Factor VIII complex combined with XTEN and Von Willebrand protein: Factor and its applications.

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

FACTOR VIII COMPLEX WITH XTEN AND VON WILLEBRAND FACTORPROTEIN, AND USES THEREOFBACKGROUND OF THE INVENTION

[0001] Haemophilia A is a bleeding disorder caused by defects in the geneencoding 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 ~ 1.7 fold compared with AD V ATE® in hemophilia A patients. See Id. Therefore, the half-life increases, despite minor improvements, may indicate the presence of other Tl / 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.

[0002] 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 October 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.

[0003] 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 themaintenance 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 200kD) and a light chain (MW 73kD). 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.

[0004] Due to the frequent dosing and inconvenience caused by the dosingschedule, 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

[0005] The present invention is directed to a chimeric protein comprising (i) a vonWillebrand 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 twopolypeptide 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.

[0006] In one embodiment, the chimeric protein of the invention comprises a formula comprising:(a) V-X-FVIII,(b) FVIII-X-V,(c) V-X:FVIII,(d) X-V:FVIII,(e) FVIILV-X, or(f) FVIILX-V,wherein V comprises a VWF fragment,X comprises one or more XTEN sequences, andFVIII 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.

[0007] In another embodiment, the chimeric protein further comprises (iv) an immunoglobulin (Ig) constant region or a portion thereof (also indicated as Fl 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.

[0008] 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 (Fl). The second Ig constant region or a portion thereof (F2) and the first Ig constant region or a portion thereof (Fl) 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 FVIIIprotein 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.

[0009] In some embodiments, the chimeric protein comprises a formulacomprising:(g) V-L2-X-L1-F1 :FVm-L3-F2;GO V-L2-X-L1-F1 :F2-L3-FVIII;(i) F 1 -L 1 -X-L2-V : FVIII-L3 -F2;0)F 1 -L 1 -X-L2-V:F2-L3 -FVIII;(k) V-L2-X-L 1 -F 1 -L4-FVIII-L3 -F2;(1) F2-L3-FVIII-L4-F 1 -L 1 -X-L2-V;(m) FVIII-L3-F2-L4-V-L2-X-L1-F1; or(n) F 1 -L 1 -X-L2-V-L4-F2-L3-FVIII,wherein V comprises a VWF fragment,each of LI, 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,Fl 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.

[0010] The present invention is also directed to a chimeric protein comprising (i) aFVIII 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 insertedimmediately 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 cancomprise 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.In other aspects, a chimeric protein comprises (i) a FVIII protein, (ii) anXTEN 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:(1) FVIII(X1 )-L 1 -F 1 : V-L2-X2-L3-F2;(2) FVIII(X1 )-L 1 -F 1 :F2-L3-X2-L2-V;(3) F 1 -L 1 -FVIII(X 1 ) : V-L2-X2-L3 -F2;(4) F 1 -L 1 -FVIII(X 1 ) :F2-L3 -X2-L2-V;(5) FVIII(X1 )-L 1 -F 1 -L4-V-L2-X2-L3-F2;(6) FVIII(X1 )-L 1 -F 1 -L4-F2-L3-X2-L2-V;(?) F 1 -L 1 -FVIII(X1 )-L4-V-L2-X2-L3-F2, or(8) F 1 -L 1 -FVIII(X1 )-L4-F2-L3-X2-L2-V,wherein FVIII(Xl) 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 LI, 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;Fl comprises an Ig constant region or a portion thereof;F2 comprises an optional additional Ig constant region or a portion thereof, andV comprises a VWF fragment;(-) is a peptide bond or one or more amino acids; and(:) comprises a covalent bond or a non-covalent bond.

[0012] One aspect of the invention is that the VWF fragment useful for thechimeric 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.

[0013] 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.

[0014] 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 theFVIII protein by an optional linker. The additional Ig constant region or a portion thereof can comprise a second Fc region.

[0015] 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.

[0016] 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 Al domain, the al acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the CI 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 Al domain and al acidic region, the al 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 CI domain, the CI domain and C2 domain, and any combinations thereof or between two domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al 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 C 1 domain, the C 1 domain and C2 domain, and any combinations thereof.

[0017] In one embodiment, the one or more insertion sites are locatedimmediately 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, 1 1 , or any combinations thereof.

[0018] 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, afirst XTEN sequence inserted within the a3 region, and a second XTEN sequence inserted within a permissive loop in the FVIII protein (e.g., Al-1 , Al-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., Al-1 , Al-2, A2-1 , A2-2, A3-1 , or A3- 2)·In certain embodiments, the one or more insertion sites for one or moreXTEN insertions are immediately downstream of one or more amino acids (numbered relative to mature FVIII sequence) selected from the group consisting of:(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 1 16, (1 1) amino acid 1 19, (12) amino acid 130,(13) amino acid 188, (14) amino acid 21 1 , (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 171 1 , (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.

[0020] In some embodiments, one XTEN is inserted in the FVIII protein. In some embodiments, two XTENs are inserted in the FVIII protein. In someembodiments, 3 XTENs are inserted in the FVIII protein.

[0021] 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.

[0022] In some embodiments, the FVIII protein is a dual chain FVIII isoform. In some embodiments, the FVIII protein is a single chain FVIII isoform.

[0023] 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 someembodiments, 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 IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 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.

[0024] 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.

[0025] 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.

[0026] In some embodiments, the FVIII protein contains at least one amino acid substitution, which induces the FVIII protein to be more stable than a FVIIIprotein 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.

[0027] 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 Dl domain, the D2 domain, or the Dl and D2 domains of VWF.

[0028] The VWF fragment may further comprise a VWF domain selected from the group consisting of the Al domain, the A2 domain, the A3 domain, the D4 domain, the Bl domain, the B2 domain, the B3 domain, the CI 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 Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al 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.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 (Gly4Ser)n(SEQ ID NO: 139) or S(Gly4Ser)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 (Gly4Ser)nlinker can be (Gly4Ser)3(SEQ ID NO: 63) or (Gly4Ser)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 Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase-2, Granzyme-B, TEV, Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP-20, e.g., TLDPPvSFLLPvNPNDKYEPFWEDEEK (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.

[0030] 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.

[0031] The chimeric protein of the invention can be polysialylated, pegylated, or hesylated.

[0032] The present invention is also directed to a polynucleotide or a set ofpolynucleotides 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 ofpolynucleotides 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 ofVWF.

[0033] 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 about2.5 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.

[0034] 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.

[0035] The invention is also directed to a method of preventing or inhibitingbinding 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. Thepresent 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.

[0036] 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 μg / kg to 500 mg / kg.

[0037] 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

[0038] Figure 1A-D. Schematic diagrams of VWF fragments. Fig. 1 A 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 D1D2DO3 domains in addition toalanine residues substituting cysteines at residues 336 and 379 of SEQ ID NO: 123 Fig. IB shows VWF-031, which contains the D1D2DO3 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 D1D2DO3 domains contained in pLIVE vector, and VWF-029, which is a nucleotide sequence encoding D1D2DO3 domains with two amino acid substitutions, C336A and C379A, in pLIVE vector. Fig. ID shows full-length VWF fragment comprising propeptide (the Dl and D2 domains) and mature subunits (the D', D3, Al, A2, A3, D4, Bl-3, Cl-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.

[0039] Figure 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. Figure 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). Figure 2B shows FVIII activity measured by a FVIII chromogenic assay after IV dosing of rFVIII-XTEN288 in D1D2DO3 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.

[0040] Figure 3. Schematic diagram of exemplary VWF:FVIII heterodimerconstructs. 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 Fcregion. 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.

[0041] Figure 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.

[0042] Figure 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 lOOug / mouse dose. ConstructFVIII- 170 was administered to FVIII: VWF DKO mice by HDI at 50dose. 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.

[0043] Figure 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.

[0044] Figure 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). Figure 7 A shows binding response in nanomoles of FVIII 169 and FVIIIFcdrug substance (a positive control) to immobilized hVWF. Figure 7B shows binding response of human IgGl (a negative control) to immobilized human VWF.

[0045] Figure 8. Pharmacokinetic (PK) profile of FVIII- 169 in HernA andFVIILVWF double knockout (DKO) mice. Figure 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. Figure 8B shows the PK profile of FVIII- 169 / VWF-031, FVIII- 169 / Fc, and FVIIIFc in FVIII / VWF DKO mice.

[0046] Figure 9. PK profile of FVIII-XTEN variants in D'D3 expressingFVIII / VWF DKO mice. Figure 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. Figure 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.

[0047] Figure 10. FVIII activity of FVIIIFc (hollow triangle), FVIII169:Fc (filled circle), and FVIII 169 :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). FVIII 169 isdescribed 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. FVIII 169 :VWF31 contains VWF31 in addition to the Fc dimer, FVIII 169 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.

[0048] Figure 11. Effects of Fc, XTEN, and VWF-DO3 fragments on FVIII half- life extension. BDD-FVIII (REFACTO®) (square), FVIIFc (circle), FVIII169 / Fc (triangle), and FVIII 169 / VWF031 (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.

[0049] Figure 12A-C. Effects of different XTENs in rFVIII-XTEN / VWFheterodimer in HemA mice. Figure 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- 169 / VWF-031 (filled circle), FVIII- 199 / VWF-031 (filled square), and FVIII- 195 / VWF031 (hollow square) were administered in HemA mice to measure the FVIII plasma activity. Figure 12B shows the half-life extension effect of the second XTEN insertionimmediately 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-204 / VWF031 (filled triangle), FVIII- 169 / VWF-031 (filled circle), FVIII-203 / VWF-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. Figure 12C shows the half-life extension effect of the two XTEN insertions immediately downstream of residues 18 (A 1 domain) and 745 (B domain) {i.e., FVIII-205) compared to FVIII-169 (a single XTEN insertion in the B domain) andsingle chain FVIII without any Fc regions or any XTENs (i.e., FVIII-207). Figure 12C additionally shows the half-life extension effect of three XTEN insertions incorporated immediately downstream of residues 26 (Al 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-205 / VWF-031 (filled square), FVIII-20 l / VWF-031 (inverted triangle), FVIII- 169 / VWF-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).

[0050] Figure 13. FVIII activity of rFVIII-XTEN / VWF-XTEN heterodimer inFVIII / 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 745corresponding to mature FVIII polypeptide) of FVIII.

[0051] Figure 14A-H. Schematic diagrams of various rFVIII-XTEN / VWFconstructs. 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. Figure 14B shows two polypeptide chain construct (FVIII 155 / VWF031), 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 (FVIII 199 / VWF031), 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 (FVIII201 / VWF031), the first chain comprising singlechain 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 (FVIII 169 / 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(FVIII203 / VWF031), 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(FVIII204 / VWF031), 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(FVIII205 / VWF031), 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.Figure 15. FVIII activity of rFVIII-XTEN / VWF and BDD-F VIII inFVIII / 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.

[0053] Figure 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 FVIII169 / VWF034, FVIII205 / VWF031, and BDD-FVIII. The median blood loss in ml for 200 IU / kg of FVIII169 / VWF034 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

[0054] 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.

[0055] 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 aconventional 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 ofpolynucleotides 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.

[0056] 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 singlepolynucleotide 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.

[0057] Certain proteins secreted by mammalian cells are associated with asecretory 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 (TP A) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0058] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotidesequences 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 β-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).

[0063] 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).

[0064] The term "expression" as used herein refers to a process by which apolynucleotide 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.

[0065] A "vector" refers to any vehicle for the cloning of and / or transfer of anucleic 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.

[0066] 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: lucif erase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers may also be considered to be reporters.

[0067] The term "plasmid" refers to an extra-chromosomal element oftencarrying 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] "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.

[0073] 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.

[0074] 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.

[0075] Also included in the present invention are fragments or variants ofpolypeptides, 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.

[0076] The term "VWF fragment" or "VWF fragments" used herein means anyVWF 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., preventingpremature 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.

[0077] 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 beconservatively replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0078] 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, WI 5371 1). 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 iscalculated 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.

[0079] 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.

[0080] 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 whichcorresponds 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.

[0081] 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."

[0082] 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 orprocess 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.

[0083] 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.

[0084] 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 a- phase and longer β-phase. The a-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 β-phase typically represents the catabolism of the polypeptide in the intravascular space. In someembodiments, 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 β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

[0085] 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 (-).

[0086] 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 CHI 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 examplesof 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.

[0087] 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.

[0088] 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.

[0089] 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 carboxyterminalside 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 PCl / 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.

[0090] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0091] 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.

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

[0094] Hemostatic disorder, as used herein, means a genetically inherited oracquired condition characterized by a tendency to hemorrhage, eitherspontaneously 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 bleedingtendency), and thrombasthenia of Glanzman and Naegeli (Glanzmannthrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this may increase bleeding risk.

[0095] 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.

[0096] The chimeric protein of the invention is also used for on-demandtreatment. 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.

[0097] As used herein the term "acute bleeding" refers to a bleeding episoderegardless 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.

[0098] 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, 17IU / 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 PROTEINSThe 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. TheXTEN 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.

[0100] In one embodiment, a VWF fragment is associated (or linked) with theFVIII 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 certainembodiments, 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.

[0101] 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, theVWF 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].

[0102] 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.

[0103] 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.

[0104] In certain embodiments, a chimeric protein of the invention comprises aFVIII 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.

[0105] 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

[0106] The present invention is directed to a chimeric protein comprising (i) aVWF 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.

[0107] 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., (al) 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 anotherembodiment, 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.

[0108] In certain embodiments, a chimeric protein of the invention comprises a formula comprising:(a) V-X-FVIII,(b) FVIII-X-V,(c) V-X:FVIII,(d) X-V:FVIII,(e) FVIII:V-X,(f) FVIII:X-V, or(a5) X-V-FVIII,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.In other embodiments, a chimeric protein of the invention comprises a formula comprising:(a) V(X1)-X2-FVIII,(b) FVIII-X2-V(X1),(c) V(X1):FVIII,(d) FVIII: V(X1), or(a5) X2-V(X1)-FVIII,wherein V(X1) comprises a VWF fragment and a first XTEN sequence (XI), 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.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:(bl) V-L1-X-L2-FVIII,0>2) FVIII-L2-X-L1-V,0>3) V-L1-X:FVIII,0)4) X-L1-V:FVIII,0)5) FVIII:V-L1-X,0)6) FVIII:X-L1-V,0)7) X-L1-V-L2-FVIII, or0)8) FVIII-L2-V-L1-X,wherein V comprises a VWF fragment,X comprises one or more XTEN sequences,FVIII comprises a FVIII protein,LI 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 sequencefrom 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 (bl) 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.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 (Fl 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. Inanother 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:(g) V-L2-X-L1-F1 : FVIII-L3-F2;GO V-L2-X-L1-F1 :F2-L3-FVni;(i) F-L1-X-L2-V: FVIII-L3-F2;0)F-L1-X-L2-V:F2-L3-FVIII;(k) V-L2-X-L 1 -F 1 -L4-FVIII-L3 -F2;(1) F2-L3-FVIII-L4-F 1 -L 1 -X-L2-V;(m) FVIII-L2-F2-L4-V-L2-X-L1-F1; or(n) F 1 -L 1 -X-L2-V-L4-F2-L2-F VIII,wherein V comprises a VWF fragment,each of LI 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,Fl 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.

[0112] 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.

[0113] 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, ahydrophilic 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) Fl- L1-X-L2-V-L4-F2-L2-FVIII means formula NH2-F1 -LI -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.In one embodiment, either or both of the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "Fl") 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 "Fl") 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. Fl or F2 can comprise an Fc region or an FcRn binding partner. In other embodiments, either or both of Fl and F2 linked to the VWF fragment and / or the FVIII protein form a covalent bond {e.g., a disulfide bond) between Fl 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, Fl and F2 are identical or different. Non-limiting examples of Fl and F2 can be selected from the group consisting of a CHI 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, Fl, F2, or bothcomprise at least one CHI 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, Fl, 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, Fl, 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 thecombination 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 Fl and a second Fc region or a second FcRn binding partner for F2. In other embodiments, Fl is linked to the VWF fragment by a linker, and / or F2 is linked to the FVIII protein by a linker. In some embodiments, Fl 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.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 orN-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.

[0116] 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.

[0117] 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 constantregion 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.

[0118] 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 specificembodiment, the second linker is a thrombin cleavable linker. The linkers useful in the invention are described elsewhere herein.(2) FVIII, XTEN, and Fc

[0119] 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 aportion 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).

[0120] The present invention is also directed to a chimeric protein comprising (i) aFVIII 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 "Fl "), 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 certainembodiments, 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.

[0121] In other aspects, the chimeric protein is a single polypeptide chaincomprising 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 regionor a portion thereof, wherein the single polypeptide chain is processed after expression by an intracellular enzyme and becomes two polypeptide chains.

[0122] In one embodiment, the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "Fl ") 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 "Fl") is an Fc region or an FcRn binding partner described elsewhere herein.

[0123] In other embodiments, the additional Ig constant region or a portionthereof (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.

[0124] 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.

[0125] In some embodiments, either or both of the Ig constant region or a portion thereof ("F" or "Fl" 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 CHI 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 CHI 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 thereofand 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.

[0126] The chimeric protein of the invention can have an extended half-life of theFVIII 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

[0127] In one aspect, a chimeric protein of the present invention comprises (i) aFVIII 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 A3domain 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.

[0128] In another aspect, a chimeric protein comprises (i) a FVIII protein, (ii) anXTEN 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).

[0129] 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.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 sequencesuch 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.In other aspects, a chimeric protein comprises a formula comprising(1) FVIII(X1)-L1-F1 :V-L2-X2-L3-F2;(2) FVIII(X1)-L1-F1 :F2-L3-X2-L2-V;(3) F 1 -L 1 -FVIII(X 1 ) : V-L2-X2-L3 -F2;(4) F 1 -L 1 -FVIII(X 1 ) :F2-L3 -X2-L2-V;(5) FVIII(X1 )-L 1 -F 1 -L4-V-L2-X2-L3-F2;(6) FVIII(X1 )-L 1 -F 1 -L4-F2-L3-X2-L2-V;(?) F 1 -L 1 -FVIII(X1 )-L4-V-L2-X2-L3-F2, or(8) F 1 -L 1 -FVIII(X1 )-L4- F2-L3-X2-L2-V, wherein FVIII(Xl) 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 LI, 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;Fl 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. 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., acleavable linker, which is further linked to the second Ig constant region or a portion thereof, and wherein the first polypeptide chain and the secondpolypeptide 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).

[0133] 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.

[0134] 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 thesecond 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.

[0135] 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.

[0136] 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 withinor 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.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.

[0138] 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

[0139] 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 (a-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 Al domain (which binds to platelet GPIb- receptor, heparin, and / or possibly collagen), the A3 domain (which binds to collagen), the CI domain (in which the RGD domain binds to platelet integrin llbp3 when this is activated), and the "cysteine knot" domain at the C-terminalend of the protein (which VWF shares with platelet-derived growth factor(PDGF), transforming growth factor-β (TGFP) and β-human chorionicgonadotropin (PHCG).

[0140] 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.

[0141] The 2813 monomer amino acid sequence for human VWF is reported asAccession Number NP 000543.2 in Genbank. The nucleotide sequence encoding the human VWF is reported as Accession Number NM_000552.3_ inGenbank. 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 1. VWF SequencesVWF domains Amino acid SequenceVWF Signal Peptide MI PARFAGVL LALALILPGT LC22(Amino acids 1 to 22of SEQ ID NO: 2)VWF D1D2 region 2 3 AEGTRGRSSTARCSLFGS DFV TFDGSM(Amino acids 23 to 5 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK 763 of SEQ ID NO: 2) RVSLSVYLGE FFDIHLFV G1 01 TVTQGDQRVS MPYASKGLYL ETEAGYYKLSGEAYGFVARI DGSGNFQVLL1 1 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTLTSDPYDFANS WALSSGEQWC2 01 ERASPPSSSC NISSGEMQKG LWEQCQLLKSTSVFARCHPL VDPEPFVALC2 51 EKTLCECAGG LECACPALLE YARTCAQEGMVLYGWTDHSA CSPVCPAGME3 01 YRQCVSPCAR TCQSLHINEM CQERCVDGCSCPEGQLLDEG LCVESTECPC3 51 VHSGKRYPPG TSLSRDCNTC ICRNSQWICSNEECPGECLV TGQSHFKSFD4 01 NRYFTFSGIC QYLLARDCQD HSFSIVIETVQCADDRDAVC TRSVTVRLPG45 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDLRIQHTVTASV RLSYGEDLQM50 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGNQGDDFLTPSG LAEPRVEDFG55 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFSEEACAVLTSP TFEACHRAVS60 PLPYLRNCRY DVCSCSDGRE CLCGALASYAAACAGRGVRV AWREPGRCEL65 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECNEACLEGCFCP PGLYMDERGD70 CVPKAQCPCY YDGEIFQPED IFSDHHTMCYCEDGFMHCTM SGVPGSLLPD AVLSSPLSHR SKR76VWF D' Domain 764 SLSCRPP MVKLVCPADNLRAEGLECTKTCONYDLECM!0: SMGCVSGCLC PPGMVRHENR CVALERCPCFHQGKEYAPGE TVKIGCNTCV CRDRKWNCTD HVCDATVWF D3 Domain 867 CSTI GMAHYLTFDGLKYLFPGECQ YVLVQDYCGS90 1 NPGTFRILVG NKGCSHPSVK CKKRV ILVEGGEIELFDGE VNVKRPMKDE95 1 THFEVVESGR YIILLLGKAL SVVWDRHLSISVVLKQTYQE KVCGLCGNFD10 01 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQCADTRKVPLD SSPATCHNNI10 51 MKQTMVDSSC RILTSDVFQD CNKLVDPEPYLDVCIYDTCS CESIGDCACF11 01 CD IAAYAHV CAQHGKVVTW RTATLCPQSCEERNLRENGY ECEWRYNSCA1 51 PACQVTCQHP EPLACPVQCV EGCHAHCPPGKILDELLQTC VDPEDCPVCE1201 KVTLNPSDPE HCQICHCDVVNLTCEACQEP■:4 oVWF Al Domain 1241 GGLVVPPTE1251 PVSPTTLYVE DISEPPLHDF YCSRLLDLVFLLDGSSRLSE AEFEVLKAFV VDMMERLRIS QKWVRVAVVE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA135: GSQVASTSEV LKYTLFQIFS KIDRPEASRIALLLMASQEP QRMSRNFVRY140: VQGLKKKKVI VIPVGIGPHA NLKQIRLIEKQAPENKAFVL SSVDELEQQRDEIVSYLCDL APEAPPPTLP PDMAQVTVG1479PGLLGVSTLGP KRNSMVLDVA FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA1600PNLVYMVTGN PASDEIKRLP GDIQVVPIGV GPNANVQELE RIGWPNAPIL IQDFETLPRE APDLVLQRCC SGEGLQIPTL SPAPDCSQPL DVILLLDGSS SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNVVPE KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNVVK LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG CMKSIEVKHS ALSVEXHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE VVTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC2601 TTCRCMVQVG VISGFKLECR KTTCNPCPLGYKEENNTGEC CGRCLPTACT2651 IQLRGGQIMT LKRDETLQDG CDTHFCKVNERGEYFWEKRV TGCPPFDEHK2701 CLAEGGKIMK IPGTCCDTCE EPECNDITARLQYVKVGSCK SEVEVDIHYC2751 QGKCASKAMY SIDINDVQDQ CSCCSPTRTEPMQVALHCTN GSVVYHEVLN2801 AMECKCSPRK CSKNucleotide Sequence (SEQ ID NO: 1)Full-length VWF 1 ATGATTCCTG CCAGATTTGCCGGGGTGCTG CTTGCTCTGG CCCTCATTTT51 GCCAGGGACC CTTTGTGCAGAAGGAACTCG CGGCAGGTCA TCCACGGCCC101 GATGCAGCCT TTTCGGAAGTGACTTCGTCA ACACCTTTGA TGGGAGCATG151 TACAGCTTTG CGGGATACTGCAGTTACCTC CTGGCAGGGG GCTGCCAGAA201 ACGCTCCTTC TCGATTATTGGGGACTTCCA GAATGGCAAG AGAGTGAGCC251 TCTCCGTGTA TCTTGGGGAATTTTTTGACA TCCATTTGTT TGTCAATGGT301 ACCGTGACAC AGGGGGACCAAAGAGTCTCC ATGCCCTATG CCTCCAAAGG351 GCTGTATCTA GAAACTGAGGCTGGGTACTA CAAGCTGTCC GGTGAGGCCT401 ATGGCTTTGT GGCCAGGATCGATGGCAGCG GCAACTTTCA AGTCCTGCTG451 TCAGACAGAT ACTTCAACAAGACCTGCGGG CTGTGTGGCA ACTTTAACAT501 CTTTGCTGAA GATGACTTTATGACCCAAGA AGGGACCTTG ACCTCGGACC551 CTTATGACTT TGCCAACTCATGGGCTCTGA GCAGTGGAGA ACAGTGGTGT601 GAACGGGCAT CTCCTCCCAGCAGCTCATGC AACATCTCCT CTGGGGAAAT651 GCAGAAGGGC CTGTGGGAGCAGTGCCAGCT TCTGAAGAGC ACCTCGGTGT701 TTGCCCGCTG CCACCCTCTGGTGGACCCCG AGCCTTTTGT GGCCCTGTGT751 GAGAAGACTT TGTGTGAGTGTGCTGGGGGG CTGGAGTGCG CCTGCCCTGC801 CCTCCTGGAG TACGCCCGGACCTGTGCCCA GGAGGGAATG GTGCTGTACG851 GCTGGACCGA CCACAGCGCGTGCAGCCCAG TGTGCCCTGC TGGTATGGAG901 TATAGGCAGT GTGTGTCCCCTTGCGCCAGG ACCTGCCAGA GCCTGCACAT951 CAATGAAATG TGTCAGGAGCGATGCGTGGA TGGCTGCAGC TGCCCTGAGG1001 GACAGCTCCT GGATGAAGGCCTCTGCGTGG AGAGCACCGA GTGTCCCTGC1051 GTGCATTCCG GAAAGCGCTACCCTCCCGGC ACCTCCCTCT CTCGAGACTG1101 CAACACCTGC ATTTGCCGAAACAGCCAGTG GATCTGCAGC AATGAAGAAT1151 GTCCAGGGGA GTGCCTTGTCACTGGTCAAT CCCACTTCAA GAGCTTTGAC1201 AACAGATACT TCACCTTCAGTGGGATCTGC CAGTACCTGC TGGCCCGGGA1251 TTGCCAGGAC CACTCCTTCTCCATTGTCAT TGAGACTGTC CAGTGTGCTG1301 ATGACCGCGA CGCTGTGTGCACCCGCTCCG TCACCGTCCG GCTGCCTGGC1351 CTGCACAACA GCCTTGTGAAACTGAAGCAT GGGGCAGGAG TTGCCATGGA1401 TGGCCAGGAC ATCCAGCTCCCCCTCCTGAA AGGTGACCTC CGCATCCAGC1451 ATACAGTGAC GGCCTCCGTGCGCCTCAGCT ACGGGGAGGA CCTGCAGATG1501 GACTGGGATG GCCGCGGGAGGCTGCTGGTG AAGCTGTCCC CCGTCTATGC1551 CGGGAAGACC TGCGGCCTGTGTGGGAATTA CAATGGCAAC CAGGGCGACG1601 ACTTCCTTAC CCCCTCTGGGCTGGCRGAGC CCCGGGTGGA GGACTTCGGG1651 AACGCCTGGA AGCTGCACGGGGACTGCCAG GACCTGCAGA AGCAGCACAG1701 CGATCCCTGC GCCCTCAACCCGCGCATGAC CAGGTTCTCC GAGGAGGCGT1751 GCGCGGTCCT GACGTCCCCCACATTCGAGG CCTGCCATCG TGCCGTCAGC1801 CCGCTGCCCT ACCTGCGGAACTGCCGCTAC GACGTGTGCT CCTGCTCGGA1851 CGGCCGCGAG TGCCTGTGCGGCGCCCTGGC CAGCTATGCC GCGGCCTGCG1901 CGGGGAGAGG CGTGCGCGTCGCGTGGCGCG AGCCAGGCCG CTGTGAGCTG1951 AACTGCCCGA AAGGCCAGGTGTACCTGCAG TGCGGGACCC CCTGCAACCT2001 GACCTGCCGC TCTCTCTCTTACCCGGATGA GGAATGCAAT GAGGCCTGCC2051 TGGAGGGCTG CTTCTGCCCCCCAGGGCTCT ACATGGATGA GAGGGGGGAC2101 TGCGTGCCCA AGGCCCAGTGCCCCTGTTAC TATGACGGTG AGATCTTCCA2151 GCCAGAAGAC ATCTTCTCAGACCATCACAC CATGTGCTAC TGTGAGGATG2201 GCTTCATGCA CTGTACCATGAGTGGAGTCC CCGGAAGCTT GCTGCCTGAC2251 GCTGTCCTCA GCAGTCCCCTGTCTCATCGC AGCAAAAGGA GCCTATCCTG2301 TCGGCCCCCC ATGGTCAAGCTGGTGTGTCC CGCTGACAAC CTGCGGGCTG2351 AAGGGCTCGA GTGTACCAAAACGTGCCAGA ACTATGACCT GGAGTGCATG2401 AGCATGGGCT GTGTCTCTGGCTGCCTCTGC CCCCCGGGCA TGGTCCGGCA2451 TGAGAACAGA TGTGTGGCCCTGGAAAGGTG TCCCTGCTTC CATCAGGGCA2501 AGGAGTATGC CCCTGGAGAAACAGTGAAGA TTGGCTGCAA CACTTGTGTC2551 TGTCGGGACC GGAAGTGGAACTGCACAGAC CATGTGTGTG ATGCCACGTG2601 CTCCACGATC GGCATGGCCCACTACCTCAC CTTCGACGGG CTCAAATACC2651 TGTTCCCCGG GGAGTGCCAGTACGTTCTGG TGCAGGATTA CTGCGGCAGT2701 AACCCTGGGA CCTTTCGGATCCTAGTGGGG AATAAGGGAT GCAGCCACCC2751 CTCAGTGAAA TGCAAGAAACGGGTCACCAT CCTGGTGGAG GGAGGAGAGA2801 TTGAGCTGTT TGACGGGGAGGTGAATGTGA AGAGGCCCAT GAAGGATGAG2851 ACTCACTTTG AGGTGGTGGAGTCTGGCCGG TACATCATTC TGCTGCTGGG2901 CAAAGCCCTC TCCGTGGTCTGGGACCGCCA CCTGAGCATC TCCGTGGTCC2951 TGAAGCAGAC ATACCAGGAGAAAGTGTGTG GCCTGTGTGG GAATTTTGAT3001 GGCATCCAGA ACAATGACCTCACCAGCAGC AACCTCCAAG TGGAGGAAGA3051 CCCTGTGGAC TTTGGGAACTCCTGGAAAGT GAGCTCGCAG TGTGCTGACA3101 CCAGAAAAGT GCCTCTGGACTCATCCCCTG CCACCTGCCA TAACAACATC3151 ATGAAGCAGA CGATGGTGGATTCCTCCTGT AGAATCCTTA CCAGTGACGT3201 CTTCCAGGAC TGCAACAAGCTGGTGGACCC CGAGCCATAT CTGGATGTCT3251 GCATTTACGA CACCTGCTCCTGTGAGTCCA TTGGGGACTG CGCCTGCTTC3301 TGCGACACCA TTGCTGCCTATGCCCACGTG TGTGCCCAGC ATGGCAAGGT3351 GGTGACCTGG AGGACGGCCACATTGTGCCC CCAGAGCTGC GAGGAGAGGA3401 ATCTCCGGGA GAACGGGTATGAGTGTGAGT GGCGCTATAA CAGCTGTGCA3451 CCTGCCTGTC AAGTCACGTGTCAGCACCCT GAGCCACTGG CCTGCCCTGT3501 GCAGTGTGTG GAGGGCTGCCATGCCCACTG CCCTCCAGGG AAAATCCTGG3551 ATGAGCTTTT GCAGACCTGCGTTGACCCTG AAGACTGTCC AGTGTGTGAG3601 GTGGCTGGCC GGCGTTTTGCCTCAGGAAAG AAAGTCACCT TGAATCCCAG3651 TGACCCTGAG CACTGCCAGATTTGCCACTG TGATGTTGTC AACCTCACCT3701 GTGAAGCCTG CCAGGAGCCGGGAGGCCTGG TGGTGCCTCC CACAGATGCC3751 CCGGTGAGCC CCACCACTCTGTATGTGGAG GACATCTCGG AACCGCCGTT3801 GCACGATTTC TACTGCAGCAGGCTACTGGA CCTGGTCTTC CTGCTGGATG3851 GCTCCTCCAG GCTGTCCGAGGCTGAGTTTG AAGTGCTGAA GGCCTTTGTG3901 GTGGACATGA TGGAGCGGCTGCGCATCTCC CAGAAGTGGG TCCGCGTGGC3951 CGTGGTGGAG TACCACGACGGCTCCCACGC CTACATCGGG CTCAAGGACC4001 GGAAGCGACC GTCAGAGCTGCGGCGCATTG CCAGCCAGGT GAAGTATGCG4051 GGCAGCCAGG TGGCCTCCACCAGCGAGGTC TTGAAATACA CACTGTTCCA4101 AATCTTCAGC AAGATCGACCGCCCTGAAGC CTCCCGCATC GCCCTGCTCC4151 TGATGGCCAG CCAGGAGCCCCAACGGATGT CCCGGAACTT TGTCCGCTAC4201 GTCCAGGGCC TGAAGAAGAAGAAGGTCATT GTGATCCCGG TGGGCATTGG4251 GCCCCATGCC AACCTCAAGCAGATCCGCCT CATCGAGAAG CAGGCCCCTG4301 AGAACAAGGC CTTCGTGCTGAGCAGTGTGG ATGAGCTGGA GCAGCAAAGG4351 GACGAGATCG TTAGCTACCTCTGTGACCTT GCCCCTGAAG CCCCTCCTCC4401 TACTCTGCCC CCCGACATGGCACAAGTCAC TGTGGGCCCG GGGCTCTTGG4451 GGGTTTCGAC CCTGGGGCCCAAGAGGAACT CCATGGTTCT GGATGTGGCG4501 TTCGTCCTGG AAGGATCGGACAAAATTGGT GAAGCCGACT TCAACAGGAG4551 CAAGGAGTTC ATGGAGGAGGTGATTCAGCG GATGGATGTG GGCCAGGACA4601 GCATCCACGT CACGGTGCTGCAGTACTCCT ACATGGTGAC CGTGGAGTAC4651 CCCTTCAGCG AGGCACAGTCCAAAGGGGAC ATCCTGCAGC GGGTGCGAGA4701 GATCCGCTAC CAGGGCGGCAACAGGACCAA CACTGGGCTG GCCCTGCGGT4751 ACCTCTCTGA CCACAGCTTCTTGGTCAGCC AGGGTGACCG GGAGCAGGCG4801 CCCAACCTGG TCTACATGGTCACCGGAAAT CCTGCCTCTG ATGAGATCAA4851 GAGGCTGCCT GGAGACATCCAGGTGGTGCC CATTGGAGTG GGCCCTAATG4901 CCAACGTGCA GGAGCTGGAGAGGATTGGCT GGCCCAATGC CCCTATCCTC4951 ATCCAGGACT TTGAGACGCTCCCCCGAGAG GCTCCTGACC TGGTGCTGCA5001 GAGGTGCTGC TCCGGAGAGGGGCTGCAGAT CCCCACCCTC TCCCCTGCAC5051 CTGACTGCAG CCAGCCCCTGGACGTGATCC TTCTCCTGGA TGGCTCCTCC5101 AGTTTCCCAG CTTCTTATTTTGATGAAATG AAGAGTTTCG CCAAGGCTTT5151 CATTTCAAAA GCCAATATAGGGCCTCGTCT CACTCAGGTG TCAGTGCTGC5201 AGTATGGAAG CATCACCACCATTGACGTGC CATGGAACGT GGTCCCGGAG5251 AAAGCCCATT TGCTGAGCCTTGTGGACGTC ATGCAGCGGG AGGGAGGCCC5301 CAGCCAAATC GGGGATGCCTTGGGCTTTGC TGTGCGATAC TTGACTTCAG5351 AAATGCATGG TGCCAGGCCGGGAGCCTCAA AGGCGGTGGT CATCCTGGTC5401 ACGGACGTCT CTGTGGATTCAGTGGATGCA GCAGCTGATG CCGCCAGGTC5451 CAACAGAGTG ACAGTGTTCCCTATTGGAAT TGGAGATCGC TACGATGCAG5501 CCCAGCTACG GATCTTGGCAGGCCCAGCAG GCGACTCCAA CGTGGTGAAG5551 CTCCAGCGAA TCGAAGACCTCCCTACCATG GTCACCTTGG GCAATTCCTT5601 CCTCCACAAA CTGTGCTCTGGATTTGTTAG GATTTGCATG GATGAGGATG5651 GGAATGAGAA GAGGCCCGGGGACGTCTGGA CCTTGCCAGA CCAGTGCCAC5701 ACCGTGACTT GCCAGCCAGATGGCCAGACC TTGCTGAAGA GTCATCGGGT5751 CAACTGTGAC CGGGGGCTGAGGCCTTCGTG CCCTAACAGC CAGTCCCCTG5801 TTAAAGTGGA AGAGACCTGTGGCTGCCGCT GGACCTGCCC CTGYGTGTGC5851 ACAGGCAGCT CCACTCGGCACATCGTGACC TTTGATGGGC AGAATTTCAA5901 GCTGACTGGC AGCTGTTCTTATGTCCTATT TCAAAACAAG GAGCAGGACC5951 TGGAGGTGAT TCTCCATAATGGTGCCTGCA GCCCTGGAGC AAGGCAGGGC6001 TGCATGAAAT CCATCGAGGTGAAGCACAGT GCCCTCTCCG TCGAGSTGCA6051 CAGTGACATG GAGGTGACGGTGAATGGGAG ACTGGTCTCT GTTCCTTACG6101 TGGGTGGGAA CATGGAAGTCAACGTTTATG GTGCCATCAT GCATGAGGTC6151 AGATTCAATC ACCTTGGTCACATCTTCACA TTCACTCCAC AAAACAATGA6201 GTTCCAACTG CAGCTCAGCCCCAAGACTTT TGCTTCAAAG ACGTATGGTC6251 TGTGTGGGAT CTGTGATGAGAACGGAGCCA ATGACTTCAT GCTGAGGGAT6301 GGCACAGTCA CCACAGACTGGAAAACACTT GTTCAGGAAT GGACTGTGCA6351 GCGGCCAGGG CAGACGTGCCAGCCCATCCT GGAGGAGCAG TGTCTTGTCC6401 CCGACAGCTC CCACTGCCAGGTCCTCCTCT TACCACTGTT TGCTGAATGC6451 CACAAGGTCC TGGCTCCAGCCACATTCTAT GCCATCTGCC AGCAGGACAG6501 TTGCCACCAG GAGCAAGTGTGTGAGGTGAT CGCCTCTTAT GCCCACCTCT6551 GTCGGACCAA CGGGGTCTGCGTTGACTGGA GGACACCTGA TTTCTGTGCT6601 ATGTCATGCC CACCATCTCTGGTCTACAAC CACTGTGAGC ATGGCTGTCC6651 CCGGCACTGT GATGGCAACGTGAGCTCCTG TGGGGACCAT CCCTCCGAAG6701 GCTGTTTCTG CCCTCCAGATAAAGTCATGT TGGAAGGCAG CTGTGTCCCT6751 GAAGAGGCCT GCACTCAGTGCATTGGTGAG GATGGAGTCC AGCACCAGTT6801 CCTGGAAGCC TGGGTCCCGGACCACCAGCC CTGTCAGATC TGCACATGCC6851 TCAGCGGGCG GAAGGTCAACTGCACAACGC AGCCCTGCCC CACGGCCAAA6901 GCTCCCACGT GTGGCCTGTGTGAAGTAGCC CGCCTCCGCC AGAATGCAGA6951 CCAGTGCTGC CCCGAGTATGAGTGTGTGTG TGACCCAGTG AGCTGTGACC7001 TGCCCCCAGT GCCTCACTGTGAACGTGGCC TCCAGCCCAC ACTGACCAAC7051 CCTGGCGAGT GCAGACCCAACTTCACCTGC GCCTGCAGGA AGGAGGAGTG7101 CAAAAGAGTG TCCCCACCCTCCTGCCCCCC GCACCGTTTG CCCACCCTTC7151 GGAAGACCCA GTGCTGTGATGAGTATGAGT GTGCCTGCAA CTGTGTCAAC7201 TCCACAGTGA GCTGTCCCCTTGGGTACTTG GCCTCAACCG CCACCAATGA7251 CTGTGGCTGT ACCACAACCACCTGCCTTCC CGACAAGGTG TGTGTCCACC7301 GAAGCACCAT CTACCCTGTGGGCCAGTTCT GGGAGGAGGG CTGCGATGTG7351 TGCACCTGCA CCGACATGGAGGATGCCGTG ATGGGCCTCC GCGTGGCCCA7401 GTGCTCCCAG AAGCCCTGTGAGGACAGCTG TCGGTCGGGC TTCACTTACG7451 TTCTGCATGA AGGCGAGTGCTGTGGAAGGT GCCTGCCATC TGCCTGTGAG7501 GTGGTGACTG GCTCACCGCGGGGGGACTCC CAGTCTTCCT GGAAGAGTGT7551 CGGCTCCCAG TGGGCCTCCCCGGAGAACCC CTGCCTCATC AATGAGTGTG7601 TCCGAGTGAA GGAGGAGGTCTTTATACAAC AAAGGAACGT CTCCTGCCCC7651 CAGCTGGAGG TCCCTGTCTGCCCCTCGGGC TTTCAGCTGA GCTGTAAGAC7701 CTCAGCGTGC TGCCCAAGCTGTCGCTGTGA GCGCATGGAG GCCTGCATGC7751 TCAATGGCAC TGTCATTGGGCCCGGGAAGA CTGTGATGAT CGATGTGTGC7801 ACGACCTGCC GCTGCATGGTGCAGGTGGGG GTCATCTCTG GATTCAAGCT7851 GGAGTGCAGG AAGACCACCTGCAACCCCTG CCCCCTGGGT TACAAGGAAG7901 AAAATAACAC AGGTGAATGTTGTGGGAGAT GTTTGCCTAC GGCTTGCACC7951 ATTCAGCTAA GAGGAGGACAGATCATGACA CTGAAGCGTG ATGAGACGCT8001 CCAGGATGGC TGTGATACTCACTTCTGCAA GGTCAATGAG AGAGGAGAGT8051 ACTTCTGGGA GAAGAGGGTCACAGGCTGCC CACCCTTTGA TGAACACAAG8101 TGTCTTGCTG AGGGAGGTAAAATTATGAAA ATTCCAGGCA CCTGCTGTGA8151 CACATGTGAG GAGCCTGAGTGCAACGACAT CACTGCCAGG CTGCAGTATG8201 TCAAGGTGGG AAGCTGTAAGTCTGAAGTAG AGGTGGATAT CCACTACTGC8251 CAGGGCAAAT GTGCCAGCAAAGCCATGTAC TCCATTGACA TCAACGATGT8301 GCAGGACCAG TGCTCCTGCTGCTCTCCGAC ACGGACGGAG CCCATGCAGG8351 TGGCCCTGCA CTGCACCAATGGCTCTGTTG TGTACCATGA GGTTCTCAAT8401 GCCATGGAGT GCAAATGCTCCCCCAGGAAG TGCAGCAAGT GAThe VWF fragment as used herein can be a VWF fragment comprising aain and a D3 domain of VWF, wherein the VWF fragment binds to FactorVIII (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 A 1 domain, an A2 domain, an A3 domain, a Dl domain, a D2 domain, a D4 domain, a Bl domain, a B2 domain, a B3 domain, a CI domain, a C2 domain, a CK domain, one or more fragments thereof, and any combinations thereof. In oneembodiment, a VWF fragment comprises, consists essentially of, or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al domains of VWF or fragments thereof. 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.In one embodiment, the VWF fragment binds to or is associated with aFVIII 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 clearancepathway 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.

[0144] 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 D l , 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.

[0145] In some embodiments, a VWF fragment of the invention consistsessentially of or consists of (1) the D'D3 domain, the D1D'D3 domain, D2D'D3 domain, or D1D2DO3 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 SEQID 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.

[0146] 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.

[0147] 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.

[0148] In still other embodiments, a VWF fragment of the present inventioncomprises, consists essentially of, or consists of an amino acid sequence corresponding to the D' domain, D3 domain, and Al 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.

[0149] 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 Al domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a Bl domain, (6) a B2 domain, (7) a B3 domain, (8) a CI 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 CI domain, (13) a CK domain, a C2 domain, a CI domain, a B3 domain, (14) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, (15) a CK domain, a C2 domain, a CIdomain, a B3 domain, a B2 domain, and a Bl domain, (16) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, and a D4 domain, (17) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, a D4 domain, and an A3 domain, (18) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, a D4 domain, an A3 domain, and an A2 domain, (19) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, a D4 domain, an A3 domain, an A2 domain, and an Al domain, and (20) any combinations thereof.

[0150] 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 April 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 Al domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL-4 module, CI module, C2 module, C3 module, C4 module, C5 module, C5 module, C6 module, and any combinations thereof.

[0151] 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.

[0152] In certain embodiments, a VWF fragment of the invention forms amultimer, 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.

[0153] 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

[0154] 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.

[0155] 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 certainembodiments, 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.

[0156] 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 Al .

[0157] 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 2A. XTEN Sequence Motifs of 12 Amino Acids and Motif FamiliesMotif I'aniih · MOTH SKQl KM l.AD GESPGGSSGSESAD GSEGSSGPGESSAD GSSESGSSEGGPAD GSGGEPSESGSSAE, AM GSPAGSPTSTEEAE, AM, AQ GSEPATSGSETPAE, AM, AQ GTSESATPESGPAE, AM, AQ GTSTEPSEGSAPAF, AM GSTSESPSGTAPAF, AM GTSTPESGSASPAF, AM GTSPSGESSTAPAF, AM GSTSSTAESPGPAG, AM GTPGSGTASSSPAG, AM GSSTPSGATGSPAG, AM GSSPSASTGTGPAG, AM GASPGTSSTGSPAQ GEPAGSPTSTSEAQ GTGEPSSTPASEAQ GSGPSTESAPTEAQ GSETPSGPSETAAQ GPSETSTSEPGAAQ GSPSEPTEGTSABC GSGASEPTSTEPBC GSEPATSGTEPSBC GTSEPSTSEPGABC GTSTEPSEPGSABD GSTAGSETSTEABD GSETATSGSETABD GTSESATSESGABD GTSTEASEGSAS• Denotes individual motif sequences that, when used together in various permutations, results in a "family sequence"

[0158] XTEN can have varying lengths for insertion into or linkage to FVIII orVWF. 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.

[0159] In some embodiments, the XTEN sequence used in the invention is at least60%, 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, AE1 140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1 140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US 2010- 0239554 Al .

[0160] 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 IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 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), AG 144 (SEQ ID NO: 38), and any combinations thereof.

[0161] 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 2 A 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).

[0162] 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 IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 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), AG 144 (SEQ ID NO: 38), and any combinations thereof. In a specificembodiment, the XTEN sequence is AE288. The amino acid sequences for certain XTEN sequences of the invention are shown in Table 2B.TABLE 2B. XTEN SequencesXTEN Amino Acid SequenceAE42 GAPGSPAGSP S EEG SESA PESGPGSEPA SGSE PASSSEQ ID NO:36AE72 GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPASEQ ID NO: TSGSETPGTSESATPESGPGTSTEPSEGSAPGASS127AE144 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTESEQ ID EGTSTEPSEGNO:37 SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESAPESGPGSEPATSGSETPGTSTEPSEGSAPAE144_2A TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET(SEQ ID NO:PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES128) GPGAE144 3B SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA(SEQ ID NO:PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS129) APGAE144_4A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE(SEQ ID NO:EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS130) APGAE144_5A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG(SEQ ID NO:PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST131) EEGAE144 6B TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA(SEQ ID NO:PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS132) APGAG 144 G PGSGTASSSPGSS PSGATGSPGSS PSAS GTGPGSS PSAS GTGSEQ ID PGASPGTSSTNO:38 GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPAG144_A GAS PG SS GSPGSS PSAS GTGPGSS PSAS GTGPG PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS(SEQ ID NO:SPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGS PGASPGTSST133) GSPAG144 B GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSS PSAS TGTGPGSS PSAS TGTGPGSSTPSGATG(SEQ ID NO:SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSST134) GSPAG144_C GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG(SEQ ID NO:SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS PGASPGTSST135) GSPAG144 F GSS PSAS TGTGPGSS PSAS TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT(SEQ ID NO:GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS PGASPGTSST136) GSPAE288 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGSEQ IDPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE NO:39 TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGT S TE P SEGSAPGSE PAT S GSETPGTSE SAT PESGPGT STEPS EGSAPAE288_2 GS PAGS PT S TEEGT SE SAT PESGPGSE PATS GSETPGTSE SAT PESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS(SEQ ID NO:APGT STEPSEGSAPGTSTEPSEGSAPGS PAGS PTS TEEGT STEPSEG137) SAPGT SE SAT PE SGPGSE PATS GSETPGTSE SAT PESGPGSE PAT SGSE T PGT SE SAT PE SGPGTSTEPSEGSAPGTSE SAT PESGPGS PAGS P T S TEEGS PAGS PT S TEEGS PAGS PTS TEEGT SE SAT PESGPGT STEP SEGSAPAG288 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSEQ ID SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT NO:40GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSPSASTGTGPGAS PG S S GSPGSS PSAS GTGPG PGSGTASSSPGS S PS GATGSAE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSASEQ IDPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE NO:41 TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSPT S TEEGT SE SAT PE S GPGSE PAT SGSET PGT SE SAT PESGPGT STEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGS PT S TEEGT STEPSEGSAPGTSE SAT PES GPGSE PAT SGSET PGT SE SATPESGPGSEPAT S GSE T PGT SE SAT PESGPGT STEPSEGSAPGTSE SAT PESGPGSP AGSPTSTEEGSPAGS PT S TEEGSPAGSPTS TEEGT SE SAT PESGPGTSTEPSEGSAPAG576 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSEQ IDSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTNO:42 GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPSGATGS PGSS PSAS TGTGPGAS PGT SSTGSPGAS PGT SSTGS PGT PGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGS PGT PGSGTASSS PGS STPSGATGS PGT PGSGTASSS PGSS TPSGATGSPGSST PSGATGS PGSS PSAS TGTGPGSS PSAS TGTGPGAS PGT SSTGS PGT PGSGTASSSPGSSTPSGATGS PGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGSAE864 GS PAGSPTS TEEGT SE SAT PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSASEQ ID PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTST NO:43EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSPT S TEEGT SE SAT PE S GPGSE PAT SGSETPGTSE SAT PESGPGT STEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGS PT S TEEGT STEPSEGSAPGTSE SAT PES GPGSE PAT SGSETPGTSE SATPESGPGSEPAT S GSE T PGTSE SAT PESGPGT STEPSEGSAPGTSE SAT PESGPGSP AGSPTSTEEGSPAGS PT STEEGSPAGSPTS TEEGT SE SAT PESGPGTSTEPSEGSAPGT SESATPESGPGSE PAT S GSE T PGTSE SAT PES GPGSE PAT SGSETPGTSE SAT PESGPG TSTEPSEGSAPGS PAGS PT S TEEGT SE SAT PES GPGSE PAT SGSETPGTSE SAT PESGP GSPAGSPTSTEEGS PAGS PT S TEEGT STEPSEGSAPGTSE SAT PESGPGTSE SAT PESG PGTSESATPESGPGSE PAT S GSE TPGSE PATS GSE TPGS PAGS PTS TEEGT STEPSEGS APGTSTEPSEGSAPGSE PAT S GSE T PGTSE SAT PESGPGT STEPSEGS APAG864 GAS PGTSSTGSPGSS PSAS TGTGPGSS PSAS TGTGPGTPGSGTASSSPGSSTPSGATGSSEQ IDPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGNO:44 SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGAS PGTSSTGSPGSS PSA STGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGSSTPSGATGSPGASPGTSSTGSPGT PGSGTASSSPGS ST PSGATGSPGSST PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSS PSGATGSPGSS PSAS GTGPGSS PSAS GTGPGASPG SS GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

[0163] 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.

[0164] A variety of methods and assays can be employed to determine thephysical / 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, HP LC -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).

[0165] Additional examples of XTEN sequences that can be used according to the present invention and are disclosed in US Patent Publication Nos.2010 / 0239554 Al, 2010 / 0323956 Al, 2011 / 0046060 Al, 2011 / 0046061 Al, 2011 / 0077199 Al, or 2011 / 0172146 A 1, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2.C) Factor VIII (FVIII) Protein

[0166] "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 an2_|_ability to form a tenase complex with factor ΓΧ 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); US 6,251,632).

[0167] 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®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0168] 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.

[0169] ROTEM analysis provides information on the whole kinetics ofhaemostasis: 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 whichinfluence these interactions. This assay can provide a complete view of secondary haemostasis.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 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)Mature Factor VIII (SEQ ID NO: 4)*SFSQMSREPS R QKQFN A T'IFENDI E.KTD PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAI ESNNSlYYEMlYLiYRPQLEHSGEtiVbYYYiJSGLTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVEYDSGLDPTLFXFKKSSPLTESGGP LSLSEENNDSKLLESGLYINSQESSWGKNVSSTESGELFKGERAEGPALLTKENAL FKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPL IHDRMLMDKNA TA .,RLNHMSNKTTSSKNME^ QQKKEGPIPPDAQNPDMSFFKPL FLPESAR WIQR HGKNSLNSGGGPS PKQLVSLGPEKSVEGQNFLSEKNKVVVGKG EEYKDVGLKEMVFPSSRNLFL TNLDNLHENNTENQEKK1OEE1EKKETLIQENVV LPQ I HTVTG TKNFMKNIJ IELLS RONVEGSYDGAYAPVLQDFRSLNDSTNRTKKETAEFSKKGEEENLEGLGNG KQIVEKYACTTE^LSPNTSGQ^R^Y RGERALEQ RL PLEETELEKEYI VDD TS QWSKNMKELTPS LTQIDYNEKEKGAITQSPLS D CLT RSHSYPQANRSPLP AKVSSEPSLRPYYL YRVLFQDNSSHLPAA.S YRKKESGVOE SSHELQGAKKNNLSLA IL TLEMTGDQREVGSLGYSA TNS VTYKKVENTVLPKPDL PKTSGKVELLPKVEYYQKDLFPTETSNGSPGHLDLVEGSLLGGTEGAJKWNEANR PGKVPF1E AYESSAXYY>SKLIA:GYJAIYDN]LYPYIRAEVEDNIMVTFRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTY FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQ VTVQEFALFF IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYI MDTLPGLVMAQDQRIRWYLLSMGSNE IHS IHFSGHVFTVRKKEEYKMALYNLYP GVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF QI ASGQYGQWAPKLARLHYSGS INAWS KEPFSWIKVDLLAPMI IHGIKTQGAR QKFSSLYI SQFI IMYSLDGKKWQTYRGNS GTLMVFFGNVDSSGIKHNIFNPPI I ARYIRLHPTHYS IRS LRMELMGCDLNSCSMPLGMESKAI SDAQI ASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLI SSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLYTABLE 4. Nucleotide Sequence Encoding Full-Length FVIII (SEQ ID NO661 ATGCAAATAGAGC TCTCCACCTG721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACCAGAAGATACT ACCTGGGTGC781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGTGAGCTGCCTG TGGACGCAAG841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACCTCAGTCGTGT ACAAAAAGAC901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCTAAGCCAAGGC CACCCTGGAT961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGATACAGTGGTCA TTACACTTAA1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGTGTATCCTACT GGAAAGCTTC1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAGAAAGAAGATG ATAAAGTCTT1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAAGAGAATGGTC CAATGGCCTC1.201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTGGACCTGGTAA AAGACTTGAA1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGGAGTCTGGCCA AGGAAAAGAC1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTTGATGAAGGGA AAAGTTGGCA1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCTGCATCTGCTC GGGCCTGGCC1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTGCCAGGTCTGA TTGGATGCCA1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACCACTCCTGAAG TGCACTCAAT1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGCCAGGCGTCCT TGGAAATCTC1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGACCTTGGACAGT TTCTACTGTT1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCTTATGTCAAAG TAGACAGCTG1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAAGCGGAAGACT ATGATGATGA1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGATGACAACTCTC CTTCCTTTAT1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGGGTACATTACA TTGCTGCTGA1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCCGATGACAGAA GTTATAAAAG1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAGTACAAAAAAG TCCGATTTAT2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATTCAGCATGAAT CAGGAATCTT2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTGATTATATTTA AGAATCAAGC2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGATGTCCGTCCTT TGTATTCAAG2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCAATTCTGCCAG GAGAAATATT2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACTAAATCAGATC CTCGGTGCCT2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGATCTAGCTTCAG GACTCATTGG2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGAGGAAACCAGA TAATGTCAGA2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAACCGAAGCTGGT ACCTCACAGA2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAGCTTGAGGATC CAGAGTTCCA2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTTGATAGTTTGC AGTTGTCAGT2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATTGGAGCACAGA CTGACTTCCT2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATGGTCTATGAAG ACACACTCAC2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATGGAAAACCCAG GTCTATGGAT2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATGACCGCCTTAC TGAAGGTTTC2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGTTATGAAGATA TTTCAGCATA2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTCTCCCAGAATT CAAGACACCC3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCAGAAAATGACA TAGAGAAGAC3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATACAAAATGTCT CCTCTAGTGA3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGGCTATCCTTAT CTGATCTCCA3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCTGGAGCAATAG ACAGTAATAA3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCATCACAGTGGGG ACATGGTATT3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAA GAGAAACTGGGGACAA CTGCAGCAAC3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCAAATAATCTGA TTTCAACAAT3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTTGGCAAAAAGT CATCTCCCCT3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAATAATGATTCAA AGTTGTTAGA3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAAAATGTATCGT CAACAGAGAG3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCTTTGTTGACTA AAGATAATGC3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAAACTTCCAATA ATTCAGCAAC3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATTGAGAATAGTC CATCAGTCTG3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTGACACCTTTGA TTCATGACAG3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAATCATATGTCAA ATAAAACTAC3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAGGGCCCCATTC CACCAGATGC4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTGCCAGAATCAG CAAGGTGGAT4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAAGGCCCCAGTC CAAAGCAATT4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAATTTCTTGTCTG AGAAAAACAA4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGACTCAAAGAGA TGGTTTTTCC4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTACATGAAAATA ATACACACAA4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAAACATTAATCC AAGAGAATGT4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAATTTCATGAAGA ACCTTTTCTT4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGGGCATATGCTC CAGTACTTCA4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAGAAACACACAG CTCATTTCTC4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAATCAAACCAAGC AAATTGTAGA4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGCCAGCAGAATT TTGTCACGCA4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTAGAAGAAACAG AACTTGAAAA4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAAAACATGAAAC ATTTGACCCC4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAAGGGGCCATTA CTCAGTCTCC4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAAGCAAATAGAT CTCCATTACC4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATATATCTGACCA GGGTCCTATT4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGAAAGAAAGATT CTGGGGTCCA5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAACCTTTCTTTAG CCATTCTAAC5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTGGGGACAAGTG CCACAAATTC5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAACCAGACTTGC CCAAAACATC5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAGAAGGACCTAT TCCCTACGGA5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAAGGGAGCCTTC TTCAGGGAAC5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGAAAAGTTCCCT TTCTGAGAGT5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTGGATCCTCTTG CTTGGGATAA5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCCCAAGAGAAGT CACCAGAAAA5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATAGAAGTCACCT GGGCAAAGCA5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTCTTGAAACGCC ATCAACGGGA5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAGGATGAAAATC AGAGCCCCCG5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCTCAGAGTGGCA GTGTCCCTCA5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCCTTTACTCAGC CCTTATACCG6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATATATAAGAGCAG AAGTTGAAGA6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCCTATTCCTTCT ATTCTAGCCT6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCTAGAAAAAACT TTGTCAAGCC6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCATATGGCACCCA CTAAAGATGA6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGACCTGGAAAAAG ATGTGCACTC6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACACTGAACCCTG CTCATGGGAG6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATCTTTGATGAGA CCAAAAGCTG6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAATGGCTACATAA TGGATACACT6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGGTATCTGCTCA GCATGGGCAG6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTGTTCACTGTAC GAAAAAAAGA6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTTTTTGAGACAG TGGAAATGTT6721 ACCA CCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATTGGCGAGCATC TACATGCTGG6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAGACTCCCCTGG GAATGGCTTC6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAATATGGACAGT GGGCCCCAAA6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCACGGCATCAAGA CCCAGGGTGC7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATCATCATGTATA GTCTTGATGG7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACCTTAATGGTCT TCTTTGGCAA7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCTCCAATTATTG CTCGATACAT7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTTCGCATGGAGT TGATGGGCTG7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGTAAAGCAATAT CAGATGCACA7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACCTGGTCTCCTT CAAAAGCTCG7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAGGTGAATAATC CAAAAGAGTG7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGAGTAACTACTC AGGGAGTAAA7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATCTCCAGCAGTC AAGATGGCCA7 5 61 TCAGTGGACT CTCT T T T T TC AGAATGGCAA AGTAAAGGT TT T TCAGGGAA A CAAGACTC7 62 1 CT TCACACCT GTGGTGAACT CTCTAGACCC ACCGT TACTGACTCGCTACC T TCGAAT TCA7 68 1 CCCCCAGAGT TGGGTGCACC AGAT TGCCCT GAGGATGGAGGT TCTGGGCT GCGAGGCACA7 7 4 1 GGACCTCTAC*The underlined nucleic acids encode a signal peptide.

[0171] 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.

[0172] The sequence of native mature human FVIII is presented as SEQ ID NO:4. A native FVIII protein has the following formula: Al-al-A2-a2-B-a3-A3-Cl- C2, where Al, A2, and A3 are the structurally-related "A domains," B is the "B domain," CI and C2 are the structurally-related "C domains," and al, a2 and a3 are acidic spacer regions. Referring to the primary amino acid sequence position in SEQ ID NO:4, the Al domain of human FVIII extends from Alal to about Arg336, the al 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 Glul649 to about Argl689, the A3 domain extends from about Serl690 to about Leu2025, the CI 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."

[0173] 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 wasdeduced 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.

[0174] 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 Al and A2 domains of porcine FVIII and a chimeric FVIII with porcine Al 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.

[0175] 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 reducedimmunoreactivity. US Appl. Publ. No. 2005 / 0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

[0176] 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 aFVIII 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.

[0177] 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-Argl648 of full-length human FVIII. The other human FVIII domains are defined by the following amino acid residues: Al, residues Alal-Arg372; A2, residues Ser373-Arg740; A3, residues Serl690- Asn2019; CI, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Serl690-Tyr2332. The remaining sequence, residues Glul649-Argl689, 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 "BDDFVIII"). An example of a BDD FVIII is REFACTO®(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 5. Amino Acid Sequence of B-domain Deleted Factor VIII (BDDFVIII)BDD FVIII (SEQ ID NO: 6)ATRRY LGAVELS DY^DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASE GA^YDDQ^KHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNT GDYYEDSYEDISAYLLSKNAIEPRS S F V PVL?; NXJJ¾EITR^TLOSDG^IDYDD I sVEMKKE"D"FDI"Y"6EDE QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDE FDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF IFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNE IHS IHFSGHVFTVRKKEEYKMALYNLYPGVFE VEMLPSKAG IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGS INAWS KEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYI SQFI IM YSLDGKKWQTYRGNS GTLMVFFGNVDSSGIKHNIFNPPI IARYIRLHPTHYS IR S LRMELMGCDLNSCSMPLGMESKAI SDAQI ASSYFTNMFATWSPSKARLHLQG RSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLI SSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLYTABLE 6. Nucleotide Sequence Encoding BDD FVIII (SEQ ID NO: 7)*661 A TGCAAATAGAGCTCTCCACC TGCTTCTTTC721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATACTACCTGGGT GCAGTGGAAC781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCCTGTGGACGCA AGATTTCCTC841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGTGTACAAAAAG ACTCTGTTTG901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAGGCCACCCTGG ATGGGTCTGC961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGTCA ACAC T AAGAACA GG1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTACTGGAAAGCT TCTGAGGGAG1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGATGATAAAGTC TTCCCTGGTG1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGGTCCAATGGCC TCTGACCCAC1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGTAAAAGACTTG AATTCAGGCC1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGCCAAGGAAAAG ACACAGACCT1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGGGAAAAGTTGG CACTCAGAAA1381 CAAAGAACTC CTTGATGCAG GA AGGGATG CTGCA CTGCTCGGGCCTGG CCTAAAATGC1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCTGA GGATGC CACAGGAAAT1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGAAGTGCACTCA ATATTCCTCG1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTCCTTGGAAATC TCGCCAATAA1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACAGTTTCTACTG TTTTGTCATA1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAAAGTAGACAGC TGTCCAGAGG1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGACTATGATGAT GATCTTACTG1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTCTCCTTCCTTT ATCCAAATTC1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTACATTGCTGCT GAAGAGGAGG1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAGAAGTTATAAA AGTCAATATT1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAAAGTCCGATTT ATGGCATACA2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGAATCAGGAATC TTGGGACCTT2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATTTAAGAATCAA GCAAGCAGAC2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCCTTTGTATTCA AGGAGATTAC2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCCAGGAGAAA A TTCAAA A A2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGATCCTCGGTGC CTGACCCGCT2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTCAGGACTCATT GGCCCTCTCC2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCAGATAATGTCA GACAAGAGGA2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTGGTACCTCACA GAGAATATAC2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGATCCAGAGTTC CAAGCCTCCA2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTTGCAGTTGTCA GTTTGTTTGC2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACAGACTGACTTC CTTTCTGTCT2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGAAGACACACTC ACCCTATTCC2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCCAGGTCTATGG ATTCTGGGGT2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTTACTGAAGGTT TCTAGTTGTG2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGATATTTCAGCA TACTTGCTGA2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAACCCACCAGTC TTGAAACGCC3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCAAGAGGAAATT GACTATGATG3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACATTTATGATGAG GATGAAAATC3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTTTATTGCTGCA GTGGAGAGGC3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTACTGATGGCTCC TTTACTCAGC3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCTGGGGCCATAT ATAAGAGCAG3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGCCTCTCGTCCC TATTCCTTCT3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGTGCAACATCAT ATGGCACCCA3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTCTGATGTTGAC CTGGAAAAAG3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCACACTAACACA CTGAACCCTG3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTTTTTCACCATC TTTGATGAGA3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTGCAGGGCTCCC TGCAATATCC3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCATGCAATCAAT GGCTACATAA3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAGGATTCGATGG TATCTGCTCA3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAGTGGACATGTG TTCACTGTAC3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTATCCAGGTGTT TTTGAGACAG4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGAATGCCTTATT GGCGAGCATC4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAATAAGTGTCAG ACTCCCCTGG4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGCTTCAGGACAA TATGGACAGT4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAATCAATGCCTGG AGCACCAAGG4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAATGATTATTCAC GGCATCAAGA4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATAGTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTCCACTGGAACC TTAATGGTCT4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATATTTTTAACCCT CCAATTATTG4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCGCAGCACTCTT CGCATGGAGT4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGGAATGGAGAGT AAAGCAATAT4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATATGTTTGCCACC TGGTCTCCTT68 1 CAAAAGCTCG ACT TCACCTC CAAGGGAGGA GTAATGCCTGGAGACCTCAG GTGAATAATC7 4 1 CAAAAGAGTG GCTGCAAGTG GACT TCCAGA AGACAATGAAAGTCACAGGA GTAACTACTC8 0 1 AG G GAG AAA ATCTCTGCT T ACCAGCATGT ATGTGAAGGAGT TCCTCATC TCCAGCAGTC8 61 AAGATGGCCA TCAGTGGACT CTCT T T T T TC AGAATGGCAAAGTAAAGGT T T T TCAGGGAA92 1 ATCAAGACTC CT TCACACCT GTGGTGAACT CTCTAGACCCACCGT TACTG ACTCGCTACC98 1 T TCGAAT TCA CCCCCAGAGT TGGGTGCACC AGAT TGCCCTGAGGATGGAG GT TCTGGGCT0 4 1 GCGAGGCACA GGACCTCTAC*The underlined nucleic acids encode a signal peptide.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 US 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. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US6,228,620). In some embodiments, a B-domain-deleted Factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US Patent No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Patent no. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent no. 5,171,844; col. 2, lines 55-68, figure 2, and example 1 of U.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Patent 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. Patent no. 7,041,635; or col. 4, lines 25- 53, of U.S. Patent 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 -terminalsequences 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-linkedglycosylation 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 otherembodiments, 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., Argl313 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.In some embodiments, the FVIII has a partial B-domain. In some embodiments, the FVIII protein with a partial B-domain is FVIII 198 (SEQ ID NO: 89). FVIII198 is a partial B-domain containing single chain FVIIIFcmolecule-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.

[0180] In one embodiment, FVIII is cleaved right after arginine at amino acid1648 (in full-length Factor VIII or SEQ ID NO: 4), amino acid 754 (in theS743 / Q1638 B-domain deleted Factor VIII or SEQ ID NO: 6), or thecorresponding 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.

[0181] In other embodiments, FVIII is a single chain FVIII that has not beencleaved right after Arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 4), amino acid 754 (in the S743 / 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.

[0182] 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 50kDa Al, 43kDa A2, and 73kDa 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.

[0183] The protein having FVIII polypeptide linked to or associated with theVWF 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).

[0184] "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.

[0185] 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, 1 1 , 12, 13, 14, 15 or any combinations thereof.

[0186] The FVIII protein linked to one or more XTEN sequences can berepresented as FVIII(X), FVIII(Xl), FVIIIfa→^-X-FVIIIfc→rf ,wherein FVlll(a→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 XI comprises, consists essentially of, or consists of one or more XTEN sequences, FVUl(c→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, andd is the C-terminal amino acid residue of the FVIII protein, andwherein 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.

[0187] 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 Al domain and the A2 domain of the FVIII protein. The second portion of the FVIII protein comprises the A3 domain, the CI domain, and optionally the C2 domain. In yet other embodiments, the firstportion of the FVIII protein comprises the Al domain and A2 domain, and the second portion of the FVIII protein comprises a portion of the B domain, the A3 domain, the CI domain, and optionally the C2 domain. In still otherembodiments, the first portion of the FVIII protein comprises the Al 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 CI domain, and optionally the C2 domain. In still other embodiments, the first portion of the FVIII protein comprises the Al 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 C 1 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^i, 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 FVUl(c→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.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 Al domain, the A2 domain, the A3 domain, the B domain, the CI domain, the C2 domain, the C- terminus, or two or more combinations thereof or between two domains of the FVIII protein, which are the Al domain and al acidic region, and the al 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 C 1 domain, the CI 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 Al domain, the N-terminus and A2domain, the N-terminus and A3 domain, the N-terminus and B domain, the N- terminus and CI domain, the N-terminus and C2 domain, the N-terminus and the C -terminus, the Al and A2 domains, the Al and A3 domains, the Al and B domains, the Al and CI domains, the Al and C2 domains, the Al domain and the C-terminus, the A2 and A3 domains, the A2 and B domains, the A2 and CI domains, the A2 and C2 domains, the A2 domain and the C-terminus, the A3 and B domains, the A3 and C 1 domains, the A3 and C2 domains, the A3 domain and the C-terminus, the B and CI domains, the B and C2 domains, the B domain and the C-terminus, the CI and C2 domains, the CI 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.

[0189] 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).

[0190] 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.

[0191] 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 7: Exemplary XTEN Insertion Sites* 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.192] 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, 171 1 , 1725, 1905, or 1910, corresponding to SEQ ID NO: 4 or any combinations thereof.TABLE 8. Exemplary XTEN Insertion Ranges*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.

[0193] In other embodiments, one or more XTEN sequences are insertedimmediately 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 9. Exemplary XTEN Insertion Sites or Ranges* indicates range of insertion sites numbered relative to the amino acid number of mature human FVIII

[0194] In yet other embodiments, one or more XTENs are inserted in the Bdomain 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 betweenamino 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.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.- I l l -TABLE 10: FVIII XTEN insertion sites and construct designations* Indicates the amino acid number of the mature FVIII protein 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 Al domain comprises a permissive loop-1 (Al-1) region and a permissive loop-2 (A 1-2) 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.

[0197] In one aspect, a first permissive loop in the FVIII Al domain (Al-1) is located between beta strand 1 and beta strand 2, and a second permissive loop in the FVIII A2 domain (Al-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 Al-1 corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of SEQ IDNO: 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 Al-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.

[0198] In another embodiment, the one or more amino acids in which at least oneXTEN sequence is inserted is located within a3 domain, e.g., amino acids 1649 to 1689, corresponding to full-length mature FVIII polypeptide. In a particularembodiment, 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.In some embodiments, the one or more insertion sites for one or moreXTEN insertions are immediately downstream of one or more amino acids selected from the group consisting of:(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.In one embodiment, a FVIII protein useful for the invention comprises twoXTEN 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 11. Exemplary Insertion Sites for Two XTENsInsert i mi 1 Insert ion ~"Insertion Site Domain Insertion Site Domain1656 A3 1900 A31656 A3 1905 A31711 A3 40 Al1711 A3 26 Al1720 A3 26 Al1720 A3 40 Al1720 A3 18 Al1725 A3 26 Al1725 A3 40 Al1725 A3 18 Al1720 A3 403 A21720 A3 399 A21711 A3 403 A21720 A3 403 A21725 A3 403 A21725 A3 399 A21711 A3 403 A21900 A3 399 A21900 A3 403 A21905 A3 403 A21905 A3 399 A21910 A3 403 A2 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 immediatelydownstream 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.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 sequenceinserted 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 12. Exemplary Insertion Sites for Three XTENsIn 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 IDNO: 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 inserteddownstream 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.In other embodiments, a FVIII protein in the invention comprises fourXTEN 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 13. Exemplary Insertion Sites for Four XTENs0403 A2 1720 A3 745 B 2332 CT0403 A2 1900 A3 745 B 2332 CT0403 A2 1900 A3 745 B 2332 CT0409 A2 1900 A3 745 B 2332 CT0403 A2 1900 A3 745 B 2332 CT0403 A2 1900 A3 745 B 2332 CT0409 A2 1900 A3 745 B 2332 CT0409 A2 1905 A3 745 B 2332 CT0403 A2 1905 A3 745 B 2332 CT0403 A2 1905 A3 745 B 2332 CT0403 A2 1905 A3 745 B 2332 CT0409 A2 1905 A3 745 B 2332 CT0403 A2 1905 A3 745 B 2332 CT0409 A2 1910 A3 745 B 2332 CT0403 A2 1910 A3 745 B 2332 CT0403 A2 1910 A3 745 B 2332 CT0403 A2 1910 A3 745 B 2332 CT0403 A2 1910 A3 745 B 2332 CT1720 A3 1900 A3 745 B 2332 CT1720 A3 1905 A3 745 B 2332 CT1720 A3 1910 A3 745 B 2332 CT1720 A3 1910 A3 745 B 2332 CT0403 A2 1656 a3 1720 A3 2332 CT0403 A2 1656 a3 1900 A3 2332 CT0403 A2 1720 A3 1900 A3 2332 CT1656 a3 1720 A3 1900 A3 2332 CT0018 Al 0403 A2 1656 a3 2332 CT0018 Al 0403 A2 1720 A3 2332 CT0018 Al 0403 A2 1900 A3 2332 CT0018 Al 1656 a3 1720 A3 2332 CT0018 Al 1656 a3 1900 A3 2332 CT0018 Al 1720 A3 1900 A3 2332 CT0018 Al 0403 A2 0745 B 2332 CT0018 Al 0745 B 1720 A3 2332 CT0018 Al 0745 B 1900 A3 2332 CT0403 A2 0745 B 1720 A3 2332 CT0403 A2 0745 B 1900 A3 2332 CT0745 B 1720 A3 1900 A3 2332 CT0188 Al 1900 A3 0745 B 2332 CT0599 1900 A3 0745 B 2332 CT2068 1900 A3 0745 B 2332 CT2171 1900 A3 0745 B 2332 CT2227 1900 A3 0745 B 2332 CT2277 1900 A3 0745 B 2332 CTIn some embodiments, a FVIII protein comprises five XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequenceinserted 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 14. Exemplary Insertion Sites for Five XTENs

[0206] 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 15. Exemplary XTEN Insertion Sites for Six XTENsIn 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 betweenamino 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.

[0208] 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.

[0209] In some embodiments, a chimeric protein of the invention comprises two polypeptide sequences, a first polypeptide sequence comprising an amino acidsequence 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: 1 19), or VWF-036 (SEQ ID NO: 120).D) Ig Constant Region or a portion thereof

[0210] 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.

[0211] An Ig constant region is comprised of domains denoted CH (constant heavy) domains (CHI, 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.

[0212] 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 IgGl, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgGl is used.

[0213] 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 particularmodification 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.

[0214] 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, CA (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 January 25, 1995, which is incorporated by reference herein.

[0215] 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.

[0216] As used herein, the term "Fc region" is defined as the portion of apolypeptide 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).

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] FcRn binding partners useful in the present invention encompassmolecules 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.

[0222] 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 vivohalf-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.

[0223] Two FcRn receptors can bind a single Fc molecule. Crystallo graphic 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.

[0224] 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 106M"1, or higher than 108M"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.

[0225] 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 IgGl, 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 bindingportion. FcRn binding portions may be derived from heavy chains of any isotype, including IgGl, IgG2, IgG3 and IgG4. In one embodiment, an FcRn binding portion from an antibody of the human isotype IgGl is used. In another embodiment, an FcRn binding portion from an antibody of the human isotype IgG4 is used.

[0226] 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.

[0227] The Fc regions denoted as F, Fl, 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 IgGl, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgGl is used.

[0228] 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. FcyRI, FcyRII, or FcyRIII) or complement proteins (e.g. C lq), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, orcomplement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine residue.

[0229] 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 , W096 / 14339A1 , WO98 / 05787A1 , W098 / 23289A1 ,W099 / 51642A1 , W099 / 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 PatentPublication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765,US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056 ; or US Patents 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.

[0230] 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 byFcRn. 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 IgGl Fc (Fc γΐ) 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.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 FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68: 1632; Shieldset 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).

[0232] 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 IgGl "ELLG" to the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions will not bind to IgGl when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0233] 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).

[0234] 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 ora portion thereof can further be linked to one or more domains of CHI, 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.

[0235] 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 andaglycosylated 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.

[0236] 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.

[0237] 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., US Patents 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 bedesired 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 A FcRn "contact zone." As used herein, the term 15 A 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.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).

[0239] 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

[0240] 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.

[0241] One or more linkers can be present between any two proteins in thechimeric 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) anXTEN 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).

[0242] 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).

[0243] 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.

[0244] The linker useful in the present invention can comprise any organicmolecule. 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 900-1000amino 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 Al, 2010 / 0323956 Al, 2011 / 0046060 Al, 2011 / 0046061 Al, 2011 / 0077199 Al, or 2011 / 0172146 Al, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO2011028344 A2. In another embodiment, the linker is a PAS sequence.

[0245] The linker useful in the present invention can comprise any organicmolecule. 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.

[0246] 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)n. The linker can comprise the sequence (GGS)n. In other embodiments, the linker comprises (GGGS)n(SEQ ID NO: 57). In still other embodiments, the linker comprises the sequence (GGS)n(GGGGS)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), orGGGGSGGGGSGGGGS (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.

[0247] 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)n(SEQ ID NO: 64) where G represents glycine, S represents serine and n is an integer from 1-20.F) Cleavage Sites

[0248] 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.

[0249] 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.

[0250] 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 RR R (SEQ ID NO: 9), R RRKR (SEQ ID NO: 10), and RRRRS (SEQ ID NO: 11).

[0251] 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 Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (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., KLTRjAET (SEQ ID NO: 65)), a FXIa cleavage site (e.g, DFTRjVVG (SEQ ID NO: 66)), a FXIIa cleavage site (e.g., TMTRjIVGG (SEQ ID NO: 67)), aKallikrein cleavage site (e.g., SPFRjSTGG (SEQ ID NO: 68)), a FVIIa cleavagesite (e.g., LQVR4IVGG (SEQ ID NO: 69)), a FIXa cleavage site (e.g.,PLGR4IVGG (SEQ ID NO: 70)), a FXa cleavage site (e.g., IEGRjTVGG (SEQ ID NO: 71)), a Flla (thrombin) cleavage site (e.g, LTPRjSLLV (SEQ ID NO: 72)), a Elastase-2 cleavage site (e.g, LGPV jSGVP (SEQ ID NO: 73)), aGranzyme-B cleavage (e.g, VAGD jSLEE (SEQ ID NO: 74)), a MMP-12 cleavage site (e.g., GPAGjLGGA (SEQ ID NO: 75)), a MMP-13 cleavage site (e.g., GPAGjLRGA (SEQ ID NO: 76)), a MMP-17 cleavage site (e.g.,APLGjLRLR (SEQ ID NO: 77)), a MMP-20 cleavage site (e.g., PALP jLVAQ (SEQ ID NO: 78)), a TEV cleavage site (e.g., ENLYFQjG (SEQ ID NO: 79)), a Enterokinase cleavage site (e.g., DDDKjIVGG (SEQ ID NO: 80)), a Protease 3C (PRESCISSION™) cleavage site (e.g., LEVLFQjGP (SEQ ID NO: 81)), and a Sortase A cleavage site (e.g., LPKTjGSES) (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)).

[0252] In a specific embodiment, the cleavage site isTLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).POLYNUCLEOTIDES, VECTORS, AND HOST CELLS

[0253] 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, Fl comprises a first Ig constant region or a portion thereof, e.g., a first Fc region, LI 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 comprisinga 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.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.

[0255] 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.

[0256] 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.

[0257] A gene expression control sequence as used herein is any regulatorynucleotide 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: hypoxanthinephosphoribosyl 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.

[0258] In general, the gene expression control sequence shall include, asnecessary, 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.

[0259] 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 ofdirecting synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high ef...