i.e., FVIII-207). FIG. 12C additionally shows the half-life extension effect of three XTEN insertions incorporated immediately downstream of residues 26 (A1 domain), 1656 (A3 domain), and 1900 (A3 domain) (i.e., FVIII-201) compared to FVIII-169 (a single XTEN insertion immediately downstream of residue 745). FVIII-205NWF-031 (filled square), FVIII-201NWF-031 (inverted triangle), FVIII-169NWF-031 (filled circle), and FVIII-207 (hollow diamond) were administered to HemA mice. The FVIII plasma activity normalized to 5 min value (%) (X-axis) was measured over time in hours (Y-axis).

[0052] FIG. 13. FVIII activity of rFVIII-XTEN/VWF-XTEN heterodimer in FVIII/VWF DKO mice. FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity (X-axis) as a function of time (Y-axis) was plotted. FVIII-155 (scFVIIIFc without any XTENs) was co-expressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residue thrombin cleavable linker). The half-life of FVIII-155/VWF-034 was compared with that of FVIII-169/VWF-031, which has a AE 288 XTEN inserted into the B domain junction (immediately downstream of residue 745 corresponding to mature FVIII polypeptide) of FVIII.

[0053] FIG. 14A-H. Schematic diagrams of various rFVIII-XTEN/VWF constructs. These constructs are also described in other sections herein. FIG. 14A shows single chain B domain deleted FVIII protein (sometimes indicated herein as scBDD-FVIII). The scBDD-FVIII constructs contain two substitutions at residues 1645 and 1648 from Arg to Ala. FIG. 14B shows two polypeptide chain construct (FVIII155NWF031), the first chain comprising single chain FVIII linked to an Fc region without any XTENS and the second chain comprising the VWF D'D3 fragment linked to an Fc region. This construct is used as a control. FIG. 14C shows two polypeptide chain construct (FVIII199NWF031), the first chain comprising single chain FVIII linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 1900 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 1656 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. FIG. 14D shows two polypeptide chain construct (FVIII201NWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 26 corresponding to mature FVIII sequence, a second XTEN is inserted immediately downstream of residue 1656 corresponding to mature FVIII sequence, and a third XTEN is inserted immediately downstream of residue 1900 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. FIG. 14E shows two polypeptide chain constructs (FVIII169/VWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which an XTEN is inserted immediately downstream of residue 745 (indicated as "B") corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. FIG. 14F shows two polypeptide chain construct (FVIII203NWF031), the first chain comprising single chain FVIII protein, in which a first XTEN is inserted at residue 745 ("B") corresponding to mature FVIII sequence and a second XTEN is inserted at residue 1900 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. FIG. 14G shows two polypeptide chain construct (FVIII204NWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 403 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 745 ("B") corresponding to mature FVIII sequence, and a second chain comprising the VWF D'D3 fragment linked to an Fc region. FIG. 14H shows two polypeptide chain construct (FVIII205NWF031), the first chain comprising single chain FVIII, in which a first XTEN is inserted immediately downstream of residue 18 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 745 ("B") corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region.

[0054] FIG. 15. FVIII activity of rFVIII-XTEN/VWF and BDD-FVIII in FVIII/VWF DKO mice. FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity (X-axis) as a function of time (Y-axis) was plotted. The half-life of rFVIII-XTEN/VWF (FVIII-205/VWF-031) was compared with that of BDD-FVIII and rFVIIIFc.

[0055] FIG. 16. Efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimers in HemA mice using tail clip bleeding model. The HemA mice tail clip bleeding model was used to compare the efficacy of FVIII169NWF034, FVIII205NWF031, and BDD-FVIII. The median blood loss in ml for 200 IU/kg of FVIII169NWF034 and FVIII205/VWF031 is compared with 200 IU/kg of BDD-FVIII, 65 IU/kg of BDD-FVIII, 20 IU/kg of BDD-FVIII, and vehicle.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0056] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0057] The term "polynucleotide" or "nucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide comprises a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0058] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode a binding domain-A and a binding domain-B as described below. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding domain of the invention. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0059] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide, and are cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, e.g., a human tissue plasminogen activator (TPA) or mouse B-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0060] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0061] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0062] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0063] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0064] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0065] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0066] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0067] A "vector" refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0068] Vectors may be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers may also be considered to be reporters.

[0069] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0070] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0071] A "cloning vector" refers to a "replicon," which is a unit length of a nucleic acid that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria and expression in another, e.g., eukaryotic cells. Cloning vectors typically comprise one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

[0072] The term "expression vector" refers to a vehicle designed to enable the expression of an inserted nucleic acid sequence following insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with regulatory regions as described above.

[0073] Vectors are introduced into host cells by methods well known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter.

[0074] "Culture," "to culture" and "culturing," as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. "Cultured cells," as used herein, means cells that are propagated in vitro.

[0075] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0076] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0077] Also included in the present invention are fragments or variants of polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to polypeptide binding domains or binding molecules of the present invention include any polypeptides which retain at least some of the properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for an FVIII variant, or FVIII binding activity for the VWF fragment) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of polypeptide binding domains or binding molecules of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0078] The term "VWF fragment" or "VWF fragments" used herein means any VWF fragments that interact with FVIII and retain at least one or more properties that are normally provided to FVIII by full-length VWF, e.g., preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that could lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and/or stabilizing the FVIII heavy chain and light chain interactions.

[0079] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

[0080] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0081] As used herein, an "amino acid corresponding to" or an "equivalent amino acid" in a VWF sequence or a FVIII protein sequence is identified by alignment to maximize the identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify an equivalent amino acid in a second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0082] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or fragment, variant, or derivative thereof, which is immediately upstream of the position at which a heterologous moiety can be inserted. An "insertion site" is specified as a number, the number being the number of the amino acid in mature native FVIII (SEQ ID NO:4) to which the insertion site corresponds, which is immediately N-terminal to the position of the insertion. For example, the phrase "a3 comprises an XTEN at an insertion site which corresponds to amino acid 1656 of SEQ ID NO: 4" indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 4.

[0083] The phrase "immediately downstream of an amino acid" as used herein refers to position right next to the terminal carboxyl group of the amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position right next to the terminal amine group of the amino acid. Therefore, the phrase "between two amino acids of an insertion site" as used herein refers to a position in which an XTEN or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases "inserted immediately downstream of an amino acid" and "inserted between two amino acids of an insertion site" are used synonymously with "inserted at an insertion site."

[0084] The terms "inserted," "is inserted," "inserted into" or grammatically related terms, as used herein refers to the position of an XTEN in a chimeric polypeptide relative to the analogous position in native mature human FVIII. As used herein the terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII, and do not indicate, imply or infer any methods or process by which the chimeric polypeptide was made. For example, in reference to a chimeric polypeptide provided herein, the phrase "an XTEN is inserted into immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric polypeptide comprises an XTEN immediately downstream of an amino acid which corresponds to amino acid 745 in native mature human FVIII, e.g., bounded by amino acids corresponding to amino acids 745 and 746 of native mature human FVIII.

[0085] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, e.g., fusion of a Factor VIII domain of the invention with an Ig Fc domain. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein can further comprises a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond.

[0086] As used herein, the term "half-life" refers to a biological half-life of a particular polypeptide in vivo. Half-life may be represented by the time required for half the quantity administered to a subject to be cleared from the circulation and/or other tissues in the animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid .alpha.-phase and longer .beta.-phase. The .alpha.-phase typically represents an equilibration of the administered Fc polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide. The .beta.-phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, and thus do not have an alpha phase, but just the single beta phase. Therefore, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the .beta.-phase. The typical .beta.-phase half-life of a human antibody in humans is 21 days.

[0087] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly joined or juxtaposed to the second amino acid or nucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" means not only a fusion of a first amino acid sequence to a second amino acid sequence at the C-terminus or the N-terminus, but also includes insertion of the whole first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to a second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to a second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). The term "linked" is also indicated by a hyphen (-).

[0088] As used herein the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" means a covalent, non-peptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond except a peptide bond. For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by a disulfide bond and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bond include an ionic bond (e.g., cation-pi bond or salt bond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, low-barrier hydrogen bond, or symmetric hydrogen bond), van der Walls force, London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, stacking, entropic force, or chemical polarity.

[0089] The term "monomer-dimer hybrid" used herein refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain, which are associated with each other by a disulfide bond, wherein the first chain comprises a clotting factor, e.g., Factor VIII, and a first Fc region and the second chain comprises, consists essentially of, or consists of a second Fc region without the clotting factor. The monomer-dimer hybrid construct thus is a hybrid comprising a monomer aspect having only one clotting factor and a dimer aspect having two Fc regions.

[0090] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Certain enzymatic cleavage sites comprise an intracellular processing site. In one embodiment, a polypeptide has an enzymatic cleavage site cleaved by an enzyme that is activated during the clotting cascade, such that cleavage of such sites occurs at the site of clot formation. Exemplary such sites include, e.g., those recognized by thrombin, Factor XIa or Factor Xa. Exemplary FXIa cleavage sites include, e.g., TQSFNDFTR (SEQ ID NO: 45) and SVSQTSKLTR (SEQ ID NO: 46). Exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 47), TTKIKPR (SEQ ID NO: 48), LVPRG (SEQ ID NO: 49) and ALRPR (amino acids 1 to 5 of SEQ ID NO: 50). Other enzymatic cleavage sites are known in the art.

[0091] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide which is a target for enzymes that function after translation of the polypeptide. In one embodiment, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides prior to secretion of the protein from the cell. Examples of such processing sites include, e.g., those targeted by the PACE/furin (where PACE is an acronym for Paired basic Amino acid Cleaving Enzyme) family of endopeptidases. These enzymes are localized to the Golgi membrane and cleave proteins on the carboxyterminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein the "furin" family of enzymes includes, e.g., PCSK1 (also known as PC1/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known as PC7/LPC, PC8, or SPC7). Other processing sites are known in the art.

[0092] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0093] The term "Furin" refers to the enzymes corresponding to EC No. 3.4.21.75. Furin is subtilisin-like proprotein convertase, which is also known as PACE (Paired basic Amino acid Cleaving Enzyme). Furin deletes sections of inactive precursor proteins to convert them into biologically active proteins. During its intracellular transport, pro-peptide of VWF can be cleaved from mature VWF molecule by a Furin enzyme. In some embodiments, Furin cleaves the D1D2 from the D'D3 of VWF. In other embodiments, a nucleotide sequence encoding Furin can be expressed together with the nucleotide sequence encoding a VWF fragment so that D1D2 domains can be cleaved off intracellularly by Furin.

[0094] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0095] A "processable linker" as used herein refers to a linker comprising at least one intracellular processing site, which is described elsewhere herein.

[0096] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot. Examples of such disorders include the hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease") and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, e.g., Von Willebrand disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X or factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb, the receptor for VWF, can be defective and lead to lack of primary clot formation (primary hemostasis) and increased bleeding tendency), and thrombasthenia of Glanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this may increase bleeding risk.

[0097] The chimeric molecules of the invention can be used prophylactically. As used herein the term "prophylactic treatment" refers to the administration of a molecule prior to a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to or after surgery as a prophylactic. The chimeric protein of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

[0098] The chimeric protein of the invention is also used for on-demand treatment. The term "on-demand treatment" refers to the administration of a chimeric molecule in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, the on-demand treatment can be given to a subject when bleeding starts, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, the on-demand treatment can be given prior to activities that increase the risk of bleeding, such as contact sports.

[0099] As used herein the term "acute bleeding" refers to a bleeding episode regardless of the underlying cause. For example, a subject may have trauma, uremia, a hereditary bleeding disorder (e.g., factor VII deficiency) a platelet disorder, or resistance owing to the development of antibodies to clotting factors.

[0100] Treat, treatment, treating, as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, or the prophylaxis of one or more symptoms associated with a disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining a FVIII trough level at least about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7 IU/dL, 8 IU/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12 IU/dL, 13 IU/dL, 14 IU/dL, 15 IU/dL, 16 IU/dL, 17 IU/dL, 18 IU/dL, 19 IU/dL, or 20 IU/dL in a subject by administering a chimeric protein or a VWF fragment of the invention. In another embodiment, treating or treatment means maintaining a FVIII trough level between about 1 and about 20 IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL, about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9 and about 20 IU/dL, or about 10 and about 20 IU/dL. Treatment or treating of a disease or condition can also include maintaining FVIII activity in a subject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophiliac subject. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each person.

Chimeric Proteins

[0101] The present invention is directed to extending the half-life of a Factor VIII protein using a VWF fragment and an XTEN sequence by preventing or inhibiting a FVIII half-life limiting factor, i.e., endogenous VWF, from associating with the FVIII protein. Endogenous VWF associates with about 95% to about 98% of FVIII in non-covalent complexes. While endogenous VWF is a FVIII half-life limiting factor, endogenous VWF bound to a FVIII protein is also known to protect FVIII in various ways. For example, full length VWF (as a multimer having about 250 kDa) can protect FVIII from protease cleavage and FVIII activation, stabilize the FVIII heavy chain and/or light chain, and prevent clearance of FVIII by scavenger receptors. But, at the same time, endogenous VWF limits the FVIII half-life by preventing pinocytosis and by clearing FVIII-VWF complex from the system through the VWF clearance pathway. It is believed, while not bound by a theory, that endogenous VWF is a half-life limiting factor that prevents the half-life of a FVIII protein fused to a half-life extender from being longer than about two-fold that of wild-type FVIII. Therefore, the present invention is directed to preventing or inhibiting interaction between endogenous VWF and a FVIII protein using a VWF fragment, thereby increasing a half-life of the FVIII protein by using an XTEN sequence alone or an XTEN sequence in combination with an Ig constant region or a portion thereof. The XTEN sequence can be linked to the FVIII protein or the VWF fragment. The FVIII protein associated with the VWF fragment is thus cleared from the circulation more slowly by one or more VWF clearance receptors and then can have the full half-life extension of the XTEN sequence or the XTEN sequence in combination of the Ig constant region, as compared to wild type FVIII or a FVIII protein without the VWF fragment.

[0102] In one embodiment, a VWF fragment is associated (or linked) with the FVIII protein by a covalent or a non-covalent bond. In some instances, however, the physical blockage or chemical association (e.g., non-covalent bonding) between the VWF fragment and the FVIII protein may not be strong enough to provide a stable complex comprising the FVIII protein and the VWF fragment in the presence of endogenous VWF. For example, a VWF fragment forming a non-covalent bond with a FVIII protein without any other connections may readily be dissociated in vivo from the FVIII protein in the presence of endogenous VWF, replacing the VWF fragment (e.g., recombinant VWF, i.e., rVWF) with endogenous VWF. Therefore, the FVIII protein non-covalently bound to endogenous VWF would undergo the VWF clearance pathway and be readily cleared from the system. In order to prevent the dissociation of the VWF fragment with the FVIII protein, in some embodiments, the association or linkage between the FVIII protein and the VWF fragment is a covalent bond, e.g., a peptide bond, one or more amino acids, or a disulfide bond. In certain embodiments, the association (i.e., linkage) between the adjunct moiety and the FVIII protein is a peptide bond or a linker between the FVIII protein and the VWF fragment ("FVIII/VWF linker"). Non-limiting examples of the linker are described elsewhere herein. In some embodiments, the VWF fragment is a polypeptide comprising, consisting essentially of, or consisting of at least about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 4000 amino acids. Non-limiting examples of the VWF fragment are described elsewhere herein.

[0103] In certain embodiments, the VWF fragment chemically (e.g., non-covalently) binds to or physically blocks one or more VWF binding sites on a FVIII protein. The VWF binding site on a FVIII protein is located within the A3 domain or the C2 domain of the FVIII protein. In still other embodiments, the VWF binding site on a FVIII protein is located within the A3 domain and C2 domain. For example, the VWF binding site on a FVIII protein can correspond to amino acids 1669 to 1689 and/or 2303 to 2332 of SEQ ID NO: 4 [full-length mature FVIII].

[0104] The invention also provides a chimeric protein (comprising a FVIII protein and a VWF fragment) further comprising one or more XTEN sequences, which provide additional half-life extension properties. The one or more XTEN sequences can be inserted within the FVIII protein or the VWF fragment or linked to the N-terminus or the C-terminus of the FVIII protein or the VWF fragment. The invention also includes a FVIII protein linked to an XTEN sequence (a first half-life extending moiety) and an Ig constant region or a portion thereof (a second half-life extending moiety) so that the two half-life extending moieties extend the half-life of the FVIII protein through two different mechanisms.

[0105] In some embodiments, a chimeric protein comprises a FVIII protein linked to a first Ig constant region or a portion thereof (e.g., a first FcRn binding partner), a VWF fragment linked to a second Ig constant region or a portion thereof (e.g., a second FcRn binding partner), and one or more XTEN sequences inserted or linked to the FVIII protein or the VWF fragment, wherein the VWF fragment prevents the FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein, wherein the first and second Ig constant regions or portions thereof forms a covalent bond, e.g., a disulfide bond, and the one or more XTEN sequences extends the half-life of the FVIII protein.

[0106] In certain embodiments, a chimeric protein of the invention comprises a FVIII protein linked to a VWF fragment by an optional linker (i.e., FVIII/VWF linker) and one or more XTEN sequences inserted or linked to the FVIII protein or the VWF fragment, wherein the VWF fragment prevents the FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein and the one or more XTEN sequences extends the half-life of the FVIII protein. In one aspect, the optional linker (FVIII/VWF linker) comprises a sortase recognition motif. In another aspect, the optional linker (FVIII/VWF linker) comprises a cleavable site. Examples of the cleavage linker (i.e., linker containing one or more cleavage site) are described elsewhere herein.

[0107] The chimeric protein of the present invention includes, but is not limited to:

(1) a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, and FVIII, wherein the XTEN sequence is linked to the VWF fragment; (2) a FVIII protein, an XTEN sequence, and an Ig constant region or a portion thereof, wherein the FVIII protein is linked to an XTEN sequence and the Ig constant region or a portion thereof, or (3) a FVIII protein, an XTEN sequence, and a VWF fragment, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or N-terminus or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) of FVIII, and the VWF fragment and the FVIII protein are associated with each other.

(1) Von Willebrand Factor (VWF) Fragment Linked to XTEN, and FVIII

[0108] The present invention is directed to a chimeric protein comprising (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein (i), (ii), and (iii) are linked to or associated with each other. The VWF fragment linked to the XTEN sequence, as a part of a chimeric protein in the present invention, associates with the FVIII protein, thus preventing or inhibiting interaction between endogenous VWF and the FVIII protein. In certain embodiments, the VWF fragment, which is capable of preventing or inhibiting binding of the FVIII protein with endogenous VWF, can at the same time have at least one VWF-like FVIII protecting property. Examples of the VWF-like FVIII protecting properties include, but are not limited to, protecting FVIII from protease cleavage and FVIII activation, stabilizing the FVIII heavy chain and/or light chain, and preventing clearance of FVIII by scavenger receptors. As a result, the VWF fragment can prevent clearance of the FVIII protein through the VWF clearance pathway, thus reducing clearance of FVIII from the circulatory system. In some embodiments, the VWF fragments of the present invention bind to or are associated with a FVIII protein and/or physically or chemically block the VWF binding site on the FVIII protein. The FVIII protein associated with the VWF fragment is thus cleared from the circulation more slowly, as compared to wild type FVIII or FVIII not associated with the VWF fragment.

[0109] In one embodiment, the invention is directed to a chimeric protein comprising (i) a VWF fragment comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the XTEN sequence is linked to the VWF fragment (e.g., (a1) V-X or (a2) X-V, wherein V comprises a VWF fragment and X comprises an XTEN sequence), and the VWF fragment is linked to or associated with the FVIII protein. In another embodiment, the VWF fragment and the XTEN sequence can be linked by a linker (e.g., (a3) V-L-X or (a4) X-L-V) or a peptide bond. The linker can be a cleavable linker, e.g., a thrombin cleavable linker, which can be cleaved at the site of coagulation. In other embodiments, the VWF fragment, the XTEN sequence, and the FVIII protein are placed in a single polypeptide chain. In still other embodiments, the chimeric protein comprises two polypeptide chains, a first chain comprising the VWF fragment and the XTEN sequence and a second chain comprising the FVIII protein. In yet other embodiments, the chimeric protein comprises three polypeptide chains, a first chain comprising the VWF fragment and the XTEN sequence, a second chain comprising a light chain of FVIII and a third chain comprising a heavy chain of FVIII, wherein the first chain and the second chain are associated with each other (e.g., covalent bond, e.g., disulfide bond), and the second chain and the third chain are associated with each other (e.g., metal bond). In still other embodiments, the XTEN sequence can be linked to the N-terminus or the C-terminus of the VWF fragment or inserted immediately downstream of one or more amino acids in the VWF fragment.

[0110] In certain embodiments, a chimeric protein of the invention comprises a formula comprising:

V-X-FVIII, (a)

FVIII-X-V, (b)

V-X:FVIII, (c)

X-V:FVIII, (d)

FVIII:V-X, (e)

FVIII:X-V, or (f)

X-V-FVIII, (a5)

wherein V comprises a VWF fragment, X comprises one or more XTEN sequences, FVIII comprises a FVIII protein; (-) represents a peptide bond or one or more amino acids; and (:) is a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula V-X-FVIII means formula NH2-V-X-FVIII-COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. For example, formula V-X-FVIII can further comprise any sequences at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII unless otherwise specified. In another embodiment, the hyphen (-) indicates a peptide bond.

[0111] In other embodiments, a chimeric protein of the invention comprises a formula comprising:

V(X1)-X2-FVIII, (a)

FVIII-X2-V(X1), (b)

V(X1):FVIII, (c)

FVIII:V(X1), or (d)

X2-V(X1)-FVIII, (a5)

wherein V(X1) comprises a VWF fragment and a first XTEN sequence (X1), wherein the XTEN sequence is inserted immediately downstream of one or more amino acids in the VWF fragment, X2 comprises one or more optional XTEN sequences, FVIII comprises a FVIII protein; (-) is a peptide bond or one or more amino acids; and (:) is a chemical association or a physical association.

[0112] In some embodiments, a chimeric protein comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) a first optional linker, and (v) a second optional linker, wherein the XTEN sequence is linked to the VWF fragment and/or to the FVIII protein by the linker. In certain embodiments, a chimeric protein comprises a formula comprising:

V-L1-X-L2-FVIII, (b1)

FVIII-L2-X-L1-V, (b2)

V-L1-X:FVIII, (b3)

X-L1-V:FVIII, (b4)

FVIII:V-L1-X, (b5)

FVIII:X-L1-V, (b6)

X-L1-V-L2-FVIII, or (b7)

FVIII-L2-V-L1-X, (b8)

wherein V comprises a VWF fragment, X comprises one or more XTEN sequences, FVIII comprises a FVIII protein, L1 comprises a first optional linker, e.g., a first cleavable linker, L2 comprises a second optional linker, e.g., a second cleavable linker or an optional processable linker; (-) is a peptide bond or one or amino acids; and (:) is a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula (b1) V-L1-X-L2-FVIII means formula NH2-V-L1-X-L2-FVIII-COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. In another embodiment, the hyphen (-) indicates a peptide bond.

[0113] Another aspect of the present invention is to provide a FVIII chimeric protein having reduced or no interactions with a FVIII half-life limiting factor, e.g., endogenous VWF, and at the same time maximizing the half-life of the FVIII protein using an XTEN sequence (a first half-life extender) in combination with a second half-life extender or a moiety providing a covalent bond between the FVIII protein and the VWF fragment, e.g., an Ig constant region or a portion thereof. In one embodiment, a chimeric protein of the invention comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, and (iv) an Ig constant region or a portion thereof (also referred to herein as F), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker, (2) the VWF fragment is associated with or linked to the FVIII protein by an additional optional linker, e.g., a cleavable linker, and (3) the Ig constant region or a portion thereof is linked to the VWF fragment, the XTEN sequence, or the FVIII protein. In another embodiment, a chimeric protein of the invention comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) an Ig constant region or a portion thereof (F1 or a first Ig constant region or a portion thereof), and (v) an additional Ig constant region or a portion thereof (F2 or a second Ig constant region or a portion thereof), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker, (2) the XTEN sequence or the VWF fragment is linked to the Ig constant region or a portion thereof, (3) the FVIII is linked to the additional Ig constant region or a portion thereof, and (4) the Ig constant region or a portion thereof is associated with or linked to the additional Ig constant region or a portion thereof. In one embodiment, the association or linkage between the two Ig constant regions or a portion thereof is a covalent bond, e.g., a disulfide bond. In another embodiment, the association or linkage between the two Ig constant regions or a portion thereof is a processable linker, wherein the processable linker is intracellularly processed by a protease. For example, the chimeric protein comprises a formula comprising:

V-L2-X-L1-F1:FVIII-L3-F2; (g)

V-L2-X-L1-F1:F2-L3-FVIII; (h)

F-L1-X-L2-V: FVIII-L3-F2; (i)

F-L1-X-L2-V:F2-L3-FVIII; (j)

V-L2-X-L1-F1-L4-FVIII-L3-F2; (k)

F2-L3-FVIII-L4-F1-L1-X-L2-V; (l)

FVIII-L2-F2-L4-V-L2-X-L1-F1; or (m)

F1-L1-X-L2-V-L4-F2-L2-FVIII, (n)

wherein V comprises a VWF fragment, each of L1 and L3 comprises an optional linker, L2 comprises an optional linker, e.g., a cleavable linker, L4 is an optional linker, e.g., a processable linker, FVIII comprises a FVIII protein, X comprises one or more XTEN sequences, F1 comprises an optional Ig constant region or a portion thereof, F2 comprises an optional additional Ig constant region or a portion thereof; (-) is a peptide bond or one or more amino acids; and (:) is a chemical association or a physical association.

[0114] In some embodiments, the FVIII protein in any constructs or formulas disclosed herein can further comprises at least one, at least two, at least three, at least four, at least five, or at least six XTEN sequences, each of the XTEN sequences inserted immediately downstream of one or more amino acids in the FVIII protein or linked to the N-terminus or the C-terminus of the FVIII protein. Non-limiting examples of the XTEN insertion sites are disclosed elsewhere herein.

[0115] In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula (n) F1-L1-X-L2-V-L4-F2-L2-FVIII means formula NH2-F1-L1-X-L2-V-L4-F2-L2-FVIII-COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. In another embodiment, the hyphen (-) indicates a peptide bond.

[0116] In one embodiment, either or both of the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "F1") and the additional Ig constant region or a portion thereof (sometimes indicated herein by "F2") linked to the VWF fragment or the FVIII protein can extend the half-life of the VWF fragment, the FVIII protein, or both. In another embodiment, a pair of the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "F1") and the additional Ig constant region or a portion thereof (sometimes indicated herein by "F2"), each of which are linked to the VWF fragment and the FVIII protein, provides a bond stronger than the non-covalent bond between the FVIII protein and the VWF fragment, i.e., a covalent bond, e.g., a disulfide bond, thereby preventing endogenous VWF from replacing the VWF fragment in vivo. F1 or F2 can comprise an Fc region or an FcRn binding partner. In other embodiments, either or both of F1 and F2 linked to the VWF fragment and/or the FVIII protein form a covalent bond (e.g., a disulfide bond) between F1 and F2, thereby placing the VWF fragment and the FVIII protein in close proximity to prevent interaction of the FVIII protein with the VWF fragment. In some embodiments, F1 and F2 are identical or different. Non-limiting examples of F1 and F2 can be selected from the group consisting of a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragments, derivatives, or analogs thereof, and two or more combinations thereof. In one embodiment, F1, F2, or both comprise at least one CH1 domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or the functional fragments, derivatives, or analogs thereof. In another embodiment, F1, F2, or both comprise at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in the hinge-CH2 orientation). In other embodiments, F1, F2, or both comprise at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in the CH2-CH3 orientation.) Examples of the combination include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain, which are also known as an Fc region (or Fc domain), e.g., a first Fc region or a first FcRn binding partner for F1 and a second Fc region or a second FcRn binding partner for F2. In other embodiments, F1 is linked to the VWF fragment by a linker, and/or F2 is linked to the FVIII protein by a linker. In some embodiments, F1 and/or F2 comprises, consisting essentially of, or consisting of a hinge region. Additional non-limiting examples of the Fc regions or the FcRn binding partners are described elsewhere herein.

[0117] In certain embodiments, a chimeric protein of the invention comprises two polypeptide chains, a first polypeptide chain comprising, consisting essentially of, or consisting of a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, a first Ig constant region or a portion thereof (e.g., a first Fc region), and an optional linker between the VWF fragment and the XTEN sequence or the XTEN sequence or the first Ig constant region or a portion thereof and a second polypeptide chain comprising, consisting essentially of, or consisting of a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region). The linker between the VWF fragment and the first Ig constant region or a portion thereof can be a cleavable linker, e.g., a thrombin cleavable linker, which can be cleaved at the site of coagulation. In some embodiments, the first polypeptide chain and the second polypeptide chain are associated with each other. The association between the first chain and the second chain prevents replacement of the first chain comprising the VWF fragment with endogenous VWF in vivo. In one embodiment, the association between the first chain and the second chain can be a covalent bond. In a particular embodiment, the covalent bond is a disulfide bond. In some embodiments, the FVIII protein in the second chain further comprises one or more XTEN sequences linked to the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., at least one insertion site disclosed herein) in the FVIII protein. Non-limiting examples of the insertion sites are described elsewhere herein.

[0118] In other embodiments, a chimeric protein of the invention comprises three polypeptide chains, wherein a first polypeptide chain comprises, consists essentially of, or consists of a heavy chain of a FVIII protein, a second polypeptide chain comprises, consists essentially of, or consists of a light chain of a FVIII protein fused to a first Ig constant region or a portion thereof (e.g., a first Fc region), and a third polypeptide chain comprises, consists essentially of, or consists of a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, a second Ig constant region or a portion thereof (e.g., a second Fc region), and an optional linker between the XTEN sequence and the second Ig constant region or a portion thereof or the VWF fragment and the XTEN sequence. The linker in the third chain can be a cleavable linker, which is cleaved at the site of coagulation, e.g., a thrombin cleavage site. In some embodiments, the heavy chain FVIII or the light chain FVIII is linked to one or more XTEN sequences, which can be linked to the N-terminus, the C-terminus, or inserted within one or more insertion sites within the FVIII sequence. Non-limiting examples of the insertion sites are disclosed elsewhere herein.

[0119] In yet other embodiments, a chimeric protein of the invention comprises two polypeptide chains, a first polypeptide chain comprising, consisting essentially of, or consisting of a heavy chain of a FVIII protein and a second polypeptide chain comprising, consisting essentially of, or consisting of a light chain of a FVIII protein, a first Ig constant region or a portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker, which contains one or more protease cleavage sites comprising one or more intracellular processing sites), a VWF fragment, a second linker (e.g., a thrombin cleavable linker), an XTEN sequence, and a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the light chain of the FVIII protein is linked to the first Ig constant region or a portion thereof (e.g., the first Fc region), which is further linked to the VWF fragment by the first linker, and wherein the VWF fragment is linked to the XTEN sequence, which is further linked to the second Ig constant region or a portion thereof by the second linker. In certain embodiments, the first linker is a processable linker, and the second linker is a cleavable linker. Upon expression, the chimeric protein can be processed by an intracellular processing enzyme, which cleaves the processable linker, and thus the chimeric protein can comprise, consists essentially of, or consists of three polypeptide chains. In addition, the VWF fragment can be cleaved off at the site of coagulation due to the cleavable linker.

[0120] In certain embodiments, a chimeric protein of the invention comprises one polypeptide chain, which comprises a single chain FVIII protein, a first Ig constant region or a portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker), a VWF fragment, an XTEN sequence, a second linker (e.g., a thrombin cleavable linker), and a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the single chain FVIII protein is linked to the first Ig constant region or a portion thereof, which is also linked to the VWF fragment by the first linker, and the VWF fragment is linked to the XTEN sequence, which is further linked to the second Ig constant region or a portion thereof. In one embodiment, the VWF fragment and the XTEN sequence are linked by the second linker. In another embodiment, the XTEN sequence and the second Ig constant region or a portion thereof are linked by the second linker. In other embodiments, the second chain further comprises a third linker. The single polypeptide chain can thus comprise the VWF fragment linked to the XTEN sequence by the second linker and the XTEN linked to the second Ig constant region or a portion thereof by the third linker. The second linker and the third linker can be identical or different. In one embodiment, the first linker is a processable linker. In another embodiment, the second linker or the third linker is a cleavable linker comprising one or two cleavable sites. In a specific embodiment, the second linker is a thrombin cleavable linker. The linkers useful in the invention are described elsewhere herein.

(2) FVIII, XTEN, and Fe

[0121] A chimeric protein of the invention also comprises (i) a FVIII protein, (ii) an XTEN sequence (a first half-life extender), and (iii) an Ig constant region or a portion thereof (a second half-life extender), in which the XTEN sequence is linked to the FVIII protein by an optional linker and the Ig constant region or a portion thereof by an additional optional linker. The XTEN sequence and the Ig constant region or a portion thereof can be used together to extend half-life of the FVIII protein. In one embodiment, the chimeric protein is a monomer. In another embodiment, the chimeric protein is a dimer (a homodimer or a heterodimer).

[0122] The present invention is also directed to a chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) an Ig constant region or a portion thereof (i.e., a first Ig constant region or a portion thereof, "F," or "F1"), and (iv) an additional Ig constant region or a portion thereof (i.e., a second Ig constant region or a portion thereof or "F2"). In one embodiment, the XTEN sequence is linked to the FVIII protein at the C-terminus or the N-terminus or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites), the FVIII protein is linked to the first Ig constant region or a portion thereof, and the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are associated with or linked to each other by an optional linker. In certain aspects, the chimeric protein is a monomer-dimer hybrid, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a FVIII protein, an XTEN sequence, and a first Ig constant region or a portion thereof, and the second polypeptide chain comprises, consists essentially of, or consists of a second Ig constant region or a portion thereof without the FVIII protein and wherein the first chain and the second chain are associated with each other. The association between the Ig constant region or a portion thereof (e.g., the first Fc region) and the additional Ig constant region or a portion thereof (e.g., a second Fc region) is a chemical association or a physical association. In certain embodiments, the chemical association is a covalent bond. In other embodiments, the chemical association is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, the association is a non-peptide covalent bond. In still other embodiments, the association is a peptide bond.

[0123] In other aspects, the chimeric protein is a single polypeptide chain comprising a FVIII protein, an XTEN sequence, a first Ig constant region or a portion thereof, a linker, e.g., a processable linker, and a second Ig constant region or a portion thereof, wherein the single polypeptide chain is processed after expression by an intracellular enzyme and becomes two polypeptide chains.

[0124] In one embodiment, the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "F1") linked to the FVIII protein can extend the half-life of the FVIII protein together with the XTEN sequence. In another embodiment, the Ig constant region or a portion thereof ("F" or "F1") is an Fc region or an FcRn binding partner described elsewhere herein.

[0125] In other embodiments, the additional Ig constant region or a portion thereof (sometimes indicated herein by "F2" or a second Ig constant region or a portion thereof) associated with or linked to the first Ig constant region or a portion thereof can also extend the half-life of the FVIII protein. In other embodiments, the second Ig constant region or a portion thereof ("F2") together with the first Ig constant region or a portion thereof and the XTEN sequence can extend the half-life of the FVIII protein. The additional Ig constant region or a portion thereof can be an Fc region or an FcRn binding partner described elsewhere herein.

[0126] In certain embodiments, the second Ig constant region or a portion thereof associated with the first Ig constant region or a portion thereof is further linked to a VWF fragment described elsewhere herein and an optional XTEN sequence.

[0127] In some embodiments, either or both of the Ig constant region or a portion thereof ("F" or "F1" or a first Ig constant region or a portion thereof) and an additional Ig constant region or a portion thereof (i.e., a second Ig constant region or a portion thereof or "F2") (indicated in this paragraph as "the Ig constant regions or portion thereof") can include, but not limited to, a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragments, derivatives, or analogs thereof or two or more combinations thereof. In one embodiment, the Ig constant region or a portion thereof comprises at least one CH1 domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or the functional fragments, derivatives, or analogues thereof. In another embodiment, the Ig constant region or a portion thereof comprises at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in the hinge-CH2 orientation). In other embodiments, the Ig constant domain or portion thereof comprises at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in the CF2-CH3 orientation). Examples of the combination include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain, which are also known as an Fc region (or Fc domain), e.g., first Fc region. Additional examples of the Ig constant regions or portion thereof are described elsewhere herein.

[0128] The chimeric protein of the invention can have an extended half-life of the FVIII protein compared to wild-type FVIII. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the half-life of wild type FVIII. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

(3) FVIII, XTEN, and VWF

[0129] In one aspect, a chimeric protein of the present invention comprises (i) a FVIII protein, (ii) an XTEN sequence, and (iii) a VWF fragment comprising a D' domain and a D3 domain of VWF, wherein the FVIII protein is linked to the XTEN sequence and wherein the FVIII protein is associated with or linked to the VWF fragment. In one embodiment, the VWF fragment of the chimeric protein described herein is not capable of binding to a VWF clearance receptor. In another embodiment, the VWF fragment is capable of protecting the FVIII protein from one or more protease cleavages, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors. In other embodiments, the VWF fragment prevents or inhibits binding of endogenous VWF to the VWF binding site in the FVIII protein. The VWF binding site can be located in the A3 domain or the C2 domain of the FVIII protein or both the A3 domain and the C2 domain. In a specific embodiment, the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and/or amino acids 2303 to 2332 of SEQ ID NO: 2.

[0130] In another aspect, a chimeric protein comprises (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, which comprises a D' domain and a D3 domain of VWF, and (iv) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or the N-terminus or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein, the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof. The Ig constant region or a portion thereof useful for chimeric proteins of the invention is described elsewhere herein. In one embodiment, the Ig constant region or a portion thereof is capable of extending the half-life of a FVIII protein. In another embodiment, the Ig constant region or a portion thereof comprises a first Fc region or a first FcRn binding partner. In yet other embodiments, the Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker. In still other embodiments, the linker comprises a cleavable linker. The chimeric protein can be a single polypeptide chain, i.e., a monomer (i.e., a single chain), containing (i), (ii), (iii), and (iv) or two chains containing a first chain comprising (i) and (ii) and a second chain comprising (iii) and (iv). In other aspects, the chimeric protein is a dimer (e.g., a homodimer or a heterodimer). In one embodiment, the chimeric protein comprises two chains, each comprising (i), (ii), (iii), and (iv).

[0131] In certain embodiments, a chimeric protein comprises (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, which comprises a D' domain and a D3 domain of VWF, (iv) an Ig constant region or a portion thereof (sometimes also indicated as "F," "a first Ig constant region or a portion thereof", or "F2"), and (v) an additional Ig constant region or a portion thereof (sometimes also indicated as "F2" or "a second Ig constant region or a portion thereof"), wherein (1) the FVIII protein is linked to the XTEN sequence at the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein, (2) either the XTEN sequence or the FVIII protein is linked to the Ig constant region or a portion thereof, (3) the VWF fragment is linked to the second Ig constant region or a portion thereof, and (4) the Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof. In one embodiment, the Ig constant region or a portion thereof linked to the FVII protein or the XTEN sequence is further linked to the VWF fragment by a linker, e.g., a processable linker. In another embodiment, the additional Ig constant region or a portion thereof useful for chimeric proteins of the invention can further be linked to the FVIII protein or the Ig constant region or a portion thereof by an optional linker, e.g., a processable linker. In some embodiments, a pair of the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof, each of which are linked to the VWF fragment and the FVIII protein, provides a bond stronger than the non-covalent bond between the FVIII protein and the VWF fragment, i.e., a covalent bond, e.g., a disulfide bond, thereby preventing endogenous VWF from replacing the VWF fragment in vivo. In other embodiments, either or both of the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are capable of extending a half-life of the FVIII protein or the VWF fragment. In other embodiments, the additional Ig constant region or a portion thereof comprises a second Fc region or an FcRn binding partner. The Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof in the chimeric proteins are identical or different.

[0132] In certain embodiments, the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are associated by a chemical association or a physical association. In one embodiment, the chemical association, i.e., (:), is at least one non-peptide bond. In certain embodiments, the chemical association, i.e., (:), is a covalent bond. In other embodiments, the chemical association, i.e., (:), is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety. In some embodiments, the association between the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof can be a covalent bond, e.g., a disulfide bond, which prevents replacement the VWF fragment or the polypeptide containing the VWF fragment with endogenous VWF. Therefore, preventing interaction between the FVIII protein and endogenous VWF reduces or eliminates this half-life limiting factor for the FVIII protein, and thus the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF protein or wild-type FVIII.

[0133] In other aspects, a chimeric protein comprises a formula comprising:

FVIII(X1)-L1-F1:V-L2-X2-L3-F2; (1)

FVIII(X1)-L1-F1:F2-L3-X2-L2-V; (2)

F1-L1-FVIII(X1):V-L2-X2-L3-F2; (3)

F1-L1-FVIII(X1):F2-L3-X2-L2-V; (4)

FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2; (5)

FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V; (6)

F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (7)

F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V, (8)

wherein FVIII(X1) comprises a FVIII protein and one or more XTEN sequences, wherein the one or more XTEN sequence are linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein; each of L1, L2, or L3 comprises an optional linker, e.g., a cleavable linker; L4 is a linker, e.g., a processable linker; X2 comprises one or more optional XTEN sequences; F1 comprises an Ig constant region or a portion thereof; F2 comprises an optional additional Ig constant region or a portion thereof, and V comprises a VWF fragment; (-) is a peptide bond or one or more amino acids; and (:) comprises a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula V-X-FVIII means formula NH2-V-X-FVIII-COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. For example, formula V-X-FVIII can further comprise any sequences at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII unless otherwise specified. In another embodiment, the hyphen (-) indicates a peptide bond.

[0134] In one aspect, the chimeric protein comprises two polypeptide chains, (A) a first chain comprising (i) a single chain FVIII protein (ii) an XTEN sequence, and (iii) a first Ig constant region or a portion thereof, e.g., a first Fc region or FcRn binding partner, wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus or inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., one or more XTEN insertion sites disclosed herein) and the first Ig constant region or a portion thereof is linked to the XTEN sequence when the XTEN sequence is linked to the FVIII protein at the N-terminus or the C-terminus or the FVIII protein when the XTEN sequence is inserted within the FVIII protein, and (B) a second chain comprising (iv) a VWF fragment comprising a D' domain and a D3 domain, (v) a linker, and (vi) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein the VWF fragment is linked to the linker, e.g., a cleavable linker, which is further linked to the second Ig constant region or a portion thereof, and wherein the first polypeptide chain and the second polypeptide chain are associated with each other, e.g., a covalent bond, e.g., a disulfide bond. In one embodiment, the linker is a cleavable linker described elsewhere herein, e.g., a thrombin cleavable linker. In some embodiments, the second chain comprises one or more XTEN sequences between (iv) and (v) or (v) and (vi).

[0135] In other aspects, the chimeric protein comprises one polypeptide chain comprising (i) a single chain FVIII protein (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof, e.g., a first Fc region or a first FcRn binding partner, (iv) a first linker, (v) a VWF fragment comprising a D' domain and a D3 domain, (vi) a second linker, and (vii) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein (i) to (vii) are linked in the order or in any orders. In one embodiment, the first linker is a processable linker, which can be intracellularly processed or cleaved after expression and makes the single polypeptide chain into two polypeptide chains. In another embodiment, the second linker is a cleavable linker described herein, e.g., a thrombin cleavable linker. The XTEN sequence used herein can be linked to the FVIII protein by an optional linker at the N-terminus or the C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein.

[0136] In certain aspects, a chimeric protein comprises three polypeptide chains, (A) a first polypeptide chain comprising (i) a heavy chain of a FVIII protein and (ii) an XTEN sequence, which are linked to each other and (B) a second polypeptide chain comprising (iii) a light chain of the FVIII protein and (iv) a first Ig constant region or a portion thereof, e.g., a first Fc region or a first FcRn binding partner, which are linked to each other, and (C) a third polypeptide chain comprising (v) a VWF fragment comprising a D' domain and a D3 domain, (vi) a linker, and (vii) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein the second chain is associated with the first chain and the third chain. In one embodiment, the association between the first chain and the second chain is a chemical association or a physical association. For example, the association between the first chain and the second chain can be a metal bond. In another embodiment, the association between the second chain and the third chain is also a chemical association or a physical association, e.g., a covalent bond or a non-covalent bond. In certain embodiments, the association between the second chain and the third chain is through the two Ig constant regions or a portion thereof and is a disulfide bond. The bonding between the second chain and the third chain prevents or inhibits binding of the FVIII protein with endogenous VWF, thus preventing the FVIII protein being cleared by the VWF clearance pathway. In some embodiments, the linker is a processable linker, which is intracellularly cleaved after expression in a host cell. The XTEN sequence used herein is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein.

[0137] In certain embodiments, the VWF fragment is directly linked to the FVIII protein, which comprises one or more XTENs, by a peptide bond or a linker. As one way of linking the VWF fragment and the FVIII protein, in which one or more XTENs are inserted or linked, through a direct link (e.g. a peptide bond) or a linker, an enzymatic ligation (e.g., sortase) can be employed. For example, sortase refers to a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. For most substrates of sortase enzymes, the recognition signal consists of the motif LPXTG (Leu-Pro-any-Thr-Gly (SEQ ID NO: 51), then a highly hydrophobic transmembrane sequence, then a cluster of basic residues such as arginine. Cleavage occurs between the Thr and Gly, with transient attachment through the Thr residue to the active site Cys residue of a ligation partner, followed by transpeptidation that attaches the protein covalently to the cell wall. In some embodiments, the ligation partner contains Gly(n). In other embodiments, the chimeric protein further comprises a sortase recognition motif. In some embodiments, the VWF fragment is attached to FVIII comprising one or more XTENs inserted within or linked to using sortase mediated in vitro protein ligation.

[0138] In one embodiment, a VWF fragment linked to a sortase recognition motif by an optional linker can be fused to a FVIII protein linked to Gly(n) by a sortase, wherein n can be any integer and wherein one or more XTENs are inserted within or linked to the FVIII protein. A ligation construct comprises the VWF fragment (N-terminal portion of the construct) and the FVIII protein, in which one or more XTENs are inserted or linked (C-terminal portion of the construct), wherein the sortase recognition motif is inserted in between. Another ligation construct comprises the VWF fragment (N-terminal portion of the construct, the linker, the sortase recognition motif, and the FVIII protein, in which one or more XTENs are inserted or linked (C-terminal portion of the construct). In another embodiment, a FVIII protein linked to a sortase recognition motif by an optional linker can be fused to a VWF fragment linked to Gly(n) by a sortase, wherein n is any integer. A resulting ligation construct comprises the FVIII protein (N-terminal portion of the construct), in which one or more XTENs are inserted or linked, and the VWF fragment (C-terminal portion of the construct), wherein the sortase recognition motif is inserted in between. Another resulting ligation construct comprises the FVIII protein (N-terminal portion of the construct), in which one or more XTENs are inserted or linked, the linker, the sortase recognition motif, and the VWF fragment (C-terminal portion of the construct). In other embodiments, a VWF fragment linked to a sortase recognition motif by a first optional linker can be fused to a heterologous moiety, e.g., an immunoglobulin constant region or a portion thereof, e.g., an Fc region, linked to a thrombin cleavage site by a second optional linker. A resulting construct can comprise the VWF fragment (N-terminal portion), the first linker, the sortase recognition motif, the protease cleavage site, the second optional linker, and the heterologous moiety.

[0139] In some embodiments, the VWF fragment is associated with the FVIII protein. The association between the VWF fragment and the FVIII protein can be a chemical association or a physical association. The chemical association can be a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In yet other embodiments, the association between the FVIII protein and the VWF fragment is a physical association between two sequences, e.g., due to an additional association between the sequence having the FVIII protein and the sequence having the VWF fragment, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety.

[0140] As a result of preventing or inhibiting endogenous VWF interaction with the FVIII protein by the VWF fragment, the chimeric protein described herein have an extended half-life compared to wild-type FVIII or the corresponding chimeric protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the VWF fragment. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In a particular embodiment, the half-life of the FVIII protein is extended at least 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, or at least about 27 hours in HemA mice.

A) Von Willebrand Factor (VWF) Fragments

[0141] VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (.alpha.-granules of platelets), and subendothelian connective tissue. The basic VWF monomer is a 2813 amino acid protein. Every monomer contains a number of specific domains with a specific function, the D'/D3 domain (which binds to Factor VIII), the A1 domain (which binds to platelet GPIb-receptor, heparin, and/or possibly collagen), the A3 domain (which binds to collagen), the C1 domain (in which the RGD domain binds to platelet integrin .alpha.IIb.beta.3 when this is activated), and the "cysteine knot" domain at the C-terminal end of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-.beta. (TGF.beta.) and .beta.-human chorionic gonadotropin (.beta.HCG).

[0142] The term "a VWF fragment" as used herein includes, but is not limited to, functional VWF fragments comprising a D' domain and a D3 domain, which are capable of inhibiting binding of endogenous VWF to FVIII. In one embodiment, the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment blocks the VWF binding site on the FVIII protein, thereby inhibiting interaction of the FVIII protein with endogenous VWF. The VWF fragments include derivatives, variants, mutants, or analogues that retain these activities of VWF.

[0143] The 2813 monomer amino acid sequence for human VWF is reported as Accession Number_NP_000543.2_ in Genbank. The nucleotide sequence encoding the human VWF is reported as Accession Number _NM_000552.3_ in Genbank. The nucleotide sequence of human VWF is designated as SEQ ID NO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. Each domain of VWF is listed in Table 1.

TABLE-US-00002 TABLE 1 VWF Sequences VWF domains Amino acid Sequence VWF Signal Peptide 1 MIPARFAGVL LALALILPGT LC (Amino acids 1 to 22 22 of SEQ ID NO: 2) VWF D1D2 region 23 AEGTRGRS (Amino acids 23 to STARCSLFGS DFVNTFDGSM 763 of SEQ ID NO: 2) 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGEVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKR 763 VWF D' Domain 764 SLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDAT 866 VWF D3 Domain 867 ##STR00001## ##STR00002## 901 ##STR00003## ##STR00004## 951 ##STR00005## ##STR00006## 1001 ##STR00007## ##STR00008## 1051 ##STR00009## ##STR00010## 1101 ##STR00011## ##STR00012## 1151 ##STR00013## ##STR00014## 1201 ##STR00015## ##STR00016## 1240 VWF A1 Domain 1241 GGLVVPPTDA 1251 PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV 1301 VDMMERLRIS QKWVRVAVVE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA 1351 GSQVASTSEV LKYTLFQIFS KIDRPEASRI ALLLMASQEP QRMSRNFVRY 1401 VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1451 DEIVSYLCDL APEAPPPTLP PDMAQVTVG 1479 1480 P GLLGVSTLGP KRNSMVLDVA 1501 FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY 1551 PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA 1600 1601 PNLVYMVTGN PASDEIKRLP GDIQVVPIGV GPNANVQELE RIGWPNAPIL 1651 IQDFETLPRE APDLVLQRCC SGEGLQIPTL SPAPDCSQPL DVILLLDGSS 1701 SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNVVPE 1751 KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV 1801 TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNVVK 1851 LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH 1901 TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC 1951 TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG 2001 CMKSIEVKHS ALSVEXHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV 2051 RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD 2101 GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC 2151 HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA 2201 MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP 2251 EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK 2301 APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN 2351 PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN 2401 STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV 2451 CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE 2501 VVTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP 2551 QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC 2601 TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC CGRCLPTACT 2651 IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2701 CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC 2751 QGKCASKAMY SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSVVYHEVLN 2801 AMECKCSPRK CSK Nucleotide Sequence (SEQ ID NO: 1) Full-length VWF 1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT 51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG

851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCRGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCTGCTTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGTGTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG GGAGGCCTGG TGGTGCCTCC CACAGATGCC 3751 CCGGTGAGCC CCACCACTCT GTATGTGGAG GACATCTCGG AACCGCCGTT 3801 GCACGATTTC TACTGCAGCA GGCTACTGGA CCTGGTCTTC CTGCTGGATG 3851 GCTCCTCCAG GCTGTCCGAG GCTGAGTTTG AAGTGCTGAA GGCCTTTGTG 3901 GTGGACATGA TGGAGCGGCT GCGCATCTCC CAGAAGTGGG TCCGCGTGGC 3951 CGTGGTGGAG TACCACGACG GCTCCCACGC CTACATCGGG CTCAAGGACC 4001 GGAAGCGACC GTCAGAGCTG CGGCGCATTG CCAGCCAGGT GAAGTATGCG 4051 GGCAGCCAGG TGGCCTCCAC CAGCGAGGTC TTGAAATACA CACTGTTCCA 4101 AATCTTCAGC AAGATCGACC GCCCTGAAGC CTCCCGCATC GCCCTGCTCC 4151 TGATGGCCAG CCAGGAGCCC CAACGGATGT CCCGGAACTT TGTCCGCTAC 4201 GTCCAGGGCC TGAAGAAGAA GAAGGTCATT GTGATCCCGG TGGGCATTGG 4251 GCCCCATGCC AACCTCAAGC AGATCCGCCT CATCGAGAAG CAGGCCCCTG 4301 AGAACAAGGC CTTCGTGCTG AGCAGTGTGG ATGAGCTGGA GCAGCAAAGG 4351 GACGAGATCG TTAGCTACCT CTGTGACCTT GCCCCTGAAG CCCCTCCTCC 4401 TACTCTGCCC CCCGACATGG CACAAGTCAC TGTGGGCCCG GGGCTCTTGG 4451 GGGTTTCGAC CCTGGGGCCC AAGAGGAACT CCATGGTTCT GGATGTGGCG 4501 TTCGTCCTGG AAGGATCGGA CAAAATTGGT GAAGCCGACT TCAACAGGAG 4551 CAAGGAGTTC ATGGAGGAGG TGATTCAGCG GATGGATGTG GGCCAGGACA 4601 GCATCCACGT CACGGTGCTG CAGTACTCCT ACATGGTGAC CGTGGAGTAC 4651 CCCTTCAGCG AGGCACAGTC CAAAGGGGAC ATCCTGCAGC GGGTGCGAGA 4701 GATCCGCTAC CAGGGCGGCA ACAGGACCAA CACTGGGCTG GCCCTGCGGT 4751 ACCTCTCTGA CCACAGCTTC TTGGTCAGCC AGGGTGACCG GGAGCAGGCG 4801 CCCAACCTGG TCTACATGGT CACCGGAAAT CCTGCCTCTG ATGAGATCAA 4851 GAGGCTGCCT GGAGACATCC AGGTGGTGCC CATTGGAGTG GGCCCTAATG 4901 CCAACGTGCA GGAGCTGGAG AGGATTGGCT GGCCCAATGC CCCTATCCTC 4951 ATCCAGGACT TTGAGACGCT CCCCCGAGAG GCTCCTGACC TGGTGCTGCA 5001 GAGGTGCTGC TCCGGAGAGG GGCTGCAGAT CCCCACCCTC TCCCCTGCAC

5051 CTGACTGCAG CCAGCCCCTG GACGTGATCC TTCTCCTGGA TGGCTCCTCC 5101 AGTTTCCCAG CTTCTTATTT TGATGAAATG AAGAGTTTCG CCAAGGCTTT 5151 CATTTCAAAA GCCAATATAG GGCCTCGTCT CACTCAGGTG TCAGTGCTGC 5201 AGTATGGAAG CATCACCACC ATTGACGTGC CATGGAACGT GGTCCCGGAG 5251 AAAGCCCATT TGCTGAGCCT TGTGGACGTC ATGCAGCGGG AGGGAGGCCC 5301 CAGCCAAATC GGGGATGCCT TGGGCTTTGC TGTGCGATAC TTGACTTCAG 5351 AAATGCATGG TGCCAGGCCG GGAGCCTCAA AGGCGGTGGT CATCCTGGTC 5401 ACGGACGTCT CTGTGGATTC AGTGGATGCA GCAGCTGATG CCGCCAGGTC 5451 CAACAGAGTG ACAGTGTTCC CTATTGGAAT TGGAGATCGC TACGATGCAG 5501 CCCAGCTACG GATCTTGGCA GGCCCAGCAG GCGACTCCAA CGTGGTGAAG 5551 CTCCAGCGAA TCGAAGACCT CCCTACCATG GTCACCTTGG GCAATTCCTT 5601 CCTCCACAAA CTGTGCTCTG GATTTGTTAG GATTTGCATG GATGAGGATG 5651 GGAATGAGAA GAGGCCCGGG GACGTCTGGA CCTTGCCAGA CCAGTGCCAC 5701 ACCGTGACTT GCCAGCCAGA TGGCCAGACC TTGCTGAAGA GTCATCGGGT 5751 CAACTGTGAC CGGGGGCTGA GGCCTTCGTG CCCTAACAGC CAGTCCCCTG 5801 TTAAAGTGGA AGAGACCTGT GGCTGCCGCT GGACCTGCCC CTGYGTGTGC 5851 ACAGGCAGCT CCACTCGGCA CATCGTGACC TTTGATGGGC AGAATTTCAA 5901 GCTGACTGGC AGCTGTTCTT ATGTCCTATT TCAAAACAAG GAGCAGGACC 5951 TGGAGGTGAT TCTCCATAAT GGTGCCTGCA GCCCTGGAGC AAGGCAGGGC 6001 TGCATGAAAT CCATCGAGGT GAAGCACAGT GCCCTCTCCG TCGAGSTGCA 6051 CAGTGACATG GAGGTGACGG TGAATGGGAG ACTGGTCTCT GTTCCTTACG 6101 TGGGTGGGAA CATGGAAGTC AACGTTTATG GTGCCATCAT GCATGAGGTC 6151 AGATTCAATC ACCTTGGTCA CATCTTCACA TTCACTCCAC AAAACAATGA 6201 GTTCCAACTG CAGCTCAGCC CCAAGACTTT TGCTTCAAAG ACGTATGGTC 6251 TGTGTGGGAT CTGTGATGAG AACGGAGCCA ATGACTTCAT GCTGAGGGAT 6301 GGCACAGTCA CCACAGACTG GAAAACACTT GTTCAGGAAT GGACTGTGCA 6351 GCGGCCAGGG CAGACGTGCC AGCCCATCCT GGAGGAGCAG TGTCTTGTCC 6401 CCGACAGCTC CCACTGCCAG GTCCTCCTCT TACCACTGTT TGCTGAATGC 6451 CACAAGGTCC TGGCTCCAGC CACATTCTAT GCCATCTGCC AGCAGGACAG 6501 TTGCCACCAG GAGCAAGTGT GTGAGGTGAT CGCCTCTTAT GCCCACCTCT 6551 GTCGGACCAA CGGGGTCTGC GTTGACTGGA GGACACCTGA TTTCTGTGCT 6601 ATGTCATGCC CACCATCTCT GGTCTACAAC CACTGTGAGC ATGGCTGTCC 6651 CCGGCACTGT GATGGCAACG TGAGCTCCTG TGGGGACCAT CCCTCCGAAG 6701 GCTGTTTCTG CCCTCCAGAT AAAGTCATGT TGGAAGGCAG CTGTGTCCCT 6751 GAAGAGGCCT GCACTCAGTG CATTGGTGAG GATGGAGTCC AGCACCAGTT 6801 CCTGGAAGCC TGGGTCCCGG ACCACCAGCC CTGTCAGATC TGCACATGCC 6851 TCAGCGGGCG GAAGGTCAAC TGCACAACGC AGCCCTGCCC CACGGCCAAA 6901 GCTCCCACGT GTGGCCTGTG TGAAGTAGCC CGCCTCCGCC AGAATGCAGA 6951 CCAGTGCTGC CCCGAGTATG AGTGTGTGTG TGACCCAGTG AGCTGTGACC 7001 TGCCCCCAGT GCCTCACTGT GAACGTGGCC TCCAGCCCAC ACTGACCAAC 7051 CCTGGCGAGT GCAGACCCAA CTTCACCTGC GCCTGCAGGA AGGAGGAGTG 7101 CAAAAGAGTG TCCCCACCCT CCTGCCCCCC GCACCGTTTG CCCACCCTTC 7151 GGAAGACCCA GTGCTGTGAT GAGTATGAGT GTGCCTGCAA CTGTGTCAAC 7201 TCCACAGTGA GCTGTCCCCT TGGGTACTTG GCCTCAACCG CCACCAATGA 7251 CTGTGGCTGT ACCACAACCA CCTGCCTTCC CGACAAGGTG TGTGTCCACC 7301 GAAGCACCAT CTACCCTGTG GGCCAGTTCT GGGAGGAGGG CTGCGATGTG 7351 TGCACCTGCA CCGACATGGA GGATGCCGTG ATGGGCCTCC GCGTGGCCCA 7401 GTGCTCCCAG AAGCCCTGTG AGGACAGCTG TCGGTCGGGC TTCACTTACG 7451 TTCTGCATGA AGGCGAGTGC TGTGGAAGGT GCCTGCCATC TGCCTGTGAG 7501 GTGGTGACTG GCTCACCGCG GGGGGACTCC CAGTCTTCCT GGAAGAGTGT 7551 CGGCTCCCAG TGGGCCTCCC CGGAGAACCC CTGCCTCATC AATGAGTGTG 7601 TCCGAGTGAA GGAGGAGGTC TTTATACAAC AAAGGAACGT CTCCTGCCCC 7651 CAGCTGGAGG TCCCTGTCTG CCCCTCGGGC TTTCAGCTGA GCTGTAAGAC 7701 CTCAGCGTGC TGCCCAAGCT GTCGCTGTGA GCGCATGGAG GCCTGCATGC 7751 TCAATGGCAC TGTCATTGGG CCCGGGAAGA CTGTGATGAT CGATGTGTGC 7801 ACGACCTGCC GCTGCATGGT GCAGGTGGGG GTCATCTCTG GATTCAAGCT 7851 GGAGTGCAGG AAGACCACCT GCAACCCCTG CCCCCTGGGT TACAAGGAAG 7901 AAAATAACAC AGGTGAATGT TGTGGGAGAT GTTTGCCTAC GGCTTGCACC 7951 ATTCAGCTAA GAGGAGGACA GATCATGACA CTGAAGCGTG ATGAGACGCT 8001 CCAGGATGGC TGTGATACTC ACTTCTGCAA GGTCAATGAG AGAGGAGAGT 8051 ACTTCTGGGA GAAGAGGGTC ACAGGCTGCC CACCCTTTGA TGAACACAAG 8101 TGTCTTGCTG AGGGAGGTAA AATTATGAAA ATTCCAGGCA CCTGCTGTGA 8151 CACATGTGAG GAGCCTGAGT GCAACGACAT CACTGCCAGG CTGCAGTATG 8201 TCAAGGTGGG AAGCTGTAAG TCTGAAGTAG AGGTGGATAT CCACTACTGC 8251 CAGGGCAAAT GTGCCAGCAA AGCCATGTAC TCCATTGACA TCAACGATGT 8301 GCAGGACCAG TGCTCCTGCT GCTCTCCGAC ACGGACGGAG CCCATGCAGG 8351 TGGCCCTGCA CTGCACCAAT GGCTCTGTTG TGTACCATGA GGTTCTCAAT 8401 GCCATGGAGT GCAAATGCTC CCCCAGGAAG TGCAGCAAGT GA

[0144] The VWF fragment as used herein can be a VWF fragment comprising a D' domain and a D3 domain of VWF, wherein the VWF fragment binds to Factor VIII (FVIII) and inhibits binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment comprising the D' domain and the D3 domain can further comprise a VWF domain selected from the group consisting of an A1 domain, an A2 domain, an A3 domain, a D1 domain, a D2 domain, a D4 domain, a B1 domain, a B2 domain, a B3 domain, a C1 domain, a C2 domain, a CK domain, one or more fragments thereof, and any combinations thereof. In one embodiment, a VWF fragment comprises, consists essentially of, or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the D1, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof, (4) the D1, D2, D', and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3, and A1 domains of VWF or fragments thereof.

[0145] The VWF fragment described herein does not contain a site binding to a VWF clearance receptor. In another embodiment, the VWF fragment described herein is not amino acids 764 to 1274 of SEQ ID NO: 2. The VWF fragment of the present invention can comprise any other sequences linked to or fused to the VWF fragment. For example, a VWF fragment described herein can further comprise a signal peptide.

[0146] In one embodiment, the VWF fragment binds to or is associated with a FVIII protein. By binding to or associating with a FVIII protein, a VWF fragment of the invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy chain and light chain of FVIII, and prevents clearance of FVIII by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with a FVIII protein and blocks or prevents binding of the FVIII protein to phospholipid and activated Protein C. By preventing or inhibiting binding of the FVIII protein with endogenous, full-length VWF, the VWF fragment of the invention reduces the clearance of FVIII by VWF clearance receptors and thus extends half-life of the FVIII protein. In one embodiment, the half-life extension of a FVIII protein is thus due to the binding of or associating with the VWF fragment lacking a VWF clearance receptor binding site to the FVIII protein and shielding or protecting of the FVIII protein by the VWF fragment from endogenous VWF which contains the VWF clearance receptor binding site. The FVIII protein bound to or protected by the VWF fragment can also allow recycling of a FVIII protein. By eliminating the VWF clearance pathway receptor binding sites contained in the full length VWF molecule, the FVIII/VWF heterodimers of the invention are shielded from the VWF clearance pathway, further extending FVIII half-life.

[0147] In one embodiment, a VWF fragment of the present invention comprises the D' domain and the D3 domain of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In another embodiment, a VWF fragment comprises the D' domain and the D3 domain of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In some embodiments, a VWF fragment described herein comprises, consists essentially of, or consists of the D' domain and D3 domain of VWF, which are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In other embodiments, a VWF fragment comprises, consists essentially of, or consists of the D1, D2, D', and D3 domains at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In still other embodiments, the VWF fragment further comprises a signal peptide operably linked thereto.

[0148] In some embodiments, a VWF fragment of the invention consists essentially of or consists of (1) the D'D3 domain, the D1D'D3 domain, D2D'D3 domain, or D1D2D'D3 domain and (2) an additional VWF sequence up to about 10 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up to about 15 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about 20 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), up to about 25 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1265 of SEQ ID NO: 2), or up to about 30 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a particular embodiment, the VWF fragment comprising or consisting essentially of the D' domain and the D3 domain is neither amino acids 764 to 1274 of SEQ ID NO: 2 nor the full-length mature VWF. In some embodiments, the D1D2 domain is expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis with the D'D3 domain.

[0149] In other embodiments, the VWF fragment comprising the D'D3 domains linked to the D1D2 domains further comprises an intracellular cleavage site, e.g., (a cleavage site by PACE (furin) or PC5), allowing cleavage of the D1D2 domains from the D'D3 domains upon expression. Non-limiting examples of the intracellular cleavage site are disclosed elsewhere herein.

[0150] In yet other embodiments, a VWF fragment comprises the D' domain and the D3 domain, but does not comprise an amino acid sequence selected from the group consisting of (1) amino acids 1241 to 2813 of SEQ ID NO: 2, (2) amino acids 1270 to amino acids 2813 of SEQ ID NO: 2, (3) amino acids 1271 to amino acids 2813 of SEQ ID NO: 2, (4) amino acids 1272 to amino acids 2813 of SEQ ID NO: 2, (5) amino acids 1273 to amino acids 2813 of SEQ ID NO: 2, (6) amino acids 1274 to amino acids 2813 of SEQ ID NO: 2, and any combinations thereof.

[0151] In still other embodiments, a VWF fragment of the present invention comprises, consists essentially of, or consists of an amino acid sequence corresponding to the D' domain, D3 domain, and A1 domain, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid 764 to 1479 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In a particular embodiment, the VWF fragment is not amino acids 764 to 1274 of SEQ ID NO: 2.

[0152] In some embodiments, a VWF fragment of the invention comprises the D' domain and the D3 domain, but does not comprise at least one VWF domain selected from the group consisting of (1) an A1 domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2 domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CK domain, (11) a CK domain and C2 domain, (12) a CK domain, a C2 domain, and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, a B3 domain, (14) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, (15) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, and a B1 domain, (16) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, and a D4 domain, (17) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, and an A3 domain, (18) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, and an A2 domain, (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, an A2 domain, and an A1 domain, and (20) any combinations thereof.

[0153] In yet other embodiments, the VWF fragment comprises the D'D3 domains and one or more domains or modules. Examples of such domains or modules include, but are not limited to, the domains and modules disclosed in Zhour et al., Blood published online Apr. 6, 2012: DOI 10.1182/blood-2012-01-405134. For example, the VWF fragment can comprise the D'D3 domain and one or more domains or modules selected from the group consisting of A1 domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL-4 module, C1 module, C2 module, C3 module, C4 module, C5 module, C5 module, C6 module, and any combinations thereof.

[0154] In still other embodiments, the VWF fragment is linked to a heterologous moiety, wherein the heterologous moiety is linked to the N-terminus or the C-terminus of the VWF fragment or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein in the VWF fragment. For example, the insertion sites for the heterologous moiety in the VWF fragment can be in the D' domain, the D3 domain, or both. The heterologous moiety can be a half-life extender.

[0155] In certain embodiments, a VWF fragment of the invention forms a multimer, e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer, or the higher order multimers. In other embodiments, the VWF fragment is a monomer having only one VWF fragment. In some embodiments, the VWF fragment of the present invention can have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF fragment can include amino acid substitutions, deletions, additions, or modifications such that the VWF fragment is not capable of forming a disulfide bond or forming a dimer or a multimer. In another embodiment, the amino acid substitution is within the D' domain and the D3 domain. In a particular embodiment, a VWF fragment of the invention contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. The at least one amino acid substitution can be any amino acids that are not occurring naturally in the wild type VWF. For example, the amino acid substitution can be any amino acids other than cysteine, e.g., Isoleucine, Alanine, Leucine, Asparagine, Lysine, Aspartic acid, Methionine, Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine, Arginine, or Histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the VWF fragments from forming multimers.

[0156] In certain embodiments, the VWF fragment useful herein can be further modified to improve its interaction with FVIII, e.g., to improve binding affinity to FVIII. As a non-limiting example, the VWF fragment comprises a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO: 2 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO: 2. Residues 764 and/or 773 can contribute to the binding affinity of the VWF fragments to FVIII. In other embodiments, the VWF fragments useful for the invention can have other modifications, e.g., the protein can be pegylated, glycosylated, hesylated, or polysialylated.

B) XTEN Sequences

[0157] As used here "XTEN sequence" refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. As a chimeric protein partner, XTENs can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a VWF fragment or a FVIII sequence of the invention to create a chimeric protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments such as single-chain antibodies or Fc fragments of a light chain or a heavy chain.

[0158] In some embodiments, the XTEN sequence of the invention is a peptide or a polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or a polypeptide having greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues, or greater than 120 to about 400 residues.

[0159] The XTEN sequence of the invention can comprise one or more sequence motif of 9 to 14 amino acid residues or an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, wherein the motif comprises, consists essentially of, or consists of 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). See US 2010-0239554 A1.

[0160] In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from a single motif family selected from Table 2A, resulting in a family sequence. As used herein, "family" means that the XTEN has motifs selected only from a single motif category from Table 2A; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN not from a family motif are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or to achieve a better linkage to FVIII or VWF. In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or of the AE motif family, or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC family, or of the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 2A. These sequences can be selected to achieve desired physical/chemical characteristics, including such properties as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.

TABLE-US-00003 TABLE 2A XTEN Sequence Motifs of 12 Amino Acids and Motif Families Motif Family* MOTIF SEQUENCE AD GESPGGSSGSES AD GSEGSSGPGESS AD GSSESGSSEGGP AD GSGGEPSESGSS AE, AM GSPAGSPTSTEE AE, AM, AQ GSEPATSGSETP AE, AM, AQ GTSESATPESGP AE, AM, AQ GTSTEPSEGSAP AF, AM GSTSESPSGTAP AF, AM GTSTPESGSASP AF, AM GTSPSGESSTAP AF, AM GSTSSTAESPGP AG, AM GTPGSGTASSSP AG, AM GSSTPSGATGSP AG, AM GSSPSASTGTGP AG, AM GASPGTSSTGSP AQ GEPAGSPTSTSE AQ GTGEPSSTPASE AQ GSGPSTESAPTE AQ GSETPSGPSETA AQ GPSETSTSEPGA AQ GSPSEPTEGTSA BC GSGASEPTSTEP BC GSEPATSGTEPS BC GTSEPSTSEPGA BC GTSTEPSEPGSA BD GSTAGSETSTEA BD GSETATSGSETA BD GTSESATSESGA BD GTSTEASEGSAS Denotes individual motif sequences that, when used together in various permutations, results in a ''family sequence''

[0161] XTEN can have varying lengths for insertion into or linkage to FVIII or VWF. In one embodiment, the length of the XTEN sequence(s) is chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or function, XTEN can be short or intermediate length sequence or longer sequence that can serve as carriers. In certain embodiments, the XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN inserted into or linked to FVIII or VWF can have lengths of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues in length. In other embodiments, the XTEN sequences is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The precise length of an XTEN inserted into or linked to FVIII or VWF can vary without adversely affecting the activity of the FVIII or VWF. In one embodiment, one or more of the XTEN used herein has 36 amino acids, 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and can be selected from one or more of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD.

[0162] In some embodiments, the XTEN sequence used in the invention is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US 2010-0239554 A1.

[0163] In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combinations thereof.

[0164] In some embodiments, less than 100% of amino acids of an XTEN are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consists of the sequence motifs from Table 2A or the XTEN sequences of Table 2B. In such embodiments, the remaining amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but may be preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The content of hydrophobic amino acids in the XTEN utilized in the conjugation constructs may be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, XTEN sequences may contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: methionine (for example, to avoid oxidation), or asparagine and glutamine (to avoid desamidation).

[0165] In another embodiment, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combinations thereof. In a specific embodiment, the XTEN sequence is AE288. The amino acid sequences for certain XTEN sequences of the invention are shown in Table 2B.

TABLE-US-00004 TABLE 2B XTEN Sequences XTEN Amino Acid Sequence AE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS SEQ ID NO: 36 AE72 GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA SEQ ID NO: TSGSETPGTSESATPESGPGTSTEPSEGSAPGASS 127 AE144 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTE SEQ ID EGTSTEPSEG NO: 37 SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESA PESGPGSEPATSGSETPGTSTEPSEGSAP AE144_2A TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP (SEQ ID NO: GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET 128) PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPG AE144_3B SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP (SEQ ID NO: GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA 129) PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS APG AE144_4A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP (SEQ ID NO: GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE 130) EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APG AE144_5A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP (SEQ ID NO: GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG 131) PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEG AE144_6B TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP (SEQ ID NO: GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA 132) PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APG AG144 GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG SEQ ID PGASPGTSST NO: 38 GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS TGSPGSSPSA STGTGPGTPGSGTASSSPGSSTPSGATGSP AG144_A GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS (SEQ ID NO: PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS 133) SPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSP AG144_B GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSS (SEQ ID NO: PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATG 134) SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSP AG144_C GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS (SEQ ID NO: PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG 135) SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP AG144_F GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS (SEQ ID NO: PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT 136) GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP AE288 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET SEQ ID PPGTSESATPESG NO: 39 GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAP AE288_2 GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG (SEQ ID NO: PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS 137) APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP AG288 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SEQ ID SPGTPGSGTASS NO: 40 SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSPSASTG TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSPSAS TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS GATGS AE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE SEQ ID EGTSTEPSEGSA NO: 41 PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG576 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT SEQ ID GPGSSTPSGATG NO: 42 SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSTPSG ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT PGSGTASSSPGS STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE SEQ ID EGTSTEPSEGSA NO: 43 PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AG864 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS SEQ ID PGSSTPSGATGS NO: 44 PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGT ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSA STGTGPGTPGSG TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGSSTP SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS PGASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

[0166] In further embodiments, the XTEN sequence used in the invention affects the physical or chemical property, e.g., pharmacokinetics, of the chimeric protein of the present invention. The XTEN sequence used in the present invention can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii. In a specific embodiment, the XTEN sequence linked to a FVIII protein in this invention increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that the chimeric protein described herein stays in vivo for an increased period of time compared to wild type FVIII. In further embodiments, the XTEN sequence used in this invention increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that FVIII protein stays in vivo for an increased period of time compared to wild type FVIII.

[0167] A variety of methods and assays can be employed to determine the physical/chemical properties of proteins comprising the XTEN sequence. Such methods include, but are not limited to analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

[0168] Additional examples of XTEN sequences that can be used according to the present invention and are disclosed in US Patent Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, or WO 2011028344 A2.

C) Factor VIII (FVIII) Protein

[0169] "A FVIII protein" as used herein means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. The term a FVIII protein includes a functional fragment, variant, analog, or derivative thereof that retains the function of full-length wild-type Factor VIII in the coagulation pathway. "A FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of the FVIII functions include, but not limited to, an ability to activate coagulation, an ability to act as a cofactor for factor IX, or an ability to form a tenase complex with factor IX in the presence of Ca' and phospholipids, which then converts Factor X to the activated form Xa. The FVIII protein can be the human, porcine, canine, rat, or murine FVIII protein. In addition, comparisons between FVIII from humans and other species have identified conserved residues that are likely to be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat. No. 6,251,632).

[0170] A number of tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen testing (often by the Clauss method), platelet count, platelet function testing (often by PFA-100), TCT, bleeding time, mixing test (whether an abnormality corrects if the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM.RTM., e.g., ROTEM.RTM.) or euglobulin lysis time (ELT).

[0171] The aPTT test is a performance indicator measuring the efficacy of both the "intrinsic" (also referred to the contact activation pathway) and the common coagulation pathways. This test is commonly used to measure clotting activity of commercially available recombinant clotting factors, e.g., FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

[0172] ROTEM analysis provides information on the whole kinetics of haemostasis: clotting time, clot formation, clot stability and lysis. The different parameters in thromboelastometry are dependent on the activity of the plasmatic coagulation system, platelet function, fibrinolysis, or many factors which influence these interactions. This assay can provide a complete view of secondary haemostasis.

[0173] The FVIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified versions. Examples of human FVIII sequences (full-length) are shown below.

TABLE-US-00005 TABLE 3 Amino Acid Sequence of Full-length Factor VIII (Full-length FVIII (FVIII signal peptide underlined; FVIII heavy chain is double underlined; B domain is italicized; and FVIII light chain is in plain text) Signal Peptide: (SEQ ID NO: 3) MQIELSTCFFLCLLRFCFS Mature Factor VIII (SEQ ID NO: 4)* ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASE GAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDR DAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHT FLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHY IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGV KHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNT GDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAI DSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSS TSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGP LSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNAL FKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPL IHDRMIMDKNATALRENHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMI FLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKG EFTKDVGLKEMVFPSSRNIFLTNIDNIHENNTHNQEKKIQEEIEKKETLIQENVV LPQIHTVTGTKNFMKNIFLLSTRQNVEGSYDGAYAPVLQDFRSENDSTNRTKKHT AHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRL PLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTIIDYNEKEKGAITQSPLSDCLT RSHSIPQANRSPLPIAKVSSFPSIRPIKETRVLFQDNSSHLPAASYRKKDSGVQE SSHFLQGAKKNNISLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDL PKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANR PGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKK DTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREI TRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVER LWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTY FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQ VTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYI MDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYP GVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF QITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGAR QKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVITQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLY

TABLE-US-00006 TABLE 4 Nucleotide Sequence Encoding Full-Length FVIII (SEQ ID NO: 5)* 661 ATG CAAATAGAGC TCTCCACCTG 721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC 781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG 841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC 901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT 961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA 1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC 1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT 1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC 1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA 1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC 1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA 1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC 1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA 1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT 1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC 1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT 1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG 1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA 1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT 1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA 1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG 1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT 2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT 2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC 2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG 2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT 2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG 2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA 2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA 2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT 2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC 2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA 2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC 3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC 3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA 3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT 3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC 3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT 3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA 3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG 3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC 3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG 3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC 3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC 4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT 4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA 4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT 4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT 4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA 4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA 4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA 4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC 4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA 5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC 5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC 5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT 5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA 5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA 5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA 5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC 5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG 5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA 5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG 6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA 6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT 6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC 6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA 6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC 6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG 6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG 6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA 6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT 6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG 6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA 6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT 6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG 6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC 6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA 6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC 6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC 7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG 7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA 7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT 7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG 7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA 7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG 7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG 7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA 7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA 7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC 7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA 7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA 7741 GGACCTCTAC *The underlined nucleic acids encode a signal peptide.

[0174] FVIII polypeptides include full-length FVIII, full-length FVIII minus Met at the N-terminus, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and/or FVIII with a full or partial deletion of the B domain. In certain embodiments, FVIII variants include B domain deletions, whether partial or full deletions.

[0175] The sequence of native mature human FVIII is presented as SEQ ID NO: 4. A native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are the structurally-related "A domains," B is the "B domain," C1 and C2 are the structurally-related "C domains," and a1, a2 and a3 are acidic spacer regions. Referring to the primary amino acid sequence position in SEQ ID NO:4, the A1 domain of human FVIII extends from Ala1 to about Arg336, the a1 spacer region extends from about Met337 to about Val374, the A2 domain extends from about Ala375 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg 1648, the a3 spacer region extends from about Glu1649 to about Arg1689, the A3 domain extends from about Ser1690 to about Leu2025, the C1 domain extends from about Gly2026 to about Asn2072, and the C2 domain extends from about Ser2073 to Tyr2332. Other than specific proteolytic cleavage sites, designation of the locations of the boundaries between the domains and regions of FVIII can vary in different literature references. The boundaries noted herein are therefore designated as approximate by use of the term "about."

[0176] The human FVIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337 (1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; and U.S. Pat. No. 4,757,006). The FVIII amino acid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199. In addition, partially or fully B-domain deleted FVIII is shown in U.S. Pat. Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B-domain is replaced with the human Factor V B-domain as shown in U.S. Pat. No. 5,004,803. The cDNA sequence encoding human Factor VIII and amino acid sequence are shown in SEQ ID NOs: 4 and 5, respectively, of US Application Publ. No. 2005/0100990.

[0177] The porcine FVIII sequence is published in Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). Further, the complete porcine cDNA sequence obtained from PCR amplification of FVIII sequences from a pig spleen cDNA library has been reported in Healey, J. F., et al., Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII and a chimeric FVIII with porcine A1 and/or A2 domains substituted for the corresponding human domains were reported in WO 94/11503. U.S. Pat. No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA and deduced amino acid sequences. U.S. Pat. No. 6,458,563 discloses a B-domain-deleted porcine FVIII.

[0178] U.S. Pat. No. 5,859,204 to Lollar, J. S. reports functional mutants of FVIII having reduced antigenicity and reduced immunoreactivity. U.S. Pat. No. 6,376,463 to Lollar, J. S. also reports mutants of FVIII having reduced immunoreactivity. US Appl. Publ. No. 2005/0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

[0179] In one embodiment, the FVIII (or FVIII portion of a chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence) or a FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 3 and 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 19 of SEQ ID NO: 3 and amino acids 1 to 2332 of SEQ ID NO: 4 (with a signal sequence), wherein the FVIII has a clotting activity, e.g., activates Factor IX as a cofactor to convert Factor X to activated Factor X. The FVIII (or FVIII portion of a chimeric protein) may be identical to a FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence). The FVIII may further comprise a signal sequence.

[0180] The "B-domain" of FVIII, as used herein, is the same as the B-domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741-Arg1648 of full-length human FVIII. The other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the a3 acidic region. The locations of the boundaries for all of the domains, including the B-domains, for porcine, mouse and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B-domain-deleted factor VIII" or "BDD FVIII"). An example of a BDD FVIII is REFACTO.RTM. (recombinant BDD FVIII), which has the same sequence as the Factor VIII portion of the sequence in Table 5. (BDD FVIII heavy chain is double underlined; B domain is italicized; and BDD FVIII light chain is in plain text). A nucleotide sequence encoding the amino acid sequence set forth in Table 5 (SEQ ID NO: 7) is shown in Table 6.

TABLE-US-00007 TABLE 5 Amino Acid Sequence of B-domain Deleted Factor VIII (BDD FVIII) BDD FVIII (SEQ ID NO: 6) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASE GAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLV KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDR DAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHT FLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHY IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGV KHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNT GDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDE FDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAG IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIM YSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQG RSNAWRPQVNNPKEWLQVDFQKTMKVTGVITQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY

TABLE-US-00008 TABLE 6 Nucleotide Sequence Encoding BDD FVIII (SEQ ID NO: 7)* 661 A TGCAAATAGA GCTCTCCACC TGCTTCTTTC 721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC 781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC 841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG 901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC 961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG 1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG 1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG 1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC 1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA 1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC 1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG 1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG 1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG 4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA 4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA 4921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041 GCGAGGCACA GGACCTCTAC *The underlined nucleic acids encode a signal peptide.

[0181] A "B-domain-deleted FVIII" may have the full or partial deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In some embodiments, a B-domain-deleted FVIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and Examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No. 6,346,513). In another embodiment, a B-domain deleted Factor VIII is the S743/Q1638 B-domain deleted Factor VIII (SQ BDD FVIII) (e.g., Factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII having amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 4, i.e., SEQ ID NO: 6). In some embodiments, a B-domain-deleted FVIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. Nos. 6,060,447, 5,595,886, and 6,228,620). In some embodiments, a B-domain-deleted Factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments, a B-domain-deleted FVIII has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91/09122. In some embodiments, a B-domain-deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). A B-domain-deleted Factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988). Additional B domain deletions that are part of the invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 through 1648 (Pasek (PCT application No. 88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)). In other embodiments, BDD FVIII includes a FVIII polypeptide containing fragments of the B-domain that retain one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or optionally 943, which correspond to the amino acid sequence of the full-length FVIII sequence. Examples of the B-domain fragments include 226 amino acids or 163 amino acids of the B-domain as disclosed in Miao, H. Z., et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, S. W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011) (i.e., the first 226 amino acids or 163 amino acids of the B domain are retained). In still other embodiments, BDD FVIII further comprises a point mutation at residue 309 (from Phe to Ser) to improve expression of the BDD FVIII protein. See Miao, H. Z., et al., Blood 103(a): 3412-3419 (2004). In still other embodiments, the BDD FVIII includes a FVIII polypeptide containing a portion of the B-domain, but not containing one or more furin cleavage sites (e.g., Arg1313 and Arg 1648). See Pipe, S. W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011). Each of the foregoing deletions may be made in any FVIII sequence.

[0182] In some embodiments, the FVIII has a partial B-domain. In some embodiments, the FVIII protein with a partial B-domain is FVIII198 (SEQ ID NO: 89). FVIII198 is a partial B-domain containing single chain FVIIIFc molecule-226N6. 226 represents the N-terminus 226 amino acid of the FVIII B-domain, and N6 represents six N-glycosylation sites in the B-domain.

[0183] In one embodiment, FVIII is cleaved right after arginine at amino acid 1648 (in full-length Factor VIII or SEQ ID NO: 4), amino acid 754 (in the 5743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 6), or the corresponding arginine residue (in other variants), thereby resulting in a heavy chain and a light chain. In another embodiment, FVIII comprises a heavy chain and a light chain, which are linked or associated by a metal ion-mediated non-covalent bond.

[0184] In other embodiments, FVIII is a single chain FVIII that has not been cleaved right after Arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 4), amino acid 754 (in the 5743/Q1638 B-domain-deleted FVIII or SEQ ID NO: 6), or the corresponding Arginine residue (in other variants). A single chain FVIII may comprise one or more amino acid substitutions. In one embodiment, the amino acid substitution is at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). The amino acid substitution can be any amino acids other than arginine, e.g., isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine.

[0185] FVIII can further be cleaved by thrombin and then activated as FVIIIa, serving as a cofactor for activated Factor IX (FIXa). And the activated FIX together with activated FVIII forms a Xase complex and converts Factor X to activated Factor X (FXa). For activation, FVIII is cleaved by thrombin after three Arginine residues, at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deleted FVIII sequence), the cleavage generating FVIIIa having the 50 kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chains. In one embodiment, the FVIII protein useful for the present invention is non-active FVIII. In another embodiment, the FVIII protein is an activated FVIII.

[0186] The protein having FVIII polypeptide linked to or associated with the VWF fragment can comprise a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 6, wherein the sequence has the FVIII clotting activity, e.g., activating Factor IX as a cofactor to convert Factor X to activated Factor X (FXa).

[0187] "Hybrid" or "chimeric" polypeptides and proteins, as used herein, includes a combination of a first polypeptide chain, e.g., the VWF fragment, optionally fused to a first Ig constant region or a portion thereof, with a second polypeptide chain, e.g., a FVIII protein linked to an XTEN sequence, optionally fused to a second Ig constant region or a portion thereof, thereby forming a heterodimer. In one embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via protein-protein interactions, such as charge-charge or hydrophobic interactions. In another embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via disulfide or other covalent bond(s). Hybrids are described, for example, in US 2004/101740 and US 2006/074199. The second polypeptide may be an identical copy of the first polypeptide or a non-identical polypeptide. In one embodiment, the first polypeptide is a FVIII protein(X)-Fc fusion protein, and the second polypeptide is a polypeptide comprising, consisting essentially of, or consisting of an Fc region, wherein the first polypeptide and the second polypeptide are associated with each other. In another embodiment, the first polypeptide comprises a VWF fragment-XTEN-Fc fusion protein, and the second polypeptide comprises FVIII-Fc fusion protein, making the hybrid a heterodimer. In other embodiments, the first polypeptide comprises a VWF fragment-Fc fusion protein, and the second polypeptide comprises FVIII(X)-Fc fusion protein, making the hybrid a heterodimer. In yet other embodiments, the first polypeptide comprises a VWF fragment-XTEN-Fc fusion protein, and the second polypeptide comprises FVIII(X)-Fc fusion protein. The first polypeptide and the second polypeptide can be associated through a covalent bond, e.g., a disulfide bond, between the first Fc region and the second Fc region. The first polypeptide and the second polypeptide can further be associated with each other by binding between the VWF fragment and the FVIII protein.

[0188] A FVIII protein useful in the present invention can include FVIII having one or more additional XTEN sequences, which do not affect the FVIII coagulation activity. Such XTEN sequences can be fused to the C-terminus or N-terminus of the FVIII protein or inserted between one or more of the two amino acid residues in the FVIII protein wherein the insertions do not affect the FVIII coagulation activity or FVIII function. In one embodiment, the insertions improve pharmacokinetic properties of the FVIII protein (e.g., half-life). In another embodiment, the insertions can be multiple insertions, e.g., more than two, three, four, five, six, seven, eight, nine, or ten insertions. Examples of the insertion sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, 15 or any combinations thereof.

[0189] The FVIII protein linked to one or more XTEN sequences can be represented as FVIII(X), FVIII(X1), FVIII.sub.(a.fwdarw.b)-X-FVIII.sub.(c.fwdarw.d), wherein FVIII.sub.(a.fwdarw.b) comprises, consists essentially of, or consists of a first portion of a FVIII protein from amino acid residue "a" to amino acid residue "b"; X or X1 comprises, consists essentially of, or consists of one or more XTEN sequences, FVIII.sub.(c.fwdarw.d) comprises, consists essentially of, or consists of a second portion of a FVIII protein from amino acid residue "c" to amino acid residue "d"; a is the N-terminal amino acid residue of the first portion of the FVIII protein, b is the C-terminal amino acid residue of the first portion of the FVIII protein but is also the N-terminal amino acid residue of the two amino acids of an insertion site in which the XTEN sequence is inserted, c is the N-terminal amino acid residue of the second portion of the FVIII protein but is also the C-terminal amino acid residue of the two amino acids of an insertion site in which the XTEN sequence is inserted, and d is the C-terminal amino acid residue of the FVIII protein, and wherein the first portion of the FVIII protein and the second portion of the FVIII protein are not identical to each other and are of sufficient length together such that the FVIII protein has a FVIII coagulation activity.

[0190] In one embodiment, the first portion of the FVIII protein and the second portion of the FVIII protein are fragments of SEQ ID NO: 4 [full length mature FVIII sequence] or SEQ ID NO: 6 [B-domain deleted FVIII], e.g., N-terminal portion and C-terminal portion, respectively. In certain embodiments, the first portion of the FVIII protein comprises the A1 domain and the A2 domain of the FVIII protein. The second portion of the FVIII protein comprises the A3 domain, the C1 domain, and optionally the C2 domain. In yet other embodiments, the first portion of the FVIII protein comprises the A1 domain and A2 domain, and the second portion of the FVIII protein comprises a portion of the B domain, the A3 domain, the C1 domain, and optionally the C2 domain. In still other embodiments, the first portion of the FVIII protein comprises the A1 domain, A2 domain, and a portion of the B domain of the FVIII protein, and the second portion of the FVIII protein comprises the A3 domain, the C1 domain, and optionally the C2 domain. In still other embodiments, the first portion of the FVIII protein comprises the A1 domain, A2 domain, and a first portion of the B domain of the FVIII protein. The second portion of the FVIII protein comprises a second portion of the B domain, the A3 domain, the C1 domain, and optionally the C2 domain. In some embodiments, the two amino acids ("b" and "c") can be any one or more of the amino acid residues insertion sites shown in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15. For example, "b" can be the amino acid residue immediately upstream of the site in which one or more XTEN sequences are inserted or linked, and "c" can be the amino acid residue immediately downstream of the site in which the one or more XTEN sequences are inserted or linked. In some embodiments, "a" is the first mature amino acid sequence of a FVIII protein, and "d" is the last amino acid sequence of a FVIII protein. For example, FVIII.sub.(a.fwdarw.b) can be an amino acid sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 745 of SEQ ID NO: 6 [B domain deleted FVIII amino acid sequence] or SEQ ID NO: 4 [full length FVIII] and FVIII.sub.(c.fwdarw.d) can be amino acids 746 to 1438 of SEQ ID NO: 6 or amino acids 1641 to 2332 of SEQ ID NO: 4, respectively.

[0191] In some aspects, the insertion site in the FVIII protein is located in one or more domains of the FVIII protein, which is the N-terminus, the A1 domain, the A2 domain, the A3 domain, the B domain, the C1 domain, the C2 domain, the C-terminus, or two or more combinations thereof or between two domains of the FVIII protein, which are the A1 domain and a1 acidic region, and the a1 acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, and the A3 domain and C1 domain, the C1 domain and C2 domain, or any combinations thereof. For example, the insertion sites in which the XTEN sequence can be inserted are selected from the group consisting of the N-terminus and A1 domain, the N-terminus and A2 domain, the N-terminus and A3 domain, the N-terminus and B domain, the N-terminus and C1 domain, the N-terminus and C2 domain, the N-terminus and the C-terminus, the A1 and A2 domains, the A1 and A3 domains, the A1 and B domains, the A1 and C1 domains, the A1 and C2 domains, the A1 domain and the C-terminus, the A2 and A3 domains, the A2 and B domains, the A2 and C1 domains, the A2 and C2 domains, the A2 domain and the C-terminus, the A3 and B domains, the A3 and C1 domains, the A3 and C2 domains, the A3 domain and the C-terminus, the B and C1 domains, the B and C2 domains, the B domain and the C-terminus, the C1 and C2 domains, the C1 and the C-terminus, the C2 domain, and the C-terminus, and two or more combinations thereof. Non-limiting examples of the insertion sites are listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0192] The FVIII protein, in which the XTEN sequence is inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein or linked at the C-terminus or the N-terminus, retains the FVIII activity after linkage to or insertion by the XTEN sequence. The XTEN sequence can be inserted in the FVIII protein once or more than once, twice, three times, four times, five times, or six times such that the insertions do not affect the FVIII activity (i.e., the FVIII protein still retains the coagulation property).

[0193] The FVIII protein useful in the present invention can be linked to one or more XTEN polypeptides at the N-terminus or C-terminus of the FVIII protein by an optional linker or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein by one or more optional linkers. In one embodiment, the two amino acid residues in which the XTEN sequence is inserted or the amino acid residue to which the XTEN sequence is linked correspond to the two or one amino acid residues of SEQ ID NO: 4 [full length mature FVIII] selected from the group consisting of the residues in Table 7, Table 8, Table 9, and Table 10 and any combinations thereof.

[0194] In other embodiments, at least one XTEN sequence is inserted in any one or more XTEN insertion sites disclosed herein or any combinations thereof. In one aspect, at least one XTEN sequence is inserted in one or more XTEN insertion sites disclosed in one or more amino acids disclosed in Table 7.

TABLE-US-00009 TABLE 7 Exemplary XTEN Insertion Sites XTEN FVIII BDD Insertion Insertion Downstream FVIII No. Point* Residue Sequence Domain 1 0 (N- ATR A1 terminus) 2 3 R RYY A1 3 17 M QSD A1 4 18 Q SDL A1 5 22 G ELP A1 6 24 L PVD A1 7 26 V DAR A1 8 28 A RFP A1 9 32 P RVP A1 10 38 F PFN A1 11 40 F NTS A1 12 41 N TSV A1 13 60 N IAK A1 14 61 I AKP A1 15 65 R PPW A1 16 81 Y DTV A1 17 111 G AEY A1 18 116 D QTS A1 19 119 S QRE A1 20 120 Q REK A1 21 128 V FPG A1 22 129 F PGG A1 23 130 P GGS A1 24 182 G SLA A1 25 185 A KEK A1 26 188 K TQT A1 27 205 G KSW A1 28 210 S ETK A1 29 211 E TKN A1 30 216 L MQD A1 31 220 R DAA A1 32 222 A ASA A1 33 223 A SAR A1 34 224 S ARA A1 35 230 K MHT A1 36 243 P GLI A1 37 244 G LIG A1 38 250 R KSV A1 39 318 D GME A1 40 333 P QLR A1 42 334 Q LRM A1 43 336 R MKN a1 44 339 N NEE a1 45 345 D YDD a1 46 357 V VRF a1 47 367 S FIQ a1 48 370 S RPY a1 49 375 A KKH A2 50 376 K KHP A2 51 378 H PKT A2 52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2 55 409 S QYL A2 56 416 P QRI A2 57 434 E TFK A2 58 438 T REA A2 59 441 A IQH A2 60 442 I QHE A2 61 463 I JFK A2 62 487 Y SRR A2 63 490 R LPK A2 64 492 P KGV A2 65 493 K GVK A2 66 494 G VKH A2 67 500 D FPI A2 68 506 G EIF A2 69 518 E DGP A2 70 556 K ESV A2 71 565 Q IMS A2 72 566 I MSD A2 73 598 P AGV A2 74 599 A GVQ A2 75 603 L EDP A2 76 616 S ING A2 77 686 G LWI A2 78 713 K NTG A2 79 719 Y EDS A2 80 730 L LSK A2 81 733 K NNA A2 82 745 N PPV** B 83 1640 P PVL B 84 1652 R TTL B 85 1656 Q SDQ A3 86 1685 N QSP A3 87 1711 M SSS A3 88 1713 S SPH A3 89 1720 N RAQ A3 90 1724 S GSV A3 91 1725 G SVP A3 92 1726 S VPQ A3 93 1741 G SFT A3 94 1744 T QPL A3 95 1749 R GEL A3 96 1773 V TFR A3 97 1792 Y EED A3 98 1793 E EDQ A3 99 1796 Q RQG A3 100 1798 Q GAE A3 101 1799 G AEP A3 102 1802 P RKN A3 103 1803 R KNF A3 104 1807 V KPN A3 105 1808 K PNE A3 106 1827 K DEF A3 107 1844 E KDV A3 108 1861 N TLN A3 109 1863 L NPA A3 110 1896 E RNC A3 111 1900 R APC A3 112 1904 N IQM A3 113 1905 I QME A3 114 1910 P TFK A3 115 1920 A ING A3 116 1937 D QRI A3 117 1981 G VFE A3 118 2019 N KCQ A3 119 2020 K CQT C1 120 2044 G QWA C1 121 2068 F SWI C1 122 2073 V DLL C1

123 2090 R QKF C1 124 2092 K FSS C1 125 2093 F SSL C1 126 2111 K WQT C1 127 2115 Y RGN C1 128 2120 T GTL C1 129 2125 V FFG C1 130 2171 L NSC C1 131 2173 S CSM C2 132 2188 A QIT C2 133 2223 V NNP C2 134 2224 N NPK C2 135 2227 K EWL C2 136 2268 G HQW C2 137 2277 N GKV C2 138 2278 G KVK C2 139 2290 F TPV C2 140 2332 Y C terminus CT of FVIII *Indicates an insertion point for XTEN based on the amino acid number of mature full-length human FVIII, wherein the insertion could be either on the N- or C-terminal side of the indicated amino acid.

[0195] In some embodiments, one or more XTEN sequences are inserted within about six amino acids up or down from amino acids 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905, or 1910, corresponding to SEQ ID NO: 4 or any combinations thereof.

TABLE-US-00010 TABLE 8 Exemplary XTEN Insertion Ranges Distance XTEN FVIII BDD from Insertion Insertion Downstream FVIII insertion No. Point Residue Sequence Domain residue* 9 32 P RVP A1 -3, +6 31 220 R DAA A1 -- 34 224 S ARA A1 +5 43 336 R MKN a1 -1, +6 44 339 N NEE a1 -4, +5 52 399 V LAP A2 -6, +3 56 416 P QRI A2 +6 75 603 L EDP A2 _6 +6 85 1656 Q SDQ B -3, +6 87 1711 M SSS A3 -6, +1 91 1725 G SVP A3 +6 113 1905 I QME A3 +6 114 1910 P TFK A3 -5, +6 *Distance from insertion residue refers to the relative number of amino acids away from the N-terminus (negative numbers) or C-terminus (positive numbers) of the designated insertion residue (residue "0") where an insertion may be made. The designation "-x" refers to an insertion site which is x amino acids away on the N-terminal side of the designated insertion residue. Similarly, the designation "+x" refers to an insertion site which is x amino acids away on the C-terminal side of the designated insertion residue. For example, "-1, +2" indicates that the insertion is made at the N-terminus or C-terminus of amino acid residues denoted -1, 0, +1 or +2.

[0196] In other embodiments, one or more XTEN sequences are inserted immediately down stream of one or more amino acids corresponding to the full-length mature human FVIII selected from the group consisting of one or more insertion sites in Table 9.

TABLE-US-00011 TABLE 9 Exemplary XTEN Insertion Sites or Ranges XTEN Insertion First Insertion FVIII No. Point Range* Residue Domain 3 18-32 Q A1 8 40 F A1 18 211-224 E A1 27 336-403 R A1, A2 43 599 A A2 47 745-1640 N B 50 1656-1728 Q B, a3, A3 57 1796-1804 R A3 65 1900-1912 R A3 81 2171-2332 L C1, C2 *indicates range of insertion sites numbered relative to the amino acid number of mature human FVIII

[0197] In yet other embodiments, one or more XTENs are inserted in the B domain of FVIII. In one example, an XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 740 and 1640 is optionally not present. In another example, an XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 740 and 1690 is optionally not present. In other examples, an XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 741 and 1648 is optionally not present. In yet other examples, an XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 743 and 1638 is optionally not present. In still other examples, an XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1656 is optionally not present. In some examples, an XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1657 is optionally not present. In certain examples, an XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1667 is optionally not present. In still other examples, an XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1686 is optionally not present. In some other examples, an XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 747 and 1642 is optionally not present. In still other examples, an XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 751 and 1667 is optionally not present.

[0198] In some embodiments, one or more XTENs are inserted in one or more amino acids immediately downstream of an amino acid of an insertion site selected from the group consisting of the amino acid residues in Table 10.

TABLE-US-00012 TABLE 10 FVIII XTEN insertion sites and construct designations Up- Down- stream stream Construct Residue Residue Upstream Downstream Number Domain No.* No.* Sequence Sequence F8X-1 A1 3 4 ATR RYY F8X-2 A1 18 19 YMQ SDL F8X-3 A1 22 23 DLG ELP F8X-4 A1 26 27 LPV DAR F8X-5 A1 40 41 FPF NTS F8X-6 A1 60 61 LFN IAK F8X-7 A1 116 117 YDD QTS F8X-8 A1 130 131 VFP GGS F8X-9 A1 188 189 KEK TQT F8X-10 A1 216 217 NSL MQD F8X-11 A1 230 231 WPK MHT F8X-12 A1 333 334 EEP QLR F8X-13 A2 375 376 SVA KKH F8X-14 A2 403 404 APD DRS F8X-15 A2 442 443 EAI QHE F8X-16 A2 490 491 RRL PKG F8X-17 A2 518 519 TVE DGP F8X-18 A2 599 600 NPA GVQ F8X-19 A2 713 714 CDK NTG F8X-20 BD 745 746 SQN PPV F8X-21 BD 745 746 SQN PPV F8X-22 BD** 745 746 SQN PPV F8X-23 A3 1720 1721 APT KDE F8X-24 A3 1796 1797 EDQ RQG F8X-25 A3 1802 1803 AEP RKN F8X-26 A3 1827 1828 PTK DEF F8X-27 A3 1861 1862 HTN TLN F8X-28 A3 1896 1897 NME RNC F8X-29 A3 1900 1901 NCR APC F8X-30 A3 1904 1905 PCN TQM F8X-31 A3 1937 1938 AQD QRI F8X-32 C1 2019 2020 YSN KCQ F8X-33 C1 2068 2069 EPF SWI F8X-34 C1 2111 2112 GKK WQT F8X-35 C1 2120 2121 NST GTL F8X-36 C2 2171 2172 CDL NSC F8X-37 C2 2188 2189 SDA QIT F8X-38 C2 2227 2228 NPK EWL F8X-39 C2 2277 2278 FQN GKV F8X-40 CT 2332 NA DLY NA F8X-41 CT 2332 NA DLY NA F8X-42 A1 3 4 ATR ATR pSD0001 A2 403 404 pSD0002 A2 599 600 pSD0021 N-term 0 1 pSD0022 A1 32 33 pSD0023 A1 65 66 pSD0024 A1 81 82 pSD0025 A1 119 120 pSD0026 A1 211 212 pSD0027 A1 220 221 pSD0028 A1 224 225 pSD0029 A1 336 337 pSD0030 A1 339 340 pSD0031 A2 378 379 pSD0032 A2 399 400 pSD0033 A2 409 410 pSD0034 A2 416 417 pSD0035 A2 487 488 pSD0036 A2 494 495 pSD0037 A2 500 501 pSD0038 A2 603 604 pSD0039 A3 1656 1657 pSD0040 A3 1711 1712 pSD0041 A3 1725 1726 pSD0042 A3 1749 1750 pSD0043 A3 1905 1906 pSD0044 A3 1910 1911 pDS0062 A3 1900 1901 * Indicates the amino acid number of the mature FVIII protein

[0199] In one embodiment, the one or more XTEN insertion sites are located within one or more surface-exposed, flexible loop structure of the FVIII protein (e.g., a permissive loop). For example, at least one XTEN sequence can be inserted in each FVIII "A" domain comprising at least two "permissive loops" into which at least one XTEN polypeptide can be inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to be expressed in vivo or in vitro in a host cell. The permissive loops are regions that allow insertion of at least one XTEN sequence with, among other attributes, high surface or solvent exposure and high conformational flexibility. The A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region, the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region, the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region.

[0200] In one aspect, a first permissive loop in the FVIII A1 domain (A1-1) is located between beta strand 1 and beta strand 2, and a second permissive loop in the FVIII A2 domain (A1-2) is located between beta strand 11 and beta strand 12. A first permissive loop in the FVIII A2 domain (A2-1) is located between beta strand 22 and beta strand 23, and a second permissive loop in the FVIII A2 domain (A2-2) is located between beta strand 32 and beta strand 33. A first permissive loop in the FVIII A3 domain (A3-1) is located between beta strand 38 and beta strand 39, and a second permissive loop in the FVIII A3 (A3-2) is located between beta strand 45 and beta strand 46. In certain aspects, the surface-exposed, flexible loop structure comprising A1-1 corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of SEQ ID NO: 4, e.g., from about amino acid 18 to about amino acid 41 of SEQ ID NO: 4. In other aspects, the surface-exposed, flexible loop structure comprising A1-2 corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO: 4, e.g., from about amino acid 218 to about amino acid 229 of SEQ ID NO: 4. In yet other aspects, the surface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 of SEQ ID NO: 4, e.g. from about amino acid 397 to about amino acid 418 of SEQ ID NO: 4. In still other embodiments, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 of SEQ ID NO: 4, e.g., from about amino acid 595 to about amino acid 607 of SEQ ID NO: 4. In certain aspects the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 of SEQ ID NO: 4, e.g., from about amino acid 1711 to about amino acid 1725 of SEQ ID NO: 4. In yet other aspects, the surface-exposed, flexible loop structure comprising A3-2 corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 of SEQ ID NO: 4, e.g., from about amino acid 1899 to about amino acid 1911 of SEQ ID NO: 4.

[0201] In another embodiment, the one or more amino acids in which at least one XTEN sequence is inserted is located within a3 domain, e.g., amino acids 1649 to 1689, corresponding to full-length mature FVIII polypeptide. In a particular embodiment, an XTEN sequence is inserted between amino acids 1656 and 1657 of SEQ ID NO: 4 (full-length mature FVIII). In a specific embodiment, a FVIII protein comprising an XTEN sequence inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 further comprises a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

[0202] In some embodiments, the one or more insertion sites for one or more XTEN insertions are immediately downstream of one or more amino acids selected from the group consisting of:

TABLE-US-00013 (1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) amino acid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) amino acid 1937, (52) amino acid 2019, (53) amino acid 2068, (54) amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188, (58) amino acid 2227, (59) amino acid 2277, and (60) two or more combinations thereof.

[0203] In one embodiment, a FVIII protein useful for the invention comprises two XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site and a second XTEN inserted into a second XTEN insertion site. Non-limiting examples of the first XTEN insertion site and the second XTEN insertion site are listed in Table 11.

TABLE-US-00014 TABLE 11 Exemplary Insertion Sites for Two XTENs Insertion 1 Insertion 2 Insertion Site Domain Insertion Site Domain 745 B 2332 CT 26 A1 403 A2 40 A1 403 A2 18 A1 403 A2 26 A1 599 A2 40 A1 599 A2 18 A1 599 A2 1720 A3 1900 A3 1725 A3 1900 A3 1711 A3 1905 A3 1720 A3 1905 A3 1725 A3 1905 A3 1656 A3 26 A1 1656 A3 18 A1 1656 A3 40 A1 1656 A3 399 A2 1656 A3 403 A2 1656 A3 1725 A3 1656 A3 1720 A3 1900 A3 18 A1 1900 A3 26 A1 1900 A3 40 A1 1905 A3 18 A1 1905 A3 40 A1 1905 A3 26 A1 1910 A3 26 A1 18 A1 399 A2 26 A1 399 A2 40 A1 399 A2 18 A1 403 A2 1656 A3 1900 A3 1656 A3 1905 A3 1711 A3 40 A1 1711 A3 26 A1 1720 A3 26 A1 1720 A3 40 A1 1720 A3 18 A1 1725 A3 26 A1 1725 A3 40 A1 1725 A3 18 A1 1720 A3 403 A2 1720 A3 399 A2 1711 A3 403 A2 1720 A3 403 A2 1725 A3 403 A2 1725 A3 399 A2 1711 A3 403 A2 1900 A3 399 A2 1900 A3 403 A2 1905 A3 403 A2 1905 A3 399 A2 1910 A3 403 A2

[0204] The two XTENs inserted or linked to the FVIII protein can be identical or different. In some embodiments, a FVIII protein useful for the invention comprises two XTEN sequences inserted in the FVIII protein, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4 (the C-terminus). In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 1656, or 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 599 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1725, 1720, 1900, 1905, or 2332 corresponding to SEQ ID NO: 4. In certain embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 399 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 4. In a particular embodiment, the FVIII protein comprising two XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4, wherein the FVIII protein further has a deletion from amino acid 745 corresponding to SEQ ID NO: 4 to amino acid 1685 corresponding to SEQ ID NO: 4, a mutation or substitution at amino acid 1680 corresponding to SEQ ID NO: 4, e.g., Y1680F, a mutation or substitution at amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, or at least two mutations or substitutions at amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, and amino acid 1680 corresponding to SEQ ID NO: 4, e.g., Y1680F. In a specific embodiment, the FVIII protein comprises two XTEN sequences, a first XTEN inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 of SEQ ID NO: 4, wherein the FVIII protein further has a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

[0205] In certain embodiments, a FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN sequence, and a third XTEN sequence inserted into a third XTEN insertion site. The first, second, or third XTEN sequences can be identical or different. The first, second, and third insertion sites can be selected from the group of any one of the insertion sites disclosed herein. In some embodiments, the FVIII protein comprising three XTEN sequences can further comprise a mutation or substitution, e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A. For example, non-limiting examples of the first, second, and third XTEN insertion sites are listed in Table 12.

TABLE-US-00015 TABLE 12 Exemplary Insertion Sites for Three XTENs Insertion 1 Insertion 2 Insertion 3 Insertion Insertion Insertion Site Domain Site Domain Site Domain 26 A1 403 A2 1656 A3 26 A1 403 A2 1720 A3 26 A1 403 A2 1900 A3 26 A1 1656 A3 1720 A3 26 A1 1656 A3 1900 A3 26 A1 1720 A3 1900 A3 403 A2 1656 A3 1720 A3 403 A2 1656 A3 1900 A3 403 A2 1720 A3 1900 A3 1656 A3 1720 A3 1900 A3 745 B 1900 2332 CT 18 A1 745 B 2332 CT 26 A1 745 B 2332 CT 40 A1 745 B 2332 CT 18 A1 745 B 2332 CT 40 A1 745 B 2332 CT 403 A2 745 B 2332 CT 399 A2 745 B 2332 CT 1725 A3 745 B 2332 CT 1720 A3 745 B 2332 CT 1711 A3 745 B 2332 CT 1900 A3 745 B 2332 CT 1905 A3 745 B 2332 CT 1910 A3 745 B 2332 CT

[0206] In some embodiments, a FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence inserted downstream of amino acid 403 corresponding to SEQ ID NO: 4, and a third XTEN sequence inserted downstream of amino acid 1656, 1720, or 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or 1910 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 745 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 2332 corresponding to SEQ ID NO: 4.

[0207] In other embodiments, a FVIII protein in the invention comprises four XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third insertion site, and a fourth XTEN sequence inserted into a fourth insertion site. The first, second, third, and fourth XTEN sequences can be identical, different, or combinations thereof. In some embodiments, the FVIII protein comprising four XTEN sequences can further comprise a mutation or substitution, e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A. Non-limiting examples of the first, second, third, and fourth XTEN insertion sites are listed in Table 13.

TABLE-US-00016 TABLE 13 Exemplary Insertion Sites for Four XTENs Insertion 1 Insertion 2 Insertion 3 Insertion 4 Insertion Do- Insertion Do- Insertion Do- Insertion Do- Site main Site main Site main Site main 26 A1 403 A2 1656 a3 1720 A3 26 A1 403 A2 1656 a3 1900 A3 26 A1 403 A2 1720 A3 1900 A3 26 A1 1656 a3 1720 A3 1900 A3 403 A2 1656 a3 1720 A3 1900 A3 0040 A1 0403 A2 745 B 2332 CT 0040 A1 0403 A2 745 B 2332 CT 0018 A1 0409 A2 745 B 2332 CT 0040 A1 0409 A2 745 B 2332 CT 0040 A1 0409 A2 745 B 2332 CT 0018 A1 0409 A2 745 B 2332 CT 0040 A1 1720 A3 745 B 2332 CT 0026 A1 1720 A3 745 B 2332 CT 0018 A1 1720 A3 745 B 2332 CT 0018 A1 1720 A3 745 B 2332 CT 0018 A1 1720 A3 745 B 2332 CT 0026 A1 1720 A3 745 B 2332 CT 0018 A1 1720 A3 745 B 2332 CT 0018 A1 1900 A3 745 B 2332 CT 0018 A1 1900 A3 745 B 2332 CT 0026 A1 1900 A3 745 B 2332 CT 0040 A1 1900 A3 745 B 2332 CT 0040 A1 1905 A3 745 B 2332 CT 0018 A1 1905 A3 745 B 2332 CT 0040 A1 1905 A3 745 B 2332 CT 0026 A1 1905 A3 745 B 2332 CT 0018 A1 1905 A3 745 B 2332 CT 0018 A1 1905 A3 745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0040 A1 1910 A3 745 B 2332 CT 0026 A1 1910 A3 745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0026 A1 1910 A3 745 B 2332 CT 0040 A1 1910 A3 745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0409 A2 1720 A3 745 B 2332 CT 0403 A2 1720 A3 745 B 2332 CT 0409 A2 1720 A3 745 B 2332 CT 0403 A2 1720 A3 745 B 2332 CT 0403 A2 1720 A3 745 B 2332 CT 0403 A2 1900 A3 745 B 2332 CT 0403 A2 1900 A3 745 B 2332 CT 0409 A2 1900 A3 745 B 2332 CT 0403 A2 1900 A3 745 B 2332 CT 0403 A2 1900 A3 745 B 2332 CT 0409 A2 1900 A3 745 B 2332 CT 0409 A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3 745 B 2332 CT 0409 A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3 745 B 2332 CT 0409 A2 1910 A3 745 B 2332 CT 0403 A2 1910 A3 745 B 2332 CT 0403 A2 1910 A3 745 B 2332 CT 0403 A2 1910 A3 745 B 2332 CT 0403 A2 1910 A3 745 B 2332 CT 1720 A3 1900 A3 745 B 2332 CT 1720 A3 1905 A3 745 B 2332 CT 1720 A3 1910 A3 745 B 2332 CT 1720 A3 1910 A3 745 B 2332 CT 0403 A2 1656 a3 1720 A3 2332 CT 0403 A2 1656 a3 1900 A3 2332 CT 0403 A2 1720 A3 1900 A3 2332 CT 1656 a3 1720 A3 1900 A3 2332 CT 0018 A1 0403 A2 1656 a3 2332 CT 0018 A1 0403 A2 1720 A3 2332 CT 0018 A1 0403 A2 1900 A3 2332 CT 0018 A1 1656 a3 1720 A3 2332 CT 0018 A1 1656 a3 1900 A3 2332 CT 0018 A1 1720 A3 1900 A3 2332 CT 0018 A1 0403 A2 0745 B 2332 CT 0018 A1 0745 B 1720 A3 2332 CT 0018 A1 0745 B 1900 A3 2332 CT 0403 A2 0745 B 1720 A3 2332 CT 0403 A2 0745 B 1900 A3 2332 CT 0745 B 1720 A3 1900 A3 2332 CT 0188 A1 1900 A3 0745 B 2332 CT 0599 1900 A3 0745 B 2332 CT 2068 1900 A3 0745 B 2332 CT 2171 1900 A3 0745 B 2332 CT 2227 1900 A3 0745 B 2332 CT 2277 1900 A3 0745 B 2332 CT

[0208] In some embodiments, a FVIII protein comprises five XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, and a fifth XTEN sequence inserted into a fifth XTEN insertion site. The first, second, third, fourth, of fifth XTEN sequences can be identical, different, or combinations thereof. Non-limiting examples of the first, second, third, fourth, and fifth insertion sites are listed in Table 14.

TABLE-US-00017 TABLE 14 Exemplary Insertion Sites for Five XTENs XTEN XTEN XTEN XTEN XTEN Insertion 1 insertion 2 Insertion 3 Insertion 4 Insertion 5 0403 1656 1720 1900 2332 0018 0403 1656 1720 2332 0018 0403 1656 1900 2332 0018 0403 1720 1900 2332 0018 1656 1720 1900 2332 0018 0403 0745 1720 2332 0018 0403 0745 1900 2332 0018 0745 1720 1900 2332 0403 0745 1720 1900 2332

[0209] In certain embodiments, a FVIII protein comprises six XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, a fifth XTEN sequence inserted into a fifth XTEN insertion site, and a sixth XTEN sequence inserted into a sixth XTEN insertion site. The first, second, third, fourth, fifth, or sixth XTEN sequences can be identical, different, or combinations thereof. Examples of the six XTEN insertion sites include, but are not limited to the insertion sites listed in Table 15.

TABLE-US-00018 TABLE 15 Exemplary XTEN Insertion Sites for Six XTENs XTEN XTEN XTEN XTEN XTEN XTEN Inser- inser- Inser- Inser- Inser- Inser- tion 1 tion 2 tion 3 tion 4 tion 5 tion 6 0018 0403 1656 1720 1900 2332 0018 0403 0745 1720 1900 2332

[0210] In a particular example, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4 (full-length mature FVIII). In another example, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In some examples, a first XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In other examples, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In yet other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In still other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In certain embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4, and a fourth XTEN is inserted between 1900 and 1901 corresponding to SEQ ID NO: 4.

[0211] In a particular embodiment, an XTEN sequence is inserted between amino acids 745 and 746 of a full-length Factor VIII or the corresponding insertion site of the B-domain deleted Factor VIII.

[0212] In some embodiments, a chimeric protein of the invention comprises two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to a sequence selected from FVIII-161 (SEQ ID NO: 101), FVIII-169 (SEQ ID NO: 103), FVIII-170 (SEQ ID NO: 102), FVIII-173 (SEQ ID NO: 104); FVIII-195 (SEQ ID NO: 105); FVIII-196 (SEQ ID NO: 106), FVIII-199 (SEQ ID NO: 107), FVIII-201 (SEQ ID NO: 108); FVIII-203 (SEQ ID NO: 109), FVIII-204 (SEQ ID NO: 110), FVIII-205 (SEQ ID NO: 111), FVIII-266 (SEQ ID NO:112), FVIII-267 (SEQ ID NO: 113), FVIII-268 (SEQ ID NO: 114), FVIII-269 (SEQ ID NO: 115), FVIII-271 (SEQ ID NO: 116), or FVIII-272 (SEQ ID NO: 117) and a second polypeptide sequence comprising an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to a sequence selected from VWF031 (SEQ ID NO: 118), VWF034 (SEQ ID NO: 119), or VWF-036 (SEQ ID NO: 120).

D) Ig Constant Region or a Portion Thereof

[0213] The VWF fragment or the FVIII protein linked to an XTEN sequence in the present invention can further comprise an Ig constant region or a portion thereof. The Ig constant region or a portion thereof can improve pharmacokinetic or pharmacodynamic properties of the VWF fragment or the FVIII protein in combination with the XTEN sequence. In certain embodiments, the Ig constant region or a portion thereof extends a half-life of a molecule fused to the Ig constant region or a portion thereof.

[0214] An Ig constant region is comprised of domains denoted CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA, IgD, or IgE), the constant region can be comprised of three or four CH domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

[0215] An Ig constant region or a portion thereof for producing the chimeric protein of the present invention may be obtained from a number of different sources. In some embodiments, an Ig constant region or a portion thereof is derived from a human Ig. It is understood, however, that the Ig constant region or a portion thereof may be derived from an Ig of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover, the Ig constant region or a portion thereof may be derived from any Ig class, including IgM, IgG, IgD, IgA, and IgE, and any Ig isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgG1 is used.

[0216] A variety of the Ig constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains sequence can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Ig constant region sequences (e.g., hinge, CH2, and/or CH3 sequences, or portions thereof) can be derived from these sequences using art recognized techniques. The genetic material obtained using any of the foregoing methods may then be altered or synthesized to obtain polypeptides of the present invention. It will further be appreciated that the scope of this invention encompasses alleles, variants and mutations of constant region DNA sequences.

[0217] The sequences of the Ig constant region or a portion thereof can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. To clone a sequence of the Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., "PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplification of antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). The cloning of antibody sequences is further described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by reference herein.

[0218] An Ig constant region used herein can include all domains and the hinge region or portions thereof. In one embodiment, the Ig constant region or a portion thereof comprises CH2 domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[0219] As used herein, the term "Fc region" is defined as the portion of a polypeptide which corresponds to the Fc region of native Ig, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, the term "genetically-fused Fc region" or "single-chain Fc region" (scFc region), as used herein, refers to a synthetic dimeric Fc region comprised of Fc domains genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence).

[0220] In one embodiment, the "Fc region" refers to the portion of a single Ig heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0221] The Fc region of an Ig constant region, depending on the Ig isotype can include the CH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Fc region of an Ig bestow several desirable properties on a chimeric protein including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which are incorporated herein by reference in their entireties.

[0222] An Ig constant region or a portion thereof can be an FcRn binding partner. FcRn is active in adult epithelial tissues and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). An FcRn binding partner is a portion of an Ig that binds to FcRn.

[0223] The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other Ig classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

[0224] FcRn binding partners useful in the present invention encompass molecules that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of Igs or Ig fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[0225] Fc regions or FcRn binding partners bound to FcRn can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an Fc region or an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo half-life of these proteins. In certain embodiments, the portions of Ig constant regions are an Fc region or an FcRn binding partner that typically associates, via disulfide bonds and other non-specific interactions, with another Fc region or another FcRn binding partner to form dimers and higher order multimers.

[0226] Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginally, as an aerosol administered nasally or via a pulmonary route, or via an ocular route. In another embodiment, the chimeric protein can be administered invasively, e.g., subcutaneously, intravenously.

[0227] An FcRn binding partner region is a molecule or a portion thereof that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the Fc region. Specifically bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 10.sup.6 M.sup.-1, or higher than 10.sup.8 M.sup.-1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

[0228] In certain embodiments, a chimeric protein of the invention comprises one or more truncated Fc regions that are nonetheless sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of an Fc region that binds to FcRn (i.e., the FcRn binding portion) comprises from about amino acids 282-438 of IgG1, EU numbering (with the primary contact sites being amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus, an Fc region of the invention may comprise or consist of an FcRn binding portion. FcRn binding portions may be derived from heavy chains of any isotype, including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn binding portion from an antibody of the human isotype IgG1 is used. In another embodiment, an FcRn binding portion from an antibody of the human isotype IgG4 is used.

[0229] In another embodiment, the "Fc region" includes an amino acid sequence of an Fc domain or derived from an Fc domain. In certain embodiments, an Fc region comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain (about amino acids 216-230 of an antibody Fc region according to EU numbering), a CH2 domain (about amino acids 231-340 of an antibody Fc region according to EU numbering), a CH3 domain (about amino acids 341-438 of an antibody Fc region according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc region comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, an Fc region comprises, consists essentially of, or consists of a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In still other embodiments, an Fc region lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In a particular embodiment, an Fc region comprises or consists of amino acids corresponding to EU numbers 221 to 447.

[0230] The Fc regions denoted as F, F1, or F2 herein may be obtained from a number of different sources. In one embodiment, an Fc region of the polypeptide is derived from a human Ig. It is understood, however, that an Fc region may be derived from an Ig of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, or guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover, the polypeptide of the Fc domains or portions thereof may be derived from any Ig class, including IgM, IgG, IgD, IgA and IgE, and any Ig isotype, including IgG1, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgG1 is used.

[0231] In certain embodiments, the Fc variant confers a change in at least one effector function imparted by an Fc region comprising said wild-type Fc domain (e.g., an improvement or reduction in the ability of the Fc region to bind to Fc receptors (e.g. Fc.gamma.RI, Fc.gamma.RII, or Fc.gamma.RIII) or complement proteins (e.g. C1q), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine residue.

[0232] The Fc regions of the invention may employ art-recognized Fc variants which are known to impart a change (e.g., an enhancement or reduction) in effector function and/or FcR or FcRn binding. Specifically, a binding molecule of the invention may include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, and WO06/085967A2; US Patent Publication Nos. US2007/0231329, US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767, US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091, each of which is incorporated by reference herein. In one embodiment, the specific change (e.g., the specific substitution of one or more amino acids disclosed in the art) may be made at one or more of the disclosed amino acid positions. In another embodiment, a different change at one or more of the disclosed amino acid positions (e.g., the different substitution of one or more amino acid position disclosed in the art) may be made.

[0233] The Fc region or FcRn binding partner of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example, the following single amino acid residues in human IgG1 Fc (Fc .gamma.l) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wild type proline substituted by alanine at position number 238. As an example, a specific embodiment incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids may be substituted for the wild type amino acids at the positions specified above. Mutations may be introduced singly into Fc giving rise to more than one hundred Fc regions distinct from the native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more Fc regions. Moreover, one of the Fc region of a construct of the invention may be mutated and the other Fc region of the construct not mutated at all, or they both may be mutated but with different mutations.

[0234] Certain of the above mutations may confer new functionality upon the Fc region or FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the Fc region, and to render the Fc region incapable of binding to Fc.gamma.R1, Fc.gamma.RIIA, Fc.gamma.RIIB, and Fc.gamma.RIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased "on" rate, a decreased "off" rate or both an increased "on" rate and a decreased "off" rate. Examples of mutations believed to impart an increased affinity for FcRn include, but not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0235] Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG1 "ELLG" to the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII, which mediate various effector functions will not bind to IgG1 when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0236] In one embodiment, the Ig constant region or a portion thereof, e.g, an Fc region, is a polypeptide including the sequence PKNSSMISNTP (SEQ ID NO: 52) and optionally further including a sequence selected from HQSLGTQ (SEQ ID NO: 53), HQNLSDGK (SEQ ID NO: 54), HQNISDGK (SEQ ID NO: 55), or VISSHLGQ (SEQ ID NO: 56) (U.S. Pat. No. 5,739,277).

[0237] In another embodiment, the immunoglobulin constant region or a portion thereof comprises an amino acid sequence in the hinge region or a portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or a portion thereof. The disulfide bond by the immunoglobulin constant region or a portion thereof places the first polypeptide comprising FVIII and the second polypeptide comprising the VWF fragment together so that endogenous VWF does not replace the VWF fragment and does not bind to the FVIII. Therefore, the disulfide bond between the first immunoglobulin constant region or a portion thereof and a second immunoglobulin constant region or a portion thereof prevents interaction between endogenous VWF and the FVIII protein. This inhibition of interaction between the VWF and the FVIII protein allows the half-life of the FVIII protein to go beyond the two fold limit. The hinge region or a portion thereof can further be linked to one or more domains of CH1, CH2, CH3, a fragment thereof, and any combinations thereof. In a particular embodiment, the immunoglobulin constant region or a portion thereof is a hinge region and CH2.

[0238] In certain embodiments, the Ig constant region or a portion thereof is hemi-glycosylated. For example, the chimeric protein comprising two Fc regions or FcRn binding partners may contain a first, glycosylated, Fc region (e.g., a glycosylated CH2 region) or FcRn binding partner and a second, aglycosylated, Fc region (e.g., an aglycosylated CH2 region) or FcRn binding partner. In one embodiment, a linker may be interposed between the glycosylated and aglycosylated Fc regions. In another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all of the Fc regions are glycosylated. In other embodiments, the Fc region may be aglycosylated, i.e., none of the Fc moieties are glycosylated.

[0239] In certain embodiments, a chimeric protein of the invention comprises an amino acid substitution to an Ig constant region or a portion thereof (e.g., Fc variants), which alters the antigen-independent effector functions of the Ig constant region, in particular the circulating half-life of the protein.

[0240] Such proteins exhibit either increased or decreased binding to FcRn when compared to proteins lacking these substitutions and, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered polypeptide is desired, e.g., to treat a chronic disease or disorder (see, e.g., U.S. Pat. Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations where the starting polypeptide has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be desired include those applications in which localization the brain, kidney, and/or liver is desired. In one exemplary embodiment, the chimeric protein of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the chimeric protein of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, a protein with altered FcRn binding comprises at least one Fc region or FcRn binding partner (e.g, one or two Fc regions or FcRn binding partners) having one or more amino acid substitutions within the "FcRn binding loop" of an Ig constant region. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering) of a wild-type, full-length, Fc region. In other embodiments, an Ig constant region or a portion thereof in a chimeric protein of the invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions within the 15 {acute over (.ANG.)} FcRn "contact zone." As used herein, the term 15 {acute over (.ANG.)} FcRn "contact zone" includes residues at the following positions of a wild-type, full-length Fc moiety: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering). In other embodiments, a Ig constant region or a portion thereof of the invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions at an amino acid position corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein.

[0241] An Fc region or FcRn binding partner used in the invention may also comprise an art recognized amino acid substitution which alters the glycosylation of the chimeric protein. For example, the Fc region or FcRn binding partner of the chimeric protein linked to a VWF fragment or a FVIII protein may comprise an Fc region having a mutation leading to reduced glycosylation (e.g., N- or O-linked glycosylation) or may comprise an altered glycoform of the wild-type Fc moiety (e.g., a low fucose or fucose-free glycan).

[0242] In one embodiment, an unprocessed chimeric protein of the invention may comprise a genetically fused Fc region (i.e., scFc region) having two or more of its constituent Ig constant region or a portion thereof independently selected from the Ig constant region or a portion thereof described herein. In one embodiment, the Fc regions of a dimeric Fc region are the same. In another embodiment, at least two of the Fc regions are different. For example, the Fc regions or FcRn binding partners of the proteins of the invention comprise the same number of amino acid residues or they may differ in length by one or more amino acid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc regions or FcRn binding partners of the protein of the invention may differ in sequence at one or more amino acid positions. For example, at least two of the Fc regions or FcRn binding partners may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).

E) Linkers

[0243] The chimeric protein of the present invention further comprises one or more linkers. One type of the linkers is a cleavable linker, which can be cleaved by various proteases when administered to a subject in vivo, e.g., at a site of coagulation. In one embodiment, the cleavable linker allows cleavage of moiety, e.g., a VWF fragment, from the chimeric protein at the site of the coagulation cascade, thus allowing activated FVIII (FVIIIa) to have its FVIIIa activity. Another type of the linkers is a processable linker, which contains an intracellular cleavage site and thus can be cleaved by an intracellular processing enzyme in a host cell, allowing convenient expression of a polypeptide and formation of a chimeric protein.

[0244] One or more linkers can be present between any two proteins in the chimeric protein. In one embodiment, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence by a linker, e.g., a cleavable linker, and the XTEN sequence is further linked to the FVIII protein (i.e., V-L-X-FVIII). In another embodiment, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence, and the XTEN sequence is linked to the FVIII protein by a linker, e.g., a cleavable linker (i.e., V-X-L-FVIII).

[0245] In certain embodiments, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof (e.g., a first Fc region), (iv) a FVIII protein, and (v) a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker. The XTEN sequence can be further linked to the first Ig constant region or a portion thereof by a linker, e.g., a cleavable linker. The FVIII protein (with or without an XTEN sequence) can also be linked to the second Ig constant region or a portion thereof by an optional linker, e.g. a cleavable linker. In certain embodiments, the chimeric protein further comprises one or more linkers, e.g., processable linkers, between the first Ig constant region or a portion thereof (e.g., first Fc region) and the second Ig constant region or a portion thereof (e.g., second Fc region), between the VWF fragment and the second Ig constant region or a portion thereof, or between the FVIII protein and the first Ig constant region or a portion thereof (e.g., first Fc region).

[0246] In some embodiments, the present invention includes a chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof, and (iv) a second Ig constant region or a portion thereof, wherein the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are linked by a processable linker.

[0247] The linker useful in the present invention can comprise any organic molecule. In one embodiment, the linker comprises a polymer, e.g., polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker comprises an amino acids sequence. The linker can comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids. In one embodiment, the linker comprises an XTEN sequence. Additional examples of XTEN can be used according to the present invention and are disclosed in US Patent Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, or WO 2011028344 A2. In another embodiment, the linker is a PAS sequence.

[0248] The linker useful in the present invention can comprise any organic molecule. In one embodiment, the linker is a polymer, e.g., polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker is an amino acid sequence. The linker can comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids.

[0249] Examples of linkers are well known in the art. In one embodiment, the linker comprises the sequence Gn. The linker can comprise the sequence (GA).sub.n. The linker can comprise the sequence (GGS).sub.n. In other embodiments, the linker comprises (GGGS).sub.n(SEQ ID NO: 57). In still other embodiments, the linker comprises the sequence (GGS).sub.n(GGGGS).sub.n (SEQ ID NO: 58). In these instances, n may be an integer from 1-100. In other instances, n may be an integer from 1-20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 59), GGSGGSGGSGGSGGG (SEQ ID NO: 60), GGSGGSGGGGSGGGGS (SEQ ID NO: 61), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 62), or GGGGSGGGGSGGGGS (SEQ ID NO: 63). The linker does not eliminate or diminish the VWF fragment activity or the clotting activity of Factor VIII. Optionally, the linker enhances the VWF fragment activity or the clotting activity of Factor VIII protein, e.g., by further diminishing the effects of steric hindrance and making the VWF fragment or Factor VIII portion more accessible to its target binding site.

[0250] In one embodiment, the linker useful for the chimeric protein is 15-25 amino acids long. In another embodiment, the linker useful for the chimeric protein is 15-20 amino acids long. In some embodiments, the linker for the chimeric protein is 10-25 amino acids long. In other embodiments, the linker for the chimeric protein is 15 amino acids long. In still other embodiments, the linker for the chimeric protein is (GGGGS).sub.n (SEQ ID NO: 64) where G represents glycine, S represents serine and n is an integer from 1-20.

F) Cleavage Sites

[0251] The linker may also incorporate a moiety capable of being cleaved either chemically (e.g., hydrolysis of an ester bond), enzymatically (i.e., incorporation of a protease cleavage sequence), or photolytically (e.g., a chromophore such as 3-amino-3-(2-nitrophenyl) proprionic acid (ANP)) in order to release one molecule from another.

[0252] In one embodiment, the linker is a cleavable linker. The cleavable linkers can comprise one or more cleavage sites at the N-terminus or C-terminus or both. In another embodiment, the cleavable linker consists essentially of or consists of one or more cleavable sites. In other embodiments, the cleavable linker comprises heterologous amino acid linker sequences described herein or polymers and one or more cleavable sites.

[0253] In certain embodiments, a cleavable linker comprises one or more cleavage sites that can be cleaved in a host cell (i.e., intracellular processing sites). Non limiting examples of the cleavage site include RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), and RRRRS (SEQ ID NO: 11).

[0254] In other embodiments, a cleavable linker comprises one or more cleavage sites that are cleaved by a protease after a chimeric protein comprising the cleavable linker is administered to a subject. In one embodiment, the cleavage site is cleaved by a protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP-13, MMP-17, and MMP-20. In another embodiment, the cleavage site is selected from the group consisting of a FXIa cleavage site (e.g., KLTR.dwnarw.AET (SEQ ID NO: 65)), a FXIa cleavage site (e.g, DFTR.dwnarw.VVG (SEQ ID NO: 66)), a FXIIa cleavage site (e.g., TMTR.dwnarw.IVGG (SEQ ID NO: 67)), a Kallikrein cleavage site (e.g., SPFR.dwnarw.STGG (SEQ ID NO: 68)), a FVIIa cleavage site (e.g., LQVR.dwnarw.IVGG (SEQ ID NO: 69)), a FIXa cleavage site (e.g., PLGR.dwnarw.IVGG (SEQ ID NO: 70)), a FXa cleavage site (e.g., IEGR.dwnarw.TVGG (SEQ ID NO: 71)), a FIIa (thrombin) cleavage site (e.g, LTPR.dwnarw.SLLV (SEQ ID NO: 72)), a Elastase-2 cleavage site (e.g, LGPV.dwnarw.SGVP (SEQ ID NO: 73)), a Granzyme-B cleavage (e.g, VAGD.dwnarw.SLEE (SEQ ID NO: 74)), a MMP-12 cleavage site (e.g., GPAG.dwnarw.LGGA (SEQ ID NO: 75)), a MMP-13 cleavage site (e.g., GPAG.dwnarw.LRGA (SEQ ID NO: 76)), a MMP-17 cleavage site (e.g., APLG.dwnarw.LRLR (SEQ ID NO: 77)), a MMP-20 cleavage site (e.g., PALP.dwnarw.LVAQ (SEQ ID NO: 78)), a TEV cleavage site (e.g., ENLYFQ.dwnarw.G (SEQ ID NO: 79)), a Enterokinase cleavage site (e.g., DDDK.dwnarw.IVGG (SEQ ID NO: 80)), a Protease 3C (PRESCISSION.TM.) cleavage site (e.g., LEVLFQ.dwnarw.GP (SEQ ID NO: 81)), and a Sortase A cleavage site (e.g., LPKT.dwnarw.GSES) (SEQ ID NO: 82). In certain embodiments, the FXIa cleavage sites include, but are not limited to, e.g., TQSFNDFTR (SEQ ID NO: 83) and SVSQTSKLTR (SEQ ID NO: 84). Non-limiting exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 85), TTKIKPR (SEQ ID NO: 86), or LVPRG (SEQ ID NO: 87), and a sequence comprising, consisting essentially of, or consisting of ALRPR (SEQ ID NO: 17) (e.g., ALRPRVVGGA (SEQ ID NO: 88)).

[0255] In a specific embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).

Polynucleotides, Vectors, and Host Cells

[0256] Also provided in the invention is a polynucleotide encoding (a) a VWF fragment linked to an XTEN sequence and a FVIII protein, (b) a FVIII protein linked to an XTEN sequence and Fc, or (c) a FVIII protein linked to an XTEN sequence and a VWF fragment described herein. When a chimeric protein is a single polypeptide chain (e.g., F2-L2-X-V-L1-F1-FVIII, wherein FVIII comprises a FVIII protein, F1 comprises a first Ig constant region or a portion thereof, e.g., a first Fc region, L1 comprises a first linker, V comprises a VWF fragment, X comprises an XTEN sequence, L2 comprises a second linker, and F2 comprises a second Ig constant region or a portion thereof, e.g., a second Fc region), the invention is drawn to a single polynucleotide chain encoding the single polypeptide chain. When the chimeric protein comprises a first and a second polypeptide chains (F2-L2-X-V:FVIII-F1), the first polypeptide chain comprising a VWF fragment linked to a XTEN sequence, which is further linked to a first Ig constant region or a portion thereof (e.g., a first Fc region) by a cleavable linker (e.g., F2-L2-X-V) and the second polypeptide chain comprising a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region) (e.g, FVIII-F1), wherein the first polypeptide chain and the second polypeptide chain are associated with each other, a polynucleotide can comprise the first nucleotide sequence and the second nucleotide sequence. In one embodiment, the first polypeptide chain and the second polypeptide chain can be encoded by a single polynucleotide chain. In another embodiment, the first polypeptide chain and the second polypeptide chain are encoded by two different polynucleotides, i.e., a first nucleotide sequence and a second nucleotide sequence. In another embodiment, the first nucleotide sequence and the second nucleotide sequence are on two different polynucleotides (e.g., different vectors). In certain embodiments, the present invention is directed to a set of polynucleotides comprising a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes the VWF fragment of the chimeric protein and the second nucleotide chain encodes the FVIII protein. In some embodiments, a chimeric protein comprising two polypeptide chains or three polypeptide chains can be encoded by a single polynucleotide chain, and then processed into two or three (or more) polypeptide chains. In yet other embodiments, a chimeric protein comprising these polypeptide chains can be encoded by two or three polynucleotide chains.

[0257] In other embodiments, the set of the polynucleotides further comprises an additional nucleotide chain (e.g., a second nucleotide chain when the chimeric polypeptide is encoded by a single polynucleotide chain or a third nucleotide chain when the chimeric protein is encoded by two polynucleotide chains) which encodes a protein convertase. The protein convertase can be selected from the group consisting of proprotein convertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertase subtilisin/kexin type 7 (PCSK7 or PC5), a yeast Kex 2, proprotein convertase subtilisin/kexin type 3 (PACE or PCSK3), and two or more combinations thereof. In some embodiments, the protein convertase is PACE, PC5, or PC7. In a specific embodiment, the protein convertase is PC5 or PC7. See International Application no. PCT/US2011/043568.

[0258] As used herein, an expression vector refers to any nucleic acid construct which contains the necessary elements for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0259] Expression vectors of the invention will include polynucleotides encoding the chimeric protein described herein. In one embodiment, one or more of the coding sequences for the VWF fragment and XTEN, the FVIII protein and XTEN, or both are operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to permit each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0260] A gene expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the coding nucleic acid to which it is operably linked. The gene expression control sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus, and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

[0261] In general, the gene expression control sequence shall include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined coding nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.

[0262] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors well-known in the art. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

[0263] In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0264] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using standard molecular biology techniques to remove and/or add specific fragments of DNA.

[0265] In one insect expression system that may be used to produce the proteins of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example, the polyhedron gene) of the virus and placed under control of an ACNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (see, e.g., Smith et al. (1983) J Virol 46:584; U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

[0266] Another system which can be used to express the proteins of the invention is the glutamine synthetase gene expression system, also referred to as the "GS expression system" (Lonza Biologics PLC, Berkshire UK). This expression system is described in detail in U.S. Pat. No. 5,981,216.

[0267] In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may be used. See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415; Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.

[0268] To increase efficiency of production, the polynucleotides can be designed to encode multiple units of the protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with the appropriate enzyme) in order to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, the translation of each polypeptide encoded by the mRNA is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase the yield of polypeptides driven by a single promoter.

[0269] Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems, this can include an antibiotic resistance gene such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. Selectable markers are reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable markers is well within the level of ordinary skill in the art.

[0270] Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, U.S. Pat. No. 4,713,339).

[0271] The expression vectors can encode for tags that permit easy purification of the recombinantly produced protein. Examples include, but are not limited to, vector pUR278 (Ruther et al. (1983) EMBO J 2:1791), in which coding sequences for the protein to be expressed may be ligated into the vector in frame with the lac z coding region so that a tagged fusion protein is produced; pGEX vectors may be used to express proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (thrombin or Factor Xa protease or PRESCISSION PROTEASE.TM. (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.

[0272] The expression vector or vectors are then transfected or co-transfected into a suitable target cell, which will express the polypeptides. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host-expression vector systems may be utilized to express the proteins described herein including both prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

[0273] In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell having a definitive nucleus. Eukaryotic cells of animals include cells of vertebrates, e.g., mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells of plants specifically can include, without limitation, yeast cells. A eukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

[0274] In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell line. HEK 293 cells are available as CRL-1533 from American Type Culture Collection, Manassas, Va., and as 293-H cells, Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 from Invitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cell is a PER.C6.RTM. cell, which is a human cell line derived from retina. PER.C6.RTM. cells are available from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from American Type Culture Collection, Manassas, Va. (e.g., CHO-K1; CCL-61). In still other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from American Type Culture Collection, Manassas, Va. (e.g., CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell, which is a hybrid cell line of a HEK293 cell and a human B cell line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

[0275] In one embodiment, a plasmid including a FVIII(X)-Fc fusion coding sequence, a VWF fragment-L-Fc fusion coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

[0276] In another embodiment, a plasmid including a FVIII-Fc fusion coding sequence, a VWF fragment-XTEN-L-Fc fusion coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

[0277] In other embodiments, a plasmid including a FVIII(X)-Fc fusion coding sequence, a Fc coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

[0278] In some embodiments, a first plasmid including a FVIII(X)-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including an Fc coding sequence or a VWF fragment-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

[0279] In still other embodiments, a first plasmid including a FVIII-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a VWF fragment-XTEN-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

[0280] In yet other embodiments, a first plasmid including a FVIII(X)-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a VWF fragment-XTEN-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

[0281] In certain embodiments, a first plasmid, including a chimeric protein encoding FVIII (with or without XTEN)-F1-L1-V-XTEN-L2-F2 coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a protein convertase coding sequence and a second selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The promoters for the FVIII(X)-Fc coding sequence and the VWF-XTEN-Fc coding sequence can be different or they can be the same.

[0282] In still other embodiments, transfected cells are stably transfected. These cells can be selected and maintained as a stable cell line, using conventional techniques known to those of skill in the art.

[0283] Host cells containing DNA constructs of the protein are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the media can contain one or more selection factors. Optionally the media can contain bovine calf serum or fetal calf serum (FCS). In one embodiment, the media contains substantially no IgG. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM/F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbad, Calif.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.). Selection of a medium appropriate for the particular cell line used is within the level of those ordinary skilled in the art.

[0284] In order to co-express the two polypeptide chains of the chimeric protein, the host cells are cultured under conditions that allow expression of both chains. As used herein, culturing refers to maintaining living cells in vitro for at least a definite time. Maintaining can, but need not include, an increase in population of living cells. For example, cells maintained in culture can be static in population, but still viable and capable of producing a desired product, e.g., a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include appropriate selection of culture media, media supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing can include the use of any of various types of scale-up systems including shaker flasks, roller bottles, hollow fiber bioreactors, stirred-tank bioreactors, airlift bioreactors, Wave bioreactors, and others.

[0285] The cell culture conditions are also selected to allow association of the VWF fragment with the FVIII protein. Conditions that allow expression of the VWF fragment and/or the FVIII protein may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 media (Invitrogen, Carlsbad, Calif.) or OptiCHO media (Invitrogen, Carlsbad, Calif.) supplemented with 4 mM glutamine.

[0286] In one aspect, the present invention is directed to a method of expressing, making, or producing the chimeric protein of the invention comprising a) transfecting a host cell comprising a polynucleotide encoding the chimeric protein and b) culturing the host cell in a culture medium under a condition suitable for expressing the chimeric protein, wherein the chimeric protein is expressed.

[0287] In further embodiments, the protein product containing the VWF fragment linked to an XTEN sequence or the FVIII protein linked to an XTEN sequence is secreted into the media. Media is separated from the cells, concentrated, filtered, and then passed over two or three affinity columns, e.g., a protein A column and one or two anion exchange columns.

[0288] In certain aspects, the present invention relates to the chimeric protein produced by the methods described herein.

[0289] In vitro production allows scale-up to give large amounts of the desired altered polypeptides of the invention. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC, chromatography over DEAE-cellulose or affinity chromatography.

Pharmaceutical Composition

[0290] Compositions containing the chimeric protein of the present invention may contain a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and/or auxiliaries that facilitate processing of the active compounds into preparations designed for delivery to the site of action.

[0291] The pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous, subcutaneous, or intramuscular) by bolus injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., pyrogen free water.

[0292] Suitable formulations for parenteral administration also include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, the suspension may also contain stabilizers. Liposomes also can be used to encapsulate the molecules of the invention for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like. In some embodiments, the composition comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions comprise pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the active ingredients.

[0293] Compositions of the invention may be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the mode of administration and therapeutic application.

[0294] The composition can be formulated as a solution, micro emulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0295] The active ingredient can be formulated with a controlled-release formulation or device. Examples of such formulations and devices include implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations and devices are known in the art. See e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0296] Injectable depot formulations can be made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the polymer employed, the rate of drug release can be controlled. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Depot injectable formulations also can be prepared by entrapping the drug in liposomes or microemulsions.

[0297] Supplementary active compounds can be incorporated into the compositions. In one embodiment, the chimeric protein of the invention is formulated with another clotting factor, or a variant, fragment, analogue, or derivative thereof. For example, the clotting factor includes, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue factor (rsTF) or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamic acid.

[0298] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0299] In addition to the active compound, the liquid dosage form may contain inert ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan.

[0300] Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by E. W. Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.

[0301] For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can include suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

[0302] For buccal administration, the composition may take the form of tablets or lozenges according to conventional protocols.

[0303] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or nebulizer, with optionally a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0304] The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0305] In one embodiment, a pharmaceutical composition comprises a chimeric protein, the polynucleotide encoding the chimeric protein, the vector comprising the polynucleotide, or the host cell comprising the vector, and a pharmaceutically acceptable carrier. The FVIII protein in a chimeric protein has extended half-life compared to wild type FVIII protein or the corresponding FVIII protein without the VWF fragment. In one embodiment, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. In another embodiment, the half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0306] In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration. The parenteral administration can be intravenous or subcutaneous administration.

[0307] In other embodiments, the composition is used to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof. In still other embodiments, the subject is scheduled to undergo a surgery. In yet other embodiments, the treatment is prophylactic or on-demand.

Gene Therapy

[0308] A chimeric protein thereof of the invention can be produced in vivo in a mammal, e.g., a human patient, using a gene therapy approach to treatment of a bleeding disease or disorder selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath would be therapeutically beneficial. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This involves administration of a suitable chimeric protein-encoding nucleic acid operably linked to suitable expression control sequences. In certain embodiment, these sequences are incorporated into a viral vector. Suitable viral vectors for such gene therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, and adeno associated virus (AAV) vectors. The viral vector can be a replication-defective viral vector. In other embodiments, an adenoviral vector has a deletion in its E1 gene or E3 gene. When an adenoviral vector is used, the mammal may not be exposed to a nucleic acid encoding a selectable marker gene. In other embodiments, the sequences are incorporated into a non-viral vector known to those skilled in the art.

Methods of Using Chimeric Protein

[0309] The present invention is directed to a method of using a chimeric protein described herein to prevent or inhibit endogenous VWF binding to a FVIII protein. The present invention is also directed to a method of using a chimeric protein having a FVIII protein linked to XTEN and an Ig constant region or a portion thereof.

[0310] One aspect of the present invention is directed to preventing or inhibiting FVIII interaction with endogenous VWF by blocking or shielding the VWF binding site on the FVIII from endogenous VWF and at the same time extending half-life of the FVIII protein using an XTEN sequence in combination with an Ig constant region or a portion thereof, which can also be a half-life extender. In one embodiment, the invention is directed to a method of constructing a FVIII protein having half-life longer than wild-type FVIII. In one embodiment, an XTEN sequence inhibits or prevents interaction of a FVIII protein in a chimeric protein with endogenous VWF. In another embodiment, an Ig constant region or a portion thereof inhibits or prevents interaction of the FVIII protein with endogenous VWF. The chimeric protein useful in the method includes any one or more chimeric protein described herein.

[0311] Another aspect of the invention includes a method of administering to a subject in need thereof a chimeric protein comprising a FVIII protein having half-life longer than wild-type FVIII, wherein the method comprises administering the chimeric protein described herein to the subject.

[0312] In one embodiment, the invention is directed to a method of using an XTEN sequence and an Ig constant region or a portion thereof to extend a half-life of a FVIII protein and a VWF fragment to prevent or inhibit endogenous VWF interaction with a FVIII protein. A FVIII protein linked to an XTEN sequence (e.g., FVIII(X)) and then bound to or associated with a VWF fragment is shielded or protected from the clearance pathway of VWF and thus has reduced clearance compared to the FVIII protein not bound to the VWF fragment. The shielded FVIII protein thus has maximum extension of a half-life compared to a FVIII protein not bound to or associated with the XTEN sequence and the VWF fragment. In certain embodiments, the FVIII protein associated with or protected by a VWF fragment and linked to an XTEN sequence is not cleared by a VWF clearance receptor. In other embodiments, the FVIII protein associated with or protected by a VWF fragment and linked to an XTEN sequence is cleared from the system slower than the FVIII protein that is not associated with or protected by the VWF fragment and linked to the XTEN sequence.

[0313] In one aspect, the chimeric protein comprising the FVIII protein linked to an XTEN sequence or the FVIII protein bound to or associated with a VWF fragment linked to XTEN has reduced clearance from circulation as the VWF fragment does not contain a VWF clearance receptor binding site. The VWF fragment prevents or inhibits clearance of FVIII bound to or associated with the VWF fragment from the system through the VWF clearance pathway. The VWF fragments useful for the present invention can also provide at least one or more VWF-like FVIII protection properties that are provided by endogenous VWF. In certain embodiments, the VWF fragment or the XTEN sequence can also mask one or more FVIII clearance receptor binding site, thereby preventing clearance of FVIII by its own clearance pathway.

[0314] In some embodiments, the prevention or inhibition of a FVIII protein binding to endogenous VWF by the VWF fragment or the XTEN sequence can be in vitro or in vivo.

[0315] Also provided is a method of increasing the half-life of a FVIII protein comprising administering the chimeric protein described herein to a subject in need thereof. The half-life of non-activated FVIII bound to or associated with full-length VWF is about 12 to 14 hours in plasma. In VWD type 3, wherein there is almost no VWF in circulation, the half-life of FVIII is only about six hours, leading to symptoms of mild to moderate hemophilia A in such patients due to decreased concentrations of FVIII. The half-life of the FVIII protein linked to or associated with the VWF fragment or the XTEN sequence of the present invention can increase at least about 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.6 times, 2.7. times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, or 4.0 times higher than the half-life of the non-activated FVIII bound to or associated with full-length VWF.

[0316] In one embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or linked to an Ig constant region or a portion thereof in the chimeric protein comprising an XTEN sequence increases at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 7 times, 8 times, 9 times, or 10 times higher than the half-life of the non-activated FVIII bound to or associated with full-length VWF. In another embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or an Ig constant region or a portion thereof in the chimeric protein comprising an XTEN sequence increases about 2 to about 5 times, about 3 to about 10 times, about 5 to about 15 times, about 10 to about 20 times, about 15 to about 25 times, about 20 to about 30 times, about 25 to about 35 times, about 30 to about 40 times, about 35 to about 45 times higher than the half-life of the non-activated FVIII bound to or associated with full-length VWF or wild type FVIII. In a specific embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or linked to an Ig constant region in the chimeric protein comprising an XTEN sequence increases at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 times higher than the half-life of the wild type FVIII in a FVIII and VWF double knockout mouse.

[0317] In some embodiments, the half-life of the chimeric protein comprising the VWF fragment fused to a first Ig constant region or a portion thereof, e.g., a first Fc region and an XTEN sequence, and a FVIII protein linked to an XTEN sequence and a second Ig constant region or a portion thereof, e.g., a second Fc region, is longer than the half-life of a FVIII associated with endogenous VWF. In other embodiments, the half-life of the chimeric protein is at least about 1.5 times, 2 times, 2.5 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.5 times, or 5.0 times the half-life of wild type FVIII or a FVIII protein associated with endogenous VWF.

[0318] In some embodiments, as a result of the invention the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF fragment or wild-type FVIII. The half-life of the chimeric protein of the invention is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the half-life of a FVIII protein without the VWF fragment or wild-type FVIII. In one embodiment, the half-life of FVIII is about 1.5-fold to about 20-fold, about 1.5 fold to about 15 fold, or about 1.5 fold to about 10 fold longer than the half-life of wild-type FVIII. In another embodiment, the half-life of the FVIII is extended about 2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2-fold to about 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-fold to about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to about 10-fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-fold to about 5-fold, about 3-fold to about 4-fold, about 4-fold to about 6 fold, about 5-fold to about 7-fold, or about 6-fold to about 8 fold as compared to wild-type FVIII or a FVIII protein without the VWF fragment. In other embodiments, the half-life of the chimeric protein of the invention is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In still other embodiments, the half-life of the chimeric protein of the invention is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours to about one week, about 24 hours to about one week, about 36 hours to about one week, about 48 hours to about one week, about 60 hours to about one week, about 24 hours to about six days, about 24 hours to about five days, about 24 hours to about four days, about 24 hours to about three days, or about 24 hours to about two days.

[0319] In some embodiments, the average half-life of the chimeric protein of the invention per subject is about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about six days, about seven days (one week), about eight days, about nine days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0320] In addition, the invention provides a method of treating or preventing a bleeding disease or disorder comprising administering an effective amount of a chimeric protein. In one embodiment, the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath. In a specific embodiment, the bleeding disease or disorder is hemophilia A.

[0321] The chimeric protein comprising an XTEN sequence and an Ig constant region or a portion thereof in combination with a VWF fragment described herein, that prevents or inhibits interaction of the FVIII protein with endogenous VWF prepared by the invention, has many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent. In one embodiment, the invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of the chimeric protein.

[0322] The FVIII protein portion in the chimeric protein treats or prevents a hemostatic disorder by serving as a cofactor to Factor IX on a negatively charged phospholipid surface, thereby forming a Xase complex. The binding of activated coagulation factors to a phospholipid surface localizes this process to sites of vascular damage. On a phospholipid surface, Factor VIIIa increases the maximum velocity of Factor X activation by Factor IXa, by approximately 200,000-fold, leading to the large second burst of thrombin generation.

[0323] The chimeric protein of the invention can be used to treat any hemostatic disorder. The hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, as well as deficiencies or structural abnormalities relating to Factor VIII. In one embodiment, the hemostatic disorder is hemophilia A.

[0324] The chimeric protein of the invention can be used prophylactically to treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand's factor. In one embodiment, the hemostatic disorder is an inherited disorder. In another embodiment, the hemostatic disorder is an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an auto-immune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).

[0325] The invention also relates to methods of treating a subject that does not have a congenital hemostatic disorder, but has a secondary disease or condition resulting in acquisition of a hemostatic disorder, e.g., due to development of an anti-FVIII antibody or a surgery. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of the chimeric protein prepared by the present methods.

[0326] The present invention is also related to methods of reducing immunogenicity of FVIII or inducing less immunogenicity against FVIII comprising administering an effective amount of the chimeric proteins described herein, or the polynucleotides encoding the same.

[0327] In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to, during, or after surgery as a prophylactic regimen. The chimeric protein of the invention can be administered prior to, during, or after surgery to control an acute bleeding episode.

[0328] The chimeric protein of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding. Non limiting examples of bleeding episodes include a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, and any combinations thereof.

[0329] In prophylactic applications, one or more compositions containing the chimeric protein of the invention or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance or reduce symptoms associated with a disease or disorder. Such an amount is defined to be a "prophylactic effective dose." In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of polypeptide per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmuno conjugates and higher doses for cytotoxin-drug modified polypeptides) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0330] In some embodiments, a chimeric protein or a composition of the invention is used for on-demand treatment, which includes treatment for a bleeding episode, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (head trauma), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. The subject may be in need of surgical prophylaxis, pen-operative management, or treatment for surgery. Such surgeries include, e.g., minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

[0331] In one embodiment, the chimeric protein of the present invention is administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route. The chimeric protein comprising a VWF fragment and a FVIII protein of the present invention can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the site of bleeding or implanted into bandage/dressing. The dose of the chimeric protein will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 .mu.g/kg body weight. In one embodiment, the dosing range is 0.1-1,000 .mu.g/kg. In another embodiment, the dosing range is 0.1-500 .mu.g/kg. The protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e.g., STA-CLOT VIIa-rTF clotting assay or ROTEM clotting assay. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).

[0332] Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. All patents, publications, and articles referred to herein are expressly and specifically incorporated herein by reference.

EXAMPLES

[0333] Throughout the examples, the following materials and methods were used unless otherwise stated.

Materials and Methods

[0334] In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in electrophoresis. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., CS. H. L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Example 1: Cloning Different VWF Domains (FIG. 1)

[0335] (a) Cloning of pSYN-VWF-002

[0336] pSYN-VWF-002 contains nucleotide sequences encoding a VWF fragment, which are amino acids 1-477 of SEQ ID NO: 100. [VWF-D'D3 protein sequence] Amino acid numbering represents the mature VWF sequence without propeptide and corresponds to amino acids 764-1240 of SEQ ID NO: 2. pSYN-VWF-002 construct has the FVIII signal peptide at N-terminus, which allows proper secretion of the synthesized protein and followed by a 6.times.His tag at C-terminus, which is used for protein purification. It was synthesized by using following primer combinations:

TABLE-US-00019 ESC48-Fwd-VWF-D'D3 with VIII signal and BsiW1 site (SEQ ID NO: 90) TCGCGACGTACGGCCGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTT TCTGTGCCTTTTGCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATG ESC51-Rev-VWF D'D3 (1-477 amino acid) with 6His and Not 1 site (SEQ ID NO: 91) TGACCTCGAGCGGCCGCTCAGTGGTGATGGTGATGATGCGGCTCCTGGCA GGCTTCACAGGTGAGGTTGACAAC

[0337] A 50 .mu.l PCR reaction was carried out with ESC 48/ESC 51 primer combinations and full length VWF plasmid as the template, using the 2 step PCR amplification cycle: 94.degree. C. 2 minutes; 21 cycles of (96.degree. C. 30 seconds, 68.degree. C. 2 minute). The 1460 bp band was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned into the BsiWI and Not1 restriction sites of pcDNA 4 to generate pSYN-VWF 002.

[0338] (b) Cloning of pSYN-VWF-010 and 013

[0339] pSYN-VWF-010 was constructed using pSYN-VWF-008 and pSYN-VWF-002. pSYN-VWF-008 contains the full-length VWF sequence in pcDNA 3.1 (amino acids 1-2813 of SEQ ID NO: 2), it includes 763 amino acid propeptide (i.e., D1D2 domains) followed by remaining 2050 amino acids sequence of mature VWF. The FVIII signal peptide in pSYN-VWF-002 was replaced with D1D2 domains from pSYN-VWF-008, the resulting construct is pSYN-VWF-010. pSYN-VWF-008 has a BamH1 site at Arg907 and Not1 site at the end of coding region (after stop codon). pSYN-VWF-008 and 002 were digested with BamH1 and Not1 restriction enzymes. Inserts from pSYN-VWF-002 (1026 bp) were ligated into bamH1/Not1 digested pSYN-VWF-008 (8242 bp) to obtain pSYN-VWF-010 (D1D2D'D3: amino acid 1-1240 of SEQ ID NO: 2), a 6.times.His tag was also added at the C-terminus. In transformed cells pSYN-VWF-010 is synthesized with propeptide but due to intracellular processing the secreted products do not contain any propeptide (D1D2). Protein from VWF-010 exists as dimer.

[0340] pSYN-VWF-010 was used to generate pSYN-VWF-013 which has two point mutations at C336A and C379A corresponding to SEQ ID NO: 100 (amino acid numbering represents mature VWF sequence without D1D2 domain-VWF sequence 2). These mutations are predicted to prevent dimerization of VWF D'D3 domain.

[0341] (c) Cloning of pSYN-VWF-025 and pSYN-VWF-029

[0342] pSYN-VWF-025 contains wild type D1D2D'D3 sequences of full-length VWF in pLIVE vector, and pSYN-VWF-029 contains D1D2D'D3 sequence with C336A and C379A mutation. For cloning pSYN-VWF-025, the following primer combination was used:

TABLE-US-00020 ESC 89-fwd with Nhe1 site = (SEQ ID NO: 92) CTCACTATAGGGAGACCCAAGCTGGCTAGCCG ESC 91-rev with Sal1 = (SEQ ID NO: 93) CTGGATCCCGGGAGTCGACTCGTCAGTGGTGATGGTGATGATG

[0343] A 50 .mu.l PCR reaction was carried out with ESC 89/ESC91 primer combinations and either pSYN-VWF 010 (for pSYN-VWF-025) or pSYN-VWF 013 (for pSYN-VWF-029) plasmid as the template using the 3 step PCR amplification cycle: 94.degree. C. 2 minutes; 21 cycles of (96.degree. C.-30 seconds, 55.degree. C.-30 second, 68.degree. C.-4 minutes). The expected sized band (.about.3800 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned into the Nhe1 and Sal1 restriction sites of pLIVE-Mirus vector (Invitrogen, Carlsbad, Calif.) to generate pSYN-VWF 025 and 029.

[0344] (d) Cloning pSYN-VWF-031

[0345] pSYN-VWF-031 is a D1D2D'D3(C336A/C379A)-Fc construct which has a 48 amino acid long thrombin cleavable linker (8.times. GGGGS (SEQ ID NO 94)+thrombin site) in between the VWF D1D2D'D3(C336A/C379A) and the Fc sequences. To make this construct, VWF-Fc region was amplified from construct pSYN-FVIII-064 (refer FVIII-VWF construct below). pSYN-FVIII-VWF was digested with Xba1 and Nhe1. Resulting insert region of 4165 bp, containing the VWF fragment and Fc region was used as a template for amplifying the VWF and Fc region by primer combinations LW 22/LW23.

TABLE-US-00021 LW 22-FWD-VWF-D'D3 with FVIII signal sequence and BsiW1 site (SEQ ID NO: 95) GCGCCGGCCGTACGATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTG CCTTTTGCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATG LW 23-Rev-Fc with stop codon and Not1 site (SEQ ID NO: 96) TCATCAATGTATCTTATCATGTCTGAATTCGCGGCCGCTCATTTACC

[0346] The PCR product obtained from LW22/LW23 amplification (.about.2300 bp) was cloned in BsiW1/Not1 digested pSYN-VWF-002 to obtain pSYN-VWF-014 intermediate. pSYN-VWF-014 contains FVIII signal peptide-D'D3-20 amino acid thrombin cleavable linker followed by the Fc region.

[0347] To generate the D1D2D'D3-Fc construct, the D1D2D'D3 region was amplified from pSYN-VWF-013 using primer combination LW24/LW27 by standard PCR method.

TABLE-US-00022 LW24-Fwd-VWF D1D2D'D3 cloning oligo with BsiW1 site (SEQ ID NO: 97) GCGCCGGCCGTACGATGATTCCTGCCAGATTTGCCGGGGTG LW27-Rev-VWF D'D3 oligo with EcoRV (SEQ ID NO: 98) CCACCGCCAGATATCGGCTCCTGGCAGGCTTCACAGGTGAG

[0348] The PCR product obtained from LW22/LW23 amplification (.about.3750 bp) was cloned in BsiW1/EcoRV digested pSYN-VWF-014 to obtain pSYN-VWF-015 intermediate. The linker length between the VWF fragment and Fc region was changed to obtain pSYN-VWF-031.

TABLE-US-00023 VWF-D1D2D'D3 protein sequence 1 (SEQ ID NO: 99) 1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY ECEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP* VWF-D'D3 protein sequence 2 (SEQ ID NO: 100) 1 SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG 51 MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC 101 DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG 151 CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII 201 LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ 251 VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL 301 TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCACFCDT IAAYAHVCAQ 351 HGKVVTWRTA TLCPQSCEER NLRENGYECE WRYNSCAPAC QVTCQHPEPL 401 ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT 451 LNPSDPEHCQ ICHCDVVNLT CEACQEP

Example 2: Effects of D'D3 and XTEN Fusion on FVIII Half-Life Extension

[0349] To evaluate D'D3 FVIII half-life extension potential on rFVIII-XTEN fusion protein, a VWF D'D3 dimer was introduced into FVIII-VWF DKO mice by hydrodynamic injection of its corresponding DNA construct VWF-025 (Example 1). After D'D3 has reached the steady state expression (day5 post injection), a single dose of rFVIII-XTEN was administered by IV injection at 200 IU/kg dose. Blood samples were collected up to 120 hrs post rFVIII-XTEN dosing. Plasma FVIII activity was analyzed by a FVIII chromogenic assay. The D'D3 expression level was measured by VWF ELISA, and rFVIIIFc PK profile was analyzed using WinNonlin program.

[0350] The study results were shown in FIG. 2, and the PK parameter of rFVIII-XTEN with/without D'D3 in circulation was listed in Table 16. The D'D3 dimer further extended rFIII-XTEN t.sub.1/2 from 3.4 hr to 17.8 hr, a 5 fold increase. In addition to half-life, 5 fold of increase on MRT, 3.6 fold increases on AUC, 3.8 fold decreases on clearance were also observed.

[0351] We have observed a synergistic effect of D'D3 fragment and XTEN technology, a serial of FVIII/VWF/XTEN constructs will be evaluated for their FVIII half-life extension potential in Hemophilic animals.

TABLE-US-00024 TABLE 16 rFVIII-XTEN PK parameter with/without D'D3 in blood circulation 5 min Cl AUC_D Recovery t.sub.1/2 MRT (mL/ Vss (hr*kg*mIU/ Treatment (%) (hr) (hr) hr/kg) (mL/kg) mL/mIU) rFVIIIXTEN 80 17.8 19.3 3.5 67.4 0.29 VWF-025 rFVIIIXTEN 74 3.4 3.8 13.1 63.68 0.08 Improvement 1.1 5.2 5.1 3.8 0.9 3.6 fold

Protein Purification of FVIII-XTEN

[0352] An AE288 XTEN was inserted at the C-terminus of BDD-FVIII for this study. To purify this protein, a tangential flow filtration (TFF) step was used first to buffer exchange the conditioned media. Products in the filtrate were then captured using a strong anion exchange chromatography, and then further purified using affinity chromatography. Purity of the molecule was acceptable by HPLC-SEC and was further confirmed by western blotting. The specific activity of the molecule was comparable to B-domain deleted FVIII, as measured by aPTT assay and ELISA.

FVIII Chromogenic Assay

[0353] The FVIII activity was measured using the COATEST SP FVIII kit from DiaPharma (lot #N089019) and all incubations were performed on a 37.degree. C. plate heater with shaking.

[0354] The range of rFVIII standard was from 100 mIU/mL to 0.78 mIU/mL. A pooled normal human plasma assay control and plasma samples (diluted with 1.times. Coatest buffer) were added into Immulon 2HB 96-well plates in duplicate (25 .mu.L/well). Freshly prepared IXa/FX/Phospholipid mix (50 .mu.L), 25 .mu.L of 25 mM CaCl2, and 50 .mu.L of FXa substrate were added sequentially into each well with 5 minutes incubation between each addition. After incubating with the substrate, 25 .mu.L of 20% Acetic Acid was added to terminate the color reaction, and the absorbance of OD405 was measured with a SpectraMAX plus (Molecular Devices) instrument. Data were analyzed with SoftMax Pro software (version 5.2). The Lowest Level of Quantification (LLOQ) is 7.8 mIU/mL.

VWF ELISA:

[0355] Goat anti-human VWF antibody (Affinity purified, affinity biological, GAVWF-AP) was used as the capture antibody at 0.5 ug/well and VWF-EIA-D (Affinity Biologicals, VWF-EIA-D, 1:100 dilution) was used as the detecting antibody for the VWF ELISA. ELISA assay was performed following the standard ELISA procedure, TMB was used as the HRP substrate, PBST/1.5% BSA/0.5M NaCl buffer was used as blocking and binding buffer. The assay standard range is 100 ng to 0.78 ng, and assay's lowest limit of quantification (LLOQ) is 7.8 ng/mL.

Example 3: Plasmid Construction of XTEN Containing FVIII/VWF Constructs

[0356] (a) Cloning of pSYN-FVIII-161 (FIG. 3)

[0357] The FVIII-161 plasmid comprises a single chain Fc (scFc) scaffold with enzyme cleavage sites which are processed during synthesis in a cell. The construct has a FVIII binding domain of full-length VWF (D'D3).

[0358] Plasmid (pSYN-FVIII-161) was designed for the expression FVIII-Fc and VWF-Fc heterodimer, where the D'D3 domains to bind FVIII and prevents FVIII interaction with phospholipids and activated protein C. Protein from pSYN-FVIII-161 is expressed in the cell as a single polypeptide where the C-terminus of the FVIII-Fc subunit is linked to the N-terminus of the VWF D'D3-Fc subunit by a 6.times. (GGGGS) polypeptide linker (SEQ ID NO: 64). In addition, RRRRS (SEQ ID NO: 11) and RKRRKR (SEQ ID NO: 10) sequences were inserted at the 5' and 3' end of the polypeptide linker, respectively, for intracellular cleavage by proprotein convertases following the last Arg at each sequence. Hence, the cells can express a double chain FVIII-Fc/D'D3-Fc heterodimer where the FVIII-Fc chain has a RRRRS sequence (SEQ ID NO: 11) at the C-terminus, but the remainder of the linker sequence has been removed. An AE288 XTEN fragment immediately followed by IS{5X(GGGGS)}LVPRGSGG (SEQ ID NO: 122) polypeptide (contains thrombin cleavage site) is introduced in between the VWF domains and the Fc region to facilitate release of the VWF fragment from FVIII once the FVIII-VWF hetero-dimeric protein is activated by thrombin allowing interaction of FVIII with other clotting factors.

TABLE-US-00025 pSYN-FVIII-161 (SEQ ID NO: 101). protein sequence (FVIII sequence amino acid position 1-1457; underlined region represents Fc region; curvy underline represents cleavable linker in between first Fc and VWF fragment; double underlined region represents VWF fragment; bold region represents cleavable linker in between VWF fragment and Fc. 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE 801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 851 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1001 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI 1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1101 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1651 ##STR00017## 1701 ##STR00018## DNLRAEGLEC 1751 TKTCQNYDLE CMSMGCVSGC LCPPGMVRHE NRCVALERCP CFHQGKEYAP 1801 GETVKIGCNT CVCRDRKWNC TDHVCDATCS TIGMAHYLTF DGLKYLFPGE 1851 CQYVLVQDYC GSNPGTFRIL VGNKGCSHPS VKCKKRVTIL VEGGEIELFD 1901 GEVNVKRPMK DETHFEVVES GRYIILLLGK ALSVVWDRHL SISVVLKQTY 1951 QEKVCGLCGN FDGIQNNDLT SSNLQVEEDP VDFGNSWKVS SQCADTRKVP 2001 LDSSPATCHN NIMKQTMVDS SCRILTSDVF QDCNKLVDPE PYLDVCIYDT 2051 CSCESIGDCA AFCDTIAAYA HVCAQHGKVV TWRIATLCPQ SCEERNLREN 2101 GYEAEWRYNS CAPACQVTCQ HPEPLACPVQ CVEGCHAHCP PGKILDELLQ 2151 TCVDPEDCPV CEVAGRRFAS GKKVTLNPSD PEHCQICHCD VVNLTCEACQ 2201 ##STR00019## GSEPATSGSE 2251 TPGTSESATP ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP 2301 GSEPATSGSE TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGT 2351 STEPSEGSAP GTSESATPES GPGTSESATP ESGPGTSESA TPESGPGSEP 2401 ATSGSETPGS EPATSGSETP GSPAGSPTST EEGTSTEPSE GSAPGTSTEP 2451 SEGSAPGSEP ATSGSETPGT SESATPESGP GTSTEPSEGS APDSGGGGSG 2501 GGGSGGGGSG GGGSGGGGSL VPRGSGGDKT HTCPPCPAPE LLGGPSVFLF 2551 PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE 2601 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP 2651 REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 2701 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL 2751 SPGK

[0359] (b) Cloning of pSYN-FVIII-168, 175, 172 and 174 (FIG. 4A-4D)

[0360] pSYN-FVIII-168, 172, 174 and 175 are derivatives of pSYN-FVIII-161. R1645A/R1648A mutations were introduced into pSYN-FVIII-161 to form pSYN-FVIII-168, which produces a SC-FVIII isoform, and an AE288 XTEN was directly fused into the C-terminus of FVIII-HC for further half-life extension. To construct pSYN-FVIII-175, the D'D3 codon sequence was remove form pSYN-FVIII-168 for evaluation of the effect of Fc and XTEN technology on FVIII half-life extension.

[0361] To construct pSYN-FVIII-172, the AE288 XTEN fragment was directly fused into the C-terminus of FVIII-HC for further half-life extension, and the D'D3 codon sequence was removed from pSYN-FVIII-172 to form pSYN-FVIII-174 for evaluation of the effect of Fc and XTEN technology on FVIII half-life extension.

[0362] (c) Cloning of pSYN-FVIII-170 (FIG. 4E)

[0363] pSYN-FVIII-170 was constructed to evaluate the effect of XTEN and D'D3 fragment on FVIII half-life extension. The codon sequence VWF-D1D2D'D3 fragment and BDD-FVIII were introduced into the 5' and 3' end of expression casket, an AE288 XTEN codon sequence which followed by a 35 aa thrombin cleavable linker was used to connect the VWF and FVIII molecule. After intra cellular processing, the secreted protein comprises a polypeptide contains the D'D3 fragment of mature VWF molecule which is linked to the N-terminus of mature BDD-FVIII by an AE288 XTEN/35 aa thrombin cleavable linker.

TABLE-US-00026 pSYN-FVIII-170 protein sequence (SEQ ID NO: 102) 1 SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG 51 MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC 101 DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG 151 CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII 201 LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ 251 VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL 301 TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCAAFCDT IAAYAHVCAQ 351 HGKVVTWRTA TLCPQSCEER NLRENGYEAE WRYNSCAPAC QVTCQHPEPL 401 ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT 451 LNPSDPEHCQ ICHCDVVNLT CEACQEPISG TSESATPESG PGSEPATSGS 501 ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG TSTEPSEGSA 551 PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG 601 SPAGSPTSTE EGSPAGSPTS TEEGTSTEPS EGSAPGTSES ATPESGPGTS 651 ESATPESGPG TSESATPESG PGSEPATSGS ETPGSEPATS GSETPGSPAG 701 SPTSTEEGTS TEPSEGSAPG TSTEPSEGSA PGSEPATSGS ETPGTSESAT 751 PESGPGTSTE PSEGSAPDSG GGGSGGGGSG GGGSGGGGSG GGGSLVPRGS 801 GGASATRRYY LGAVELSWDY MQSDLGELPV DARFPPRVPK SFPFNTSVVY 851 KKTLFVEFTD HLFNIAKPRP PWMGLLGPTI QAEVYDTVVI TLKNMASHPV 901 SLHAVGVSYW KASEGAEYDD QTSQREKEDD KVFPGGSHTY VWQVLKENGP 951 MASDPLCLTY SYLSHVDLVK DLNSGLIGAL LVCREGSLAK EKTQTLHKFI 1001 LLFAVFDEGK SWHSETKNSL MQDRDAASAR AWPKMHTVNG YVNRSLPGLI 1051 GCHRKSVYWH VIGMGTTPEV HSIFLEGHTF LVRNHRQASL EISPITFLTA 1101 QTLLMDLGQF LLFCHISSHQ HDGMEAYVKV DSCPEEPQLR MKNNEEAEDY 1151 DDDLTDSEMD VVRFDDDNSP SFIQIRSVAK KHPKTWVHYI AAEEEDWDYA 1201 PLVLAPDDRS YKSQYLNNGP QRIGRKYKKV RFMAYTDETF KTREAIQHES 1251 GILGPLLYGE VGDTLLIIFK NQASRPYNIY PHGITDVRPL YSRRLPKGVK 1301 HLKDFPILPG EIFKYKWTVT VEDGPTKSDP RCLTRYYSSF VNMERDLASG 1351 LIGPLLICYK ESVDQRGNQI MSDKRNVILF SVFDENRSWY LTENIQRFLP 1401 NPAGVQLEDP EFQASNIMHS INGYVFDSLQ LSVCLHEVAY WYILSIGAQT 1451 DFLSVFFSGY TFKHKMVYED TLTLFPFSGE TVFMSMENPG LWILGCHNSD 1501 FRNRGMTALL KVSSCDKNTG DYYEDSYEDI SAYLLSKNNA IEPRSFSQNP 1551 PVLKRHQREI TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS 1601 FQKKTRHYFI AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD 1651 GSFTQPLYRG ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI 1701 SYEEDQRQGA EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD 1751 VDLEKDVHSG LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY 1801 FTENMERNCR APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI 1851 RWYLLSMGSN ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP 1901 SKAGIWRVEC LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS 1951 GQYGQWAPKL ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR 2001 QKFSSLYISQ FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF 2051 NPPIIARYIR LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI 2101 TASSYFTNMF ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV 2151 TGVTTQGVKS LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF 2201 TPVVNSLDPP LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LY

Example 4: Hydrodynamic Injection of XTEN Containing FVIIIF/VWF Constructs in FVIII and VWF Deficient Mice

[0364] The XTEN containing DNA constructs in FIGS. 3 and 4 have combined 2-3 half-life extension elements together. To evaluate their FVIII half-life extension potential, a selective group of DNA constructs in FIG. 3 and FIG. 4 were introduced into FVIII/VWF double knockout (DKO) mice by Hydrodynamic injection (HDI) at 100 ug/mouse dose. Blood samples were then collected by retro orbital blood collection at 24 hr post HDI. The post HDI plasma FVIII activity was analyzed by FVIII chromogenic assay, and results were listed in Table 17 and FIG. 5. Compared to wild type BDD-FVIII, all XTEN containing DNA constructs yield significantly higher FVIII plasma activity at 24 hr post HDI, indicating the corresponding molecules had significant longer circulating protein half-life than BDD-FVIII. The application of the combination of those half-life extending elements was further evaluated in Hemophilic animals.

TABLE-US-00027 TABLE 17 FVIII plasma activity 24 hr post HDI in FVIII/VWF DKO mice DNA BDD- FVIII- FVIII- BDD- FVIII- Construct FVIII FVIII-161 168 172 FVIII 170 DNA Dose 100 100 100 100 50 50 (.mu.g/mouse) FVIII 219 .+-. 2446 .+-. 2209 .+-. 1671 .+-. 197 .+-. 399 .+-. Activity 72 1012 609 223 21 30 (mU/mL)

Hydrodynamic Injection:

[0365] Hydrodynamic Injection is an efficient and safe non-viral gene delivery method to the liver in small animals, such as mice and rats. It was originally described as a rapid injection of a naked plasmid DNA/saline solution free of endotoxin at a tenth volume of the animal's body weight in about 5-7 seconds. The naked plasmid DNA contains the gene of interest and the liver produced in a tenth volume of the animal's body weight. The targeted protein is produced in the liver from the injected DNA and can be detected within 24 hours post-injection. Plasma samples were then collected to study the therapeutic property of the expressed protein.

[0366] For all the hydrodynamic injections that were performed herein, 2 ml of plasmid DNA in 0.9% sterile saline solution was delivered via intravenous tail vein injection within about 4-7 seconds to mice weighing 20-35 grams. The mice were closely monitored for the first couple of hours until the normal activity resumed. After the blood samples were collected via retro orbital blood collection, plasma samples were then obtained and stored at -80.degree. C. for further analysis.

Example 5: Plasmid Construction of Co-Transfection System for FVIIIFc-VWF Heterodimer Contain XTEN Insertions (FIG. 6)

[0367] To increase the protein production yield, two co-transfection systems were generated for protein production, which contains three DNA constructs. The first DNA construct encoded a FVIII-Fc fusion protein in which a AE288 XTEN fragment was directly fuse to the C-terminus of the FVIII heavy chain and followed by either a wild type FVIII light chain fragment (pSYN-FVIII-173, FIG. 6B) or a FVIII light chain fragment with R1645A/R1648A mutations (pSYN-FVIII-169, FIG. 6A), the FVIII light chain was then directly fused to a single Fc fragment. The second DNA construct is pSYN-VWF-031 which encoding a D'D3-Fc fusion protein (Example 1). HEK293F cells were transfected with the two plasmid along with a third plasmid (PC5) at 80:15:5 ratio. The synthesized proteins were secreted as FVIII (XTEN) Fc/D'D3Fc heterodimer and D'D3Fc dimer and the FVIII (XTEN) Fc/D'D3Fc heterodimer was separated from the D'D3Fc dimer by protein purification.

TABLE-US-00028 pSYN-FVIII-169 mature Protein sequence (SEQ ID NO: 103): 1 ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL 51 FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA 101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD 151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA 201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR 251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL 301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL 351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL 401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG 451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD 501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP 551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG 601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS 651 VFFSGYTFKH KMVYEDTLIL FPFSGETVFM SMENPGLWIL GCHNSDFRNR 701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGAPGT 751 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE 801 SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT 851 SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE 901 GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE 951 TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP 1001 GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP PVLKRHQAEI 1051 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI 1101 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG 1151 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA 1201 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG 1251 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR 1301 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN 1351 ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC 1401 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL 1451 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ 1501 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR 1551 LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF 1601 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS 1651 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP 1701 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP CPAPELLGGP 1751 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK 1801 TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK 1851 AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 1901 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ 1951 KSLSLSPGK pSYN-FVIII-173 mature Protein sequencing (SEQ ID NO: 104): 1 ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL 51 FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA 101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD 151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA 201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR 251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL 301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL 351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL 401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG 451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD 501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP 551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG 601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS 651 VFFSGYTFKH KMVYEDTLIL FPFSGETVFM SMENPGLWIL GCHNSDFRNR 701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGAPGT 751 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE 801 SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT 851 SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE 901 GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE 951 TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP 1001 GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP PVLKRHQREI 1051 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI 1101 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG 1151 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA 1201 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG 1251 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR 1301 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN 1351 ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC 1401 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL 1451 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ 1501 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR 1551 LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF 1601 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS 1651 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP 1701 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP CPAPELLGGP 1751 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK 1801 TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK 1851 AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 1901 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ 1951 KSLSLSPGK

Example 6. Protein Purification for FVIII-169/VWF-031 and FVIII-173/VWF-031

[0368] A tangential flow filtration (TFF) step was used to buffer exchange the clarified conditioned media. The FVIII-169NWF-031 or FVIII-173NWF-031 heterodimer was then purified using a two-step chromatography process. A weak anion exchange resin was used, followed by affinity chromatography. The final purified product had acceptable purity by SEC-HPLC. The specific activity was compatible to B-domain deleted FVIII, as measured by FVIII chromogenic assay and A280 concentration. Purity and the presence of each moiety of this molecule were confirmed by SDS-PAGE and western blotting.

Example 7. Evaluation the VWF Binding Ability of FVIII-169/VWF-031 by Octet Assay

[0369] The VWF binding ability of FVIII-169/VWF-031 was obtained by Bio-Layer Interferometry (BLI) based measurements (Octet assay) at 25.degree. C. with a ForteBio Octet 384 instrument, using Tris binding buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 5 mM CaCl.sub.2)). The Octet assay for determining FVIII binding was based on the hydrophobic immobilization of Human von Willebrand Factor (Haematologic Technologies Catalog No. HCVWF-0191) onto the APS Biosensor, then followed by the binding of 1.0% Bovine Serum Albumin (Jackson ImmunoResearch Catalog No. 001-000-161). Briefly, hvWF (20 .mu.g/mL) was diluted in Tris buffer and loaded across APS Biosensors for 600 sec, yielding approximately 3.0-3.5 nm binding on the reaction probes. Control APS probes were loaded with 1.0% BSA in the absence of hvWF for reference subtraction. After loading, all probes were incubated in Tris buffer for 300 sec to establish a new baseline. Subsequently, biosensor probes were incubated in solutions of FVIII-XTEN 169 or FVIIIFc Drug Substance (0, 0.6, 2, 6, 20, 60, 200, 600 IU/mL) for 5 min at room temperature, followed by a 5 min dissociation step. Using the Octet data analysis software, the binding response (nm) was derived from the subtracted data (Reaction probe minus Reference probe). No binding to immobilized VWF was detected for FVIII-169/VWF-031 (FIG. 7), indicating a complete shielding of FVIII from full length VWF molecule by the D'D3 fragment.

Example 8. FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO Mice

[0370] The PK profile of FVIII-169/VWF-031 was tested in HemA and FVIII/VWF DKO mice to evaluate the ability of the D'D3 fragment to shield the FVIII moiety from the endogenous VWF. HemA or FVIII/VWF DKO mice were treated with a single intravenous dose of FVIII-169/VWF-031 at 200 IU/kg, plasma samples were then collected at 5 min, 8 hr, 24 hr, 48 hr and 72 hours post dosing. The FVIII activity of plasma sample was tested by FVIII chromogenic assay, and half-life of FVIII-169/VWF-031 was calculated using WinNonlin program.

[0371] Complete inhibition of the constructs' binding to immobilized VWF was demonstrated by biolayer interferometry (FIG. 7) for FVIII-169/VWF-031. This indicates the D'D3 fragment in the molecule had successfully blocked the FVIII binding to native VWF molecules, therefor similar half-life of FVIII-169/VWF-031 was predicted in the two different mouse strains. As shown in FIG. 8A and Table 18, as expected, FVIII-169/VWF-031 had similar PK profile in both HemA and FVIII/VWF DKO mice, which has demonstrated that the half-life of FVIIIFc/VWF heterodimer is independent from the half-life of endogenous VWF. The separation of the FVIIIFc/VWF heterodimer half-life from the endogenous VWF half-life, eliminated the FVIII extension ceiling and opened the possibility of further extending FVIII half-life beyond the 2 fold half-life limit imposed by endogenous VWF.

TABLE-US-00029 TABLE 18 FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO mice Cl AUC Mouse Recovery t.sub.1/2 MRT (mL/hr/ Vss (hr*kg*mIU/ Strain (%) (hr) (hr) kg) (mL/kg) mL/mIU) FVIII/VWF 69 17.94 20.1 4.06 81.69 0.2461 DKO HemA 83 16.65 18.44 3.57 85.72 0.28

[0372] The FVIII protecting ability of the XTEN insertion and D'D3 fragment was evaluated by comparing the half-life of FVIII-169/VWF-031 with FVIII-169/Fc and FVIIIFc in FVIII/VWF DKO mice. After a single IV administration, blood samples were collected at 5 min, 8 hr, 24 hr, 48 hr and 72 hr for FVIII-169/VWF-031, 5 min, 8 hr, 24 hr, 32 hr, 48 hr for FVIII-169/Fc and at 5 min, 1, 2, 4, 6 and 8 hrs for FVIIIFc. The FVIII activity of plasma sample was tested by FVIII chromogenic assay, and half-life of FVIII-155/VWF-031 was calculated using WinNonlin program.

[0373] The study results were summarized in FIG. 8B and Table 19, rFVIIIFc has a 1.6 hr half-life in DKO mice due to the loss of VWF protection. When an XTEN insertion was introduced into the FVIIIFc molecule, the resulting FVIII-169/Fc molecule has a 7 hr half-life, a 4 fold half-life extension by the XTEN insertion. Finally, when D'D3 fragment was incorporated into the molecule to form FVIII-169/VWF-031, a 17 hr half-life was observed, another 2.5 fold further increase by the D'D3 fragment. In addition of the half-life improvement, improved Mean residency time (MRT), Clearance (C1) and AUC were also observed as shown in Table 19.

[0374] FVIII-169/VWF-031 has achieved 17-18 hr t.sub.1/2 in both HemA and FVIII/VWF DKO mice, which is the upper limit of the t.sub.1/2 extension ceiling that imposed by VWF clearance. More t.sub.1/2 extension elements can be further incorporated into this molecule, such as a second XTEN insertion within FVIII. The synergistic effect of D'D3 fragment and XTEN insertions provided the possibility of the complete protection for FVIII from its clearance pathway, a final breakthrough of the 2 fold FVIII t.sub.1/2 extension limit might be achieved by the FVIIIFc/XTEN/VWF variants.

TABLE-US-00030 TABLE 19 FVIII-169/VWF-031 PK in FVIII/VWF DKO mice AUC/D Re- Cl (hr*kg* Mouse covery t.sub.1/2 MRT (mL/hr/ Vss mIU/mL/ Strain Treatment (%) (hr) (hr) kg) (mL/kg) mIU) FVIII/ rFVIIIFc 35 1.6 2.1 57.7 120.2 0.0173 VWF rFVIII- 77 7.0 6.2 6.4 39.2 0.1573 DKO 169/Fc rFVIII- 69 17.9 20.1 4.1 81.7 0.2461 169/ VWF-031

Example 9: FVIII-XTEN Variants Cell Media Concentrate PK in D'D3 Expressing FVIII/VWF DKO Mice

[0375] The ability of D'D3 fragment to extend the t.sub.1/2 of FVII-XTEN was evaluated in the D'D3 expressing FVIII/VWF DKO mouse model (described in example 2). In this study, instead of using VWF-025 to introduce the D'D3 dimer into the circulation, VWF-029 construct was used to introduce the D'D3 monomer into the circulation. To prepare FVIII-XTEN variants protein, a small scale (50-100 mL) transient transfection culture media was prepared, at day 4 post transfection, cell culture was harvested and concentrated to reach 10-20 IU/mL of FVIII activity range which is suitable for PK study. The concentrated cell media were then used for standard PK study in FVIII/VWF DKO mice with or without D'D3 in the circulation.

[0376] Total of 6 FVIII-XTEN variants that contains 1-3 XTEN insertions were tested in the system, their t.sub.1/2 were summarized in Table 20 and data from representative variants were plotted in FIG. 9A.

[0377] Longer half-life was observed for all the FVIII-XTEN variants with the presents of D'D3 fragment in the circulation (Table 20), which demonstrated the D'D3 protection for FVIII-XTEN from its clearance pathways. Furthermore, when compared to its 14 hr half-life in HemA mice, LSD0055.021 has a 20.4 hr t.sub.1/2 in D'D3 expressing DKO mice (FIG. 9B, Table 20), indicates the final breakthrough of the 2 fold half-life extension ceiling for FVIII molecules. By further modify the FVIII(XTEN)/VWF molecule, we could potentially achieve even longer FVIII t.sub.1/2, and provide HemA patients a FVIII protein that only requires once weekly or less frequent dosing regimen.

TABLE-US-00031 TABLE 20 FVIII-XTEN t.sub.1/2 in D'D3 expressing FVIII/VWF DKO mice # of t.sub.1/2 (hr) t.sub.1/2 XTEN t.sub.1/2 (hr) pLIVE- (hr) FVIII-XTEN inser- Insertion XTEN DKO D'D3/ HemA ID tions sites size mice DKO mice mice pSD-0013 1 CT 144 3.3 7.9 LSD0003.009 2 B*/CT 144/288 9.7 16.4 LSD0038.015 2 1656/26 144/144 7.8 17.2 LSD0049.002 3 18/B*/ 144/144/ 12.6 17.5 CT 288 LSD0051.002 3 403/B*/ 144/144/ 11.1 19.9 CT 288 LSD0055.021 3 1900/B*/ 144/144/ 16 20.4 14 CT 288 *B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

Example 10: Stability of VWF- and XTEN-Containing FVIII Variants in FVIII/VWF Double Knockout (DKO) Plasma

[0378] Plasma stability of rFVIIIFc protein variants was tested in FVIII/VWF double knockout (DKO) mouse plasma. For the stability assay, HEK293 cells were co-transfected with plasmids directing the expression of rFVIIIFc or FVIII-169 (rFVIIIFc with 288 AE XTEN inserted at the B-domain junction) and plasmids directing the expression of either IgG-Fc or VWF-031 (VWF D'D3 region fused to IgG-Fc). At day four post-transfection, cell culture media was harvested and concentrated to 30 IU/mL based on FVIII chromogenic activity. Concentrated cell culture medium was then added into DKO mouse plasma to yield a FVIII activity of 5 IU/mL and incubated at 37.degree. C. Aliquots were collected at different time points for activity measurement by chromogenic assay. Activity at each time point was measured in duplicate, and the average activity was plotted as a function of time. The activity of FVIIIFc, a dual chain (dc) FVIII molecule in which heavy and light chains are held together by non-covalent interaction, decreases with time in DKO mouse plasma (FIG. 10). The activity of FVIII-169:Fc, which contains a 288 AE XTEN insertion at the B-domain junction, decays at a reduced rate relative to rFVIIIFc, indicating that enhanced stability is conferred by the XTEN insertion. Given that VWF has been proposed to enhance the stability of FVIII in vivo, we evaluated the plasma stability of FVIII-169:VWF-031. This heterodimeric molecule, in which the FVIII element and the VWF D'D3 element are fused to respective hemi-domains of Fc, exhibited additional plasma stability relative to FVIII-169:Fc, indicating that the VWF D'D3 domain and XTEN have a synergistic effect on the plasma stability of rFVIIIFc. Example 11: The effect on FVIII half-life of Fc fusion, XTEN insertion and the D'D3 fragment of VWF.

[0379] To assess the effect of Fc fusion, XTEN insertion and D'D3 fragment of VWF on the half-life of FVIII, the pharmacokinetic properties of B domain deleted recombinant FVIII (rBDD-FVIII), rFVIIIFc, FVIII-169:Fc and FVIII-169:VWF-031 were evaluated in FVIII/VWF double knockout (DKO) mice.

[0380] DKO mice were treated with a single intra venous administration of 200 IU/kg of FVIII proteins, and plasma samples were collected at designated time points as indicated in FIG. 11. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay and half-life was calculated using the WinNonlin-Phoenix program. The pharmacokinetic parameters of the tested molecules are listed in Table 21. The time regression curve of plasma FVIII activity for each FVIII variants were plotted in FIG. 11.

[0381] Unmodified BDD-FVIII had a half-life of 0.23 hr in DKO mice, the FVIIIFc fusion protein has an extended half-life of 1.66 hr in DKO mice due to the recycling of FVIIIFc protein through the Fc:FcRn interaction. When a 288 residue of AEXTEN polypeptide was incorporated into the B domain region of FVIII within the FVIIIFc molecule, the half-life of the resulting FVIII169/Fc protein was further extended to 7.41 hr in DKO mice. Finally, with the addition of the D'D3 domain of VWF, the half-life of FVIII169NWF031 heterodimer has reached 17.9 hr in DKO mice (FIG. 11, Table 21). In addition of the half-life, all of the other PK parameters also improved proportionally with the addition of each element (Table 21). FVIII can tolerate multiple half-life extension elements, and this synergistic effect of the three elements on FVIII half-life extension, enabled the further improvement of the half-life of FVIII-XTEN VWF heterodimers.

TABLE-US-00032 TABLE 21 PK parameters of FVIII variants XTEN Insertions Cl AUC_D FVIII XTEN T.sub.1/2 MRT (mL/hr/ Vss kg*hr/ FVIII Isoform Site Length (hr) (hr) kg) (mL/kg) mL BDD-FVIII dc 0.23 0.24 407.72 97.42 0.0025 FVIIIFc dc 1.66 2.06 62.66 128.82 0.0161 FVIII169/Fc se B* AE288 7.41 6.67 6.24 41.61 0.1603 FVIII169/VWF031 sc B* AE288 17.94 20.1 4.06 81.69 0.2463 *B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

Example 12: Pharmacokinetic Properties of Different FVIII-XTEN VWF Heterodimers

[0382] To evaluate the combined effect of the VWF-D'D3 fragment and XTEN insertions on the FVIII half-life, the pharmacokinetic properties of FVIII-XTEN-Fc:VWF-Fc heterodimers were tested in HemA mice and compared to those of the single chain isoform of BDD-FVIII (scBDD-FVIII) and FVIII-169:VWF-031 (example 10). Seven new FVIII-XTEN-Fc constructs were generated (protein sequences were listed in Table 24). Schematic diagrams of those constructs are shown in FIG. 14A-H. FVIII-195 and FVIII-199, respectively, are the FVIII dual chain and single chain isoforms that each contains two XTEN insertions at positions 1900 and 1656. FVIII-196 and FVIII-201, respectively, are the FVIII dual chain and single chain isoforms that each contains three XTEN insertions at positions 26, 1656 and 1900. FVIII-203, -204 and -205 are sc-FVIIIFc molecules with two XTEN insertions at the B domain junction and at positions 1900, 403 or 18, respectively. Each FVIII-XTEN-Fc construct was co-expressed with VWF-031 in HEK293 cells to produce FVIII-XTEN-Fc/VWF heterodimeric proteins. At day four post-transfection, cell culture medium was harvested and either concentrated to 20 IU/mL based on FVIII chromogenic activity (FVIII-195:VWF-031, FVIII-196:VWF-031, FVIII-199:VWF-031, FVIII-203:VWF-031 and FVIII-204:VWF-031) or purified (scBDD-FVIII, FVIII-169:VWF-031, FVIII-201:VWF-031 and FVIII-205:VWF-031). Having demonstrated the complete intra-molecular shielding of FVIII molecule from the endogenous VWF by the D'D3 fragment in the FVIII-XTEN-Fc:VWF-Fc heterodimer (FVIII-169:VWF-031, Example 5), HemA mice was chosen for the PK evaluations. Purified protein or concentrated cell culture medium was administered to 8-12 week-old HemA mice by intravenous administration at a dose of 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 8 hr, 16 hr, 24 hr, 32 hr, 48 hr, 72 hr and 96 hr post-dosing. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay and half-life was calculated using the WinNonlin-Phoenix program. The pharmacokinetic parameters of the tested molecules are listed in Table 22. The plasma FVIII activities at selected time points for FVIII-XTEN-Fc/VWF-Fc variants were plotted in FIGS. 12A-C.

[0383] When XTEN was inserted into positions 1900 and 1656 (FVIII-195, FVIII-199), moderate improvement in half-life was observed for the scFVIII isoform (FVIII-199:VWF-031) compared to FVIII-169:VWF-031. However, the dcFVIII isoform exhibited a shorter half-life than did FVIII-169:VWF-031, indicating that the single chain isoform might be significantly more stable than the corresponding dual chain isoform (Table 22 and FIG. 12A). When a third XTEN insertion was incorporated into FVIII-199 at position 26, the half-life of the resulting molecule FVIII-201:VWF-031 had reached 24.6 hr, which represents greater than a threefold half-life improvement relative to scBDD-FVIII (Table 22 and FIG. 12C). We have also tested the half-life extension effect of the second XTEN insertion at position 403 (A2 domain), 1900 (A3 domain) and 18 (A1 domain) each in combination with the B domain XTEN insertion. While the addition of the A2 or A3 XTEN insertion did not confer an additional half-life benefit (Table 22, FIG. 12b), the addition of the A1 insertion further extended the half-life of the FVIII-XTEN-Fc:VWF-Fc heterodimer to 29.4 hr (Table 22, FIG. 12C), which is greater than threefold longer than that of scBDD-FVIII.

[0384] When XTENs were incorporated into the FVIIIFc/VWF heterodimer construct, degree of half-life improvement of the resulting molecules was variable, and no obvious correlation was observed between half-lives and either the site or number of XTEN insertion, suggesting that the half-life of the FVIII-XTEN-Fc/VWF heterodimer is determined by the integrity of the whole molecule rather than by the number or placement of XTEN insertions.

[0385] The 24.6 hr and 29.4 hr half-lives observed for FVIII-XTEN-Fc:VWF-Fc heterodimers clearly exceeded the 1.6- to 2-fold limitation on FVIII half-life extension. If this finding translates for HemA patients, it will allow once-weekly or less frequent dosing for FVIII prophylaxis.

TABLE-US-00033 TABLE 22 PK parameters of FVIII-XTEN-Fc/VWF-Fc heterodimers Cl AUC_D FVIII XTEN Insertions T.sub.1/2 MRT (mL/hr/ Vss kg*hr/ FVIII Isoform Site XTEN Length (hr) (hr) kg) (mL/kg) mL scBDD-FVIII sc 7.16 10.16 4.38 44.44 0.23 FVIII169/VWF031 sc B* AE288 16.65 18.44 3.57 65.79 0.28 FVIII195/VWF031 dc 1656/1900 AG144/ 12.56 13.88 9.04 125.48 0.11 AE144 FV111199/VWF031 sc 1656/1900 AG144/ 18.57 20.09 6.24 125.28 0.16 AE144 FVIII201/VWF031 sc 26/1656/ AG144/AG144/ 24.63 33.67 1.9 63.97 0.53 1900 AE144 FVIII203/VWF031 sc 403/B* AE144/AE288 15.52 18 3.65 65.61 0.27 FVIII204/VWF031 sc 1900/B* AE144/AE288 16.3 20.63 2.87 59.14 0.35 FVIII205/VWF031 sc 18/B* AE144/AE288 29.4 37.06 1.82 67.39 0.55 *B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

[0386] In addition to incorporating XTEN into the FVIII molecule, we also evaluated the potential half-life extension benefit of incorporating XTEN as a linker between the D'D3 and Fc fragment. FVIII-155 (scFVIIIFc) was co-expressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residue thrombin cleavable linker) in HEK293 cells. At day 4 post-transfection, cell culture medium was harvested and concentrated to 20 IU/mL based on FVIII activity assay. FVIII/VWF DKO mice were dosed with concentrated cell culture media at 200 IU/10 mL/kg with a single intravenous injection. Plasma samples were collected at 5 min, 8 hr, 24 hr, 48 hr, 72 hr and 96 hr post-dosing. The FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity as a function of time was plotted (FIG. 13). FVIII-155/VWF-034 exhibited the same improvement in half-life as FVIII-169/VWF-031, which has AE 288 XTEN inserted into the B domain junction of FVIII, as illustrated by the over lapping regression curves for the two molecules (FIG. 13). The demonstration that XTEN insertion into the VWF-Fc polypeptide confers half-life improvement of a magnitude similar to that conferred by XTEN insertion at the B domain junction of the FVIII polypeptide suggests that further half-life improvement may be possible in a heterodimeric molecule in which intra-molecular XTEN insertion in the FVIII polypeptide is combined with inter-domain XTEN insertion between the VWF and Fc elements of the VWF-Fc polypeptide.

Example 13A: Pharmacokinetic Properties of Additional FVIII-XTEN VWF Heterodimers

[0387] In addition to the FVIII-XTEN VWF heterodimers that were listed in Table 22, FVIII-XTEN VWF heterodimers containing different composition of XTEN insertions, single chain and dual chain version of FVIII (Table 23A) are either tested or will be tested in HemA for their pharmacokinetic properties. Various FVIII constructs (Table 23B) and VWF constructs (Table 23C) are also disclosed below. HemA mice will be treated with a single dose of intravenous administration of the heterodimer proteins at 200 IU/10 mL/kg. Plasma samples will then be collected at 5 min, 24, 48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma samples will be analyzed by FVIII chromogenic assay and half-life will be calculated using the WinNonlin-Phoenix program. The protein sequences of the listed heterodimers were listed in Table 25.

TABLE-US-00034 TABLE 23A Plausible FVIII-XTEN-Fc:VVVF-Fc heterodimer combinations for PK and activity improvement. pSYN pSYN VWF- pSYN VWF- pSYN VWF-015 031 034 ** VWF-036 pSYN FVIII -- t.sub.1/2 8.7 hr To be tested -- 010 DKO mice pSYN FVIII t.sub.1/2 6.3 hr t.sub.1/2 10.8 hr t.sub.1/2 18.6 hr t.sub.1/2 13.3 hr 155 DKO mice HemA mice HemA mice HemA mice pSYN FVIII -- t.sub.1/2 16.7 hr t.sub.1/2 31.1 hr -- 169 ** HemA mice HemA mice pSYN FVIII -- t.sub.1/2 15.2 hr t.sub.1/2 28.9 hr To be tested 173 ** DKO mice HemA mice pSYN FVIII -- t.sub.1/2 29.4 hr t.sub.1/2 32.4 hr t.sub.1/2 29.7 hr 205 HemA mice HemA mice HemA mice pSYN FVIII -- t.sub.1/2 24.5 hr t.sub.1/2 27.4 hr -- 266 HemA mice HemA mice pSYN FVII -- t.sub.1/2 23.0 hr t.sub.1/2 25.7 hr 267 HemA mice HemA mice pSYN FVIII -- To be tested To be tested To be tested 268 Dual chain To be tested To be tested To be tested isoform of pSYN FVIII 268 ** Length of XTEN can be changed to 72, 144, 288, 324, 333, 576, or 864.

TABLE-US-00035 TABLE 23B FVIII Constructs: pSYN FVIII dual chain FVIIIFc 010 pSYN FVIII Single chain FVIIIFc with 288 AE XTEN in B- 169 domain pSYN FVIII dual chain FVIIIFc with 288 AE XTEN in B- 173 domain pSYN FVIII dual chain FVIIIFc with two 144 XTENs at amino 195 acid 1656 and 1900 pSYN FVIII dual chain FVIIIFc with three 144 XTENs at amino 196 acid 26, 1656 and 1900 pSYN FVIII Single chain FVIIIFc with two 144 XTENs at amino 199 acid 1656 and 1900 pSYN FVIII Single chain FVIIIFc with three 144 XTENs at 201 amino acid 26, 1656 and 1900 pSYN FVIII Single chain FVIIIFc with 144 AE XTEN at amino 203 acid 1900 and 288 AE XTEN in B-domain pSYN FVIII Single chain FVIIIFc with 144 AE XTEN at amino 204 acid 403 and 288 AE XTEN in B-domain pSYN FVIII Single chain FVIIIFc with 144 AE XTEN at amino 205 acid 18 and 288 AE XTEN in B-domain pSYN FVIII Single chain FVIII (no Fc, no XTEN) 207 pSYN FVIII Single chain FVIIIFc with 42 AE XTEN at amino 266 acid 18 and 288 AE XTEN in B-domain pSYN FVIII Single chain FVIIIFc with 72 AE XTEN at amino 267 acid 18 and 288 AE XTEN in B-domain pSYN FVIII Single chain FVIIIFc with 144 AE XTEN at amino 268 acid 18 pSYN FVIII Single chain FVIIIFc with 72 AE XTEN at amino 269 acid 18 pSYN FVIII Single chain FVIIIFc with 42 AE XTEN at amino 271 acid 18 pSYN FVIII Single chain FVIII with 144 AE XTEN at amino 272 acid 18 and 288 AE XTEN in B-domain (no Fc)

TABLE-US-00036 TABLE 23C VWF Constructs: pSYN VWF-D1D2D'D3- 48aa long thrombin cleavable GS VWF031 linker-Fc with C1099A/C1142A pSYN VWF-D1D2D'D3- 288AE XTEN +35aa long thrombin VWF034 cleavable GS linker-Fc with C1099A/C1142A pSYN VWF-D1D2D'D3- 72aa long thrombin cleavable GSl VWF035 inker-Fc with C1099A/C1142A pSYN VWF-D1D2D'D3- 98aa long thrombin cleavable GS VWF036 linker-Fc with C1099A/C1142A pSYN VWF-D1D2D'D3 with 288 AE XTEN in D3 and 48aa VWF041 long thrombin cleavable GS linker after D3-Fc with C1099A/C1142A

Example 13B: Pharmacokinetic Properties of Additional FVIII-XTEN VWF Heterodimers

[0388] FVIII-XTEN VWF heterodimers were tested in HemA mice for their pharmacokinetic properties. The heterodimers tested are FVIII169NWF034, FVIII205NWF034, FVIII205NWF036 and FVII1266NWF031. HemA mice were administered with a single intravenous dose of various heterodimer proteins at 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 24, 48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay, and half-lives were calculated using the WinNonlin-Phoenix program. The PK results are shown below in Table 24.

TABLE-US-00037 TABLE 24 Additional FVIII-XTEN_VWF - PK in HemA Mice Fold of t.sub.1/2 AUC_D increase 5 min Cl Vss (hr*kg* vs recovery HL MRT (mL/hr/ (mL/ mIU/ scBDD- Treatment (%) (hr) (hr) kg) kg) mL/mIU) FVIII ScBDD- 7.16 10.16 4.83 44.44 0.23 -- FVIII FVIII169/ 76 31.1 34.57 1.73 59.77 0.58 4.3 VWF034 FVIII205/ 68 32.41 39.79 1.55 61.73 0.64 4.6 VWF034 FVIII205/ 74 29.71 36.35 1.61 58.43 0.62 4.1 VWF036 FVIII266/ 66 24.45 22.75 2.67 60.83 0.37 3.4 VWF031

TABLE-US-00038 pSYNFVIII 010 nucleotide sequence-(Dual chain FVIIIFc) (SEQ ID NO: 125) 1 ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG 51 CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG 101 ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT 151 CCTAGAGTGC CAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAA 201 GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC GCTAAGCCAA 251 GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT 301 GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT 351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG 401 ATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT 451 GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC 501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG 551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA 601 GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT 651 TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT 701 CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG 751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG 801 CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG 851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT 901 CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC 951 ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC 1001 ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG 1051 GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA 1101 TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT GATGACAACT 1151 CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT 1201 TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT 1251 AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG 1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC 1351 ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT 1401 CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT 1451 TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT 1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT 1551 GAAGGATTTT CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG 1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC 1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT 1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC 1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG 1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC 1851 AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC 1901 ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG 1951 CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT 2001 CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG 2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101 ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG 2151 GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA 2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG 2251 AGTAAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACCCACCAGT 2301 CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT CAGTCAGATC 2351 AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA 2401 GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA 2451 AAAGAAAACA CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT 2501 ATGGGATGAG TAGCTCCCCA CATGTTCTAA GAAACAGGGC TCAGAGTGGC 2551 AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC CAGGAATTTA CTGATGGCTC 2601 CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT TTGGGACTCC 2651 TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC 2701 AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA 2751 TGAGGAAGAT CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC 2801 CTAATGAAAC CAAAACTTAC TTTTGGAAAG TGCAACATCA TATGGCACCC 2851 ACTAAAGATG AGTTTGACTG CAAAGCCTGG GCTTATTTCT CTGATGTTGA 2901 CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT CTGGTCTGCC 2951 ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA 3001 TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC 3051 TGAAAATATG GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG 3101 ATCCCACTTT TAAAGAGAAT TATCGCTTCC ATGCAATCAA TGGCTACATA 3151 ATGGATACAC TACCTGGCTT AGTAATGGCT CAGGATCAAA GGATTCGATG 3201 GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT ATTCATTTCA 3251 GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG 3301 TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA 3351 AGCTGGAATT TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG 3401 GGATGAGCAC ACTTTTTCTG GTGTACAGCA ATAAGTGTCA GACTCCCCTG 3451 GGAATGGCTT CTGGACACAT TAGAGATTTT CAGATTACAG CTTCAGGACA 3501 ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT TCCGGATCAA 3551 TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG 3601 TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA 3651 GTTCTCCAGC CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG 3701 GGAAGAAGTG GCAGACTTAT CGAGGAAATT CCACTGGAAC CTTAATGGTC 3751 TTCTTTGGCA ATGTGGATTC ATCTGGGATA AAACACAATA TTTTTAACCC 3801 TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT TATAGCATTC 3851 GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC 3901 ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC 3951 TTCATCCTAC TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC 4001 GACTTCACCT CCAAGGGAGG AGTAATGCCT GGAGACCTCA GGTGAATAAT 4051 CCAAAAGAGT GGCTGCAAGT GGACTTCCAG AAGACAATGA AAGTCACAGG 4101 AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG TATGTGAAGG 4151 AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT 4201 CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC 4251 TGTGGTGAAC TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC 4301 ACCCCCAGAG TTGGGTGCAC CAGATTGCCC TGAGGATGGA GGTTCTGGGC 4351 TGCGAGGCAC AGGACCTCTA CGACAAAACT CACACATGCC CACCGTGCCC 4401 AGCTCCAGAA CTCCTGGGCG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC 4451 CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG 4501 GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA 4551 CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA 4601 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 4651 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC 4701 CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC 4751 AGGTGTACAC CCTGCCCCCA TCCCGGGATG AGCTGACCAA GAACCAGGTC 4801 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA 4851 GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG 4901 TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 4951 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA 5001 GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA 5051 AATGA pSYNFVIII 010 protein sequence-(Dual chain FVIIIFc) (SEQ ID NO: 126) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE 801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 851 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1001 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI

1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1101 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1651 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

Example 14: A New Class of Coagulation Factor VIII Molecules with Greater than Three-Fold Half-Life Extension in Hemophilia a Mice

[0389] The new class of FVIII molecules was designed to contain two polypeptides; one that consists of a single chain B-domain deleted (BDD) FVIII with XTEN inserted at one or more locations within the FVIII sequence, and one that is composed of the D'D3 region of VWF. Each polypeptide was also recombinantly fused to the Fc region of IgG1 to enable the D'D3 region to be correctly aligned to bind the FVIII moiety. The resulting FVIII variants were expressed in HEK 293 cells by transient transfection, and purified from the conditioned media. FVIII activity was evaluated by FVIII chromogenic assay and the pharmacokinetic properties were assessed in both FVIII knockout (HemA) and FVIII/VWF double knock-out (DKO) mice.

[0390] Incorporating XTEN and D'D3 region of VWF into rFVIII led to the uncoupling of the clearance of the fusion proteins from endogenous VWF while extending their circulating half-life. FVIII in this fusion configuration is completely shielded from interacting with VWF, as measured by biolayer interferometry (Octet) analysis. Consistent with this, their pharmacokinetic profiles in HemA and DKO mice were found to be identical, indicating that their clearance rate in mice was effectively disconnected from VWF. Optimization of XTEN length and the locations for inserting XTEN identified a subset of the proteins that have exceeded the VWF limitation (16 hours), reaching a circulating half-life of up to 30 hours in HemA mice representing a 4-fold improvement over BDD-FVIII. Importantly, these proteins maintained their functionality, as judged by FVIII chromogenic assay.

[0391] The VWF dependency has set a fundamental limitation for half-life of therapeutic FVIII. Uncoupling FVIII from VWF clearance pathways while extending half-life by the fusion of D'D3 region of VWF and XTEN has generated a novel FVIII molecule with a 4-fold half-life extension. This is the first report of an engineered FVIII that has exceeded the half-life limitation observed through industry-wide efforts in development of long-lasting FVIII, representing a potentially significant advancement in prophylactic treatment of hemophilia A.

TABLE-US-00039 TABLE 25 Protein sequences of FVIII-XTEN-Fc and VWF-Fc constructs FVIII 195 protein sequence (dual chain FVIIIFc with two 144 AE XTENs at amino acid 1656 and 1900) (SEQ ID NO: 105) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLIR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG TASSSPGASP 801 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 851 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA 901 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE 951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1151 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1201 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1251 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1301 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1351 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1401 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1451 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1501 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1551 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1601 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1651 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1701 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1751 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1801 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* FVIII 196 protein sequence (dual chain FVIIIFc with three 144 AE XTENs at amino add 26, 1656 and 1900) (SEQ ID NO: 106) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVGAPGS 51 SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGASPGT SSTGSPGSST 101 PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 151 TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG SPASSDARFP 201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 901 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG TASSSPGASP 951 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 1001 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA 1051 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE 1101 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1151 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1201 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1251 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1301 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1351 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1401 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1451 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1501 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1551 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1601 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1651 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1701 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1751 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1801 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1851 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1901 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1951 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 2001 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 2051 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 2101 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* FVIII 199 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 1656 and 1900) (SEO ID NO: 107) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG TASSSPGASP 801 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 851 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA 901 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE 951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1151 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1201 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1251 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1301 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1351 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1401 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1451 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1501 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1551 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1601 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1651 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1701 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1751 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1801 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* FVIII 201 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 26, 1656 & 1900) (SEQ ID NO: 108) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVGAPGS 51 SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGASPGT SSTGSPGSST 101 PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 151 TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG SPASSDARFP 201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 901 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG TASSSPGASP 951 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 1001 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA 1051 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE 1101 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1151 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1201 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1251 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1301 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1351 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1401 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1451 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1501 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1551 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1601 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1651 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1701 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1751 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1801 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1851 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1901 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1951 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 2001 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 2051 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 2101 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* FVIII 203 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 1900) (SEQ ID NO: 109) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE 801 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 851 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 901 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 951 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1001 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1051 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1101 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1151 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1201 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1251 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1301 IFDETKSWYF TENMERNCRG APTSESATPE SGPGSEPATS GSETPGTSES 1351 ATPESGPGSE PATSGSETPG TSESATPESG PGTSTEPSEG SAPGTSESAT 1401 PESGPGSPAG SPTSTEEGSP AGSPTSTEEG SPAGSPTSTE EGTSESATPE 1451 SGPGTSTEPS EGSAPGASSA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK* FVIII 204 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 403) (SEQ ID NO: 110) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDGAPTSTEP SEGSAPGSPA GSPTSTEEGT 451 STEPSEGSAP GTSTEPSEGS APGTSESATP ESGPGTSTEP SEGSAPGTSE 501 SATPESGPGS EPATSGSETP GTSTEPSEGS APGTSTEPSE GSAPGTSESA 551 TPESGPGTSE SATPESGPGA SSDRSYKSQY LNNGPQRIGR KYKKVRFMAY 601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE 951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK* FVIII 205 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 18) (SEQ ID NO: 111) 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG 51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG 101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS 151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP 201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE 951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK* pSYN FVIII 266 protein sequence (FVIII Fc with 42 AE-XTEN at amino acid 18 and 288 AE XTEN in B-domain) SEQ ID NO: 112 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST 51 EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR FPPRVPKSFP 101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK 151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ 201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT 251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN 301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS 351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN 401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE 451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR 501 EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR 551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM 601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE 651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI 701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI 751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP 801 RSFSQNGAPG TSESATPESG PGSEPATSGS ETPGTSESAT PESGPGSEPA 851 TSGSETPGTS ESATPESGPG TSTEPSEGSA PGSPAGSPTS TEEGTSESAT 901 PESGPGSEPA TSGSETPGTS ESATPESGPG SPAGSPTSTE EGSPAGSPTS 951 TEEGTSTEPS EGSAPGTSES ATPESGPGTS ESATPESGPG TSESATPESG 1001 PGSEPATSGS ETPGSEPATS GSETPGSPAG SPTSTEEGTS TEPSEGSAPG 1051 TSTEPSEGSA PGSEPATSGS ETPGTSESAT PESGPGTSTE PSEGSAPASS 1101 PPVLKRHQAE ITRTTLQSDQ EEIDYDDTIS VEMKKEDFDI YDEDENQSPR 1151 SFQKKTRHYF IAAVERLWDY GMSSSPHVLR NRAQSGSVPQ FKKVVFQEFT 1201 DGSFTQPLYR GELNEHLGLL GPYIRAEVED NIMVTFRNQA SRPYSFYSSL 1251 ISYEEDQRQG AEPRKNFVKP NETKTYFWKV QHHMAPTKDE FDCKAWAYFS 1301 DVDLEKDVHS GLIGPLLVCH TNTLNPAHGR QVTVQEFALF FTIFDETKSW 1351 YFTENMERNC RAPCNIQMED PTFKENYRFH AINGYIMDTL PGLVMAQDQR 1401 IRWYLLSMGS NENIHSIHFS GHVFTVRKKE EYKMALYNLY PGVFETVEML 1451 PSKAGIWRVE CLIGEHLHAG MSTLFLVYSN KCQTPLGMAS GHIRDFQITA 1501 SGQYGQWAPK LARLHYSGSI NAWSTKEPFS WIKVDLLAPM IIHGIKTQGA 1551 RQKFSSLYIS QFIIMYSLDG KKWQTYRGNS TGTLMVFFGN VDSSGIKHNI 1601 FNPPIIARYI RLHPTHYSIR STLRMELMGC DLNSCSMPLG MESKAISDAQ 1651 ITASSYFTNM FATWSPSKAR LHLQGRSNAW RPQVNNPKEW LQVDFQKTMK 1701 VTGVTTQGVK SLLTSMYVKE FLISSSQDGH QWTLFFQNGK VKVFQGNQDS 1751 FTPVVNSLDP PLLTRYLRIH PQSWVHQIAL RMEVLGCEAQ DLYDKTHTCP 1801 PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW 1851 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA 1901 LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI 1951 AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV 2001 MHEALHNHYT QKSLSLSPGK * pSYN FVIII 267 protein sequence (FVIII Fc with 72 AE-XTEN at amino acid 18 and 288 AE XTEN in B-domain) SEQ ID NO: 113 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG 51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG 101 TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS VVYKKTLFVE 151 FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS HPVSLHAVGV 201 SYWKASEGAE YDDQTSQREK EDDKVFPGGS HTYVWQVLKE NGPMASDPLC 251 LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH KFILLFAVFD 301 EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP GLIGCHRKSV 351 YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF LTAQTLLMDL 401 GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA EDYDDDLTDS 451 EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW DYAPLVLAPD 501 DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ HESGILGPLL 551 YGEVGDTLLI IFKNQASRPY NIYPHGITDV RPLYSRRLPK GVKHLKDFPI 601 LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL ASGLIGPLLI 651 CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR FLPNPAGVQL 701 EDPEFQASNI MHSINGYVFD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF 751 SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRGMT 801 ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS QNGAPGTSES 851 ATPESGPGSE PATSGSETPG TSESATPESG PGSEPATSGS ETPGTSESAT 901 PESGPGTSTE PSEGSAPGSP AGSPTSTEEG TSESATPESG PGSEPATSGS 951 ETPGTSESAT PESGPGSPAG SPTSTEEGSP AGSPTSTEEG TSTEPSEGSA 1001 PGTSESATPE SGPGTSESAT PESGPGTSES ATPESGPGSE PATSGSETPG 1051 SEPATSGSET PGSPAGSPTS TEEGTSTEPS EGSAPGTSTE PSEGSAPGSE 1101 PATSGSETPG TSESATPESG PGTSTEPSEG SAPASSPPVL KRHQAEITRT 1151 TLQSDQEEID YDDTISVEMK KEDFDIYDED ENQSPRSFQK KTRHYFIAAV 1201 ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV VFQEFTDGSF TQPLYRGELN 1251 EHLGLLGPYI RAEVEDNIMV TFRNQASRPY SFYSSLISYE EDQRQGAEPR 1301 KNFVKPNETK TYFWKVQHHM APTKDEFDCK AWAYFSDVDL EKDVHSGLIG 1351 PLLVCHTNTL NPAHGRQVTV QEFALFFTIF DETKSWYFTE NMERNCRAPC

1401 NIQMEDPTFK ENYRFHAING YIMDTLPGLV MAQDQRIRWY LLSMGSNENI 1451 HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF ETVEMLPSKA GIWRVECLIG 1501 EHLHAGMSTL FLVYSNKCQT PLGMASGHIR DFQITASGQY GQWAPKLARL 1551 HYSGSINAWS TKEPFSWIKV DLLAPMIIHG IKTQGARQKF SSLYISQFII 1601 MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS GIKHNIFNPP IIARYIRLHP 1651 THYSIRSTLR MELMGCDLNS CSMPLGMESK AISDAQITAS SYFTNMFATW 1701 SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD FQKTMKVTGV TTQGVKSLLT 1751 SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF QGNQDSFTPV VNSLDPPLLT 1801 RYLRIHPQSW VHQIALRMEV LGCEAQDLYD KTHTCPPCPA PELLGGPSVF 1851 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 1901 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 1951 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY 2001 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 2051 SLSPGK* pSYN FVIII 268 protein sequence (FVIII Fc with 144 AE-XTEN at amino acid 18) SEQ ID NO: 114 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG 51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG 101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS 151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP 201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 901 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QSDQEEIDYD DTISVEMKKE 951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1151 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI 1201 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1251 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1301 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1351 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1401 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1451 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1501 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1551 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1601 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1651 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1701 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1751 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1801 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* pSYN FVIII 269 protein sequence (FVIII Fc with 72 AE-XTEN at amino acid 18) SEQ ID NO: 115 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG 51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG 101 TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS VVYKKTLFVE 151 FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS HPVSLHAVGV 201 SYWKASEGAE YDDQTSQREK EDDKVFPGGS HTYVWQVLKE NGPMASDPLC 251 LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH KFILLFAVFD 301 EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP GLIGCHRKSV 351 YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF LTAQTLLMDL 401 GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA EDYDDDLTDS 451 EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW DYAPLVLAPD 501 DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ HESGILGPLL 551 YGEVGDTLLI IFKNQASRPY NIYPHGITDV RPLYSRRLPK GVKHLKDFPI 601 LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL ASGLIGPLLI 651 CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR FLPNPAGVQL 701 EDPEFQASNI MHSINGYVFD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF 751 SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRGMT 801 ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS QNPPVLKRHQ 851 AEITRTTLQS DQEEIDYDDT ISVEMKKEDF DIYDEDENQS PRSFQKKTRH 901 YFIAAVERLW DYGMSSSPHV LRNRAQSGSV PQFKKVVFQE FTDGSFTQPL 951 YRGELNEHLG LLGPYIRAEV EDNIMVTFRN QASRPYSFYS SLISYEEDQR 1001 QGAEPRKNFV KPNETKTYFW KVQHHMAPTK DEFDCKAWAY FSDVDLEKDV 1051 HSGLIGPLLV CHTNTLNPAH GRQVTVQEFA LFFTIFDETK SWYFTENMER 1101 NCRAPCNIQM EDPTFKENYR FHAINGYIMD TLPGLVMAQD QRIRWYLLSM 1151 GSNENIHSIH FSGHVFTVRK KEEYKMALYN LYPGVFETVE MLPSKAGIWR 1201 VECLIGEHLH AGMSTLFLVY SNKCQTPLGM ASGHIRDFQI TASGQYGQWA 1251 PKLARLHYSG SINAWSTKEP FSWIKVDLLA PMIIHGIKTQ GARQKFSSLY 1301 ISQFIIMYSL DGKKWQTYRG NSTGTLMVFF GNVDSSGIKH NIFNPPIIAR 1351 YIRLHPTHYS IRSTLRMELM GCDLNSCSMP LGMESKAISD AQITASSYFT 1401 NMFATWSPSK ARLHLQGRSN AWRPQVNNPK EWLQVDFQKT MKVTGVTTQG 1451 VKSLLTSMYV KEFLISSSQD GHQWTLFFQN GKVKVFQGNQ DSFTPVVNSL 1501 DPPLLTRYLR IHPQSWVHQI ALRMEVLGCE AQDLYDKTHT CPPCPAPELL 1551 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH 1601 NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 1651 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG 1701 QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 1751 YTQKSLSLSP GK* pSYNFVIII 271 protein sequence (FVIII Fc with 42 AE-XTEN at amino acid 18) SEQ ID NO: 116 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST 51 EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR FPPRVPKSFP 101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK 151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ 201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT 251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN 301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS 351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN 401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE 451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR 501 EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR 551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM 601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE 651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI 701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI 751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP 801 RSFSQNPPVL KRHQAEITRT TLQSDQEEID YDDTISVEMK KEDFDIYDED 851 ENQSPRSFQK KTRHYFIAAV ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV 901 VFQEFTDGSF TQPLYRGELN EHLGLLGPYI RAEVEDNIMV TFRNQASRPY 951 SFYSSLISYE EDQRQGAEPR KNFVKPNETK TYFWKVQHHM APTKDEFDCK 1001 AWAYFSDVDL EKDVHSGLIG PLLVCHTNTL NPAHGRQVTV QEFALFFTIF 1051 DETKSWYFTE NMERNCRAPC NIQMEDPTFK ENYRFHAING YIMDTLPGLV 1101 MAQDQRIRWY LLSMGSNENI HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF 1151 ETVEMLPSKA GIWRVECLIG EHLHAGMSTL FLVYSNKCQT PLGMASGHIR 1201 DFQITASGQY GQWAPKLARL HYSGSINAWS TKEPFSWIKV DLLAPMIIHG 1251 IKTQGARQKF SSLYISQFII MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS 1301 GIKHNIFNPP IIARYIRLHP THYSIRSTLR MELMGCDLNS CSMPLGMESK 1351 AISDAQITAS SYFTNMFATW SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD 1401 FQKTMKVTGV TTQGVKSLLT SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF 1451 QGNQDSFTPV VNSLDPPLLT RYLRIHPQSW VHQIALRMEV LGCEAQDLYD 1501 KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP 1551 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC 1601 KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG 1651 FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 1701 VFSCSVMHEA LHNHYTQKSL SLSPGK* pSYN FVIII protein sequence 272 ( FVIII with 144 AE XTEN at amino

acid 18 and 244 AE XTEN in B-domain-no Fc) SEQ ID NO: 117 1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG 51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG 101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS 151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP 201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE 951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901 Y* pSYN VWF 031 protein sequence (VWF D1D2D'D3-48 aa long thrombin cleavable GS linker-Fc) SEQ ID NO: 118 1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVILNPSDPE HCQICHCDVV NLTCEACQEP ISGGGGSGGG 1251 GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP 1301 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 1351 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 1401 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 1451 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 1501 HNHYTQKSLS LSPGK* pSYN VWF 034 protein sequence (VWF D1D2D'D3-288AE XTEN-35 aa long thrombin cleavable GS linker-Fc) SEQ ID NO: 119 1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGTSESATP 1251 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES 1301 GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP 1351 GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT 1401 SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP 1451 ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTSTEPSE GSAPGSEPAT 1501 SGSETPGTSE SATPESGPGT STEPSEGSAP DIGGGGGSGG GGSLVPRGSG 1551 GDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1601 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 1651 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 1701 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 1751 GNVFSCSVMH EALHNHYTQK SLSLSPGK* pSYN VWF 036 protein sequence (VWF D1D2D'D-98 aa long thrombin cleavable GS linker-Fc) SEQ ID NO: 120 1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGGGGSGGG 1251 GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG 1301 GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP 1351 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 1401 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 1451 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 1501 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 1551 HNHYTQKSLS LSPGK* pSYN Fc-015 protein sequence (IgG-Fc domain) SEQ ID NO: 121 1 METDTLLLWV LLLWVPGSTG DKTHTCPPCP APELLGGPSV FLFPPKPKDT 51 LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY 101 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 151 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 201 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

Example 15: FVIII-XTEN-Fc:VWF-Fc Heterodimers have Maintained Normal FVIII Specific Activity as Compared to Wild Type BDD-FVIII

[0392] The FVIII specific activity of FVIII-XTEN-Fc:VWF-Fc heterodimers were determined. Heterodimers were purified using a two-step chromatography process. A weak anion exchange resin was used, followed by affinity chromatography. The final purified product had acceptable purity by SEC-HPLC. The specific activity was compared to B-domain deleted FVIII (BDD-FVIII), as measured by FVIII chromogenic assay and A280 concentration. The data are presented in Table 26. All tested molecules had demonstrated comparable FVIII specific activities to BDD-FVIII. Purity and the presence of each moiety of the molecules were confirmed by SDS-PAGE and western blotting.

TABLE-US-00040 TABLE 26 FVIII specific activity of FVIII-XTEN-Fc:VWF-Fc heterodimers FVIII FVIII FVIII FVIII FVIII FVIII 207 FVIII-66 155/ 155/ 169/ 205/ 169/ Construct scBDDFVIII dcBDDFVIII) vWF31 vWF39 vWF31 vWF31 vWF34 Measured 1473 1592 1534 1796 1511 1345 1505 Specific Activity (IU/nmol)

[0393] The half-lives of rFVIII-XTEN/D'D3 and BDD-FVIII were compared in HemA Mice (FIG. 15; Table 27). As FIG. 15 shows, rFVIII-XTEN/D'D3 achieved a half-life that was four fold longer than the half-life achieved by BDD-FVIII.

TABLE-US-00041 TABLE 27 rFVIII-XTEN/D'D3 and BDD-FVIII in HemA mice 5 minutes Cl AUC_D Recovery HL MRT (mL/hr/ Vss (hr*kg* Treatment (%) (hr) (hr) kg) (mL/kg) mIU/mL/mIU) BDD-FVIII 89 7.6 11 4.5 49.2 0.22 rFVIIIFc 78 16 20 2.9 57.8 0.35 rFVIII- 86 30 36 1.8 63.4 0.57 XTEN/D'D3

Example 16: FVIII-XTEN-Fc:VWF-Fc Heterodimer's Potency (FVIII Activity) in Hemostasis as Measured by One Stage aPTT Assay

[0394] The potency of FVIII-XTEN-Fc:VWF-Fc heterodimers in hemostasis was evaluated by their FVIII specific aPTT activity as summarized in Table 28. As demonstrated by Table 28, while the addition of the VWF D'D3 fragment and the insertion of XTEN into the intra-domains of FVIII reduce the FVIII specific aPTT activity of the heterodimers (as indicated by the FVIII155NWF031 data and the FVIII205NWF031 data), XTEN insertions in the FVIII B domain region or C-terminus of the VWF D'D3 fragment have no negative effect on the FVIII specific aPTT activity (as indicated by the FVIII169NWF031 data and the FVIII169NWF034 data). Compared to dual-chain BDD-FVIII (dcBDD-FVIII), FVIII155NWF031, FVIII169NWF031, FVIII169NWF034 and VWF205NWF031 showed reduction of specific aPTT activity by 2.5-fold, 2.8-fold, 2.6-fold and 5.5-fold, respectively.

TABLE-US-00042 TABLE 28 FVIII specific aPTT activity of FVIII-XTEN-Fc:VWF-Fc heterodimers FVIII 207 FVIII-66 FVIII FVIII FVIII FVIII scBDD- dcBDD- 155/ 169/ 205/ 169/ Construct FVIII FVIII VWF31 VWF31 VWF31 VWF34 Measured 818 .+-. 1188 .+-. 448 .+-. 416 .+-. 214 .+-. 436 .+-. Specific 153 213 111 70 38 189 aPTT Activity (IU/nmol)

FVIII Specific aPTT Assay

[0395] FVIII variants were diluted with aPTT buffer (0.15 M NaCl, 0.05 M Tris-HCl, 1% BSA, pH 7.4) to the linear assay range (200-1.6 mU/mL). 50 .mu.L of diluted samples or standards were sequentially mixed with 50 .mu.L of 37.degree. C. naive human HemA pooled plasma, 50 .mu.L of 37.degree. C. aPTT reagent (ACTIN.RTM. FSL activated cephaloplastin reagent--Dade Behring, reference #B4219-2) and incubated at 37.degree. C. for 4 minutes. 50 .mu.l of 20 mM CaCl.sub.2) (Dade Behring [reference #ORFO37]) was then added to the reaction mixture to start the clotting reactions. Using the clotting time of each sample (the length of time from the addition of CaCl.sub.2) until the onset of clot formation), the aPTT activity was calculated against the standard that was generated with the 8.sup.th international standard FVIII concentrate. Specific aPTT activity was calculated against the protein concentration of each molecule that measured by OD280.

Example 17: In Vivo Efficacy of FVIII-XTEN-Fc:VWF-Fc Heterodimer in HemA Mice Tail Clip Bleeding Model

[0396] To further access the hemostasis potency of the heterodimers, the acute efficacy of FVIII169NWF034 and FVIII205NWF031 was evaluated in comparison with BDD-FVIII in the HemA mice Tail clip bleeding model. HemA mice were treated with a single IV injection of BDD-FVIII at 200, 65 and 20 IU/kg to generate the post tail clip injury blood loss control level. Blood loss from mice treated with 200 IU/kg of FVIII169NWF034 or FVIII205NWF031 was compared to that of the BDD-FVIII treated control group mice to estimate their potency on hemostasis. Vehicle treated animals were used to generate blood loss baseline for the model. As shown in FIG. 16, significant reduction in blood loss was observed from all FVIII treatment groups compared to that of the vehicle treated animals (p<0.05). Both FVIII169NWF034 and FVIII205NWF031 are efficacious in the HemA mice Tail Clip model. Compared to BDD-FVIII, about 3 fold lower potency was observed for FVIII169NWF034, as demonstrated by the similar blood loss reduction achieved by 65 IU/kg BDD-FVIII and 200 IU/kg FVIII169NWF034. As for FVIII205NWF034, a 10 fold potency reduction has been observed, as demonstrated by the similar blood loss reduction achieved by 20 IU/kg BDD-FVIII and 200 IU/kg FVIII205NWF031.

[0397] Even though FVII169NWF034 and FVIII205NWF031 had similar specific FVIII chromogenic activity compared to rBDD-FVIII, their FVIII aPTT activity and in vivo potency were both reduced due to the modifications of the molecules. Those data indicate that the aPTT activity of a FVIII molecule is a more accurate measurement on predicating its in vivo potency on hemostasis than the FVIII chromogenic activity.

HemA Mice Tail Clip Bleeding Model

[0398] 8-10 weeks old male HemA mice were used for the study. Prior to tail clip injury, mice were anesthetized with a 50 mg/kg Ketamine/0.5 mg/kg Dexmedetomidine cocktail and placed on a 37.degree. C. heating pad to help maintain the body temperature. The tails of the mice were then be immersed in 37.degree. C. water for 10 minutes to dilate the lateral vein. After vein dilation, rFVIII or vehicle solution were injected via the tail vein and 5 min later, the distal 1 cm of the tail was cut off using a #11 scalpel with straight edge. The shed blood was collected into 13 ml of 37.degree. C. warm saline for 30 minutes and the mice were then euthanized while still under anesthesia by bilateral thoracotomy. Blood loss was quantified gravimetrically by weight change of the blood collection tubes before and after blood was collected in gram, which translated into milliliter (mL) of blood loss volume (1 g weight change=1 mL blood loss).

[0399] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0400] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0401] All patents and publications cited herein are incorporated by reference herein in their entirety.

Sequence CWU 1

1

17118442DNAHomo sapiens 1atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa gagctttgac 1200aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa aggtgacctc 1440cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620ctggcrgagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgcctgcttc 3300tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct gtatgtggag 3780gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga cctggtcttc 3840ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa ggcctttgtg 3900gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc cgtggtggag 3960taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc gtcagagctg 4020cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac cagcgaggtc 4080ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc ctcccgcatc 4140gccctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt tgtccgctac 4200gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg gccccatgcc 4260aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc cttcgtgctg 4320agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct ctgtgacctt 4380gcccctgaag cccctcctcc tactctgccc cccgacatgg cacaagtcac tgtgggcccg 4440gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct ggatgtggcg 4500ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag caaggagttc 4560atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt cacggtgctg 4620cagtactcct acatggtgac cgtggagtac cccttcagcg aggcacagtc caaaggggac 4680atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa cactgggctg 4740gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg ggagcaggcg 4800cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa gaggctgcct 4860ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca ggagctggag 4920aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct cccccgagag 4980gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat ccccaccctc 5040tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga tggctcctcc 5100agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt catttcaaaa 5160gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag catcaccacc 5220attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct tgtggacgtc 5280atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc tgtgcgatac 5340ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt catcctggtc 5400acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc caacagagtg 5460acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg gatcttggca 5520ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct ccctaccatg 5580gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag gatttgcatg 5640gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga ccagtgccac 5700accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt caactgtgac 5760cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga agagacctgt 5820ggctgccgct ggacctgccc ctgygtgtgc acaggcagct ccactcggca catcgtgacc 5880tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt tcaaaacaag 5940gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc aaggcagggc 6000tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagstgca cagtgacatg 6060gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa catggaagtc 6120aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca catcttcaca 6180ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt tgcttcaaag 6240acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat gctgagggat 6300ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca gcggccaggg 6360cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc ccactgccag 6420gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc cacattctat 6480gccatctgcc agcaggacag ttgccaccag gagcaagtgt gtgaggtgat cgcctcttat 6540gcccacctct gtcggaccaa cggggtctgc gttgactgga ggacacctga tttctgtgct 6600atgtcatgcc caccatctct ggtctacaac cactgtgagc atggctgtcc ccggcactgt 6660gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg ccctccagat 6720aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg cattggtgag 6780gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc ctgtcagatc 6840tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc cacggccaaa 6900gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga ccagtgctgc 6960cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt gcctcactgt 7020gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa cttcacctgc 7080gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc gcaccgtttg 7140cccacccttc ggaagaccca gtgctgtgat gagtatgagt gtgcctgcaa ctgtgtcaac 7200tccacagtga gctgtcccct tgggtacttg gcctcaaccg ccaccaatga ctgtggctgt 7260accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat ctaccctgtg 7320ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga ggatgccgtg 7380atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg tcggtcgggc 7440ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc tgcctgtgag 7500gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt cggctcccag 7560tgggcctccc cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa ggaggaggtc 7620tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg cccctcgggc 7680tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga gcgcatggag 7740gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat cgatgtgtgc 7800acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct ggagtgcagg 7860aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac aggtgaatgt 7920tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca gatcatgaca 7980ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa ggtcaatgag 8040agaggagagt acttctggga gaagagggtc acaggctgcc caccctttga tgaacacaag 8100tgtcttgctg agggaggtaa aattatgaaa attccaggca cctgctgtga cacatgtgag 8160gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaaggtggg aagctgtaag 8220tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa agccatgtac 8280tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac acggacggag 8340cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga ggttctcaat 8400gccatggagt gcaaatgctc ccccaggaag tgcagcaagt ga 844222813PRTHomo sapiensmisc_feature(1)..(22)VWF Signal Peptidemisc_feature(23)..(763)VWF D1D2 regionmisc_feature(764)..(866)VWF D'Domainmisc_feature(867)..(1240)VWF D3 Domainmisc_feature(1241)..(1479)VWF A1 Domainmisc_feature(2016)..(2016)Xaa can be any naturally occurring amino acid 2Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile1 5 10 15Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys65 70 75 80Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly145 150 155 160Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu225 230 235 240Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met305 310 315 320Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp385 390 395 400Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu465 470 475 480Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln545 550 555 560Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val625 630 635 640Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp705 710 715 720Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met785 790 795 800Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly865 870 875 880Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg945 950 955 960Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025

1030 1035Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060 1065Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln 1100 1105 1110His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln 1115 1120 1125Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu 1130 1135 1140Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180 1185Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly 1190 1195 1200Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr 1235 1240 1245Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser 1250 1255 1260Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu 1265 1270 1275Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe 1280 1285 1290Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg 1295 1300 1305Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 1310 1315 1320Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser 1325 1330 1335Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln 1340 1345 1350Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile 1355 1360 1365Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu 1370 1375 1380Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val 1385 1390 1395Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro 1400 1405 1410Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile 1415 1420 1425Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val 1430 1435 1440Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys 1445 1450 1455Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met 1460 1465 1470Ala Gln Val Thr Val Gly Pro Gly Leu Leu Gly Val Ser Thr Leu 1475 1480 1485Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val Ala Phe Val Leu 1490 1495 1500Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn Arg Ser Lys 1505 1510 1515Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly Gln Asp 1520 1525 1530Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr Val 1535 1540 1545Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln 1550 1555 1560Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr 1565 1570 1575Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser 1580 1585 1590Gln Gly Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr 1595 1600 1605Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile 1610 1615 1620Gln Val Val Pro Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu 1625 1630 1635Leu Glu Arg Ile Gly Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp 1640 1645 1650Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Arg 1655 1660 1665Cys Cys Ser Gly Glu Gly Leu Gln Ile Pro Thr Leu Ser Pro Ala 1670 1675 1680Pro Asp Cys Ser Gln Pro Leu Asp Val Ile Leu Leu Leu Asp Gly 1685 1690 1695Ser Ser Ser Phe Pro Ala Ser Tyr Phe Asp Glu Met Lys Ser Phe 1700 1705 1710Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile Gly Pro Arg Leu Thr 1715 1720 1725Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr Thr Ile Asp Val 1730 1735 1740Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu Ser Leu Val 1745 1750 1755Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly Asp Ala 1760 1765 1770Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His Gly Ala 1775 1780 1785Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr Asp Val 1790 1795 1800Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg Ser Asn 1805 1810 1815Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr Asp Ala 1820 1825 1830Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser Asn Val 1835 1840 1845Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val Thr Leu 1850 1855 1860Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val Arg Ile 1865 1870 1875Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp Val Trp 1880 1885 1890Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro Asp Gly 1895 1900 1905Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg Gly Leu 1910 1915 1920Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val Glu Glu 1925 1930 1935Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr Gly Ser 1940 1945 1950Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe Lys Leu 1955 1960 1965Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu Gln Asp 1970 1975 1980Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly Ala Arg 1985 1990 1995Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala Leu Ser 2000 2005 2010Val Glu Xaa His Ser Asp Met Glu Val Thr Val Asn Gly Arg Leu 2015 2020 2025Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn Val Tyr 2030 2035 2040Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly His Ile 2045 2050 2055Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln Leu Ser 2060 2065 2070Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly Ile Cys 2075 2080 2085Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly Thr Val 2090 2095 2100Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val Gln Arg 2105 2110 2115Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys Leu Val 2120 2125 2130Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu Phe Ala 2135 2140 2145Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala Ile Cys 2150 2155 2160Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val Ile Ala 2165 2170 2175Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val Cys Val Asp Trp 2180 2185 2190Arg Thr Pro Asp Phe Cys Ala Met Ser Cys Pro Pro Ser Leu Val 2195 2200 2205Tyr Asn His Cys Glu His Gly Cys Pro Arg His Cys Asp Gly Asn 2210 2215 2220Val Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe Cys Pro 2225 2230 2235Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu Glu Ala 2240 2245 2250Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln Phe Leu 2255 2260 2265Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys Thr Cys 2270 2275 2280Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys Pro Thr 2285 2290 2295Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg Leu Arg 2300 2305 2310Gln Asn Ala Asp Gln Cys Cys Pro Glu Tyr Glu Cys Val Cys Asp 2315 2320 2325Pro Val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu Arg Gly 2330 2335 2340Leu Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro Asn Phe 2345 2350 2355Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser Pro Pro 2360 2365 2370Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr Gln Cys 2375 2380 2385Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser Thr Val 2390 2395 2400Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn Asp Cys 2405 2410 2415Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val Cys Val His 2420 2425 2430Arg Ser Thr Ile Tyr Pro Val Gly Gln Phe Trp Glu Glu Gly Cys 2435 2440 2445Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala Val Met Gly Leu 2450 2455 2460Arg Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser Cys Arg 2465 2470 2475Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys Gly Arg 2480 2485 2490Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro Arg Gly 2495 2500 2505Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp Ala Ser 2510 2515 2520Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val Lys Glu 2525 2530 2535Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro Gln Leu Glu 2540 2545 2550Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys Lys Thr Ser 2555 2560 2565Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala Cys Met 2570 2575 2580Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met Ile Asp 2585 2590 2595Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val Ile Ser 2600 2605 2610Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro Cys Pro 2615 2620 2625Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys Gly Arg 2630 2635 2640Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly Gln Ile 2645 2650 2655Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly Cys Asp Thr 2660 2665 2670His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys 2675 2680 2685Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys Leu Ala 2690 2695 2700Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys Asp Thr 2705 2710 2715Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu Gln Tyr 2720 2725 2730Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp Ile His 2735 2740 2745Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser Ile Asp 2750 2755 2760Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro Thr Arg 2765 2770 2775Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly Ser Val 2780 2785 2790Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys Ser Pro 2795 2800 2805Arg Lys Cys Ser Lys 2810319PRTHomo sapiens 3Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser42332PRTHomo sapiens 4Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe

Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490 1495 1500Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735 1740Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1835 1840 1845Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1850 1855 1860Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915 1920Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925 1930 1935Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1970 1975 1980Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035 2040Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 2045 2050 2055Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 2075 2080 2085Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090 2095 2100Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 2170 2175Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2180 2185 2190Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 2210 2215 2220Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290 2295Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315 2320 2325Gln Asp Leu Tyr 233057053DNAHomo sapiens 5atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280ttctcccaga attcaagaca ccctagcact aggcaaaagc aatttaatgc caccacaatt 2340ccagaaaatg acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa 2400atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc tactccacat 2460gggctatcct tatctgatct ccaagaagcc aaatatgaga ctttttctga tgatccatca 2520cctggagcaa tagacagtaa taacagcctg tctgaaatga cacacttcag gccacagctc 2580catcacagtg gggacatggt atttacccct gagtcaggcc tccaattaag attaaatgag 2640aaactgggga caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca 2700tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac tgataataca 2760agttccttag gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta 2820tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt gagtgaagaa 2880aataatgatt caaagttgtt agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940aaaaatgtat cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt aaagacaaac 3060aaaacttcca ataattcagc aactaataga aagactcaca ttgatggccc atcattatta 3120attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180gtgacacctt tgattcatga cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240aatcatatgt caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360ttgccagaat cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg 3420caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540ggactcaaag agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat 3600ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660gaaacattaa tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag 3720aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg ttcatatgac 3780ggggcatatg ctccagtact tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840aagaaacaca cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc tcctaataca 3960agccagcaga attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca 4020ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac ccagtggtcc 4080aaaaacatga aacatttgac cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140aaaggggcca ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct 4200caagcaaata gatctccatt acccattgca aaggtatcat catttccatc tattagacct 4260atatatctga ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat 4320agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380aacctttctt tagccattct aaccttggag atgactggtg atcaaagaga ggttggctcc 4440ctggggacaa gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560cagaaggacc tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620gaagggagcc ttcttcaggg aacagaggga gcgattaagt

ggaatgaagc aaacagacct 4680ggaaaagttc cctttctgag agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740ttggatcctc ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa 4800tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860aacgcttgtg aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980gtcttgaaac gccatcaacg ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040attgactatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 5100gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 5220gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 5280tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 5400ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 5460cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 5520catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 5640acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 5700atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 5820aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 5880tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 5940gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 6000gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 6120cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 6180caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 6240tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 6300cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 6420accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 6480cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 6540cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 6600agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 6660acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 6960ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 7020gaggttctgg gctgcgaggc acaggacctc tac 705361438PRTArtificial SequenceBDDD FVIII 6Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His 740 745 750Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile 755 760 765Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp 770 775 780Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys785 790 795 800Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly 805 810 815Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser 820 825 830Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser 835 840 845Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 850 855 860Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr865 870 875 880Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile 885 890 895Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe 900 905 910Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His 915 920 925Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 930 935 940Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro945 950 955 960Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln 965 970 975Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr 980 985 990Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro 995 1000 1005Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg 1010 1015 1020Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu 1025 1030 1035Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met 1040 1045 1050Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val 1055 1060 1065Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn 1070 1075 1080Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys 1085 1090 1095Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His 1100 1105 1110Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln 1115 1120 1125Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile 1130 1135 1140Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg 1145 1150 1155Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro 1160 1165 1170Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His 1175 1180 1185Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr 1190 1195 1200Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 1205 1210 1215Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe 1220 1225 1230Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro 1235 1240 1245Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser 1250 1255 1260Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn 1265 1270 1275Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp 1280 1285 1290Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr 1295 1300 1305Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn 1310 1315 1320Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val 1325 1330 1335Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly 1340 1345 1350Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile 1355 1360 1365Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn 1370 1375 1380Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro 1385 1390 1395Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg 1400 1405 1410Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu 1415 1420 1425Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1430 143574371DNAArtificial SequenceBDDD FVIII 7atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280ttctctcaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata

3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta c 4371825PRTArtificial Sequencecleavage site 8Thr Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pro Asn Asp Lys Tyr1 5 10 15Glu Pro Phe Trp Glu Asp Glu Glu Lys 20 2594PRTArtificial Sequencecleavage site 9Arg Arg Arg Arg1106PRTArtificial Sequencecleavage site 10Arg Lys Arg Arg Lys Arg1 5115PRTArtificial Sequencecleavage site 11Arg Arg Arg Arg Ser1 5129PRTArtificial Sequencecleavage site 12Thr Gln Ser Phe Asn Asp Phe Thr Arg1 51310PRTArtificial Sequencecleavage site 13Ser Val Ser Gln Thr Ser Lys Leu Thr Arg1 5 101410PRTArtificial Sequencecleavage site 14Asp Phe Leu Ala Glu Gly Gly Gly Val Arg1 5 10157PRTArtificial Sequencecleavage site 15Thr Thr Lys Ile Lys Pro Arg1 5165PRTArtificial Sequencecleavage site 16Leu Val Pro Arg Gly1 5175PRTArtificial Sequencecleavage site 17Ala Leu Arg Pro Arg1 5187PRTArtificial Sequencecleavage site 18Lys Leu Thr Arg Ala Glu Thr1 5197PRTArtificial Sequencecleavage site 19Asp Phe Thr Arg Val Val Gly1 5208PRTArtificial Sequencecleavage site 20Thr Met Thr Arg Ile Val Gly Gly1 5218PRTArtificial Sequencecleavage site 21Ser Pro Phe Arg Ser Thr Gly Gly1 5228PRTArtificial Sequencecleavage site 22Leu Gln Val Arg Ile Val Gly Gly1 5238PRTArtificial Sequencecleavage site 23Pro Leu Gly Arg Ile Val Gly Gly1 5248PRTArtificial Sequencecleavage site 24Ile Glu Gly Arg Thr Val Gly Gly1 5258PRTArtificial Sequencecleavage site 25Leu Thr Pro Arg Ser Leu Leu Val1 5268PRTArtificial Sequencecleavage site 26Leu Gly Pro Val Ser Gly Val Pro1 5278PRTArtificial Sequencecleavage site 27Val Ala Gly Asp Ser Leu Glu Glu1 5288PRTArtificial Sequencecleavage site 28Gly Pro Ala Gly Leu Gly Gly Ala1 5298PRTArtificial Sequencecleavage site 29Gly Pro Ala Gly Leu Arg Gly Ala1 5308PRTArtificial Sequencecleavage site 30Ala Pro Leu Gly Leu Arg Leu Arg1 5318PRTArtificial Sequencecleavage site 31Pro Ala Leu Pro Leu Val Ala Gln1 5327PRTArtificial Sequencecleavage site 32Glu Asn Leu Tyr Phe Gln Gly1 5338PRTArtificial Sequencecleavage site 33Asp Asp Asp Lys Ile Val Gly Gly1 5348PRTArtificial Sequencecleavage site 34Leu Glu Val Leu Phe Gln Gly Pro1 5358PRTArtificial Sequencecleavage site 35Leu Pro Lys Thr Gly Ser Glu Ser1 53642PRTArtificial SequenceAE42 36Gly Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly1 5 10 15Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 20 25 30Thr Ser Gly Ser Glu Thr Pro Ala Ser Ser 35 4037143PRTArtificial SequenceAE144 37Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu1 5 10 15Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly 20 25 30Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 35 40 45Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 50 55 60Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly65 70 75 80Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 85 90 95Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu 100 105 110Ser Ala Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 115 120 125Glu Thr Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 130 135 14038144PRTArtificial SequenceAG144 38Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr1 5 10 15Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr 20 25 30Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 35 40 45Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro 50 55 60Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala65 70 75 80Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 85 90 95Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro 100 105 110Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala 115 120 125Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro 130 135 14039288PRTArtificial SequenceAE288 39Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro1 5 10 15Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 20 25 30Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 35 40 45Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 50 55 60Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr65 70 75 80Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 85 90 95Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 100 105 110Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr 115 120 125Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 130 135 140Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu145 150 155 160Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro 165 170 175Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 180 185 190Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro 195 200 205Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr 210 215 220Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro225 230 235 240Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 245 250 255Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 260 265 270Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 275 280 28540288PRTArtificial SequenceAG288 40Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser1 5 10 15Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr 20 25 30Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser 35 40 45Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser 50 55 60Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr65 70 75 80Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser 85 90 95Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser 100 105 110Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser 115 120 125Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 130 135 140Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser145 150 155 160Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser 165 170 175Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly 180 185 190Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser 195 200 205Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly 210 215 220Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly225 230 235 240Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser 245 250 255Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr 260 265 270Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser 275 280 28541576PRTArtificial SequenceAE576 41Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu1 5 10 15Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu 20 25 30Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 35 40 45Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 50 55 60Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro65 70 75 80Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 85 90 95Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala 100 105 110Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro 115 120 125Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 130 135 140Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala145 150 155 160Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu 165 170 175Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 180 185 190Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 195 200 205Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 210 215 220Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro225 230 235 240Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 245 250 255Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 260 265 270Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 275 280 285Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu 290 295 300Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly305 310 315 320Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 325 330 335Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 340 345 350Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu 355 360 365Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 370 375 380Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr385 390 395 400Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 405 410 415Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 420 425 430Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 435 440 445Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 450 455 460Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro465 470 475 480Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 485 490 495Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 500 505 510Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 515 520 525Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 530 535 540Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro545 550 555 560Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 565 570 57542576PRTArtificial SequenceAG576 42Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser1 5 10 15Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser 20 25 30Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly 35 40 45Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser 50 55 60Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser65 70 75 80Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 85 90 95Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro 100 105 110Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser 115 120 125Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 130 135 140Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser145 150 155 160Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr 165 170 175Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 180 185 190Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser 195 200 205Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly 210 215 220Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser225 230 235 240Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro 245 250 255Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly 260 265 270Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser 275 280 285Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser 290 295 300Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser305 310 315 320Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 325 330 335Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser 340 345 350Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser 355 360 365Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 370 375 380Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro385 390 395 400Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly 405 410 415Ala Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser 420 425 430Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro 435 440 445Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly 450 455 460Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser465 470 475 480Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser 485 490 495Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser 500

505 510Ser Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser 515 520 525Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser 530 535 540Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser545 550 555 560Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser 565 570 57543864PRTArtificial SequenceAE864 43Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu1 5 10 15Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu 20 25 30Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 35 40 45Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 50 55 60Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro65 70 75 80Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 85 90 95Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala 100 105 110Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro 115 120 125Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 130 135 140Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala145 150 155 160Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu 165 170 175Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 180 185 190Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 195 200 205Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 210 215 220Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro225 230 235 240Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 245 250 255Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 260 265 270Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 275 280 285Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu 290 295 300Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly305 310 315 320Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 325 330 335Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 340 345 350Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu 355 360 365Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 370 375 380Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr385 390 395 400Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 405 410 415Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 420 425 430Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 435 440 445Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 450 455 460Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro465 470 475 480Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 485 490 495Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 500 505 510Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 515 520 525Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 530 535 540Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro545 550 555 560Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 565 570 575Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 580 585 590Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 595 600 605Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 610 615 620Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr625 630 635 640Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 645 650 655Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 660 665 670Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 675 680 685Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr 690 695 700Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu705 710 715 720Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu 725 730 735Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro 740 745 750Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 755 760 765Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro 770 775 780Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr785 790 795 800Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 805 810 815Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 820 825 830Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 835 840 845Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 850 855 86044864PRTArtificial SequenceAG864 44Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro1 5 10 15Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr 20 25 30Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 35 40 45Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 50 55 60Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser65 70 75 80Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 85 90 95Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly 100 105 110Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 115 120 125Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 130 135 140Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr145 150 155 160Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 165 170 175Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 180 185 190Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 195 200 205Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr 210 215 220Gly Thr Gly Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro225 230 235 240Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro 245 250 255Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 260 265 270Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 275 280 285Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro 290 295 300Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser305 310 315 320Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 325 330 335Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly 340 345 350Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 355 360 365Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 370 375 380Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr385 390 395 400Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 405 410 415Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 420 425 430Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr 435 440 445Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 450 455 460Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro465 470 475 480Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro 485 490 495Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 500 505 510Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 515 520 525Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro 530 535 540Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser545 550 555 560Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 565 570 575Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr 580 585 590Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala 595 600 605Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro 610 615 620Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr625 630 635 640Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 645 650 655Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 660 665 670Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro 675 680 685Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala 690 695 700Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro705 710 715 720Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro 725 730 735Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser 740 745 750Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 755 760 765Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 770 775 780Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser785 790 795 800Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 805 810 815Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr 820 825 830Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 835 840 845Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 850 855 860459PRTArtificial Sequencecleavage site 45Thr Gln Ser Phe Asn Asp Phe Thr Arg1 54610PRTArtificial Sequencecleavage site 46Ser Val Ser Gln Thr Ser Lys Leu Thr Arg1 5 104710PRTArtificial Sequencecleavage site 47Asp Phe Leu Ala Glu Gly Gly Gly Val Arg1 5 10487PRTArtificial Sequencecleavage site 48Thr Thr Lys Ile Lys Pro Arg1 5495PRTArtificial Sequencecleavage site 49Leu Val Pro Arg Gly1 5505PRTArtificial Sequencecleavage site 50Ala Leu Arg Pro Arg1 5515PRTArtificial Sequencesortase recognition motifmisc_feature(3)..(3)Xaa can be any naturally occurring amino acid 51Leu Pro Xaa Thr Gly1 55211PRTArtificial Sequencepolypeptide 52Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro1 5 10537PRTArtificial Sequencepolypeptide 53His Gln Ser Leu Gly Thr Gln1 5548PRTArtificial Sequencepolypeptide 54His Gln Asn Leu Ser Asp Gly Lys1 5558PRTArtificial Sequencepolypeptide 55His Gln Asn Ile Ser Asp Gly Lys1 5568PRTArtificial Sequencepolypeptide 56Val Ile Ser Ser His Leu Gly Gln1 5574PRTArtificial SequencelinkerREPEAT(1)..(4)Repeat from 1 to 100 times 57Gly Gly Gly Ser1588PRTArtificial SequencelinkerREPEAT(1)..(3)May be repeated 1 to 100 timesREPEAT(4)..(8)May be repeated 1 to 100 times 58Gly Gly Ser Gly Gly Gly Gly Ser1 5597PRTArtificial Sequencelinker 59Ser Gly Gly Ser Gly Gly Ser1 56015PRTArtificial Sequencelinker 60Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly1 5 10 156116PRTArtificial Sequencelinker 61Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 156218PRTArtificial Sequencelinker 62Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly1 5 10 15Gly Ser6315PRTArtificial Sequencelinker 63Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15645PRTArtificial SequencelinkerREPEAT(1)..(5)May be repeated 1-20 times 64Gly Gly Gly Gly Ser1 5657PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 65Lys Leu Thr Arg Ala Glu Thr1 5667PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 66Asp Phe Thr Arg Val Val Gly1 5678PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage Site 67Thr Met Thr Arg Ile Val Gly Gly1 5688PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 68Ser Pro Phe Arg Ser Thr Gly Gly1 5698PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 69Leu Gln Val Arg Ile Val Gly Gly1 5708PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 70Pro Leu Gly Arg Ile Val Gly Gly1 5718PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 71Ile Glu Gly Arg Thr Val Gly Gly1 5728PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)cleavage site 72Leu Thr Pro Arg Ser Leu Leu Val1 5738PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)cleavage site 73Leu Gly Pro Val Ser Gly Val Pro1 5748PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 74Val Ala Gly Asp Ser Leu Glu Glu1 5758PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 75Gly Pro Ala Gly Leu Gly Gly Ala1 5768PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 76Gly Pro Ala Gly Leu Arg Gly Ala1 5778PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 77Ala Pro Leu Gly Leu Arg Leu Arg1 5788PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 78Pro Ala Leu Pro Leu Val Ala Gln1 5797PRTArtificial Sequencecleavage siteMISC_FEATURE(6)..(7)Cleavage site 79Glu Asn Leu Tyr Phe Gln Gly1 5808PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 80Asp Asp Asp Lys Ile Val Gly Gly1 5818PRTArtificial Sequencecleavage siteMISC_FEATURE(6)..(7)Cleavage site 81Leu Glu Val Leu Phe Gln Gly Pro1 5828PRTArtificial Sequencecleavage siteMISC_FEATURE(4)..(5)Cleavage site 82Leu Pro Lys Thr Gly Ser Glu Ser1

5839PRTArtificial Sequencecleavage site 83Thr Gln Ser Phe Asn Asp Phe Thr Arg1 58410PRTArtificial Sequencecleavage site 84Ser Val Ser Gln Thr Ser Lys Leu Thr Arg1 5 108510PRTArtificial Sequencecleavage site 85Asp Phe Leu Ala Glu Gly Gly Gly Val Arg1 5 10867PRTArtificial Sequencecleavage site 86Thr Thr Lys Ile Lys Pro Arg1 5875PRTArtificial Sequencecleavage site 87Leu Val Pro Arg Gly1 58810PRTArtificial Sequencecleavage site 88Ala Leu Arg Pro Arg Val Val Gly Gly Ala1 5 10891896PRTArtificial SequenceFVIII 198 89Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 755 760 765Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp 770 775 780Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys785 790 795 800Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser 805 810 815Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr 820 825 830Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn 835 840 845Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly 850 855 860Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu865 870 875 880Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys 885 890 895Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn 900 905 910Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met 915 920 925Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys 930 935 940Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu945 950 955 960Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu 965 970 975Ser Ser Trp Gly Lys Asn Val Ser Ser Glu Ile Thr Arg Thr Thr Leu 980 985 990Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu 995 1000 1005Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln 1010 1015 1020Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala 1025 1030 1035Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His 1040 1045 1050Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys 1055 1060 1065Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro 1070 1075 1080Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro 1085 1090 1095Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 1100 1105 1110Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser 1115 1120 1125Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe 1130 1135 1140Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His 1145 1150 1155His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala 1160 1165 1170Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 1175 1180 1185Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala 1190 1195 1200His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr 1205 1210 1215Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu 1220 1225 1230Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr 1235 1240 1245Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met 1250 1255 1260Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg 1265 1270 1275Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile 1280 1285 1290His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr 1295 1300 1305Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val 1310 1315 1320Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu 1325 1330 1335Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 1340 1345 1350Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His 1355 1360 1365Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp 1370 1375 1380Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala 1385 1390 1395Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu 1400 1405 1410Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln 1415 1420 1425Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser 1430 1435 1440Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly 1445 1450 1455Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys 1460 1465 1470His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu 1475 1480 1485His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu 1490 1495 1500Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu 1505 1510 1515Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe 1520 1525 1530Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His 1535 1540 1545Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro 1550 1555 1560Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr 1565 1570 1575Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 1580 1585 1590Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp 1595 1600 1605Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn 1610 1615 1620Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu 1625 1630 1635Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln 1640 1645 1650Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu 1655 1660 1665Tyr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 1670 1675 1680Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 1685 1690 1695Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 1700 1705 1710Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 1715 1720 1725Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 1730 1735 1740Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 1745 1750 1755His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 1760 1765 1770Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 1775 1780 1785Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 1790 1795 1800Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 1805 1810 1815Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 1820 1825 1830Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 1835 1840 1845Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 1850 1855 1860Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 1865 1870 1875Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 1880 1885 1890Pro Gly Lys 18959099DNAArtificial SequenceESC48- Fwd - VWF-D'D3 with VIII signal and BsiW1 site 90tcgcgacgta cggccgccac catgcaaata gagctctcca cctgcttctt tctgtgcctt 60ttgcgattct gctttagcct atcctgtcgg ccccccatg 999174DNAArtificial SequenceESC51- Rev- VWF D'D3 (1-477 amino acid) with 6His and Not 1 site 91tgacctcgag cggccgctca gtggtgatgg tgatgatgcg gctcctggca ggcttcacag 60gtgaggttga caac 749232DNAArtificial SequenceESC 89-fwd with Nhe1site 92ctcactatag ggagacccaa gctggctagc cg 329343DNAArtificial SequenceESC 91-rev with Sal1 93ctggatcccg ggagtcgact cgtcagtggt gatggtgatg atg 439440PRTArtificial Sequencelinker 94Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser Gly Gly Gly Gly Ser 35 409592DNAArtificial SequenceLW 22-FWD-VWF-D'D3 with FVIII signal sequence and BsiW1 site 95gcgccggccg tacgatgcaa atagagctct ccacctgctt ctttctgtgc cttttgcgat 60tctgctttag cctatcctgt cggcccccca tg 929647DNAArtificial SequenceLW 23-Rev- Fc with stop codon and Not1 site 96tcatcaatgt atcttatcat gtctgaattc gcggccgctc atttacc 479741DNAArtificial SequenceLW24- Fwd- VWF D1D2D'D3 cloning oligo with BsiW1 site 97gcgccggccg tacgatgatt cctgccagat ttgccggggt g 419841DNAArtificial SequenceLW27-Rev-VWF D'D3 oligo with EcoRV 98ccaccgccag atatcggctc ctggcaggct tcacaggtga g 41991240PRTArtificial SequenceVWF-D1D2D'D3 protein sequence 1 99Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile1 5 10 15Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys65 70 75 80Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly145 150 155 160Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220Cys Gln Leu Leu Lys Ser Thr Ser Val Phe

Ala Arg Cys His Pro Leu225 230 235 240Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met305 310 315 320Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp385 390 395 400Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu465 470 475 480Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln545 550 555 560Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val625 630 635 640Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp705 710 715 720Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met785 790 795 800Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly865 870 875 880Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg945 950 955 960Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060 1065Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln 1100 1105 1110His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln 1115 1120 1125Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu 1130 1135 1140Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180 1185Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly 1190 1195 1200Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230Cys Glu Ala Cys Gln Glu Pro 1235 1240100477PRTArtificial SequenceVWF-D'D3 protein sequence 2 100Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp1 5 10 15Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys65 70 75 80Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr 85 90 95Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys145 150 155 160Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln225 230 235 240Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val305 310 315 320Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345 350Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu385 390 395 400Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro465 470 4751012754PRTArtificial SequencepSYN-FVIII-161 101Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu785 790 795 800Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His865 870 875 880Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp945 950 955 960Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu

965 970 975Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1010 1015 1020Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1025 1030 1035Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1040 1045 1050Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1055 1060 1065Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1070 1075 1080Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120 1125Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1145 1150 1155Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1160 1165 1170Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1175 1180 1185Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1250 1255 1260Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1265 1270 1275Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1280 1285 1290Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1295 1300 1305Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360 1365Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1385 1390 1395Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1400 1405 1410Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1415 1420 1425Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1445 1450 1455Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1460 1465 1470Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1475 1480 1485Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1490 1495 1500Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1505 1510 1515Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1520 1525 1530Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1535 1540 1545Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 1550 1555 1560Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1565 1570 1575Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1580 1585 1590Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 1595 1600 1605Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1610 1615 1620Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1625 1630 1635Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1640 1645 1650Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1655 1660 1665Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1670 1675 1680Lys Arg Arg Arg Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1685 1690 1695Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1700 1705 1710Ser Gly Gly Gly Gly Ser Arg Lys Arg Arg Lys Arg Ser Leu Ser 1715 1720 1725Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu 1730 1735 1740Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp 1745 1750 1755Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 1760 1765 1770Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg 1775 1780 1785Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr 1790 1795 1800Val Lys Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp 1805 1810 1815Asn Cys Thr Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly 1820 1825 1830Met Ala His Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro 1835 1840 1845Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr Cys Gly Ser Asn 1850 1855 1860Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly Cys Ser His 1865 1870 1875Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly 1880 1885 1890Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro 1895 1900 1905Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr 1910 1915 1920Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 1925 1930 1935His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys 1940 1945 1950Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp 1955 1960 1965Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe 1970 1975 1980Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys 1985 1990 1995Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 2000 2005 2010Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp 2015 2020 2025Val Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu 2030 2035 2040Asp Val Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp 2045 2050 2055Cys Ala Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys 2060 2065 2070Ala Gln His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys 2075 2080 2085Pro Gln Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu 2090 2095 2100Ala Glu Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr 2105 2110 2115Cys Gln His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu 2120 2125 2130Gly Cys His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu 2135 2140 2145Leu Gln Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val 2150 2155 2160Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro 2165 2170 2175Ser Asp Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn 2180 2185 2190Leu Thr Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Thr Ser Glu 2195 2200 2205Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser 2210 2215 2220Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 2225 2230 2235Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 2240 2245 2250Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 2255 2260 2265Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro 2270 2275 2280Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 2285 2290 2295Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 2300 2305 2310Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala 2315 2320 2325Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro 2330 2335 2340Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser 2345 2350 2355Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 2360 2365 2370Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu 2375 2380 2385Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser 2390 2395 2400Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu 2405 2410 2415Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly 2420 2425 2430Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 2435 2440 2445Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser 2450 2455 2460Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 2465 2470 2475Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Asp 2480 2485 2490Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 2495 2500 2505Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg 2510 2515 2520Gly Ser Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 2525 2530 2535Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 2540 2545 2550Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 2555 2560 2565Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 2570 2575 2580Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 2585 2590 2595Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 2600 2605 2610Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 2615 2620 2625Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 2630 2635 2640Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 2645 2650 2655Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 2660 2665 2670Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 2675 2680 2685Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 2690 2695 2700Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 2705 2710 2715Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 2720 2725 2730Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 2735 2740 2745Ser Leu Ser Pro Gly Lys 27501022242PRTArtificial SequencepSYN-FVIII-170 protein sequence 102Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp1 5 10 15Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys65 70 75 80Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr 85 90 95Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys145 150 155 160Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln225 230 235 240Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val305 310 315 320Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Ala 325 330 335Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345 350Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu Trp Arg Tyr Asn 370 375 380Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu385 390 395 400Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly465 470 475 480Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 485 490 495Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 500 505 510Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 515 520 525Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu 530 535 540Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser545 550 555 560Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 565 570 575Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser 580 585 590Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 595 600 605Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly 610 615 620Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser625 630 635 640Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu

645 650 655Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 660 665 670Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala 675 680 685Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 690 695 700Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly705 710 715 720Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala 725 730 735Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 740 745 750Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Asp 755 760 765Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 770 775 780Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser785 790 795 800Gly Gly Ala Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu 805 810 815Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala 820 825 830Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val 835 840 845Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn 850 855 860Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile865 870 875 880Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala 885 890 895Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala 900 905 910Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu 915 920 925Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val 930 935 940Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr945 950 955 960Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 965 970 975Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys 980 985 990Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu 995 1000 1005Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp 1010 1015 1020Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val 1025 1030 1035Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His 1040 1045 1050Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro 1055 1060 1065Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 1070 1075 1080Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu 1085 1090 1095Thr Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe 1100 1105 1110Cys His Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val 1115 1120 1125Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn 1130 1135 1140Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu 1145 1150 1155Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile 1160 1165 1170Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 1175 1180 1185Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val 1190 1195 1200Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn 1205 1210 1215Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 1220 1225 1230Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His 1235 1240 1245Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp 1250 1255 1260Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn 1265 1270 1275Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg 1280 1285 1290Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu 1295 1300 1305Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 1310 1315 1320Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser 1325 1330 1335Ser Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly 1340 1345 1350Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn 1355 1360 1365Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe 1370 1375 1380Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe 1385 1390 1395Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln 1400 1405 1410Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 1415 1420 1425Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile 1430 1435 1440Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser 1445 1450 1455Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 1460 1465 1470Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn 1475 1480 1485Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn 1490 1495 1500Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn 1505 1510 1515Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr 1520 1525 1530Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln 1535 1540 1545Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1550 1555 1560Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1565 1570 1575Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1580 1585 1590Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1595 1600 1605Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1610 1615 1620Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1625 1630 1635Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1640 1645 1650Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1655 1660 1665Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1670 1675 1680Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1685 1690 1695Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1700 1705 1710Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1715 1720 1725Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1730 1735 1740Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1745 1750 1755Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1760 1765 1770Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1775 1780 1785Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1790 1795 1800Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1805 1810 1815Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1820 1825 1830Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1835 1840 1845Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1850 1855 1860His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1865 1870 1875Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1880 1885 1890Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1895 1900 1905Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 1910 1915 1920Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 1925 1930 1935Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1940 1945 1950Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 1955 1960 1965Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 1970 1975 1980Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 1985 1990 1995Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2000 2005 2010Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2015 2020 2025Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2030 2035 2040Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2045 2050 2055Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2060 2065 2070Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2075 2080 2085Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2090 2095 2100Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2105 2110 2115Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 2120 2125 2130Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2135 2140 2145Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 2150 2155 2160Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2165 2170 2175His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2180 2185 2190Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2195 2200 2205Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2210 2215 2220Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2225 2230 2235Gln Asp Leu Tyr 22401031959PRTArtificial SequencepSYN-FVIII-169 mature Protein sequence 103Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly Thr Ser Glu 740 745 750Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly 755 760 765Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 770 775 780Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu785 790 795 800Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu 805 810 815Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 820 825 830Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 835 840 845Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala

Thr Pro 850 855 860Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu865 870 875 880Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr 885 890 895Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 900 905 910Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 915 920 925Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 930 935 940Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly945 950 955 960Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 965 970 975Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 980 985 990Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser Gly 995 1000 1005Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1010 1015 1020Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Ala Ser 1025 1030 1035Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu Ile Thr Arg Thr 1040 1045 1050Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1055 1060 1065Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1070 1075 1080Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1085 1090 1095Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1100 1105 1110Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1115 1120 1125Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1130 1135 1140Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1145 1150 1155Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1160 1165 1170Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1175 1180 1185Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1190 1195 1200Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1205 1210 1215Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1220 1225 1230Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1235 1240 1245Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1250 1255 1260Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1265 1270 1275Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1280 1285 1290Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1295 1300 1305Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1310 1315 1320Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1325 1330 1335Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1340 1345 1350His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1355 1360 1365Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1370 1375 1380Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1385 1390 1395Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 1400 1405 1410Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 1415 1420 1425Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1430 1435 1440Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 1445 1450 1455Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 1460 1465 1470Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 1475 1480 1485Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 1490 1495 1500Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 1505 1510 1515Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 1520 1525 1530Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 1535 1540 1545Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 1550 1555 1560Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 1565 1570 1575Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1580 1585 1590Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 1595 1600 1605Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 1610 1615 1620Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 1625 1630 1635Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 1640 1645 1650Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 1655 1660 1665His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 1670 1675 1680Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 1685 1690 1695Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 1700 1705 1710Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 1715 1720 1725Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1730 1735 1740Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1745 1750 1755Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 1760 1765 1770Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 1775 1780 1785Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 1790 1795 1800Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 1805 1810 1815Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 1820 1825 1830Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 1835 1840 1845Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 1850 1855 1860Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 1865 1870 1875Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 1880 1885 1890Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 1895 1900 1905Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 1910 1915 1920Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 1925 1930 1935Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 1940 1945 1950Ser Leu Ser Pro Gly Lys 19551041959PRTArtificial SequencepSYN-FVIII-173 mature Protein 104Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly Thr Ser Glu 740 745 750Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly 755 760 765Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 770 775 780Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu785 790 795 800Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu 805 810 815Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 820 825 830Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 835 840 845Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 850 855 860Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu865 870 875 880Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr 885 890 895Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 900 905 910Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 915 920 925Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 930 935 940Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly945 950 955 960Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 965 970 975Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 980 985 990Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser Gly 995 1000 1005Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1010 1015 1020Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Ala Ser 1025 1030 1035Ser Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1040 1045 1050Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1055 1060 1065Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1070 1075 1080Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1085 1090 1095Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1100 1105 1110Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1115 1120 1125Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1130 1135 1140Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1145 1150 1155Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1160 1165 1170Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1175 1180 1185Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1190 1195 1200Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1205 1210 1215Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1220 1225 1230Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1235 1240 1245Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1250 1255 1260Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1265 1270 1275Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1280 1285 1290Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1295 1300 1305Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1310 1315 1320Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1325 1330 1335Arg Ile Arg Trp Tyr Leu Leu Ser Met

Gly Ser Asn Glu Asn Ile 1340 1345 1350His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1355 1360 1365Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1370 1375 1380Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1385 1390 1395Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 1400 1405 1410Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 1415 1420 1425Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 1430 1435 1440Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 1445 1450 1455Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 1460 1465 1470Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 1475 1480 1485Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 1490 1495 1500Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 1505 1510 1515Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 1520 1525 1530Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 1535 1540 1545Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 1550 1555 1560Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 1565 1570 1575Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 1580 1585 1590Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 1595 1600 1605Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 1610 1615 1620Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 1625 1630 1635Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 1640 1645 1650Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 1655 1660 1665His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 1670 1675 1680Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 1685 1690 1695Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 1700 1705 1710Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 1715 1720 1725Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1730 1735 1740Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1745 1750 1755Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 1760 1765 1770Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 1775 1780 1785Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 1790 1795 1800Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 1805 1810 1815Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 1820 1825 1830Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 1835 1840 1845Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 1850 1855 1860Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 1865 1870 1875Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 1880 1885 1890Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 1895 1900 1905Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 1910 1915 1920Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 1925 1930 1935Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 1940 1945 1950Ser Leu Ser Pro Gly Lys 19551051984PRTArtificial SequenceFVIII 195 protein sequence 105Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Gly Ala Pro 770 775 780Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro785 790 795 800Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 805 810 815Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 820 825 830Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly 835 840 845Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 850 855 860Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro865 870 875 880Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr 885 890 895Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 900 905 910Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 915 920 925Ala Ser Ser Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser 930 935 940Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn945 950 955 960Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala 965 970 975Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val 980 985 990Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val 995 1000 1005Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1010 1015 1020Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1025 1030 1035Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln 1040 1045 1050Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu 1055 1060 1065Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1070 1075 1080Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met 1085 1090 1095Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 1100 1105 1110Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1115 1120 1125Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly 1130 1135 1140Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 1145 1150 1155Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1160 1165 1170Cys Arg Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1175 1180 1185Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr 1190 1195 1200Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 1205 1210 1215Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 1220 1225 1230Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala 1235 1240 1245Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser 1250 1255 1260Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 1265 1270 1275Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr 1280 1285 1290Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1295 1300 1305Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ala 1310 1315 1320Ser Ser Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1325 1330 1335Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1340 1345 1350Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1355 1360 1365Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1370 1375 1380Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1385 1390 1395Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1400 1405 1410Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1415 1420 1425Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1430 1435 1440Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1445 1450 1455Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1460 1465 1470Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1475 1480 1485Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1490 1495 1500Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1505 1510 1515Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1520 1525 1530Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1535 1540 1545Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1550 1555 1560Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1565 1570 1575Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1580 1585 1590Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1595 1600 1605Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1610 1615 1620Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1625 1630 1635Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1640 1645 1650Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1655 1660 1665Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1670 1675 1680Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1685 1690 1695Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1700 1705 1710Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1715 1720 1725Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1730 1735 1740Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1745 1750 1755Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1760 1765 1770Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1775 1780 1785Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1790 1795 1800Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1805 1810 1815Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1820

1825 1830Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1835 1840 1845Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 1850 1855 1860Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1865 1870 1875Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1880 1885 1890Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 1895 1900 1905Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1910 1915 1920Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1925 1930 1935Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1940 1945 1950Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1955 1960 1965Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1970 1975 1980Lys1062134PRTArtificial SequenceFVIII 196 protein sequence 106Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Gly Ala Pro 35 40 45Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro 50 55 60Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser65 70 75 80Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 85 90 95Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 100 105 110Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser 115 120 125Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 130 135 140Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr145 150 155 160Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 165 170 175Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 180 185 190Ala Ser Ser Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro 195 200 205Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr 210 215 220Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu225 230 235 240Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr 245 250 255Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val 260 265 270Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser 275 280 285Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr 290 295 300Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro305 310 315 320Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp 325 330 335Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser 340 345 350Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe 355 360 365Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 370 375 380Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met385 390 395 400His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly 405 410 415Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr 420 425 430Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 435 440 445Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr 450 455 460Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His465 470 475 480Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp 485 490 495Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala 500 505 510Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg 515 520 525Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala 530 535 540Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu545 550 555 560Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr 565 570 575Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr 580 585 590Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg 595 600 605Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly 610 615 620Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg625 630 635 640Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr 645 650 655Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 660 665 670Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 675 680 685Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser 690 695 700Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu705 710 715 720Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met 725 730 735Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg 740 745 750Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala 755 760 765Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His 770 775 780Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu785 790 795 800His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 805 810 815Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val 820 825 830Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe 835 840 845Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser 850 855 860Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys865 870 875 880Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser 885 890 895Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser 900 905 910Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 915 920 925Thr Leu Gln Gly Ala Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser 930 935 940Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala945 950 955 960Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr 965 970 975Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr 980 985 990Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala 995 1000 1005Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly 1010 1015 1020Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 1025 1030 1035Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser 1040 1045 1050Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala 1055 1060 1065Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Ala Ser Ser Ser Asp 1070 1075 1080Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys 1085 1090 1095Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro 1100 1105 1110Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val 1115 1120 1125Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu 1130 1135 1140Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val 1145 1150 1155Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1160 1165 1170Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1175 1180 1185Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln 1190 1195 1200Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu 1205 1210 1215Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1220 1225 1230Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met 1235 1240 1245Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 1250 1255 1260Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1265 1270 1275Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly 1280 1285 1290Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 1295 1300 1305Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1310 1315 1320Cys Arg Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1325 1330 1335Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr 1340 1345 1350Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 1355 1360 1365Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 1370 1375 1380Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala 1385 1390 1395Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser 1400 1405 1410Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 1415 1420 1425Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr 1430 1435 1440Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1445 1450 1455Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ala 1460 1465 1470Ser Ser Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1475 1480 1485Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1490 1495 1500Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1505 1510 1515Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1520 1525 1530Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1535 1540 1545Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1550 1555 1560Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1565 1570 1575Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1580 1585 1590Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1595 1600 1605Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1610 1615 1620Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1625 1630 1635Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1640 1645 1650Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1655 1660 1665Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1670 1675 1680Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1685 1690 1695Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1700 1705 1710Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1715 1720 1725Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1730 1735 1740Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1745 1750 1755Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1760 1765 1770Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1775 1780 1785Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1790 1795 1800Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1805 1810 1815Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1820 1825 1830Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1835 1840 1845Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1850 1855 1860Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1865 1870 1875Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1880 1885 1890Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1895 1900 1905Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1910 1915 1920Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1925 1930 1935Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1940 1945 1950Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1955 1960 1965Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1970 1975 1980Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1985 1990 1995Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 2000 2005 2010Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 2015 2020 2025Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 2030 2035 2040Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 2045 2050 2055Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 2060 2065 2070Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 2075 2080 2085Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 2090 2095 2100Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 2105 2110 2115Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 2120 2125 2130Lys1071984PRTArtificial SequenceFVIII 199 protein sequence 107Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val

Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765Lys Arg His Gln Ala Glu Ile Thr Arg Thr Thr Leu Gln Gly Ala Pro 770 775 780Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro785 790 795 800Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 805 810 815Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 820 825 830Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly 835 840 845Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 850 855 860Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro865 870 875 880Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr 885 890 895Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 900 905 910Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 915 920 925Ala Ser Ser Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser 930 935 940Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn945 950 955 960Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala 965 970 975Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val 980 985 990Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val 995 1000 1005Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1010 1015 1020Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1025 1030 1035Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln 1040 1045 1050Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu 1055 1060 1065Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1070 1075 1080Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met 1085 1090 1095Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 1100 1105 1110Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1115 1120 1125Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly 1130 1135 1140Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 1145 1150 1155Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1160 1165 1170Cys Arg Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1175 1180 1185Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr 1190 1195 1200Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 1205 1210 1215Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 1220 1225 1230Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala 1235 1240 1245Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser 1250 1255 1260Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 1265 1270 1275Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr 1280 1285 1290Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1295 1300 1305Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ala 1310 1315 1320Ser Ser Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1325 1330 1335Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1340 1345 1350Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1355 1360 1365Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1370 1375 1380Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1385 1390 1395Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1400 1405 1410Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1415 1420 1425Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1430 1435 1440Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1445 1450 1455Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1460 1465 1470Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1475 1480 1485Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1490 1495 1500Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1505 1510 1515Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1520 1525 1530Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1535 1540 1545Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1550 1555 1560Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1565 1570 1575Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1580 1585 1590Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1595 1600 1605Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1610 1615 1620Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1625 1630 1635Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1640 1645 1650Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1655 1660 1665Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1670 1675 1680Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1685 1690 1695Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1700 1705 1710Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1715 1720 1725Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1730 1735 1740Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1745 1750 1755Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1760 1765 1770Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1775 1780 1785Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1790 1795 1800Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1805 1810 1815Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1820 1825 1830Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1835 1840 1845Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 1850 1855 1860Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1865 1870 1875Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1880 1885 1890Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 1895 1900 1905Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1910 1915 1920Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1925 1930 1935Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1940 1945 1950Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1955 1960 1965Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1970 1975 1980Lys1082134PRTArtificial SequenceFVIII 201 protein sequence 108Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Gly Ala Pro 35 40 45Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro 50 55 60Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser65 70 75 80Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 85 90 95Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 100 105 110Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser 115 120 125Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 130 135 140Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr145 150 155 160Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 165 170 175Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 180 185 190Ala Ser Ser Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro 195 200 205Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr 210 215 220Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu225 230 235 240Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr 245 250 255Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val 260 265 270Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser 275 280 285Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr 290 295 300Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro305 310 315 320Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp 325 330 335Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser 340 345 350Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe 355 360 365Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 370 375 380Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met385 390 395 400His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly 405 410 415Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr 420 425 430Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 435 440 445Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr 450 455 460Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His465 470 475 480Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp 485 490 495Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala 500 505 510Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg 515 520 525Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala 530 535 540Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu545 550 555 560Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr 565 570 575Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr 580 585 590Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg 595 600 605Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly 610

615 620Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg625 630 635 640Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr 645 650 655Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 660 665 670Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 675 680 685Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser 690 695 700Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu705 710 715 720Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met 725 730 735Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg 740 745 750Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala 755 760 765Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His 770 775 780Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu785 790 795 800His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 805 810 815Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val 820 825 830Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe 835 840 845Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser 850 855 860Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys865 870 875 880Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser 885 890 895Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser 900 905 910Gln Asn Pro Pro Val Leu Lys Arg His Gln Ala Glu Ile Thr Arg Thr 915 920 925Thr Leu Gln Gly Ala Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser 930 935 940Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala945 950 955 960Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr 965 970 975Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr 980 985 990Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala 995 1000 1005Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly 1010 1015 1020Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 1025 1030 1035Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser 1040 1045 1050Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala 1055 1060 1065Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Ala Ser Ser Ser Asp 1070 1075 1080Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys 1085 1090 1095Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro 1100 1105 1110Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val 1115 1120 1125Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu 1130 1135 1140Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val 1145 1150 1155Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1160 1165 1170Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1175 1180 1185Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln 1190 1195 1200Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu 1205 1210 1215Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1220 1225 1230Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met 1235 1240 1245Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 1250 1255 1260Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1265 1270 1275Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly 1280 1285 1290Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 1295 1300 1305Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1310 1315 1320Cys Arg Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1325 1330 1335Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr 1340 1345 1350Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 1355 1360 1365Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 1370 1375 1380Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala 1385 1390 1395Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser 1400 1405 1410Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 1415 1420 1425Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr 1430 1435 1440Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 1445 1450 1455Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ala 1460 1465 1470Ser Ser Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1475 1480 1485Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1490 1495 1500Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1505 1510 1515Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1520 1525 1530Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1535 1540 1545Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1550 1555 1560Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1565 1570 1575Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1580 1585 1590Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1595 1600 1605Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1610 1615 1620Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1625 1630 1635Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1640 1645 1650Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1655 1660 1665Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1670 1675 1680Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1685 1690 1695Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1700 1705 1710Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1715 1720 1725Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1730 1735 1740Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1745 1750 1755Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1760 1765 1770Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1775 1780 1785Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1790 1795 1800Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1805 1810 1815Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1820 1825 1830Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1835 1840 1845Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1850 1855 1860Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1865 1870 1875Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1880 1885 1890Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1895 1900 1905Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1910 1915 1920Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1925 1930 1935Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1940 1945 1950Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1955 1960 1965Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1970 1975 1980Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1985 1990 1995Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 2000 2005 2010Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 2015 2020 2025Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 2030 2035 2040Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 2045 2050 2055Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 2060 2065 2070Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 2075 2080 2085Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 2090 2095 2100Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 2105 2110 2115Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 2120 2125 2130Lys1092128PRTArtificial SequenceFVIII 203 protein sequence 109Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly 755 760 765Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 770 775 780Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu785 790 795 800Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 805 810 815Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu 820 825 830Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 835 840 845Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 850 855 860Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser865 870 875 880Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 885 890 895Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly 900 905 910Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser 915 920 925Ala Thr Pro Glu Ser Gly Pro Gly Thr

Ser Glu Ser Ala Thr Pro Glu 930 935 940Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly945 950 955 960Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala 965 970 975Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 980 985 990Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 995 1000 1005Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 1010 1015 1020Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr 1025 1030 1035Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser 1040 1045 1050Ala Pro Ala Ser Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu 1055 1060 1065Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1070 1075 1080Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1085 1090 1095Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1100 1105 1110Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1115 1120 1125Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 1130 1135 1140Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 1145 1150 1155Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1160 1165 1170His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1175 1180 1185Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1190 1195 1200Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1205 1210 1215Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr 1220 1225 1230Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu 1235 1240 1245Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 1250 1255 1260Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His 1265 1270 1275Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln 1280 1285 1290Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1295 1300 1305Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Gly Ala Pro Thr 1310 1315 1320Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala 1325 1330 1335Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 1340 1345 1350Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr 1355 1360 1365Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr 1370 1375 1380Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser 1385 1390 1395Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr 1400 1405 1410Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu 1415 1420 1425Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr 1430 1435 1440Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu 1445 1450 1455Pro Ser Glu Gly Ser Ala Pro Gly Ala Ser Ser Ala Pro Cys Asn 1460 1465 1470Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1475 1480 1485Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1490 1495 1500Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1505 1510 1515Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1520 1525 1530Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1535 1540 1545Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 1550 1555 1560Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 1565 1570 1575Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 1580 1585 1590Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala 1595 1600 1605Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His 1610 1615 1620Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 1625 1630 1635Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 1640 1645 1650Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser 1655 1660 1665Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 1670 1675 1680Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1685 1690 1695Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 1700 1705 1710Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 1715 1720 1725Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 1730 1735 1740Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 1745 1750 1755Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 1760 1765 1770Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1775 1780 1785Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 1790 1795 1800Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 1805 1810 1815Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 1820 1825 1830Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys 1835 1840 1845Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 1850 1855 1860Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His 1865 1870 1875Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 1880 1885 1890Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro 1895 1900 1905Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 1910 1915 1920Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 1925 1930 1935Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 1940 1945 1950Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 1955 1960 1965Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 1970 1975 1980Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 1985 1990 1995Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 2000 2005 2010Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 2015 2020 2025Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 2030 2035 2040Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 2045 2050 2055Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 2060 2065 2070Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 2075 2080 2085Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 2090 2095 2100Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 2105 2110 2115Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 2120 21251102128PRTArtificial SequenceFVIII 204 protein sequence 110Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Gly Ala Pro Thr Ser Thr Glu Pro Ser Glu 420 425 430Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu 435 440 445Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 450 455 460Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro465 470 475 480Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 485 490 495Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 500 505 510Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Thr Glu Pro Ser Glu 515 520 525Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 530 535 540Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu545 550 555 560Ser Ala Thr Pro Glu Ser Gly Pro Gly Ala Ser Ser Asp Arg Ser Tyr 565 570 575Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr 580 585 590Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg 595 600 605Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly 610 615 620Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg625 630 635 640Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr 645 650 655Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 660 665 670Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 675 680 685Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser 690 695 700Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu705 710 715 720Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met 725 730 735Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg 740 745 750Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala 755 760 765Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His 770 775 780Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu785 790 795 800His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 805 810 815Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val 820 825 830Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe 835 840 845Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser 850 855 860Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys865 870 875 880Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser 885 890 895Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser 900 905 910Gln Asn Gly Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 915 920 925Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser 930 935 940Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser945 950 955 960Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 965 970 975Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro 980 985 990Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr 995 1000 1005Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu 1010 1015 1020Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1025 1030 1035Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 1040 1045 1050Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser 1055 1060 1065Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1070 1075 1080Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1085 1090 1095Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 1100 1105 1110Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser 1115 1120 1125Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr 1130 1135 1140Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 1145 1150 1155Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 1160 1165 1170Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr 1175 1180 1185Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser 1190 1195 1200Ala Pro Ala Ser Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu 1205 1210 1215Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1220 1225 1230Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1235 1240 1245Tyr Asp Glu Asp

Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1250 1255 1260Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1265 1270 1275Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 1280 1285 1290Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 1295 1300 1305Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1310 1315 1320His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1325 1330 1335Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1340 1345 1350Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1355 1360 1365Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr 1370 1375 1380Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu 1385 1390 1395Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 1400 1405 1410Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His 1415 1420 1425Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln 1430 1435 1440Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1445 1450 1455Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1460 1465 1470Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1475 1480 1485Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1490 1495 1500Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1505 1510 1515Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1520 1525 1530Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1535 1540 1545Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 1550 1555 1560Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 1565 1570 1575Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 1580 1585 1590Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala 1595 1600 1605Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His 1610 1615 1620Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 1625 1630 1635Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 1640 1645 1650Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser 1655 1660 1665Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 1670 1675 1680Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1685 1690 1695Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 1700 1705 1710Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 1715 1720 1725Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 1730 1735 1740Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 1745 1750 1755Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 1760 1765 1770Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1775 1780 1785Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 1790 1795 1800Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 1805 1810 1815Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 1820 1825 1830Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys 1835 1840 1845Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 1850 1855 1860Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His 1865 1870 1875Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 1880 1885 1890Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro 1895 1900 1905Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 1910 1915 1920Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 1925 1930 1935Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 1940 1945 1950Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 1955 1960 1965Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 1970 1975 1980Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 1985 1990 1995Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 2000 2005 2010Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 2015 2020 2025Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 2030 2035 2040Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 2045 2050 2055Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 2060 2065 2070Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 2075 2080 2085Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 2090 2095 2100Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 2105 2110 2115Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 2120 21251112128PRTArtificial SequenceFVIII 205 protein sequence 111Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu 35 40 45Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 50 55 60Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala65 70 75 80Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 85 90 95Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 100 105 110Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser 115 120 125Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 130 135 140Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly145 150 155 160Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 165 170 175Ser Pro Thr Ser Thr Glu Glu Gly Ala Ser Ser Ser Asp Leu Gly Glu 180 185 190Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro 195 200 205Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr 210 215 220Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu225 230 235 240Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr 245 250 255Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val 260 265 270Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser 275 280 285Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr 290 295 300Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro305 310 315 320Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp 325 330 335Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser 340 345 350Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe 355 360 365Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 370 375 380Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met385 390 395 400His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly 405 410 415Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr 420 425 430Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 435 440 445Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr 450 455 460Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His465 470 475 480Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp 485 490 495Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala 500 505 510Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg 515 520 525Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala 530 535 540Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu545 550 555 560Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr 565 570 575Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr 580 585 590Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg 595 600 605Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly 610 615 620Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg625 630 635 640Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr 645 650 655Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 660 665 670Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 675 680 685Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser 690 695 700Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu705 710 715 720Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met 725 730 735Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg 740 745 750Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala 755 760 765Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His 770 775 780Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu785 790 795 800His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 805 810 815Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val 820 825 830Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe 835 840 845Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser 850 855 860Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys865 870 875 880Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser 885 890 895Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser 900 905 910Gln Asn Gly Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 915 920 925Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser 930 935 940Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser945 950 955 960Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 965 970 975Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro 980 985 990Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr 995 1000 1005Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu 1010 1015 1020Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1025 1030 1035Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 1040 1045 1050Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser 1055 1060 1065Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1070 1075 1080Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1085 1090 1095Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 1100 1105 1110Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser 1115 1120 1125Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr 1130 1135 1140Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 1145 1150 1155Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 1160 1165 1170Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr 1175 1180 1185Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser 1190 1195 1200Ala Pro Ala Ser Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu 1205 1210 1215Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1220 1225 1230Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1235 1240 1245Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1250 1255 1260Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1265 1270 1275Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 1280 1285 1290Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 1295 1300 1305Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1310 1315 1320His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1325 1330 1335Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1340 1345 1350Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1355 1360 1365Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr 1370 1375 1380Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu 1385 1390 1395Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 1400 1405 1410Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His 1415 1420 1425Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln 1430 1435 1440Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1445 1450 1455Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1460 1465 1470Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1475 1480 1485Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1490 1495 1500Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1505 1510 1515Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1520 1525 1530Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1535 1540 1545Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 1550 1555 1560Ile Trp Arg Val Glu

Cys Leu Ile Gly Glu His Leu His Ala Gly 1565 1570 1575Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 1580 1585 1590Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala 1595 1600 1605Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His 1610 1615 1620Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 1625 1630 1635Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 1640 1645 1650Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser 1655 1660 1665Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 1670 1675 1680Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1685 1690 1695Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 1700 1705 1710Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 1715 1720 1725Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 1730 1735 1740Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 1745 1750 1755Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 1760 1765 1770Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1775 1780 1785Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 1790 1795 1800Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 1805 1810 1815Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 1820 1825 1830Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys 1835 1840 1845Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 1850 1855 1860Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His 1865 1870 1875Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 1880 1885 1890Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro 1895 1900 1905Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 1910 1915 1920Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 1925 1930 1935Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 1940 1945 1950Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 1955 1960 1965Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 1970 1975 1980Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 1985 1990 1995Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 2000 2005 2010Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 2015 2020 2025Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 2030 2035 2040Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 2045 2050 2055Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 2060 2065 2070Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 2075 2080 2085Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 2090 2095 2100Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 2105 2110 2115Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 2120 21251122020PRTArtificial SequencepSYN FVIII 266 protein sequence 112Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 35 40 45Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 50 55 60Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Ala Ser Ser Ser65 70 75 80Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro 85 90 95Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe 100 105 110Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro 115 120 125Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr 130 135 140Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His145 150 155 160Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp 165 170 175Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly 180 185 190Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met 195 200 205Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp 210 215 220Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys225 230 235 240Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe 245 250 255Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu 260 265 270Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala 275 280 285Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro 290 295 300Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly305 310 315 320Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr 325 330 335Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile 340 345 350Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu 355 360 365Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr 370 375 380Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn385 390 395 400Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met 405 410 415Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile 420 425 430Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala 435 440 445Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp 450 455 460Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile465 470 475 480Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr 485 490 495Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro 500 505 510Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn 515 520 525Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val 530 535 540Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys545 550 555 560Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val 565 570 575Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg 580 585 590Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu 595 600 605Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly 610 615 620Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe625 630 635 640Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu 645 650 655Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser 660 665 670Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu 675 680 685Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly 690 695 700Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys705 710 715 720His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly 725 730 735Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly 740 745 750Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys 755 760 765Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr 770 775 780Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro785 790 795 800Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly Thr Ser Glu Ser Ala Thr 805 810 815Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr 820 825 830Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu 835 840 845Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr 850 855 860Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala865 870 875 880Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser 885 890 895Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser 900 905 910Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 915 920 925Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro 930 935 940Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser945 950 955 960Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 965 970 975Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser 980 985 990Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser 995 1000 1005Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu 1010 1015 1020Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly 1025 1030 1035Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 1040 1045 1050Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser 1055 1060 1065Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1070 1075 1080Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Ala 1085 1090 1095Ser Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu Ile Thr Arg 1100 1105 1110Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr 1115 1120 1125Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu 1130 1135 1140Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His 1145 1150 1155Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 1160 1165 1170Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val 1175 1180 1185Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser 1190 1195 1200Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly 1205 1210 1215Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met 1220 1225 1230Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 1235 1240 1245Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro 1250 1255 1260Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp 1265 1270 1275Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys 1280 1285 1290Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val 1295 1300 1305His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr 1310 1315 1320Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala 1325 1330 1335Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr 1340 1345 1350Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met 1355 1360 1365Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn 1370 1375 1380Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp 1385 1390 1395Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 1400 1405 1410Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys 1415 1420 1425Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val 1430 1435 1440Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg 1445 1450 1455Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr 1460 1465 1470Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 1475 1480 1485Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln 1490 1495 1500Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly 1505 1510 1515Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys 1520 1525 1530Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln 1535 1540 1545Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile 1550 1555 1560Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly 1565 1570 1575Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser 1580 1585 1590Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg 1595 1600 1605Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu 1610 1615 1620Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 1625 1630 1635Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala 1640 1645 1650Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys 1655 1660 1665Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln 1670 1675 1680Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr 1685 1690 1695Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu 1700 1705 1710Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 1715 1720 1725Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val 1730 1735 1740Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu 1745 1750 1755Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser 1760 1765 1770Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu 1775 1780 1785Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1790 1795 1800Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1805 1810 1815Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 1820 1825 1830Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 1835 1840 1845Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 1850 1855 1860Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 1865 1870 1875Leu Thr

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 1880 1885 1890Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 1895 1900 1905Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 1910 1915 1920Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 1925 1930 1935Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 1940 1945 1950Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 1955 1960 1965Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 1970 1975 1980Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 1985 1990 1995Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 2000 2005 2010Leu Ser Leu Ser Pro Gly Lys 2015 20201132056PRTArtificial SequencepSYN FVIII 267 protein sequence 113Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu 35 40 45Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 50 55 60Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala65 70 75 80Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 85 90 95Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 100 105 110Ala Ser Ser Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro 115 120 125Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys 130 135 140Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys145 150 155 160Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu 165 170 175Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro 180 185 190Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly 195 200 205Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys 210 215 220Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu225 230 235 240Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu 245 250 255Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala 260 265 270Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr 275 280 285Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser 290 295 300Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala305 310 315 320Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn 325 330 335Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp 340 345 350His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu 355 360 365Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu 370 375 380Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu385 390 395 400Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly 405 410 415Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu 420 425 430Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr 435 440 445Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser 450 455 460Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val465 470 475 480His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val 485 490 495Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly 500 505 510Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr 515 520 525Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly 530 535 540Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile545 550 555 560Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly 565 570 575Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val 580 585 590Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr 595 600 605Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg 610 615 620Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu625 630 635 640Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val 645 650 655Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu 660 665 670Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile 675 680 685Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu 690 695 700Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp705 710 715 720Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile 725 730 735Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly 740 745 750Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe 755 760 765Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu 770 775 780Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr785 790 795 800Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr 805 810 815Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn 820 825 830Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly Thr Ser 835 840 845Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser 850 855 860Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly865 870 875 880Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser 885 890 895Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser 900 905 910Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu 915 920 925Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu 930 935 940Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr945 950 955 960Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu 965 970 975Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser 980 985 990Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr 995 1000 1005Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1010 1015 1020Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1025 1030 1035Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro 1040 1045 1050Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro 1055 1060 1065Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser 1070 1075 1080Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 1085 1090 1095Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 1100 1105 1110Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser 1115 1120 1125Glu Gly Ser Ala Pro Ala Ser Ser Pro Pro Val Leu Lys Arg His 1130 1135 1140Gln Ala Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu 1145 1150 1155Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp 1160 1165 1170Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 1175 1180 1185Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu 1190 1195 1200Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg 1205 1210 1215Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln 1220 1225 1230Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu 1235 1240 1245Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu 1250 1255 1260Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg 1265 1270 1275Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln 1280 1285 1290Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu 1295 1300 1305Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr 1310 1315 1320Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 1325 1330 1335Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 1340 1345 1350Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val 1355 1360 1365Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr 1370 1375 1380Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala 1385 1390 1395Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr 1400 1405 1410Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 1415 1420 1425Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser 1430 1435 1440Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His 1445 1450 1455Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr 1460 1465 1470Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser 1475 1480 1485Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu 1490 1495 1500His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys 1505 1510 1515Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln 1520 1525 1530Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala 1535 1540 1545Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu 1550 1555 1560Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1565 1570 1575His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu 1580 1585 1590Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys 1595 1600 1605Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe 1610 1615 1620Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn 1625 1630 1635Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr 1640 1645 1650Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 1655 1660 1665Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser 1670 1675 1680Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala 1685 1690 1695Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser 1700 1705 1710Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln 1715 1720 1725Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln 1730 1735 1740Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu 1745 1750 1755Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln 1760 1765 1770Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr 1775 1780 1785Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu 1790 1795 1800Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1805 1810 1815Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His 1820 1825 1830Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 1835 1840 1845Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 1850 1855 1860Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 1865 1870 1875Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 1880 1885 1890His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 1895 1900 1905Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 1910 1915 1920Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 1925 1930 1935Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 1940 1945 1950Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 1955 1960 1965Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 1970 1975 1980Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 1985 1990 1995Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 2000 2005 2010Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 2015 2020 2025Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 2030 2035 2040His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 2045 2050 20551141834PRTArtificial SequencepSYN FVIII 268 protein sequence 114Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu 35 40 45Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 50 55 60Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala65 70 75 80Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 85 90 95Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 100 105 110Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser 115 120 125Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 130 135 140Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly145 150 155 160Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 165 170 175Ser Pro Thr Ser Thr Glu Glu Gly Ala Ser Ser Ser Asp Leu Gly Glu 180 185 190Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro 195 200 205Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr 210 215 220Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu225 230 235 240Leu Gly

Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr 245 250 255Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val 260 265 270Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser 275 280 285Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr 290 295 300Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro305 310 315 320Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp 325 330 335Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser 340 345 350Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe 355 360 365Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 370 375 380Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met385 390 395 400His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly 405 410 415Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr 420 425 430Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 435 440 445Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr 450 455 460Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His465 470 475 480Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp 485 490 495Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala 500 505 510Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg 515 520 525Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala 530 535 540Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu545 550 555 560Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr 565 570 575Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr 580 585 590Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg 595 600 605Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly 610 615 620Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg625 630 635 640Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr 645 650 655Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 660 665 670Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 675 680 685Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser 690 695 700Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu705 710 715 720Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met 725 730 735Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg 740 745 750Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala 755 760 765Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His 770 775 780Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu785 790 795 800His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 805 810 815Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val 820 825 830Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe 835 840 845Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser 850 855 860Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys865 870 875 880Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser 885 890 895Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser 900 905 910Gln Asn Pro Pro Val Leu Lys Arg His Gln Ala Glu Ile Thr Arg Thr 915 920 925Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser 930 935 940Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn945 950 955 960Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala 965 970 975Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val 980 985 990Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val 995 1000 1005Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1010 1015 1020Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1025 1030 1035Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln 1040 1045 1050Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu 1055 1060 1065Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1070 1075 1080Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met 1085 1090 1095Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe 1100 1105 1110Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1115 1120 1125Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly 1130 1135 1140Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 1145 1150 1155Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1160 1165 1170Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1175 1180 1185Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1190 1195 1200Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1205 1210 1215Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1220 1225 1230Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1235 1240 1245Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1250 1255 1260Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1265 1270 1275Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1280 1285 1290Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1295 1300 1305Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1310 1315 1320Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1325 1330 1335Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1340 1345 1350Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1355 1360 1365Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1370 1375 1380Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1385 1390 1395Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1400 1405 1410Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1415 1420 1425Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1430 1435 1440Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1445 1450 1455Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1460 1465 1470Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1475 1480 1485Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1490 1495 1500Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1505 1510 1515Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1520 1525 1530Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1535 1540 1545Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1550 1555 1560Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1565 1570 1575Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1580 1585 1590Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1595 1600 1605Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1610 1615 1620Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1625 1630 1635Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1640 1645 1650Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1655 1660 1665Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1670 1675 1680Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1685 1690 1695Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 1700 1705 1710Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1715 1720 1725Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1730 1735 1740Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 1745 1750 1755Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1760 1765 1770Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1775 1780 1785Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1790 1795 1800Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1805 1810 1815Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1820 1825 1830Lys1151762PRTArtificial SequencepSYN FVIII 269 protein sequence 115Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu 35 40 45Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 50 55 60Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala65 70 75 80Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 85 90 95Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 100 105 110Ala Ser Ser Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro 115 120 125Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys 130 135 140Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys145 150 155 160Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu 165 170 175Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro 180 185 190Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly 195 200 205Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys 210 215 220Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu225 230 235 240Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu 245 250 255Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala 260 265 270Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr 275 280 285Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser 290 295 300Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala305 310 315 320Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn 325 330 335Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp 340 345 350His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu 355 360 365Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu 370 375 380Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu385 390 395 400Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly 405 410 415Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu 420 425 430Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr 435 440 445Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser 450 455 460Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val465 470 475 480His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val 485 490 495Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly 500 505 510Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr 515 520 525Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly 530 535 540Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile545 550 555 560Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly 565 570 575Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val 580 585 590Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr 595 600 605Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg 610 615 620Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu625 630 635 640Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val 645 650 655Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu 660 665 670Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile 675 680 685Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu 690 695 700Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp705 710 715 720Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile 725 730 735Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly 740 745 750Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe 755 760 765Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu 770 775 780Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr785 790 795 800Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr 805 810 815Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn 820 825 830Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg 835 840 845His Gln Ala Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu 850

855 860Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe865 870 875 880Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys 885 890 895Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 900 905 910Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly 915 920 925Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly 930 935 940Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly945 950 955 960Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 965 970 975Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu 980 985 990Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn 995 1000 1005Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln 1010 1015 1020His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 1025 1030 1035Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly 1040 1045 1050Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro 1055 1060 1065Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe 1070 1075 1080Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met 1085 1090 1095Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro 1100 1105 1110Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile 1115 1120 1125Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile 1130 1135 1140Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser 1145 1150 1155Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu 1160 1165 1170Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr 1175 1180 1185Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 1190 1195 1200Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu 1205 1210 1215Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly 1220 1225 1230His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln 1235 1240 1245Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn 1250 1255 1260Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 1265 1270 1275Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg 1280 1285 1290Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr 1295 1300 1305Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr 1310 1315 1320Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile 1325 1330 1335Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg 1340 1345 1350Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu 1355 1360 1365Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met 1370 1375 1380Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr 1385 1390 1395Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu 1400 1405 1410His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn 1415 1420 1425Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val 1430 1435 1440Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met 1445 1450 1455Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln 1460 1465 1470Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly 1475 1480 1485Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro 1490 1495 1500Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 1505 1510 1515Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp 1520 1525 1530Leu Tyr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 1535 1540 1545Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 1550 1555 1560Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 1565 1570 1575Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 1580 1585 1590Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 1595 1600 1605Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 1610 1615 1620Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 1625 1630 1635Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 1640 1645 1650Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 1655 1660 1665Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 1670 1675 1680Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 1685 1690 1695Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 1700 1705 1710Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 1715 1720 1725Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 1730 1735 1740His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 1745 1750 1755Ser Pro Gly Lys 17601161726PRTArtificial SequencepSYNFVIII 271 protein sequence 116Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 35 40 45Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 50 55 60Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Ala Ser Ser Ser65 70 75 80Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro 85 90 95Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe 100 105 110Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro 115 120 125Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr 130 135 140Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His145 150 155 160Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp 165 170 175Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly 180 185 190Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met 195 200 205Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp 210 215 220Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys225 230 235 240Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe 245 250 255Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu 260 265 270Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala 275 280 285Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro 290 295 300Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly305 310 315 320Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr 325 330 335Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile 340 345 350Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu 355 360 365Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr 370 375 380Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn385 390 395 400Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met 405 410 415Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile 420 425 430Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala 435 440 445Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp 450 455 460Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile465 470 475 480Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr 485 490 495Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro 500 505 510Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn 515 520 525Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val 530 535 540Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys545 550 555 560Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val 565 570 575Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg 580 585 590Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu 595 600 605Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly 610 615 620Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe625 630 635 640Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu 645 650 655Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser 660 665 670Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu 675 680 685Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly 690 695 700Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys705 710 715 720His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly 725 730 735Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly 740 745 750Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys 755 760 765Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr 770 775 780Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro785 790 795 800Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Ala Glu 805 810 815Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp 820 825 830Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp 835 840 845Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His 850 855 860Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser865 870 875 880Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln 885 890 895Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln 900 905 910Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro 915 920 925Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn 930 935 940Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu945 950 955 960Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro 965 970 975Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro 980 985 990Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 995 1000 1005Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 1010 1015 1020Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val 1025 1030 1035Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr 1040 1045 1050Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala 1055 1060 1065Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr 1070 1075 1080Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly 1085 1090 1095Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser 1100 1105 1110Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His 1115 1120 1125Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr 1130 1135 1140Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser 1145 1150 1155Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu 1160 1165 1170His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys 1175 1180 1185Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln 1190 1195 1200Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala 1205 1210 1215Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu 1220 1225 1230Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile 1235 1240 1245His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu 1250 1255 1260Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys 1265 1270 1275Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe 1280 1285 1290Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn 1295 1300 1305Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr 1310 1315 1320Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu 1325 1330 1335Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser 1340 1345 1350Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala 1355 1360 1365Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser 1370 1375 1380Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln 1385 1390 1395Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln 1400 1405 1410Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu 1415 1420 1425Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln 1430 1435 1440Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr 1445 1450 1455Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu 1460 1465 1470Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met 1475 1480 1485Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His 1490 1495 1500Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 1505 1510 1515Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 1520 1525 1530Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

Val Ser His Glu 1535 1540 1545Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 1550 1555 1560His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 1565 1570 1575Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 1580 1585 1590Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 1595 1600 1605Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 1610 1615 1620Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 1625 1630 1635Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 1640 1645 1650Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 1655 1660 1665Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 1670 1675 1680Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 1685 1690 1695Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 1700 1705 1710His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1715 1720 17251171901PRTArtificial SequencepSYN FVIII protein sequence 272 117Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu 35 40 45Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 50 55 60Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala65 70 75 80Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 85 90 95Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 100 105 110Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser 115 120 125Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 130 135 140Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly145 150 155 160Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 165 170 175Ser Pro Thr Ser Thr Glu Glu Gly Ala Ser Ser Ser Asp Leu Gly Glu 180 185 190Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro 195 200 205Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr 210 215 220Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu225 230 235 240Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr 245 250 255Leu Lys Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val 260 265 270Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser 275 280 285Gln Arg Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr 290 295 300Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro305 310 315 320Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp 325 330 335Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser 340 345 350Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe 355 360 365Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 370 375 380Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met385 390 395 400His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly 405 410 415Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr 420 425 430Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg 435 440 445Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr 450 455 460Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His465 470 475 480Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp 485 490 495Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala 500 505 510Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg 515 520 525Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala 530 535 540Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu545 550 555 560Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr 565 570 575Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr 580 585 590Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg 595 600 605Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly 610 615 620Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg625 630 635 640Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr 645 650 655Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 660 665 670Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp 675 680 685Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser 690 695 700Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu705 710 715 720Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met 725 730 735Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg 740 745 750Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala 755 760 765Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His 770 775 780Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu785 790 795 800His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 805 810 815Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val 820 825 830Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe 835 840 845Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser 850 855 860Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys865 870 875 880Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser 885 890 895Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser 900 905 910Gln Asn Gly Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 915 920 925Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser 930 935 940Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser945 950 955 960Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly 965 970 975Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro 980 985 990Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr 995 1000 1005Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu 1010 1015 1020Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1025 1030 1035Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 1040 1045 1050Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser 1055 1060 1065Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1070 1075 1080Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 1085 1090 1095Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro 1100 1105 1110Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser 1115 1120 1125Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr 1130 1135 1140Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 1145 1150 1155Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro 1160 1165 1170Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr 1175 1180 1185Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser 1190 1195 1200Ala Pro Ala Ser Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu 1205 1210 1215Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1220 1225 1230Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1235 1240 1245Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1250 1255 1260Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1265 1270 1275Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 1280 1285 1290Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 1295 1300 1305Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1310 1315 1320His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1325 1330 1335Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1340 1345 1350Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1355 1360 1365Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr 1370 1375 1380Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu 1385 1390 1395Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu 1400 1405 1410Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His 1415 1420 1425Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln 1430 1435 1440Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1445 1450 1455Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1460 1465 1470Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1475 1480 1485Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1490 1495 1500Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1505 1510 1515Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1520 1525 1530Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 1535 1540 1545Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 1550 1555 1560Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 1565 1570 1575Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 1580 1585 1590Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala 1595 1600 1605Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His 1610 1615 1620Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 1625 1630 1635Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile 1640 1645 1650Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser 1655 1660 1665Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 1670 1675 1680Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 1685 1690 1695Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 1700 1705 1710Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 1715 1720 1725Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 1730 1735 1740Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 1745 1750 1755Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 1760 1765 1770Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 1775 1780 1785Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 1790 1795 1800Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 1805 1810 1815Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 1820 1825 1830Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys 1835 1840 1845Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val 1850 1855 1860Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His 1865 1870 1875Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu 1880 1885 1890Gly Cys Glu Ala Gln Asp Leu Tyr 1895 19001181515PRTArtificial SequencepSYN VWF 031 protein sequence 118Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile1 5 10 15Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys65 70 75 80Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly145 150 155 160Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu225 230 235 240Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met305 310 315 320Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345

350Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp385 390 395 400Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu465 470 475 480Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln545 550 555 560Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val625 630 635 640Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp705 710 715 720Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met785 790 795 800Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly865 870 875 880Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg945 950 955 960Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060 1065Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln 1100 1105 1110His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln 1115 1120 1125Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu 1130 1135 1140Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180 1185Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly 1190 1195 1200Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Gly Gly Gly Ser Gly 1235 1240 1245Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1250 1255 1260Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser 1265 1270 1275Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 1280 1285 1290Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1295 1300 1305Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 1310 1315 1320Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 1325 1330 1335Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 1340 1345 1350Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 1355 1360 1365Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 1370 1375 1380Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 1385 1390 1395Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 1400 1405 1410Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 1415 1420 1425Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 1430 1435 1440Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 1445 1450 1455Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 1460 1465 1470Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 1475 1480 1485Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 1490 1495 1500Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1505 1510 15151191778PRTArtificial SequencepSYN VWF 034 protein sequence 119Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile1 5 10 15Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys65 70 75 80Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly145 150 155 160Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu225 230 235 240Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met305 310 315 320Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp385 390 395 400Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu465 470 475 480Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln545 550 555 560Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val625 630 635 640Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp705 710 715 720Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met785 790 795 800Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly865 870 875 880Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg945 950 955 960Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060 1065Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln 1100 1105 1110His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln 1115 1120 1125Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu 1130 1135 1140Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180 1185Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly 1190 1195 1200Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Thr Ser Glu Ser Ala 1235 1240 1245Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 1250 1255 1260Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 1265 1270 1275Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser

1280 1285 1290Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro 1295 1300 1305Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 1310 1315 1320Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 1325 1330 1335Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser 1340 1345 1350Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser 1355 1360 1365Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser 1370 1375 1380Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 1385 1390 1395Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser 1400 1405 1410Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala 1415 1420 1425Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 1430 1435 1440Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 1445 1450 1455Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser 1460 1465 1470Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro 1475 1480 1485Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 1490 1495 1500Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 1505 1510 1515Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Asp Ile Gly 1520 1525 1530Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly 1535 1540 1545Ser Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1550 1555 1560Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1565 1570 1575Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 1580 1585 1590Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 1595 1600 1605Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 1610 1615 1620Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 1625 1630 1635Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 1640 1645 1650Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 1655 1660 1665Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 1670 1675 1680Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 1685 1690 1695Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 1700 1705 1710Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 1715 1720 1725Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 1730 1735 1740Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 1745 1750 1755Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 1760 1765 1770Leu Ser Pro Gly Lys 17751201565PRTArtificial SequencepSYN VWF 036 protein sequence 120Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile1 5 10 15Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys65 70 75 80Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly145 150 155 160Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu225 230 235 240Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met305 310 315 320Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp385 390 395 400Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu465 470 475 480Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln545 550 555 560Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val625 630 635 640Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp705 710 715 720Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met785 790 795 800Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly865 870 875 880Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg945 950 955 960Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060 1065Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln 1100 1105 1110His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln 1115 1120 1125Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu 1130 1135 1140Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180 1185Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly 1190 1195 1200Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Gly Gly Gly Ser Gly 1235 1240 1245Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1250 1255 1260Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1265 1270 1275Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1280 1285 1290Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1295 1300 1305Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu 1310 1315 1320Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1325 1330 1335Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1340 1345 1350Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 1355 1360 1365Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 1370 1375 1380Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 1385 1390 1395Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 1400 1405 1410Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 1415 1420 1425Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 1430 1435 1440Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 1445 1450 1455Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 1460 1465 1470Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 1475 1480 1485Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 1490 1495 1500Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 1505 1510 1515Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 1520 1525 1530Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 1535 1540 1545Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 1550 1555 1560Gly Lys 1565121247PRTArtificial SequencepSYN Fc-015 protein sequence 121Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 24512233PRTArtificial Sequencepolypeptide 122Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser Gly 20 25 30Gly1231240PRTArtificial SequenceVWF-D1D2D'D3 123Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile1 5 10 15Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys65 70 75 80Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90

95Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly145 150 155 160Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu225 230 235 240Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met305 310 315 320Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp385 390 395 400Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu465 470 475 480Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln545 550 555 560Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val625 630 635 640Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp705 710 715 720Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met785 790 795 800Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly865 870 875 880Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg945 950 955 960Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn 1010 1015 1020Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln 1040 1045 1050Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe 1055 1060 1065Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln 1100 1105 1110His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln 1115 1120 1125Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu 1130 1135 1140Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln 1145 1150 1155His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys 1160 1165 1170His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln 1175 1180 1185Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly 1190 1195 1200Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230Cys Glu Ala Cys Gln Glu Pro 1235 1240124477PRTArtificial SequenceVWF-D'D3 124Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp1 5 10 15Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys65 70 75 80Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr 85 90 95Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys145 150 155 160Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln225 230 235 240Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val305 310 315 320Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345 350Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu385 390 395 400Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro465 470 4751255055DNAArtificial SequencepSYNFVIII 010 125atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280ttctctcaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta cgacaaaact 4380cacacatgcc caccgtgccc agctccagaa ctcctgggcg gaccgtcagt cttcctcttc 4440cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 4500gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 4560gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 4620agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 4680tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 4740cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 4800agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 4860aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgttggactc cgacggctcc 4920ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 4980tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 5040tctccgggta aatga

50551261684PRTArtificial SequencepSYNFVIII 010 126Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu785 790 795 800Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His865 870 875 880Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp945 950 955 960Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1010 1015 1020Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1025 1030 1035Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1040 1045 1050Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1055 1060 1065Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1070 1075 1080Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120 1125Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1145 1150 1155Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1160 1165 1170Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1175 1180 1185Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1250 1255 1260Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1265 1270 1275Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1280 1285 1290Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1295 1300 1305Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360 1365Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1385 1390 1395Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1400 1405 1410Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1415 1420 1425Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1445 1450 1455Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1460 1465 1470Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1475 1480 1485Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 1490 1495 1500Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1505 1510 1515Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1520 1525 1530Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 1535 1540 1545Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 1550 1555 1560Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1565 1570 1575Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1580 1585 1590Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 1595 1600 1605Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1610 1615 1620Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1625 1630 1635Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1640 1645 1650Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1655 1660 1665Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1670 1675 1680Lys12778PRTArtificial SequenceAE72 XTEN 127Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser1 5 10 15Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala 20 25 30Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu 35 40 45Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr 50 55 60Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ala Ser Ser65 70 75128144PRTArtificial SequenceAE144_2A 128Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly1 5 10 15Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly 20 25 30Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 35 40 45Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu 50 55 60Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu65 70 75 80Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 85 90 95Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu 100 105 110Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 115 120 125Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 130 135 140129144PRTArtificial SequenceAE144_3B 129Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser1 5 10 15Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser 20 25 30Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 35 40 45Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu 50 55 60Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly65 70 75 80Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 85 90 95Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu 100 105 110Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 115 120 125Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 130 135 140130144PRTArtificial SequenceAE144_4A 130Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala1 5 10 15Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 20 25 30Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 35 40 45Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu 50 55 60Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu65 70 75 80Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly 85 90 95Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly 100 105 110Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu 115 120 125Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 130 135 140131144PRTArtificial SequenceAE144_5A 131Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala1 5 10 15Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 20 25 30Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly 35 40 45Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu 50 55 60Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser65 70 75 80Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 85 90 95Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser 100 105 110Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser 115 120 125Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly 130 135 140132144PRTArtificial SequenceAE144_6B 132Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser1 5 10 15Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 20 25 30Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly 35 40 45Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala 50 55 60Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser65 70 75 80Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 85

90 95Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala 100 105 110Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu 115 120 125Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly 130 135 140133144PRTArtificial SequenceAG144_A 133Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro1 5 10 15Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr 20 25 30Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 35 40 45Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 50 55 60Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser65 70 75 80Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 85 90 95Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly 100 105 110Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 115 120 125Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 130 135 140134144PRTArtificial SequenceAG144_B 134Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr1 5 10 15Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 20 25 30Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro 35 40 45Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 50 55 60Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr65 70 75 80Gly Thr Gly Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro 85 90 95Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro 100 105 110Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 115 120 125Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 130 135 140135144PRTArtificial SequenceAG144_C 135Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro1 5 10 15Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser 20 25 30Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 35 40 45Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly 50 55 60Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser65 70 75 80Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 85 90 95Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr 100 105 110Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 115 120 125Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 130 135 140136144PRTArtificial SequenceAG144_F 136Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro1 5 10 15Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser 20 25 30Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 35 40 45Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro 50 55 60Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser65 70 75 80Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro 85 90 95Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr 100 105 110Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala 115 120 125Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro 130 135 140137288PRTArtificial SequenceAE288_2 137Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu1 5 10 15Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly 20 25 30Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro 35 40 45Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 50 55 60Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu65 70 75 80Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 85 90 95Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr 100 105 110Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr 115 120 125Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 130 135 140Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro145 150 155 160Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro 165 170 175Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro 180 185 190Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr 195 200 205Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro 210 215 220Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu225 230 235 240Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala 245 250 255Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro 260 265 270Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro 275 280 28513820PRTArtificial Sequencelinker 138Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser 201395PRTArtificial SequencelinkerREPEAT(1)..(5)repeat 1 to 10 times 139Gly Gly Gly Gly Ser1 51406PRTArtificial SequencelinkerREPEAT(2)..(6)Repeat 1 to 10 times 140Ser Gly Gly Gly Gly Ser1 514112PRTArtificial SequenceAD 141Gly Glu Ser Pro Gly Gly Ser Ser Gly Ser Glu Ser1 5 1014212PRTArtificial SequenceAD 142Gly Ser Glu Gly Ser Ser Gly Pro Gly Glu Ser Ser1 5 1014312PRTArtificial SequenceAD 143Gly Ser Ser Glu Ser Gly Ser Ser Glu Gly Gly Pro1 5 1014412PRTArtificial SequenceAD 144Gly Ser Gly Gly Glu Pro Ser Glu Ser Gly Ser Ser1 5 1014512PRTArtificial SequenceAE, AM 145Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu1 5 1014612PRTArtificial SequenceAE, AM, AQ 146Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro1 5 1014712PRTArtificial SequenceAE, AM, AQ 147Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro1 5 1014812PRTArtificial SequenceAE, AM, AQ 148Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro1 5 1014912PRTArtificial SequenceAF, AM 149Gly Ser Thr Ser Glu Ser Pro Ser Gly Thr Ala Pro1 5 1015012PRTArtificial SequenceAF, AM 150Gly Thr Ser Thr Pro Glu Ser Gly Ser Ala Ser Pro1 5 1015112PRTArtificial SequenceAF, AM 151Gly Thr Ser Pro Ser Gly Glu Ser Ser Thr Ala Pro1 5 1015212PRTArtificial SequenceAF, AM 152Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro1 5 1015312PRTArtificial SequenceAG, AM 153Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro1 5 1015412PRTArtificial SequenceAG, AM 154Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro1 5 1015512PRTArtificial SequenceAG, AM 155Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro1 5 1015612PRTArtificial SequenceAG, AM 156Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro1 5 1015712PRTArtificial SequenceAQ 157Gly Glu Pro Ala Gly Ser Pro Thr Ser Thr Ser Glu1 5 1015812PRTArtificial SequenceAQ 158Gly Thr Gly Glu Pro Ser Ser Thr Pro Ala Ser Glu1 5 1015912PRTArtificial SequenceAQ 159Gly Ser Gly Pro Ser Thr Glu Ser Ala Pro Thr Glu1 5 1016012PRTArtificial SequenceAQ 160Gly Ser Glu Thr Pro Ser Gly Pro Ser Glu Thr Ala1 5 1016112PRTArtificial SequenceAQ 161Gly Pro Ser Glu Thr Ser Thr Ser Glu Pro Gly Ala1 5 1016212PRTArtificial SequenceAQ 162Gly Ser Pro Ser Glu Pro Thr Glu Gly Thr Ser Ala1 5 1016312PRTArtificial SequenceBC 163Gly Ser Gly Ala Ser Glu Pro Thr Ser Thr Glu Pro1 5 1016412PRTArtificial SequenceBC 164Gly Ser Glu Pro Ala Thr Ser Gly Thr Glu Pro Ser1 5 1016512PRTArtificial SequenceBC 165Gly Thr Ser Glu Pro Ser Thr Ser Glu Pro Gly Ala1 5 1016612PRTArtificial SequenceBC 166Gly Thr Ser Thr Glu Pro Ser Glu Pro Gly Ser Ala1 5 1016712PRTArtificial SequenceBD 167Gly Ser Thr Ala Gly Ser Glu Thr Ser Thr Glu Ala1 5 1016812PRTArtificial SequenceBD 168Gly Ser Glu Thr Ala Thr Ser Gly Ser Glu Thr Ala1 5 1016912PRTArtificial SequenceBD 169Gly Thr Ser Glu Ser Ala Thr Ser Glu Ser Gly Ala1 5 1017012PRTArtificial SequenceBD 170Gly Thr Ser Thr Glu Ala Ser Glu Gly Ser Ala Ser1 5 1017135PRTArtificial Sequencelinker 171Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly1 5 10 15Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly 20 25 30Ser Gly Gly 35

Claims

1. A chimeric protein comprising (i) a von Willebrand Factor (VWF) protein comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein.

2. A chimeric protein comprising a formula, which comprises: V-X-FVIII, (a) FVIII-X-V, (b) V-X:FVIII, (c) X-V:FVIII, (d) FVIII:V-X, or (e) FVIII:X-V, (f) wherein V comprises a VWF fragment, X comprises one or more XTEN sequences, FVIII comprises a FVIII protein; (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond or a non-covalent bond.

3. The chimeric protein of claim 1, wherein the XTEN sequence is linked to the FVIII protein by a linker.

4-12. (canceled)

13. The chimeric protein of claim 1, further comprising a first Ig constant region or a portion thereof.

14-16. (canceled)

17. The chimeric protein of claim 13, wherein the chimeric protein comprises a second Ig constant region, wherein the second Ig constant region is linked to the VWF fragment by a linker.

18-19. (canceled)

20. The chimeric protein of claim 17, wherein the second Ig constant region or a portion thereof is associated with the first Ig constant region or a portion thereof.

21. The chimeric protein of claim 17, wherein the second Ig constant region or a portion thereof is associated with the first Ig constant region or a portion thereof by a covalent bond.

22-67. (canceled)

68. The chimeric protein of claim 1, wherein the FVIII protein is linked to an XTEN sequence at the C-terminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein or any combinations thereof.

69. (canceled)

70. The chimeric protein of claim 1, wherein the FVIII protein comprises one or more domains of FVIII selected from the group consisting of an A1 domain, a1 acidic region, an A2 domain, a2 acidic region, a B domain, an A3 domain, a3 acidic region, a C1 domain, a C2 domain, one or more fragments thereof, and any combinations thereof.

71-74. (canceled)

75. The chimeric protein of claim 1, wherein the FVIII protein comprises a B domain or a portion thereof.

76-89. (canceled)

90. The chimeric protein of claim 1, wherein the amino acid sequence of the D' domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2.

91. The chimeric protein of claim 1, wherein the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, or 99%, identical to amino acids 867 to 1240 of SEQ ID NO: 2.

92-95. (canceled)

96. The chimeric protein of claim 91, wherein the VWF fragment contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2.

97-119. (canceled)

120. A polynucleotide or a set of polynucleotides encoding the chimeric protein of claim 1.

121. (canceled)

122. A vector comprising the polynucleotide or set of polynucleotides of claim 120 and one or more promoter operably linked to the polynucleotide or the set of polynucleotides.

123. (canceled)

124. A host cell comprising the polynucleotide of or set of polynucleotides of claim 120.

125-126. (canceled)

127. A pharmaceutical composition comprising the chimeric protein of claim 1, and a pharmaceutically acceptable carrier.

128-137. (canceled)

138. A method of extending or increasing half-life of a FVIII protein in a subject, wherein the method comprises administering an effective amount of the chimeric protein of claim 1 to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases half-life of the FVIII protein.

139-142. (canceled)

143. A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein of claim 1, wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath.

144-147. (canceled)

148. A method of making a chimeric protein, comprising transfecting a host cell with the polynucleotide or set of polynucleotides of claim 120 and expressing the chimeric protein in the host cell.

149-165. (canceled)