Factor viii chimeric proteins and uses thereof

Abstract

To provide an FVIII formulation requiring less frequent administration, i.e., an FVIII formulation with a half-life longer than 1.5 to 2 times the half-life limitation.SOLUTION: The present invention provides a chimeric protein comprising (i) a first polypeptide which comprises FVIII fused to a first immunoglobulin (Ig) constant region or a portion thereof and (ii) a second polypeptide which comprises a von Willebrand factor (VWF) protein comprising a D' domain and a D3 domain of VWF fused to a second Ig constant region or a portion thereof by an XTEN sequence in between, where the XTEN sequence contains less than 288 amino acid residues, and where the first polypeptide is linked to or associated with the second polypeptide. Certain embodiments include the chimeric protein as described herein, where the XTEN sequence in the second polypeptide consists of an amino acid sequence having a length between 12 amino acids and 287 amino acids.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the Sequence Listing submitted electronically as an ASCII text file (Name: 2159_441PC02_SequenceListing_ST25.txt; Size: 823,500 bytes; Created: Jan. 9, 2015) are incorporated herein by reference in their entirety. [Background technology]

[0002] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding coagulation factor VIII (FVIII) and affects 1-2 in every 10,000 live male births. Graw et al., Nat. Rev. Genet. 6(6):488-501 (2005). Patients with hemophilia A can be treated with infusions of purified or recombinantly produced FVIII. However, all commercially available FVIII preparations are known to have half-lives of approximately 8-12 hours, necessitating frequent intravenous administration to patients. See Weiner, MA and Cairo, MS, Pediatric Hematology Secrets, Lee, MT, 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 attempted to extend the half-life of FVIII. For example, approaches in development to extend the half-life of coagulation factors include PEGylation, glycoPEGylation, and conjugation with albumin. See Dumont et al., Blood. 119(13):3024-3030 (published online January 13, 2012). However, regardless of the protein engineering used, long-acting FVIII preparations currently in development have been reported to have limited half-lives of only about 1.5-2 hours in preclinical animal models. See supra. Consistent results have been demonstrated in humans, for example, rFVIIIFc was reported to improve half-life by about 1.7-fold compared to Advate® in patients with hemophilia A. See supra. Thus, an increase in half-life, despite only a modest improvement, may indicate the presence of other T1 / 2 rate-limiting factors. See Liu, T. et al., 2007, ISTH meeting, abstract #PM-035; Henrik, A. et al., 2011, ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011, ISTH meeting abstract #P-WE-131.

[0003] Plasma von Willebrand factor (VWF) has a half-life of approximately 16 hours (range 13-18 hours). Goudemand, J. et al., J. Thromb. Haemost. 2005;3:2219-27. The half-life of VWF can be influenced by a number of factors: the glycosylation pattern in VWF, ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motifs-13), and various mutations.

[0004] In plasma, 95-98% of FVIII circulates in a tight, non-covalent complex with full-length VWF. The formation of this complex is important for the maintenance of adequate 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). Full-length wild-type FVIII exists predominantly as a heterodimer with a heavy chain (MW 200 kD) and a light chain (MW 73 kD). Upon activation of FVIII by proteolysis at positions 372 and 740 of the heavy chain and at position 1689 of the light chain, the FVIII-bound VWF is released from the activated FVIII. Activated FV Factor III, together with activated factor IX, calcium, and phospholipids (the "tenase complex"), induces the activation of factor X, generating large amounts of thrombin. Thrombin then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form soluble fibrin polymers. Thrombin also activates factor XIII, which, together with calcium, helps to crosslink and stabilize the soluble fibrin polymers, forming crosslinked (insoluble) fibrin. Activated FVIII is rapidly cleared from the circulation by proteolysis. Due to the inconvenience caused by frequent administration and dosing schedules, there remains a need to develop FVIII formulations that require less frequent administration, i.e. FVIII formulations that have a half-life longer than 1.5-2 times the half-life limit. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Graw et al., Nat. Rev. Genet. 6(6):488-501 (2005) [Non-Patent Document 2] Dumont et al., Blood. 119(13):3024-3030 [Non-Patent Document 3] Goudemand, J et al., J. Thromb. Haemost.2005;3:2219-27 [Non-Patent Document 4] Lenting et al., Blood. 92(11):3983-96 (1998) [Non-Patent Document 5] Lenting et al., J. Thromb. Haemost. 5(7):1353-60 (2007) Summary of the Invention [Means for solving the problem]

[0006] The invention provides chimeric proteins comprising (i) a first polypeptide comprising factor VIII ("FVIII") fused to a first immunoglobulin ("Ig") constant region or portion thereof, and (ii) a second polypeptide comprising a von Willebrand factor ("VWF") protein comprising the D' and D3 domains of VWF fused to a second Ig constant region or portion thereof by an intermediate XTEN sequence, wherein the XTEN sequence contains fewer than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide. Certain embodiments include chimeric proteins as described herein, wherein the XTEN sequence in the second polypeptide consists of an amino acid sequence having a length between 12 amino acids and 287 amino acids.

[0007] Also disclosed are chimeric proteins as described herein, wherein the chimeric proteins comprise a first polypeptide and a second polypeptide, and exhibit a longer half-life than a corresponding fusion protein, wherein the second polypeptide of the fusion protein comprises an XTEN sequence containing at least 288 amino acids. Some embodiments include an XTEN sequence AE288 containing at least 288 amino acids. In some embodiments, AE288 is SEQ ID NO:8.

[0008] Also disclosed are chimeric proteins as described herein, wherein the XTEN sequence of the second polypeptide contains about 36, about 42, about 72, or about 144 amino acids. In some embodiments, the XTEN sequence of the second polypeptide is selected from AE42, AE72, AE144, AG42, AG72, or AG144.

[0009] Some embodiments include a chimeric protein as described herein, wherein the XTEN sequence of the second polypeptide is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO No. 55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63.

[0010] In certain embodiments, the first polypeptide further comprises a second XTEN sequence that links the FVIII protein with the first Ig constant region or a portion thereof. Also disclosed is a chimeric protein as described herein, wherein the first polypeptide comprises a third XTEN sequence that is inserted into one or more insertion sites in the FVIII protein. In some embodiments, the first polypeptide further comprises a second XTEN sequence that is inserted into one or more insertion sites in the FVIII protein. In certain embodiments, the first polypeptide comprises a third XTEN sequence that links the FVIII protein with the first Ig constant region or a portion thereof.

[0011] Also disclosed are chimeric proteins as described herein, wherein the second XTEN sequence, the third XTEN sequence, or the second and third XTEN sequences are each independently selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. In some embodiments, the second XTEN sequence, the third XTEN sequence, or the second and third XTEN sequences are each independently selected from SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17; SEQ ID NO:54; SEQ ID NO:19; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:15; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63. In certain embodiments, the second XTEN sequence, the third XTEN sequence, or both the second and third XTEN sequences are each independently AE288 or AG288. In some embodiments, the XTEN sequence in the second polypeptide is fused to the second Ig constant region or a portion thereof by a linker. In certain embodiments, the linker is a cleavable linker.

[0012] Some embodiments include a chimeric protein as described herein, wherein the linker is cleavable by a protease selected from Factor XIa, Factor XIIa, kallikrein, Factor VIIa, Factor IXa, Factor Xa, Factor IIa (thrombin), elastase-2, granzyme-B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. In some embodiments, the linker is cleavable by Factor IIa (thrombin).

[0013] Also disclosed are chimeric proteins as described herein, wherein the linker is selected from the group consisting of RRRR (SEQ ID NO:102), RKRRKR (SEQ ID NO:103), RRRRS (SEQ ID NO:104), TQSFNDFTR (SEQ ID NO:1), SVSQTSKLTR (SEQ ID NO:3), DFLAEGGGVR (SEQ ID NO:4), TTKIKPR (SEQ ID NO:5), LVPRG (SEQ ID NO:6), ALRPR (SEQ ID NO:7), KLTRAET (SEQ ID NO:121), DFTRVVG (SEQ ID NO:122), TMTRIVGG (SEQ ID NO:123), SPFRSTGG (SEQ ID NO:124), LQVRIVGG (SEQ ID NO:125), PLGRIVGG (SEQ ID NO:126), IEGRTV GG (SEQ ID NO: 127), LTPRSLLV (SEQ ID NO: 128), LGPVSGVP (SEQ ID NO: 129), VAGDSLEE (SEQ ID NO: 130), GPAGLGGA (SEQ ID NO: 131), GPAGLRGA (SEQ ID NO: 132), APLGLRLR (SEQ ID NO: 133), PALPLVAQ (SEQ ID NO: 134), ENLYFQG (SEQ ID NO: 135), DDDKIVGG (SEQ ID NO: 136), LEVLFQGP (SEQ ID NO: 137), LPKTGSES (SEQ ID NO: 138), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), and IEPRSFS (SEQ ID NO: 194). In one embodiment, the linker comprises TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). In certain embodiments, the cleavage site comprises the amino acid sequence of LVPRG (SEQ ID NO: 6). In other embodiments, the cleavage site comprises the amino acid sequence of IEPRSFS (SEQ ID NO: 194). In yet other embodiments, the cleavage site comprises the amino acid sequence of IEPRSFS (SEQ ID NO: 194), and the cleavage site is not the full-length a2 region of FVIII. In some embodiments, the cleavage site comprises a fragment of the a2 region of FVIII comprising at least the sequence IEPRSFS (SEQ ID NO: 200). In other embodiments, the cleavage site comprises a fragment of the a2 region of FVIII comprising at least the sequence IEPRS (SEQ ID NO: 200), and the cleavage site is not the full-length a2 region. In certain embodiments, the cleavage site is cleavable in a thrombin cleavage assay as provided herein or as known in the art.

[0014] Some embodiments include a chimeric protein as described herein, wherein the first Ig constant region or portion thereof comprises a first Fc region and / or the second Ig constant region or portion thereof comprises a second Fc region. In some embodiments, the first Ig constant region or portion thereof and the second Ig constant region or portion thereof extend the half-life of the chimeric protein. In some embodiments, the first polypeptide and the second polypeptide are fused by a linker. In certain embodiments, the first polypeptide and the second polypeptide are fused by a processable linker. In some embodiments, the first Ig constant region or portion thereof is associated with the second Ig constant region or portion thereof. In certain embodiments, the first Ig constant region or portion thereof is covalently associated with the second Ig constant region or portion thereof. In some embodiments, the covalent bond is a disulfide bond.

[0015] Also disclosed are chimeric proteins comprising each of the following formulas (a) through (hh): (a) FVIII-F1:F2-L2-X-L1-V; (b) FVIII-F1: V-L1-X-L2-F2; (c) F1-FVIII:F2-L2-X-L1-V; (d) F1-FVIII: V-L1-X-L2-F2; (e) FVIII-X2-F1:F2-L2-X1-L1-V; (f) FVIII-X2-F1: V-L1-X1-L2-F2; (g)FVIII(X2)-F1:F2-L2-X1-L1-V; (h)FVIII(X2)-F1:V-L1-X1-L2-F2; (i) F1-X2-F1:F2-L2-X1-L1-V; (j) F1-X2-F1: V-L1-X1-L2-F2; (k)V-L1-X-L2-F2-L3-FVIII-L4-F1; (l)V-L1-X-L2-F2-L3-F1-L4-FVIII;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (m)F1-L4-FVIII-L3-F2-L2-X-L1-V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (n)FVIII-L4-F1-L3-F2-L2-X-L1-V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (o)FVIII-L4-F1-L3-V-L1-X-L2-F2;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (p)FVIII-L4-F1-L3-F2-L2-X-L1-V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (q)F2-L2-X-L1-V-L3-F1-L4-FVIII;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (r)F2-L2-X-L1-V-L3-FVIII-L4-F1;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (s)V-L1-X1-L2-F2-L3-FVIII(X2)-L4-F1;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (t)V-L1-X1-L2-F2-L3-F1-L4-FVIII(X2);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (u)F1-L4-FVIII(X2)-L3-F2-L2-X1-L1-V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (v)F-L4-FVIII(X2)-L3-V-L1-X1-L2-F2;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (w)FVIII(X2)-L4-F1-L3-V-L1-X1-L2-F2;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (x)FVIII(X2)-L4-F1-L3-F2-L2-X1-L1-V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (y)F2-L2-X1-L1-V-L3-F1-L4-FVIII(X2);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (z)F2-L2-X1-L1-V-L3-FVIII(X2)-L4-F1;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (aa)V-L1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (bb)V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (cc)F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (dd)F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (ee)FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (ff)FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V;<h2 style=";text-align:left;direction:ltr"> (gg)F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or (hh)F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2; wherein V is a VWF protein comprising a D' domain and a D3 domain, X or X1 is a first XTEN sequence containing less than 288 amino acids, X2 is a second XTEN sequence, FVIII comprises a FVIII protein, FVIII(X2) comprises a FVIII protein having a second XTEN sequence inserted into one or more insertion sites in the FVIII protein, F1 is a first Ig constant region or a portion thereof, F2 is a second Ig constant region or a portion thereof, L1, L2, L3, L4 or L5 is an optional linker, (-) is a peptide bond, and (:) is a covalent or non-covalent bond.

[0016] Some embodiments include a chimeric protein as described herein, wherein X or X1 consists of an amino acid sequence between 12 amino acids and 287 amino acids in length.

[0017] In certain embodiments, the chimeric protein as described herein exhibits a longer half-life than a corresponding chimeric protein comprising a formula except that X or X1 is AE288. In some embodiments, AE288 is SEQ ID NO:8.

[0018] Some embodiments include chimeric proteins as described herein, wherein X or X1 in the formula contains about 36, about 42, about 72, or about 144 amino acids. In certain embodiments, X or X1 in the formula is selected from AE42, AE72, AE144, AG42, AG72, or AG144. In some embodiments, X or X1 in the formula is selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63. In certain embodiments, X2 comprises an amino acid sequence having a length of at least about 36 amino acids, at least about 42 amino acids, at least about 144 amino acids, at least about 288 amino acids, at least about 576 amino acids, at least about 864 amino acids. In certain embodiments, X2 is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. In some embodiments, X2 is selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:17; SEQ ID NO:54; SEQ ID NO:19; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:15; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63. In certain embodiments, X2 is AE288 or AG288.

[0019] Also disclosed are chimeric proteins as described herein comprising X or X1 and / or X2 that exhibit increased half-life relative to chimeric proteins not comprising X or X1 and / or X2. In some embodiments, L1 and / or L2 are cleavable linkers. In certain embodiments, L4 and / or L5 are cleavable linkers. In certain embodiments, the linker is a protease selected from Factor XIa, Factor XIIa, kallikrein, Factor VIIa, Factor IXa, Factor Xa, Factor IIa (thrombin), elastase-2, granzyme-B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. In some embodiments, the linker is cleavable by Factor IIa (thrombin).

[0020] Some embodiments include a chimeric protein as described herein, wherein the linker is RRRR (SEQ ID NO:102), RKRRKR (SEQ ID NO:103), RRRRS (SEQ ID NO:104), TQSFNDFTR (SEQ ID NO:2), SVSQTSKLTR (SEQ ID NO:3), DFLAEGGGVR (SEQ ID NO:4), TTKIKPR (SEQ ID NO:5), LVPRG (SEQ ID NO:6), ALRPR (SEQ ID NO:7), KLTRAET (SEQ ID NO:121), DFTRVVG (SEQ ID NO:122), TMTRIVGG (SEQ ID NO:123), SPFRSTGG (SEQ ID NO:124), LQVRIVGG (SEQ ID NO:125), LQVRIVGG (SEQ ID NO:126), LQVRIVGG (SEQ ID NO:127), LQVRIVGG (SEQ ID NO:128), LQVRIVGG (SEQ ID NO:129), LQVRIVGG (SEQ ID NO:130), LQVRIVGG (SEQ ID NO:131), LQVRIVGG (SEQ ID NO:132), LQVRIVGG (SEQ ID NO:133), LQVRIVGG (SEQ ID NO:134), LQVRIVGG (SEQ ID NO:135), LQVRIVGG (SEQ ID NO:136), LQVRIVGG (SEQ ID NO:137), LQVRIVGG (SEQ ID NO:138), LQVRIVGG (SEQ ID NO:139), LQVRIVGG (SEQ ID NO:140), LQVRIVGG (SEQ ID NO:141), LQVRIVGG (SEQ ID NO:142), LQVRIVGG (SEQ ID NO:143), LQVRIVGG (SEQ ID NO:144), LQVRIVGG (SEQ ID NO:145), LQVRIV 25), PLGRIVGG (SEQ ID NO: 126), IEGRTVGG (SEQ ID NO: 127), LTPRSLLV (SEQ ID NO: 128), LGPVSGVP (SEQ ID NO: 129), VAGDSLEE (SEQ ID NO: 130), GPAGLGGA (SEQ ID NO: 131), GPAGLRGA (SEQ ID NO: 132), APLGLRLR (SEQ ID NO: 133), PALPLVAQ (SEQ ID NO: 134), ENLYFQG (SEQ ID NO: 135), DDDKIVGG (SEQ ID NO: 136), LEVLFQGP (SEQ ID NO: 137), and LPKTGSES (SEQ ID NO: 138). In some embodiments, the linker comprises TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). In certain embodiments, the linker comprises the amino acid sequence of LVPRG (SEQ ID NO: 6). In some embodiments, the linker comprises the a1 region of FVIII, the a2 region of FVIII, the a3 region of FVIII, or any combination thereof. In certain embodiments, the linker comprises a fragment of the a2 region of FVIII. The fragment of the a2 region can optionally comprise the sequence DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88). In yet other embodiments, even smaller fragments of the a2 region of FVIII can be used, including a fragment having the sequence IEPRSFS (SEQ ID NO: 194). In one particular embodiment, the linker comprises the amino acid sequence of IEPRSFS (SEQ ID NO: 194). In another embodiment, the linker comprises the amino acid sequence of IEPRSFS (SEQ ID NO: 194), where the linker is not the full-length a2 region of FVIII.

[0021] Also disclosed are chimeric proteins as described herein, wherein the a2 region of FVIII comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, or 100% identical to either ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106) or DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88). In some embodiments, the a1 region comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, or 100% identical to ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107). In certain embodiments, the a3 region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 108). In some embodiments, F1 comprises a first Fc region and / or F2 comprises a second Fc region.

[0022] Some embodiments include chimeric proteins as described herein, wherein the chimeric protein comprising F1 and F2 exhibits a longer half-life than the chimeric protein not comprising F1 and F2. In certain embodiments, L3 is a processable linker. In some embodiments, the VWF protein is associated with the FVIII protein by a non-covalent bond. In some embodiments, the half-life of the chimeric protein is extended compared to the VWF protein and / or the FVIII protein not comprising an XTEN sequence, or compared to wild-type FVIII. In certain embodiments, the half-life of the chimeric protein is extended compared to the VWF protein or the FVIII protein not comprising an XTEN sequence, or compared to wild-type FVIII. The lengthening is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer.

[0023] Also disclosed is a chimeric protein as described herein, wherein the half-life of the chimeric protein is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In some embodiments, the half-life of the chimeric protein is about 40 hours in HemA mice. In certain embodiments, the VWF protein does not substantially bind to the VWF clearance receptor. In some embodiments, the VWF protein can protect the FVIII protein from one or more protease cleavage, can protect the FVIII protein from activation, can stabilize the heavy and / or light chains of the FVIII protein, or can prevent clearance of the FVIII protein by one or more scavenger receptors.

[0024] Some embodiments include a chimeric protein as described herein, wherein the VWF protein inhibits or prevents endogenous VWF from binding to the FVIII protein by shielding or blocking the VWF binding site on the FVIII protein. In certain embodiments, the VWF binding site is located in the A3 domain or the C2 domain, or both the A3 domain and the C2 domain of the FVIII protein. In some embodiments, the VWF binding site comprises an amino acid sequence corresponding to amino acids 1669-1689 and 2303-2332 of SEQ ID NO: 65. In some embodiments, the first Ig constant region or portion thereof and the second Ig constant region or portion thereof are the same or different. In certain embodiments, the FVIII protein is linked to and / or inserted with at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five XTEN sequences, or at least six XTEN sequences.

[0025] Also disclosed are chimeric proteins as described herein, wherein the FVIII protein comprises one or more domains of FVIII selected from the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the B domain, the A3 domain, the a3 acidic region, the C1 domain, the C2 domain, one or more fragments thereof, and any combination thereof.

[0026] Also disclosed are chimeric proteins as described herein, wherein one or more insertion sites in the FVIII protein are within one or more domains of FVIII selected from the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the C1 domain, the C2 domain, and any combination thereof; or between one or more domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof; or between one or more domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof. In some embodiments, the one or more insertion sites in the FVIII protein are located between two domains of the FVIII protein selected from the group consisting of the amino acid residues in Table 7, Table 8, Table 9, and Table 10. In certain embodiments, the insertion site in the FVIII protein is located immediately downstream of amino acid 745 corresponding to the mature FVIII protein (SEQ ID NO:65). In some embodiments, the insertion site in the FVIII protein is located immediately downstream of residues 1656 and 1900 corresponding to the mature FVIII protein (SEQ ID NO:65). In some embodiments, the insertion site in the FVIII protein is immediately downstream of residues 26, 1656, and 1900 corresponding to the mature FVIII protein (SEQ ID NO:65). In certain embodiments, the insertion site in the FVIII protein is immediately downstream of residues 403 and 745 corresponding to the mature FVIII protein (SEQ ID NO:65). In some embodiments, the insertion site in the FVIII protein is immediately downstream of residues 745 and 1900, which correspond to the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion site in the FVIII protein is immediately downstream of residues 18 and 745, which correspond to the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the FVIII protein is a dual-chain FVIII isoform. In some embodiments, the FVIII protein is a single-chain FVIII isoform. In certain embodiments, the FVIII protein comprises a B domain or a portion thereof. In some embodiments, the FVIII protein is an SQB domain deleted FVIII.

[0027] Some embodiments include chimeric proteins as described herein, wherein the single chain FVIII isoform contains at least one amino acid substitution at residue 1648, residue 1645, or both, corresponding to full-length mature factor VIII polypeptide (SEQ ID NO:65), or at residues corresponding to residues 754, residue 751, or both, of SQ BDD factor VIII (SEQ ID NO:67). In certain embodiments, the amino acid substitution is an amino acid other than arginine. In some embodiments, the dual chain FVIII isoform comprises a first chain comprising a heavy chain of FVIII and a second chain comprising a light chain of FVIII, wherein the heavy and light chains are associated with each other by metal binding. In certain embodiments, the D' domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764-866 of SEQ ID NO:21. In some embodiments, the D3 domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 867-1240 of SEQ ID NO: 21. In certain embodiments, the VWF protein is monomeric.

[0028] Also disclosed are chimeric proteins as described herein, which comprise at least two VWF proteins, at least three VWF proteins, at least four VWF proteins, at least five VWF proteins, or at least six VWF proteins. In certain embodiments, the VWF protein comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764-1240 of SEQ ID NO:21. In some embodiments, the VWF protein consists essentially of, or consists of, amino acids 764-1240 of SEQ ID NO:21. In certain embodiments, the VWF protein 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:21. In some embodiments, the VWF protein contains an amino acid other than cysteine ​​substituted for a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO:21. In certain embodiments, the VWF protein further comprises the D1 domain, the D2 domain, or the D1 and D2 domains of VWF.

[0029] Some embodiments include a chimeric protein as described herein, wherein the VWF protein further comprises an A1 domain, an A2 domain, an A3 domain, a D4 domain, or a combination thereof. The VWF domains include VWF domains selected from the group consisting of the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, and any combination thereof.

[0030] Also disclosed are chimeric proteins as described herein, wherein the VWF protein consists essentially of or consists of (1) the D' and D3 domains of VWF or fragments thereof, (2) the D1, D' and D3 domains of VWF or fragments thereof, (3) the D2, D' and D3 domains of VWF or fragments thereof, (4) the D1, D2, D' and D3 domains of VWF or fragments thereof, or (5) the D1, D2, D', D3 and A1 domains of VWF or fragments thereof.

[0031] Some embodiments include a chimeric protein as described herein, wherein the VWF protein further comprises a signal peptide of VWF or FVIII operably linked to the VWF protein.

[0032] Also disclosed are chimeric proteins as described herein, wherein one or more of the linkers 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 acid residues. In some embodiments, one or more of the linkers have a length of about 1 to about 2000 amino acid residues. In certain embodiments, one or more of the linkers comprises a Gly / Ser peptide. 4 Ser) n (SEQ ID NO: 94) or S(Gly 4 Ser) n (SEQ ID NO: 164), where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In certain embodiments, (Gly 4 Ser) n The linker is (Gly 4 Ser) 3 (SEQ ID NO: 100) or (Gly 4 Ser) 4 (SEQ ID NO: 165). In some embodiments, the linker comprises 20 amino acids, 35 amino acids, 48 ​​amino acids, 73 amino acids, or 95 amino acids. In certain embodiments, the cleavable linker is SGGGGSGGGGSGGGGGSGGGGGSGGGGSLVPRGSGG (SEQ ID NO: 166).

[0033] In some embodiments, a chimeric protein as described herein is polysialylated, PEGylated, or HESylated.

[0034] Also disclosed are chimeric proteins as described herein, wherein the first polypeptide is selected from the group consisting of FVIII161 (SEQ ID NO:69), FVIII169 (SEQ ID NO:70), FVIII173 (SEQ ID NO:72), FVIII195 (SEQ ID NO:73), FVIII196 (SEQ ID NO:74), FVIII199 (SEQ ID NO:75), FVIII201 (SEQ ID NO:76), FVIII203 (SEQ ID NO:77), FVIII204 (SEQ ID NO:78), FVIII205 (SEQ ID NO:79), FVIII266 (SEQ ID NO:80), FVIII270 (SEQ ID NO:81), FVIII271 (SEQ ID NO:82), FVIII272 (SEQ ID NO:83), FVIII273 (SEQ ID NO:84), FVIII274 (SEQ ID NO:85), FVIII275 (SEQ ID NO:86), FVIII276 (SEQ ID NO:87), FVIII278 (SEQ ID NO:89), FVIII280 (SEQ ID NO:90), FVIII281 (SEQ ID NO:91), FVIII282 (SEQ ID NO:92), FVIII283 (SEQ ID NO:93), FVIII284 (SEQ ID NO:94), FVIII285 (SEQ ID NO:95), FVIII286 (SEQ ID NO:96), FVIII287 (SEQ ID NO:97), FVIII288 (SEQ ID NO:98), FVIII289 (SEQ ID NO:100), FVIII300 (SEQ ID NO:101), FVIII301 (SEQ ID NO:102), FVIII302 (SEQ ID NO:103), FVIII303 In some embodiments, the first polypeptide comprises FVIII169 (SEQ ID NO:70) and the second polypeptide comprises VWF057 (SEQ ID NO:152). In other embodiments, the first polypeptide comprises FVIII169 (SEQ ID NO:70) and the second polypeptide comprises VWF057 (SEQ ID NO:152). In other embodiments, the first polypeptide comprises FVIII169 (SEQ ID NO:70) and the second polypeptide comprises VWF057 (SEQ ID NO:152). The peptide comprises FVIII169 (SEQ ID NO: 70) and the second polypeptide comprises VWF059 (SEQ ID NO: 197). In yet another embodiment, the first polypeptide comprises FVIII169 (SEQ ID NO: 70) and the second polypeptide comprises VWF062 (SEQ ID NO: 199). In some embodiments, the chimeric protein is effective to prevent and / or stop bleeding in a subject in need thereof.

[0035] Also disclosed are polynucleotides or sets of polynucleotides that encode the chimeric proteins as described herein. In some embodiments, the polynucleotides as described herein further comprise polynucleotide strands that encode PC5 or PC7.

[0036] Some embodiments include a vector comprising a polynucleotide as described herein and one or more promoters operably linked to the polynucleotide or set of polynucleotides.

[0037] In some embodiments, a vector as described herein further comprises an additional vector comprising a polynucleotide sequence encoding PC5 or PC7.

[0038] Also disclosed are host cells comprising the polynucleotides or vectors as described herein. In some embodiments, the host cells are mammalian cells. In certain embodiments, the mammalian cells are selected from HEK293 cells, CHO cells, and BHK cells.

[0039] Also disclosed is a pharmaceutical composition comprising a chimeric protein, a polynucleotide, a vector or a host cell as described herein and a pharma- ceutically acceptable carrier. In some embodiments, the chimeric protein has an extended half-life compared to wild-type FVIII protein. In certain embodiments, the half-life of the chimeric 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.

[0040] Some embodiments include a composition as described herein, wherein the half-life of the chimeric protein is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In certain embodiments, the half-life of the chimeric protein is about 40 hours in HemA mice. In some embodiments, the compositions as described herein are administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. In certain embodiments, the parenteral administration is intravenous administration or subcutaneous administration.

[0041] In some embodiments, the compositions as described herein are used to treat a bleeding disorder or condition in a subject in need thereof. In certain embodiments, the bleeding disorder or condition is a bleeding coagulopathy, hemarthrosis, muscle bleed, oral bleed, bleeding, bleeding into muscle, oral bleed, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, or bleeding in the iliopsoas sheath. In some embodiments, the subject is scheduled to undergo surgery. In certain embodiments, the treatment is prophylactic or on-demand.

[0042] Also disclosed is a method of extending or increasing the half-life of a chimeric protein, comprising administering to a subject in need thereof an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition as described herein, wherein a VWF protein, an XTEN sequence, a first Ig constant region or portion thereof, or a second Ig constant region or portion thereof increases the half-life of the chimeric protein.

[0043] Some embodiments include a method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition as described herein, wherein the bleeding disease or disorder is selected from the group consisting of bleeding coagulopathy, hemarthrosis, muscle bleeds, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and iliopsoas sheath. In some embodiments, the subject is an animal. In certain embodiments, the animal is a human. In some embodiments, the subject has hemophilia A. In certain embodiments, the treatment is prophylactic or on demand. In some embodiments, the effective amount is between 0.1 μg / kg and 500 mg / kg.

[0044] Also disclosed are methods as described herein, wherein a chimeric protein, polynucleotide, vector, host cell, or composition as described herein is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. In certain embodiments, parenteral administration is selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration.

[0045] Some embodiments include a method of making the chimeric protein, comprising transfecting one or more host cells with a polynucleotide or vector as described herein and expressing the chimeric protein in the host cells. In some embodiments, the method as described herein further comprises isolating the chimeric protein. In certain embodiments, the chimeric protein is effective in stopping bleeding and / or preventing bleeding in a subject. The present invention provides, for example, the following items. (Item 1) A chimeric protein comprising: (i) a first polypeptide comprising a factor VIII ("FVIII") protein fused to a first immunoglobulin ("Ig") constant region or portion thereof; and (ii) a second polypeptide comprising a von Willebrand factor ("VWF") protein comprising the D' and D3 domains of VWF fused to a second Ig constant region or portion thereof by an intermediate XTEN sequence, wherein the XTEN sequence contains fewer than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide. (Item 2) 2. The chimeric protein according to item 1, wherein the XTEN sequence in the second polypeptide is an amino acid sequence having a length of between 12 amino acids and 287 amino acids. (Item 3) 3. The chimeric protein according to item 1 or 2, wherein the chimeric protein exhibits a longer half-life than a corresponding fusion protein comprising a first polypeptide and a second polypeptide, wherein the second polypeptide of the fusion protein comprises an XTEN sequence containing at least 288 amino acids. (Item 4) 4. The chimeric protein according to item 3, wherein the XTEN sequence containing at least 288 amino acids is AE288. (Item 5) 5. The chimeric protein according to item 4, wherein AE288 is SEQ ID NO: 8. (Item 6) 6. The chimeric protein according to any one of items 1 to 5, wherein the XTEN sequence of the second polypeptide contains about 36, about 42, about 72, or about 144 amino acids. (Item 7) 7. The chimeric protein of item 6, wherein the XTEN sequence of the second polypeptide is selected from AE42, AE72, AE144, AG42, AG72, or AG144. (Item 8) 8. The chimeric protein of item 7, wherein the XTEN sequence of the second polypeptide is selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; SEQ ID NO:63. (Item 9) 9. The chimeric protein according to any one of items 1 to 8, wherein the first polypeptide further comprises a second XTEN sequence linking the FVIII protein to the first Ig constant region or a part thereof. (Item 10) 10. The chimeric protein according to item 9, wherein the first polypeptide comprises a third XTEN sequence inserted at one or more insertion sites within the FVIII protein. (Item 11) 9. The chimeric protein according to any one of items 1 to 8, wherein the first polypeptide further comprises a second XTEN sequence inserted at one or more insertion sites within the FVIII protein. (Item 12) 12. The chimeric protein of item 11, wherein the first polypeptide comprises a third XTEN sequence linking the FVIII protein to the first Ig constant region or a portion thereof. (Item 13) 13. The chimeric protein according to any one of items 9 to 12, wherein the second XTEN sequence, the third XTEN sequence, or the second and third XTEN sequences are each independently selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. (Item 14) 14. The chimeric protein of item 13, wherein the second XTEN sequence, the third XTEN sequence, or the second and third XTEN sequences are each independently selected from SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17; SEQ ID NO:54; SEQ ID NO:19; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:15; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63. (Item 15) 15. The chimeric protein of item 13 or 14, wherein the second XTEN sequence, the third XTEN sequence, or both the second and third XTEN sequences are each independently AE288 or AG288. (Item 16) 16. The chimeric protein according to any one of items 1 to 15, wherein the XTEN sequence in the second polypeptide is fused to the second Ig constant region or a part thereof by a linker. (Item 17) 17. The chimeric protein according to item 16, wherein the linker is a cleavable linker. (Item 18) 18. The chimeric protein according to item 17, wherein the linker is cleavable by a protease selected from factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, granzyme-B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. (Item 19) 19. The chimeric protein according to item 18, wherein the linker is cleavable by factor IIa (thrombin). (Item 20) The linker is selected from the group consisting of RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: 103), RRRRS (SEQ ID NO: 104), TQSFNDFTR (SEQ ID NO: 2), SVSQTSKLTR (SEQ ID NO: 3), DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), KLTRAET (SEQ ID NO: 121), DFTRVVG (SEQ ID NO: 122), TMTRIVGG (SEQ ID NO: 123), SPFRSTGG (SEQ ID NO: 124), LQVRIVGG (SEQ ID NO: 125), PLGRIVGG (SEQ ID NO: 126), IEGRT 18. The chimeric protein of item 17, comprising one or more cleavage sites comprising an amino acid sequence selected from VGG (SEQ ID NO: 127), LTPRSLLV (SEQ ID NO: 128), LGPVSGVP (SEQ ID NO: 129), VAGDSLEE (SEQ ID NO: 130), GPAGLGGA (SEQ ID NO: 131), GPAGLRGA (SEQ ID NO: 132), APLGLRLR (SEQ ID NO: 133), PALPLVAQ (SEQ ID NO: 134), ENLYFQG (SEQ ID NO: 135), DDDKIVGG (SEQ ID NO: 136), LEVLFQGP (SEQ ID NO: 137), and LPKTGSES (SEQ ID NO: 138). (Item 21) 18. The chimeric protein of item 17, wherein the linker comprises TLDPRSFLLRNPNDKYEPFWEDEEK (sequence number 146). (Item 22) 21. The chimeric protein according to item 20, wherein the cleavage site comprises the amino acid sequence of LVPRG (SEQ ID NO: 6). (Item 23) 23. The chimeric protein according to any one of items 1 to 22, wherein the first Ig constant region or a portion thereof comprises a first Fc region and / or the second Ig constant region or a portion thereof comprises a second Fc region. (Item 24) 24. The chimeric protein according to item 23, wherein the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof extend the half-life of the chimeric protein. (Item 25) 25. The chimeric protein according to any one of items 1 to 24, wherein the first polypeptide and the second polypeptide are fused via a linker. (Item 26) 26. The chimeric protein according to item 25, wherein the first polypeptide and the second polypeptide are fused by a processable linker. (Item 27) 25. The chimeric protein according to any one of items 1 to 24, wherein the first Ig constant region or a part thereof is associated with a second Ig constant region or a part thereof. (Item 28) 28. The chimeric protein according to item 27, wherein the first Ig constant region or a portion thereof is covalently associated with the second Ig constant region or a portion thereof. (Item 29) 29. The chimeric protein according to item 28, wherein the covalent bond is a disulfide bond. (Item 30) A chimeric protein having the following formulas (a) to (hh): (a) FVIII-F1:F2-L2-X-L1-V; (b) FVIII-F1: V-L1-X-L2-F2; (c) F1-FVIII:F2-L2-X-L1-V; (d) F1-FVIII: V-L1-X-L2-F2; (e) FVIII-X2-F1:F2-L2-X1-L1-V; (f) FVIII-X2-F1: V-L1-X1-L2-F2; (g)FVIII(X2)-F1:F2-L2-X1-L1-V; (h)FVIII(X2)-F1:V-L1-X1-L2-F2; (i) F1-X2-F1:F2-L2-X1-L1-V; (j) F1-X2-F1: V-L1-X1-L2-F2; (k)V-L1-X-L2-F2-L3-FVIII-L4-F1; (l)V-L1-X-L2-F2-L3-F1-L4-FVIII; (m)F1-L4-FVIII-L3-F2-L2-X-L1-V; (n)FVIII-L4-F1-L3-F2-L2-X-L1-V; (o)FVIII-L4-F1-L3-V-L1-X-L2-F2; (p)FVIII-L4-F1-L3-F2-L2-X-L1-V; (q)F2-L2-X-L1-V-L3-F1-L4-FVIII; (r)F2-L2-X-L1-V-L3-FVIII-L4-F1; (s)V-L1-X1-L2-F2-L3-FVIII(X2)-L4-F1; (t)V-L1-X1-L2-F2-L3-F1-L4-FVIII(X2); (u)F1-L4-FVIII(X2)-L3-F2-L2-X1-L1-V; (v)F-L4-FVIII(X2)-L3-V-L1-X1-L2-F2; (w)FVIII(X2)-L4-F1-L3-V-L1-X1-L2-F2; (x)FVIII(X2)-L4-F1-L3-F2-L2-X1-L1-V; (y)F2-L2-X1-L1-V-L3-F1-L4-FVIII(X2); (z)F2-L2-X1-L1-V-L3-FVIII(X2)-L4-F1; (aa)V-L1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1; (bb)V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII; (cc)F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V; (dd)F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2; (ee)FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1; (ff)FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V; (gg)F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or (hh)F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2; wherein V is a VWF protein comprising a D' domain and a D3 domain, X or X1 is a first XTEN sequence containing less than 288 amino acids, X2 is a second XTEN sequence, FVIII comprises a FVIII protein, FVIII(X2) comprises a FVIII protein having a second XTEN sequence inserted into one or more insertion sites in the FVIII protein, F1 is a first Ig constant region or a portion thereof, F2 is a second Ig constant region or a portion thereof, L1, L2, L3, L4 or L5 is an optional linker, (-) is a peptide bond, and (:) is a covalent or non-covalent bond. (Item 31) 31. The chimeric protein according to item 30, wherein X or X1 consists of an amino acid sequence having a length of between 12 amino acids and 287 amino acids. (Item 32) Compared to the corresponding chimeric protein containing the formula except that X or X1 is AE288. 32. The chimeric protein according to item 30 or 31, which exhibits a long half-life in all cases. (Item 33) 33. The chimeric protein of item 32, wherein AE288 is SEQ ID NO:8. (Item 34) 34. The chimeric protein according to any one of items 30 to 33, wherein X or X1 in the formula contains about 36, about 42, about 72 or about 144 amino acids. (Item 35) 35. The chimeric protein of claim 34, wherein X or X1 in the formula is selected from AE42, AE72, AE144, AG42, AG72, or AG144. (Item 36) The chimeric protein of claim 34, wherein X or X1 in the formula is selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63. (Item 37) 37. The chimeric protein according to any one of Items 30 to 36, wherein X2 comprises an amino acid sequence having a length of at least about 36 amino acids, at least about 42 amino acids, at least about 144 amino acids, at least about 288 amino acids, at least about 576 amino acids, or at least about 864 amino acids. (Item 38) 38. The chimeric protein according to item 37, wherein X2 is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. (Item 39) 38. The chimeric protein according to item 37, wherein X2 is selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:17; SEQ ID NO:54; SEQ ID NO:19; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:15; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63. (Item 40) 38. The chimeric protein according to item 37, wherein X2 is AE288 or AG288. (Item 41) 41. The chimeric protein according to any one of items 30 to 40, wherein the chimeric protein comprising X or X1 and / or X2 exhibits a longer half-life than a chimeric protein not comprising X or X1 and / or X2. (Item 42) 42. The chimeric protein according to any one of items 30 to 41, wherein L1 and / or L2 is a cleavable linker. (Item 43) 42. The chimeric protein according to any one of items 30 to 41, wherein L4 and / or L5 is a cleavable linker. (Item 44) 44. The chimeric protein according to item 42 or 43, wherein the linker is cleavable by a protease selected from factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, granzyme-B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. (Item 45) 44. The chimeric protein according to item 42 or 43, wherein the linker is cleavable by factor IIa (thrombin). (Item 46) The linker is selected from the group consisting of RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: 103), RRRRS (SEQ ID NO: 104), TQSFNDFTR (SEQ ID NO: 1), SVSQTSKLTR (SEQ ID NO: 3), DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), KLTRAET (SEQ ID NO: 121), DFTRVVG (SEQ ID NO: 122), TMTRIVGG (SEQ ID NO: 123), SPFRSTGG (SEQ ID NO: 124), LQVRIVGG (SEQ ID NO: 125), PLGRIVGG (SEQ ID NO: 126), IEGRTVGG 44. The chimeric protein according to item 42 or 43, comprising one or more cleavage sites comprising an amino acid sequence selected from among (SEQ ID NO: 127), LTPRSLLV (SEQ ID NO: 128), LGPVSGVP (SEQ ID NO: 129), VAGDSLEE (SEQ ID NO: 130), GPAGLGGA (SEQ ID NO: 131), GPAGLRGA (SEQ ID NO: 132), APLGLRLR (SEQ ID NO: 133), PALPLVAQ (SEQ ID NO: 134), ENLYFQG (SEQ ID NO: 135), DDDKIVGG (SEQ ID NO: 136), LEVLFQGP (SEQ ID NO: 137), and LPKTGSES (SEQ ID NO: 138). (Item 47) 44. The chimeric protein according to item 42 or 43, wherein the linker comprises TLDPRSFLLRNPNDKYEPFWEDEEK (sequence number 146). (Item 48) 44. The chimeric protein according to item 42 or 43, wherein the linker comprises the amino acid sequence of LVPRG (SEQ ID NO: 6). (Item 49) Item 50. The chimeric protein according to item 42 or 43, wherein the linker comprises the a1 region of FVIII, the a2 region of FVIII, the a3 region of FVIII, or any combination thereof. 50. The chimeric protein of item 49, wherein the a2 region of FVIII comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106). (Item 51) 50. The chimeric protein of item 49, wherein the a1 region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107). (Item 52) 50. The chimeric protein of item 49, wherein the a3 region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 108). (Item 53) 53. The chimeric protein according to any one of items 30 to 52, wherein F1 comprises a first Fc region and / or F2 comprises a second Fc region. (Item 54) 54. The chimeric protein according to any one of items 30 to 53, wherein the chimeric protein comprising F1 and F2 exhibits a longer half-life than a chimeric protein not comprising F1 and F2. (Item 55) 55. The chimeric protein according to any one of items 30 to 54, wherein L3 is a processable linker. (Item 56) 56. The chimeric protein according to any one of items 1 to 55, wherein the VWF protein is non-covalently associated with the FVIII protein. (Item 57) 57. The chimeric protein according to any one of items 1 to 56, wherein the half-life of the chimeric protein is extended compared to a FVIII protein not containing a VWF protein and / or an XTEN sequence, or compared to a wild-type FVIII. (Item 58) 58. The chimeric protein of item 57, wherein the half-life of the chimeric protein is extended by 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 VWF protein or a FVIII protein that does not contain an XTEN sequence, or than wild-type FVIII. (Item 59) 58. The chimeric protein of item 57, wherein the half-life of the chimeric protein 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. (Item 60) 58. The chimeric protein according to item 57, wherein the half-life of the chimeric protein is about 40 hours in a HemA mouse. (Item 61) 61. The chimeric protein according to any one of items 1 to 60, wherein the VWF protein does not substantially bind to a VWF clearance receptor. (Item 62) 62. The chimeric protein according to any one of items 1 to 61, wherein the VWF protein is capable of protecting the FVIII protein from one or more protease cleavage, is capable of protecting the FVIII protein from activation, is capable of stabilizing the heavy chain and / or the light chain of the FVIII protein, or is capable of preventing clearance of the FVIII protein by one or more scavenger receptors. (Item 63) 63. The chimeric protein according to any one of items 1 to 62, wherein the VWF protein inhibits or prevents endogenous VWF from binding to the FVIII protein by masking or blocking the VWF binding site on the FVIII protein. (Item 64) 64. The chimeric protein according to item 63, wherein the VWF binding site is located in the A3 domain or in the C2 domain or in both the A3 and C2 domains of the FVIII protein. (Item 65) 64. The chimeric protein according to Item 63, wherein the VWF-binding site comprises an amino acid sequence corresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO: 65. (Item 66) 66. The chimeric protein according to any one of items 1 to 65, wherein the first Ig constant region or a part thereof and the second Ig constant region or a part thereof are identical or different. (Item 67) 67. Any one of items 1 to 66, wherein the FVIII protein is linked to and / or inserted with at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five XTEN sequences or at least six XTEN sequences. The chimeric protein according to claim 1. (Item 68) 68. The chimeric protein according to any one of items 1 to 67, wherein the FVIII protein comprises one or more domains of FVIII selected from the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the B domain, the A3 domain, the a3 acidic region, the C1 domain, the C2 domain, one or more fragments thereof, and a combination thereof. (Item 69) 69. The chimeric protein according to any one of items 10 to 68, wherein the one or more insertion sites in the FVIII protein are located within one or more domains of FVIII selected from the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the B domain, the a3 acidic region, the C1 domain, the C2 domain, and any combination thereof, or between one or more domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof, or between two domains of the FVIII protein selected from the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof. (Item 70) 69. The chimeric protein according to any one of items 10 to 68, wherein the one or more insertion sites in the FVIII protein are one or more amino acids selected from the group consisting of the amino acid residues in Table 7, Table 8, Table 9, and Table 10. (Item 71) 72. The chimeric protein according to claim 70, wherein the insertion site in the FVIII protein is located immediately downstream of amino acid 745, which corresponds to the mature FVIII protein (SEQ ID NO: 65). 69. The chimeric protein according to any one of items 10 to 68, wherein the insertion site in the FVIII protein is located immediately downstream of residues 1656 and 1900 corresponding to the mature FVIII protein (SEQ ID NO: 65). (Item 73) 69. The chimeric protein according to any one of items 10 to 68, wherein the insertion sites in the FVIII protein are immediately downstream of residues 26, 1656 and 1900 corresponding to the mature FVIII protein (SEQ ID NO: 65). (Item 74) 69. The chimeric protein according to any one of items 10 to 68, wherein the insertion site in the FVIII protein is immediately downstream of residues 403 and 745 corresponding to the mature FVIII protein (SEQ ID NO: 65). (Item 75) 69. The chimeric protein according to any one of items 10 to 68, wherein the insertion site in the FVIII protein is immediately downstream of residues 745 and 1900 corresponding to the mature FVIII protein (SEQ ID NO: 65). (Item 76) 69. The chimeric protein according to any one of items 10 to 68, wherein the insertion sites in the FVIII protein are immediately downstream of residues 18 and 745 corresponding to the mature FVIII protein (SEQ ID NO: 65). (Item 77) 77. The chimeric protein according to any one of items 1 to 76, wherein the FVIII protein is a dual-chain FVIII isoform. (Item 78) 77. The chimeric protein according to any one of items 1 to 76, wherein the FVIII protein is a single-chain FVIII isoform. (Item 79) 79. The chimeric protein according to any one of items 1 to 78, wherein the FVIII protein comprises a B domain or a part thereof. (Item 80) 80. The chimeric protein according to item 79, wherein the FVIII protein is an SQ B domain deleted FVIII. (Item 81) 80. The chimeric protein according to item 78 or 79, wherein the single-chain FVIII isoform contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645 or both residues corresponding to the full-length mature factor VIII polypeptide (SEQ ID NO: 65), or at a residue corresponding to residue 754, residue 751 or both residues of SQ BDD factor VIII (SEQ ID NO: 67). (Item 82) Item 83. The chimeric protein according to Item 81, wherein the amino acid substitution is an amino acid other than arginine. 78. The chimeric protein according to item 77, wherein the dual chain FVIII isoform comprises a first chain comprising a heavy chain of FVIII and a second chain comprising a light chain of FVIII, wherein the heavy chain and the light chain are associated with each other by metal binding. (Item 84) 84. The chimeric protein according to any one of Items 1 to 83, wherein the D' domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 866 of SEQ ID NO: 21. (Item 85) 85. The chimeric protein according to any one of items 1 to 84, wherein the D3 domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 21. (Item 86) 86. The chimeric protein according to any one of items 1 to 85, wherein the VWF protein is a monomer. (Item 87) 87. The chimeric protein according to any one of items 1 to 86, comprising at least two VWF proteins, at least three VWF proteins, at least four VWF proteins, at least five VWF proteins, or at least six VWF proteins. (Item 88) 88. The chimeric protein according to any one of items 1 to 87, wherein the VWF protein comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 21. (Item 89) 89. The chimeric protein according to item 88, wherein the VWF protein consists essentially of or consists of amino acids 764 to 1240 of SEQ ID NO: 21. (Item 90) 89. The chimeric protein according to any one of items 1 to 89, wherein the VWF protein 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: 21. (Item 91) 80. The chimeric protein according to any one of items 1 to 89, wherein the VWF protein contains an amino acid other than cysteine ​​substituted with a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 21. (Item 92) 92. The chimeric protein according to any one of items 1 to 91, wherein the VWF protein further comprises the D1 domain, the D2 domain or the D1 and D2 domains of VWF. (Item 93) 93. The chimeric protein according to any one of items 1 to 92, wherein the VWF protein further comprises a VWF domain selected from the group consisting of A1 domain, A2 domain, A3 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, and any combination thereof. (Item 94) 93. The chimeric protein according to any one of items 1 to 92, wherein the VWF protein essentially consists of or consists of (1) the D' and D3 domains of VWF or fragments thereof, (2) the D1, D' and D3 domains of VWF or fragments thereof, (3) the D2, D' and D3 domains of VWF or fragments thereof, (4) the D1, D2, D' and D3 domains of VWF or fragments thereof, or (5) the D1, D2, D', D3 and A1 domains of VWF or fragments thereof. (Item 95) 95. The chimeric protein according to any one of items 1 to 94, wherein the VWF protein further comprises a signal peptide of VWF or FVIII operably linked to the VWF protein. (Item 96) 96. The chimeric protein according to any one of items 16 to 95, wherein one or more of the linkers has 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 acid residues. (Item 97) 96. The chimeric protein according to any one of items 16 to 95, wherein one or more of the linkers has a length of about 1 to about 2000 amino acid residues. (Item 98) 98. The chimeric protein according to item 96 or 97, wherein one or more of the linkers comprises a Gly / Ser peptide. (Item 99) Gly / Ser peptide (Gly 4 Ser) n (SEQ ID NO: 94) or S(Gly 4 Ser) n 99. The chimeric protein according to item 98, having the formula: (SEQ ID NO: 164), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. (Item 100) (Gly 4 Ser) n The linker is (Gly 4 Ser) 3 (SEQ ID NO: 100) or (Gly 4 Ser) 4 99. The chimeric protein according to item 99, wherein the chimeric protein is (SEQ ID NO: 165). (Item 101) 101. The chimeric protein according to any one of items 16 to 100, wherein the linker comprises 20 amino acids, 35 amino acids, 48 ​​amino acids, 73 amino acids, or 95 amino acids. (Item 102) The chimeric protein according to any one of Items 17 to 29 and 42 to 101, wherein the cleavable linker is SGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGG (SEQ ID NO: 166). (Item 103) 103. The chimeric protein according to any one of items 1 to 102, which is polysialylated, PEGylated or HESylated. (Item 104) The first polypeptide is selected from the group consisting of FVIII161 (SEQ ID NO: 69), FVIII169 (SEQ ID NO: 70), FVIII173 (SEQ ID NO: 72), FVIII195 (SEQ ID NO: 73), FVIII196 (SEQ ID NO: 74), FVIII199 (SEQ ID NO: 75), FVIII201 (SEQ ID NO: 76), FVIII203 (SEQ ID NO: 77), FVIII204 (SEQ ID NO: 78), FVIII205 (SEQ ID NO: 79), FVIII266 (SEQ ID NO: 80), FVIII267 (SEQ ID NO: 81), FVII 21. The chimeric protein of any one of items 1 to 20, comprising a second polypeptide that is at least about 80%, 90%, 95%, 99% or 100% identical to VWF057 (SEQ ID NO: 152), I268 (SEQ ID NO: 82), FVIII269 (SEQ ID NO: 83), FVIII271 (SEQ ID NO: 84), FVIII272 (SEQ ID NO: 85) or FVIII282 (SEQ ID NO: 159), and wherein the second polypeptide is at least about 80%, 90%, 95%, 99% or 100% identical to VWF057 (SEQ ID NO: 152). (Item 105) 105. The chimeric protein of item 104, wherein the first polypeptide comprises FVIII169 (SEQ ID NO: 70) and the second polypeptide comprises VWF057 (SEQ ID NO: 152). (Item 106) 106. The chimeric protein according to any one of items 1 to 105, wherein the chimeric protein is effective to prevent and / or stop bleeding in a subject in need thereof. (Item 107) A polynucleotide or a set of polynucleotides encoding the chimeric protein according to any one of items 1 to 106. (Item 108) 108. The polynucleotide according to item 107, further comprising a polynucleotide chain encoding PC5 or PC7. (Item 109) 109. A vector comprising the polynucleotide of item 107 or 108 and one or more promoters operably linked to said polynucleotide or set of said polynucleotides. (Item 110) 110. The vector according to item 109, further comprising an additional vector comprising a polynucleotide chain encoding PC5 or PC7. (Item 111) A host cell comprising a polynucleotide according to item 107 or 108, or a vector according to item 109 or 110. (Item 112) 112. The host cell according to item 111, which is a mammalian cell. (Item 113) 113. The host cell according to item 112, wherein the mammalian cell is selected from HEK293 cells, CHO cells and BHK cells. (Item 114) A pharmaceutical composition comprising the chimeric protein according to any one of items 1 to 106, the polynucleotide according to item 107 or 108, the vector according to item 109 or 110, or the host cell according to any one of items 111 to 113, and a pharma- ceutically acceptable carrier. (Item 115) 15. The composition according to item 114, wherein the FVIII protein has an extended half-life compared to the wild-type FVIII protein. (Item 116) The half-life of the chimeric protein is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, 116. The composition according to item 114 or 115, wherein the prolongation is at least about 10-fold, at least about 11-fold, or at least about 12-fold. (Item 117) 16. The composition according to item 114 or 115, wherein the half-life of the chimeric protein 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. (Item 118) 116. The composition according to item 114 or 115, wherein the half-life of the chimeric protein is about 40 hours in HemA mice. (Item 119) 119. The composition according to any one of items 114 to 118, administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. (Item 120) 120. The composition according to item 119, wherein the parenteral administration is intravenous or subcutaneous administration. (Item 121) 121. The composition according to any one of items 114 to 120, for use in treating a bleeding disease or condition in a subject in need thereof. (Item 122) 122. The composition of claim 121, wherein the bleeding disease or condition is selected from the group consisting of hemorrhagic coagulopathy, hemarthrosis, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and combinations thereof. (Item 123) 123. The composition according to item 121 or 122, wherein the subject is scheduled to undergo surgery. (Item 124) 124. The composition according to any of items 114 to 123, wherein the treatment is prophylactic or on demand. (Item 125) 13. A method for extending or increasing the half-life of a chimeric protein, the method comprising administering to a subject in need thereof an effective amount of the chimeric protein according to any one of items 1 to 106, the polynucleotide according to item 107 or 108, the vector according to item 109 or 110, the host cell according to any one of items 111 to 113, or the composition according to any one of items 114 to 124, wherein the VWF protein, the XTEN sequence, the first Ig constant region or a part thereof and the second Ig constant region or a part thereof increase the half-life of the chimeric protein. (Item 126) A method for treating a bleeding disease or disorder in a subject in need thereof, comprising administering an effective amount of the chimeric protein according to any one of items 1 to 106, the polynucleotide according to item 107 or 108, the vector according to item 109 or 110, the host cell according to any one of items 111 to 113, or the composition according to any one of items 114 to 124, wherein the bleeding disease or disorder is hemorrhagic coagulopathy, hemarthrosis, muscle hemorrhage, oral hemorrhage, bleeding, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal discharge the said method for treating bleeding in the iliopsoas sheath, wherein the bleeding is selected from the group consisting of bleeding in the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. (Item 127) 127. The method according to item 125 or 126, wherein the subject is an animal. (Item 128) 128. The method according to item 127, wherein the animal is a human. (Item 129) 129. The method according to item 127 or 128, wherein the subject suffers from hemophilia A. (Item 130) 130. The method according to any one of items 126 to 129, wherein the treatment is prophylactic or on demand. (Item 131) 131. The method according to any one of items 126 to 130, wherein the effective amount is about 0.1 μg / kg to 500 mg / kg. (Item 132) The method according to any one of items 125 to 131, wherein the chimeric protein according to any one of items 1 to 106, the polynucleotide according to item 107 or 108, the vector according to item 109 or 110, the host cell according to any one of items 111 to 113, or the composition according to any one of items 114 to 124 is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. (Item 133) 133. The method of item 132, wherein parenteral administration is selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration. (Item 134) A method for producing a chimeric protein, comprising transfecting one or more host cells with a polynucleotide according to item 107 or 108 or a vector according to item 109 or 110, and expressing the chimeric protein in the host cells. (Item 135) 135. The method of item 134, further comprising isolating the chimeric protein. (Item 136) 136. The method of any one of items 124 to 135, wherein the chimeric protein is effective to stop and / or prevent bleeding in a subject. (Item 137) 20. The chimeric protein according to item 19, wherein the linker comprises the a2 region of FVIII or a fragment thereof. (Item 138) 138. The chimeric protein of item 137, wherein the linker comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88). (Item 139) 20. The chimeric protein according to item 19, wherein the linker comprises the amino acid sequence IEPRSFS (SEQ ID NO: 194). (Item 140) 130. The chimeric protein according to item 138 or 139, wherein the linker is not the full-length a2 region of FVIII. (Item 141) 44. The chimeric protein according to item 42 or 43, wherein the linker comprises a fragment of the a2 region of FVIII. (Item 142) 142. The chimeric protein according to item 141, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 88, 139, 140, 141, 142, 143, 144, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, and 200. (Item 143) 143. The chimeric protein according to item 142, wherein the linker comprises the amino acid sequence IEPRSFS (SEQ ID NO: 194). (Item 144) 144. The chimeric protein according to any one of items 141 to 143, wherein the linker is not the a2 region of the full-length FVIII. (Item 145) A chimeric protein comprising: (i) a first polypeptide comprising a factor VIII ("FVIII") protein fused to a first immunoglobulin ("Ig") constant region or portion thereof, wherein the FVIII protein comprises an XTEN sequence having a length of 288 amino acids; (ii) a second polypeptide comprising a von Willebrand factor ("VWF") protein comprising the D' and D3 domains fused to a second Ig constant region or portion thereof by an intermediate 144 amino acid XTEN sequence; The chimeric protein, wherein the first polypeptide is linked or associated with the second polypeptide via the first and second constant regions. (Item 146) 146. The chimeric protein according to item 145, further comprising a cleavable linker between the 144 amino acid XTEN sequence and the second Ig constant region. (Item 147) 147. The chimeric protein according to item 146, wherein the cleavable linker is cleavable by thrombin. (Item 148) The chimeric protein according to item 146 or 147, wherein the cleavable linker has an amino acid sequence comprising IEPRSFS (SEQ ID NO: 194). 148. The chimeric protein according to item 146 or 147, wherein the cleavable linker has an amino acid sequence comprising IEPRSFS (SEQ ID NO: 194), and wherein said linker is not the full-length a2 region of FVIII. [Brief description of the drawings]

[0046] [Figure 1] Schematic diagram of a chimeric protein comprising a first polypeptide comprising a FVIII protein (A1-A2 partial or complete B-A3-C1-C2) fused to an Fc region, where the XTEN is inserted at an insertion site in the FVIII protein, and a second polypeptide comprising a VWF protein containing a D'D3 domain, an XTEN having less than 288 amino acids, a thrombin-cleavable linker, and a second Fc region. Insertion of the XTEN in the FVIII protein and / or fusion to the VWF protein extends the half-life of the chimeric protein by increasing the hydrodynamic radius and by preventing receptor-mediated clearance. The D'D3 domain of VWF prevents the interaction of FVIII with endogenous VWF, stabilizing the FVIII protein and extending the half-life of the chimeric protein. The Fc domain can covalently link the D'D3 domain to the FVIII protein and extend the half-life of the chimeric protein via the FcRn-mediated recycling pathway. The thrombin cleavable linker allows release of the D'D3 domain upon activation of FVIII and ensures correct alignment between the D'D3 domains of FVIII and VWF. [Diagram 2]Figure 1 shows a three plasmid expression system for FVIII-XTEN-Fc:D'D3-XTEN-Fc heterodimer; the first plasmid contains a nucleotide sequence encoding a single chain FVIII-XTEN-Fc in which XTEN is inserted into the B domain; the second plasmid contains a nucleotide sequence encoding D1D2D'D3-XTEN-Fc in which the XTEN sequence contains less than 288 amino acids; and the third plasmid contains a nucleotide sequence encoding the propeptide processing enzyme PACE. When the three polypeptides are expressed from the three plasmids, intracellular processing can process the D1D2 domain of VWF from the D'D3 domain. The resulting complex contains three products, the first molecule being a FVIII-XTEN / D'D3 heterodimer, the second molecule being a by-product homodimer of D'D3-XTEN-Fc, and the third molecule being another by-product, i.e., FVIII(XTEN)-Fc. [Diagram 3] Figure 1 shows the effect of adding XTEN insertion on the extension of the half-life of the heterodimer. FVIII169 comprises a B domain deleted FVIII protein fused to an Fc region, where an XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to mature full-length FVIII. FVIII205 comprises a B domain deleted FVIII protein fused to an Fc region, where an XTEN sequence (e.g., AE144) is inserted at amino acid 18 corresponding to mature full-length FVIII, and another XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to mature full-length FVIII. VWF031 comprises the D' and D3 domains of VWF fused to the Fc region by a thrombin cleavable linker (no XTEN). VWF034 comprises the D' and D3 domains of VWF fused to AE288 and an Fc region. The half-life of FVIII169 / VWF031 (inverted triangles) is 16.7 hours in HemA mice, the half-life of FVIII205 / VWF031 (circles) is 29.4 hours in HemA mice, and the half-life of FVIII169 / VWF034 (squares) is 31.1 hours in Hem mice. [Figure 4]Figure 1 shows that AE144XTEN confers better half-life extension than AE288XTEN when inserted between the D'D3 and Fc domains of VWF. For example, the half-life of VWF169 / VWF034 (squares) is 31.1 hours in HemA mice, while the half-life of FVIII169 / VWF057 (circles) is 42 hours in HemA mice. VWF057 contains the D'D3 domain and Fc region of VWF fused to AE144. [Diagram 5] 1 shows that the Fc domain is required for the extension of the half-life of chimeric protein heterodimers. When comparing the half-life of FVIII205 / VWF031 (circles) with FVIII263 / VWF050 (squares), which contains a mutation in the FcRn binding site (IHH triple mutant Fc) and therefore cannot be recycled via the FcRn pathway, in HemA mice, the half-life of FVIII263 / VWF050 (23 hours) is shorter than that of FVIII205 / VWF031 (29.4 hours), indicating that the Fc region is required for the extension of the half-life. [Figure 6A] Figure 1 shows similar acute efficacy of FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer compared to B domain deleted FVIII (SQ BDDFVIII) in HemA mouse tail clip model. Mice were administered 75 IU / kg and activity was measured by aPTT assay. SQ BDDFVIII is shown as circles while FVIII169 / VWF034 is shown as squares, FVIII169 / VWF057 is shown as diamonds and solvent is shown as inverted triangles. Detailed constructs of FVIII169, VWF034 and VWF057 are shown elsewhere herein. [Figure 6B]FIG. 1 shows acute efficacy of FVIII169 / VWF034 compared to B-domain deleted FVIII (SQ BDDFVIII) in HemA mice at a dose of 37.5 IU / kg, activity measured by aPTT assay. Median blood loss (uL) for mice in each treatment group is shown as horizontal lines, blood loss (uL) in C57 / BL6 mice is shown as open triangles, blood loss (μL) after administration of 37.5 IU / kg rBDD-FVIII is shown as open circles, blood loss (uL) after administration of 37.5 IU / kg FVIII169 / VWF034 is shown as open squares, and blood loss (uL) after administration of vehicle is shown as inverted triangles. [Figure 7] (A,B) rFVIII169 / VWF057 heterodimer provides longer protection to HemA mice in the tail vein transection bleeding model. (A) Rebleeding data in mice administered rFVIII169 / VWF057 72 hours prior to tail injury (squares), SQ BDD-FVIII 48 hours prior to tail injury (diamonds), SQ BDD-FVIII 24 hours prior to tail injury (inverted triangles) and vehicle (circles). Activity was measured by aPTT assay. X-axis indicates time and Y-axis indicates percentage of non-bleeding. (B) Corresponding survival rates in the four categories of mice shown in FIG. 7A. Mice administered 12 IU / kg FVIII169 / VWF057 72 hours prior to tail injury showed similar protection in rebleeding and similar survival compared to mice receiving SQ BDD-FVIII treatment 24 hours prior to tail injury. [Figure 8](A) Comparable rebleeding data in mice receiving rFVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer 96 hours prior to injury compared with rBDD-FVIII 24 hours prior to injury. Filled squares represent rebleeding data in mice receiving FVIII169 / VWF034 24 hours prior to injury, open squares represent rebleeding data in mice receiving FVIII169 / VWF034 96 hours prior to injury, filled diamonds represent rebleeding data in mice receiving FVIII169 / VWF057 24 hours prior to injury, open diamonds represent rebleeding data in mice receiving FVIII169 / VWF057 96 hours prior to injury, filled circles represent rebleeding data in mice receiving rBDD-FVIII 24 hours prior to injury, open circles represent rebleeding data in mice receiving rBDD-FVIII 48 hours prior to injury, and filled inverted triangles represent rebleeding data in mice receiving vehicle. The X-axis represents time and the Y-axis represents the percentage of non-bleeding. (B) Survival curves in mice receiving rFVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimers 96 hours prior to injury compared to rBDD-FVIII 24 hours prior to injury. X-axis indicates time, Y-axis indicates percentage survival. Symbols are the same as in FIG. 8A. [Figure 9]Schematic diagrams of representative FVIII-VWF heterodimers and constructs of FVIII169, FVIII286, VWF057, VWF059, and VWF062 are shown. For example, the FVIII169 construct comprises a B domain deleted FVIII protein with a R1648A substitution fused to an Fc region, where an XTEN sequence (e.g., AE288) is inserted at amino acid 745, corresponding to mature full-length FVIII (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-Fc). The FVIII286 construct comprises a B domain deleted FVIII protein with an R1648 substitution fused to an Fc region, where an XTEN sequence (e.g., AE288) is inserted at amino acid 745, corresponding to mature full-length FVIII with an additional a2 region between FVIII and Fc (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-a2-Fc). VWF057 is a VWF-Fc fusion construct comprising a VWF linker containing an LVPRG thrombin site ("LVPRG"; SEQ ID NO:6) and the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A) ​​linked to the Fc region via a GS linker ("GS"), where an XTEN sequence (i.e., AE144) is inserted between the D'D3 domain and the VWF linker (D'D3-144XTEN-GS+LVPRG-Fc). VWF059 is a VWF-Fc fusion construct comprising the D'D3 domain of VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A) ​​linked to the Fc region via the acidic region 2 (a2) of FVIII as a linker of VWF, where an XTEN sequence (i.e., AE144) is inserted between the D'D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct comprising the D'D3 domain of VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A) ​​linked to the Fc region, where an XTEN sequence (i.e., AE144) is inserted between the D'D3 domain and the Fc region (D'D3-144XTEN-Fc). [Figure 10]Schematic diagram of FVIII / VWF heterodimer constructs, e.g., FVIII169 / VWF057, FVIII169 / VWF059, FVIII169 / VWF059A, and FVIII169 / VWF073. The arrow indicates the site where an optional linker is added to introduce a thrombin cleavable site. FVIII169 / VWF057 has a linker containing LVPRG (SEQ ID NO: 6). FVIII169 / VWF059 is a nucleotide sequence that cleaves the a2 region of FVIII. [ka] FVIII169 / VWF059A contains a truncated FVIII a2 region. [ka] FVIII169 / VWF073 has a linker within the VWF073 construct (SEQ ID NO: 175) that comprises a fragment of the a2 region of FVIII consisting of IEPRSFS (SEQ ID NO: 194). [Figure 11] Shown are SDS-PAGE images following thrombin digestion of FVIII169 / VWF057 and FVIII-Fc control. (A) SDS-PAGE gel stained with anti-D3 antibody (AB96340). Arrows highlight "LCFc:D'D3-XTEN-Fc", the uncleaved full-length FVIII169 / VWF057, and "D'D3-144XTEN", the resulting fragment following thrombin cleavage. (B) SDS-PAGE gel stained with anti-HC antibody (GMA012). Arrows highlight the heavy chain ("HC") of FVIII and the A2 domain of FVIII. (C) shows an overlay of panels (A) and (B). Samples were collected at the time points indicated above each panel. Arrows point to the relevant proteins. [Figure 12]SDS-PAGE images following thrombin digestion of FVIII169 / VWF059 are shown. (A) SDS-PAGE gel stained with anti-D3 antibody (AB96340). Arrows highlight full-length uncleaved FVIII169 / VWF059 "LCFc:D'D3-XTEN-Fc" and the resulting fragment "D'D3-144XTEN" following thrombin cleavage. (B) SDS-PAGE gel stained with anti-HC antibody (GMA012). Arrows highlight full-length uncleaved FVIII169 / VWF059; D'D3-144XTEN-a3, the resulting fragment following thrombin cleavage, and "A2", the A2 domain of FVIII. (C) shows an overlay of panels (A) and (B). Samples were collected at the time points indicated above each panel. [Figure 13] Figure 1 shows the acute efficacy of HemA mice treated with FVIII169 / VWF059 (circles) compared to HemA mice treated with BDD-FVIII control (squares). Blood loss was measured following tail clip. p=0.9883 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present invention is directed to a chimeric protein comprising two polypeptides, the first polypeptide being comprises a FVIII protein fused to a first Ig constant region, and a second polypeptide comprises a VWF protein fused to a second Ig constant region or a portion thereof by an XTEN sequence, wherein the XTEN sequence contains less than 288 amino acids.

[0048] I. Definition It should 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, "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0049] Furthermore, "and / or", when used herein, should be interpreted as a specific disclosure of each of the two specified features or components with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B", it is intended to include "A and B", "A or B" (single), "A" (single) and "B" (single). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C", it is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); C (single).

[0050] Wherever an embodiment is described herein by the predicate "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0052] Units, prefixes, and symbols are shown in the form accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written in amino to carboxy orientation from left to right. The headings provided herein are not limitations of the various aspects of this disclosure, which are obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0053] The term "about" is used herein to mean approximately, roughly, approximately, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by expanding the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the recited value by, for example, a variance of 10 percent above or below (higher or lower).

[0054] The term "polynucleotide" or "nucleotide" encompasses single nucleic acids as well as multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide may include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid fragments, 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, that 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 isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid may include regulatory elements, such as, for example, a promoter, an enhancer, a ribosomal binding site, or a transcription termination signal.

[0055] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not usually translated into an amino acid, it may be considered to be part of the coding region, although adjacent sequences, e.g., promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are usually determined by a start codon at the 5' end that encodes the amino terminus of the resulting polypeptide and a translation stop codon at the 3' end that encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the invention can be present in a single polynucleotide construct, e.g., a single vector, or in separate polynucleotide constructs, e.g., separate (different) vectors. It follows that, in essence, a single vector can contain only one coding region, or can include two or more coding regions, e.g., a single vector can separately encode binding domain A and binding domain B as described below. In addition, a vector, polynucleotide or nucleic acid of the invention can encode heterologous coding regions that are fused or not fused to a nucleic acid encoding a binding domain of the invention. Heterologous coding regions include, but are not limited to, defined elements or motifs such as secretory signal peptides or heterologous functional domains.

[0056] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those skilled in the art know that signal peptides are generally fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative of that sequence retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide can be used, for example, the signal peptide of human tissue plasminogen activator (TPA) or mouse β-glucuronidase or a functional derivative thereof.

[0057] The term "downstream" when referring to a nucleotide sequence means that the nucleic acid or nucleotide sequence is located 3' to the reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence relates to a sequence following the start of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription. The term "downstream" when referring to a polypeptide sequence means that an amino acid or amino acid insertion site is located C-terminal to the reference amino acid. For example, an insertion site immediately downstream of amino acid 745 corresponding to the mature wild-type FVIII protein is , meaning that the insertion site is between amino acids 745 and 746, corresponding to the mature wild-type FVIII protein.

[0058] The term "upstream" refers to a nucleotide sequence located 5' relative to a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence relates to a coding region or sequence located 5' of the start of transcription. For example, most promoters are located upstream of the start site of transcription.

[0059] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region and that influences the transcription, RNA processing, stability, or translation of the coding region with which it is associated. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem / loop structures. If the coding region is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences will usually be located 3' to the coding sequence.

[0060] A polynucleotide encoding a gene product, e.g., a polypeptide, can include a promoter and / or transcriptional or translational control elements operably associated with one or more coding regions. In operably associated, the coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a manner as to place expression of the gene product under the influence or control of the regulatory regions. For example, a coding region and a promoter are "operably associated" if induction of promoter function results in transcription of an mRNA encoding the gene product encoded by the coding region, and if the nature of the binding between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the gene product or the ability of the DNA template to be transcribed. Other transcriptional control elements in addition to the promoter, e.g., enhancers, operators, repressors, and transcription termination signals, can also be operably associated with the coding region to direct expression of the gene product.

[0061] A variety of transcription control regions are known to those of skill in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer fragments from cytomegalovirus (immediate early promoter with intron A), simian virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock proteins, 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 lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins), as well as tissue-specific promoters and enhancers.

[0062] Similarly, various translation control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly IRES, also called internal ribosome entry site, or CITE sequence).

[0063] The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product, e.g., RNA or a polypeptide, including, but not limited to, expression of the polynucleotide as messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other polypeptide. It includes transcription into an RNA product and translation of the mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., messenger RNA resulting from transcription of a gene, or a polypeptide translated from a transcript. Gene products as 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, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0064] A "vector" refers to a vehicle for cloning and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid fragment may be ligated to provide a replicon of the ligated fragment. A "replicon" refers to a genetic element (e.g., a plasmid, a phage, a cosmid, a chromosome, a virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replicating under its own control. The term "vector" includes both viral and non-viral vehicles for introducing nucleic acid into cells in vitro, ex vivo, or in vivo. Numerous 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 fragment into a selected vector with complementary cohesive termini.

[0065] Vectors may be engineered to encode a selectable marker or reporter that provides for the selection or identification of cells that have taken up the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that have taken up and expressed other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin-regulated genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), etc. Selectable markers are also considered to be reporters.

[0066] The term "plasmid" refers to an extrachromosomal element, often carrying genes that are not part of the central metabolism of the cell, usually in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences, genomic integrating sequences, phage or nucleotide sequences, linear, circular or supercoiled, single or double stranded DNA or RNA, from any source, and many nucleotide sequences are linked or recombined into unique constructs that are capable of introducing into cells promoter fragments and DNA sequences for selected gene products, along with appropriate 3' non-translated sequences.

[0067] Eukaryotic viral vectors that can be used include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, and poxviruses, such as vaccinia viral vectors, baculoviral vectors, or herpes viral vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA / protein complexes, and biopolymers.

[0068] A "cloning vector" is a vector that replicates in turn and contains an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment can be ligated so as to bring about replication of the ligated segment. The term "replicon" refers to a unit length of nucleic acid that may be replicated in one cell type, e.g., bacteria, and expressed in another, e.g., eukaryotic cells. Cloning vectors usually contain one or more sequences that can be used for selection of cells that contain the vector and / or one or more multiple cloning sites for insertion of a nucleic acid sequence of interest.

[0069] The term "expression vector" refers to a vehicle designed to allow for the expression of an inserted nucleic acid sequence following insertion into a host cell, the inserted nucleic acid sequence being placed in operable relationship with a regulatory region as described above.

[0070] Vectors are introduced into host cells by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or a DNA vector transporter.

[0071] "Culture", "culturing" and "culturing" as used herein refer to the incubation of cells in a test tube under conditions that allow the cells to grow or divide or that allow the cells to be maintained in a living state. "Cultured cells" as used herein refers to cells that are grown in a test tube.

[0072] As used herein, the term "polypeptide" is intended to encompass multiple "polypeptides" as well as a single "polypeptide" 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, "proteins," "amino acid chains" or other terms 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 in place of or interchangeably with those terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It can be produced by methods including chemical synthesis.

[0073] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural environment. 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 purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, and partially or substantially purified by suitable techniques.

[0074] Also included in the present invention are fragments or variants of the polypeptides and combinations thereof. The term "fragment" or "variant" when referring to a polypeptide binding domain or binding molecule of the present invention includes polypeptides that retain at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for a FVIII variant, or FVIII binding activity for a VWF fragment). Polypeptide fragments include proteolytic fragments as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include the full-length naturally occurring polypeptide (mature polypeptide). Variants of the polypeptide binding domain or binding molecule of the present invention include polypeptides that retain at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for a FVIII variant, or FVIII binding activity for a VWF fragment). Fragments of the polypeptides include specific antibody fragments as discussed elsewhere herein, as well as proteolytic fragments as well as deletion fragments, but do not include the full-length naturally occurring polypeptide (mature polypeptide). Variants include fragments as described above, and also include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0075] As used herein, the term "VWF protein" or "VWF proteins" refers to a VWF fragment that interacts with FVIII and retains at least one or more properties normally provided to FVIII by full-length VWF, such as preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that would lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but cannot bind VWF-bound FVIII, and / or stabilizing the interaction of the heavy and light chains of FVIII.

[0076] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-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 conservative. In another embodiment, strings of amino acids can be conservatively replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0077] 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. As 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 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981) to find the best segment of homology between two sequences. When using BESTFIT or other sequence comparison programs to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the present invention, the parameters are naturally set such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and allow gaps in homology of up to 5% of the total number of amino acids in the reference sequence.

[0078] As used herein, a "corresponding amino acid" or "equivalent amino acid" in a VWF or FVIII protein sequence is identified by sequence comparison to maximize the identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The numbers used to identify each amino acid are based on the numbers used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0079] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or a fragment, variant, or derivative thereof, that is immediately upstream of a position where a heterologous moiety can be inserted. An "insertion site" is specified as a number, which is the number of the amino acid in mature native FVIII (SEQ ID NO:65) to which the insertion site corresponds, which is immediately N-terminal to the position of insertion. For example, the phrase "a3 contains an XTEN at an insertion site corresponding to amino acid 1656 of SEQ ID NO:65" indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO:65.

[0080] The phrase "immediately downstream of an amino acid" as used herein refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of an amino acid. Thus, the phrase "between two amino acids at the insertion site" as used herein refers to a position where 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 at the insertion site" are used synonymously with "inserted at the insertion site."

[0081] The terms "inserted," "inserted into," "inserted into," or grammatically related terms, as used herein, refer to the location of the XTEN in the chimeric polypeptide relative to the analogous location 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 the method or process by which the chimeric polypeptide was made. For example, with reference to the chimeric polypeptides provided herein, the phrase "XTEN is inserted immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric polypeptide comprises an XTEN immediately downstream of the amino acid corresponding to amino acid 745 in native mature human FVIII, e.g., bound by amino acids corresponding to amino acids 745 and 746 of native mature human FVIII.

[0082] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally associated in nature. Amino acid sequences that are normally present in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new arrangement in a fusion polypeptide, for example, in a fusion of the Factor VIII domain of the present invention with an IgFc domain. Fusion proteins are created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which peptide regions are encoded in the desired relationship. Chimeric proteins can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide or non-covalent bond.

[0083] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life may be represented by the time required for half of an amount administered to a subject to clear from the circulation and / or other tissues of the animal. When constructing a clearance curve for a given polypeptide as a function of time, the curve is usually biphasic with a rapid α-phase and a long β-phase. The α-phase usually represents the equilibrium of the administered Fc polypeptide between the intravascular and extravascular spaces and is determined in part by the size of the polypeptide. The β-phase usually represents the catabolism of the polypeptide within the vascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, so they have no α-phase, only a single β-phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of a polypeptide in the β-phase. A typical β-phase half-life of a human antibody in humans is 21 days.

[0084] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence can be directly linked to the second amino acid sequence or nucleotide sequence, or can be juxtaposed, or alternatively, an intervening sequence can covalently link the first sequence to the second sequence. The term "linked" does not only mean the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or second amino acid sequence) to two amino acids in the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for polypeptide chains) or a nucleotide or nucleotide chain (for nucleotide chains) or a chemical moiety (for both polypeptide and polynucleotide chains). The term "linker" may also be indicated by a hyphen (-).

[0085] 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" refers to a covalent, non-peptide or non-covalent bond. The association can be indicated by a colon, i.e., (:). In another embodiment, it refers to a covalent bond excluding a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 and 229 in the EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidyl bonds, agnostic bonds, bent bonds, dipolar bonds, Pi backgrounds, double bonds, triple bonds, quadruple bonds, pentad bonds, hexad bonds, conjugation, superconjugation, aromaticity, haptometricity, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation / pi bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, gold-atom bonds, intercalation, stacking, entropic forces, or chemical polarity.

[0086] As used herein, a "monomeric / dimeric hybrid" refers to a chimeric protein comprising a first and a second polypeptide chain associated with each other by disulfide bonds, where the first chain comprises a clotting factor, e.g., Factor VIII and an Fc region, and the second chain comprises, consists essentially of, or consists of a second Fc region without the clotting factor. Thus, a monomeric / dimeric hybrid construct is a hybrid comprising a monomeric embodiment with only one clotting factor and a dimeric embodiment with two Fc regions.

[0087] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Particular enzymatic cleavage sites include intracellular processing sites. In one embodiment, a polypeptide has an enzymatic cleavage site that is cleaved by an enzyme that is activated during the coagulation cascade such that cleavage of such site occurs at the site of clot formation. Exemplary such sites include, for example, those recognized by thrombin, factor XIa, or factor Xa. Exemplary cleavage sites for FXIa include, for example, TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3). ... Suitable thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), and IEPRSFS (SEQ ID NO: 194). Other enzyme cleavage sites are known in the art and are described elsewhere herein.

[0088] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide that is a target for an enzyme that functions after translation of the polypeptide. In one embodiment, such enzymes function during transport of compartments from the Golgi lumen to the trans-Golgi. Intracellular processing enzymes cleave polypeptides prior to secretion of the protein from the cell. Examples of such processing sites include, for example, those targeted by the endopeptidase PACE / Furin (PACE is an acronym for paired basic amino acid cleaving enzymes). These enzymes localize to the Golgi membrane and cleave proteins on the carboxy-terminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein, the "furin" family of enzymes includes, for example, PCSK1 (also known as PC1 / Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known as PC7 / LPC, PC8, or SPC7). Other processing sites are known in the art.

[0089] It will be understood that in constructs that include more than one processing or cleavage site, such sites may be the same or different.

[0090] The term "furin" refers to an enzyme corresponding to the EC number 3.4.21.75. Furin is a subtilisin-like proprotein convertase also known as PACE (paired basic amino acid cleaving enzyme). Furin deletes a section of the inactive precursor protein that converts it into a biologically active protein. During its intracellular transport, the propeptide of VWF can be cleaved from the mature VWF molecule by the furin enzyme. In some embodiments, furin cleaves D1D2 from D'D3 of VWF. In other embodiments, a nucleotide sequence encoding furin can be expressed together with a nucleotide sequence encoding a VWF fragment such that the D1D2 domain can be cleaved off intracellularly by furin.

[0091] It will be understood that in constructs that include more than one processing or cleavage site, such sites may be the same or different.

[0092] "Processable linker," as used herein, refers to a linker that contains at least one intracellular processing site as described elsewhere herein.

[0093] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to bleed, either spontaneously or as a result of trauma, due to an impaired ability to form fibrin clots or an inability to form fibrin clots. Examples of such disorders include hemophilia. The three major forms are hemophilia A (deficiency of factor VIII), hemophilia B (deficiency of factor IX, or "Christmas disease"), and hemophilia C (deficiency of factor XI, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand's disease, factor XI deficiency (deficiency of PTA), factor XII deficiency, defects or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII. The most common conditions are Bernard-Soulier syndrome, which is usually a defect or deficiency in GPIb. GPIb, the receptor for VWF, can be defective, resulting in a lack of primary clot formation (primary hemostasis) and an increased tendency to bleed, and Glanzman and Naegeli thrombasthenia (Glanzman thrombasthenia). In liver failure (acute and chronic types), there is insufficient production of clotting factors by the liver, which can increase the risk of bleeding.

[0094] The chimeric molecule of the present invention can be used prophylactically. As used herein, the term "prophylactic treatment" refers to administration of a molecule prior to the appearance of a bleeding episode. In one embodiment, a subject in need of a general hemostatic agent is undergoing or about to undergo surgery. The chimeric protein of the present invention can be administered prior to or after surgery as a prophylactic agent. The chimeric protein of the present invention can be administered during or after surgery to control the appearance of acute bleeding episodes. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0095] The chimeric protein of the present invention can also be used in on-demand therapy. The term "on-demand therapy" refers to the administration of chimeric molecule in response to the onset of bleeding episodes or before activities that may cause bleeding. In one embodiment, on-demand therapy can be given to a subject when bleeding begins, such as after surgery, or when bleeding is expected, such as before surgery. In another embodiment, on-demand therapy can be given prior to activities that increase the risk of bleeding, such as contact sports.

[0096] As used herein, the term "acute bleeding" refers to the occurrence of a bleeding episode unrelated to an underlying cause. For example, a subject may have trauma, uremia, a genetic bleeding disorder (e.g., factor VII deficiency), a platelet disorder, or resistance due to the development of antibodies against clotting factors.

[0097] Treat, treatment, and treating as used herein refer to, for example, reducing the severity of a disease or condition; reducing the duration of the course of a disease; ameliorating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject with a disease or condition without necessarily curing the disease or condition, or preventing one or more symptoms associated with a disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining a trough concentration of FVIII at at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL in a subject by administering a chimeric protein or VWF fragment of the present invention. In another embodiment, treating or treatment means maintaining a trough concentration of FVIII between about 1 to about 20 IU / dL, about 2 to about 20 IU / dL, about 3 to about 20 IU / dL, about 4 to about 20 IU / dL, about 5 to about 20 IU / dL, about 6 to about 20 IU / dL, about 7 to about 20 IU / dL, about 8 to about 20 IU / dL, about 9 to about 20 IU / dL, or about 10 to about 20 IU / dL. Treating or treating a disease or condition can also include maintaining FVIII activity in a subject at a level equivalent 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 of a non-hemophilic subject. The minimum trough concentration required for treatment can be determined by one or more known methods and can be adjusted (up or down) for each individual.

[0098] II. Chimeric Proteins The present invention provides a method for preventing or inhibiting the half-life limiting factor of FVIII, i.e., endogenous VWF from associating with the FVIII protein, by inhibiting the VWF fused to the XTEN sequence. The present invention aims to extend the half-life of chimeric proteins using WF proteins. Endogenous VWF associates with about 95% to about 98% of FVIII in a non-covalent complex. While endogenous VWF is a half-life limiting factor for FVIII, endogenous VWF bound to FVIII proteins is also known to protect FVIII in various ways. For example, full-length VWF (as a multimer with about 250 kDa) can protect FVIII from protease cleavage and FVIII activation, stabilize the heavy and / or light chains of FVIII, and prevent clearance of FVIII by scavenger receptors. However, at the same time, endogenous VWF limits the half-life of FVIII by preventing pinocytosis and by clearing the FVIII / VWF complex from the system via the VWF clearance pathway. Without being bound by theory, it is believed that endogenous VWF is a half-life limiting factor that prevents the half-life of a chimeric protein fused to a half-life extender from being longer than about twice the half-life of wild-type FVIII. Thus, the present invention is directed to preventing or inhibiting the interaction between endogenous VWF and FVIII protein using a VWF protein (e.g., a VWF fragment) that includes the D' and D3 domains, while increasing the half-life of the resulting FVIII protein by using an XTEN sequence in combination with an Ig constant region or a portion thereof. In particular, the present invention shows that shorter XTEN sequences (i.e., XTENs containing less than 288 amino acids, i.e., XTENs shorter than 288 amino acids) are even better at extending the half-life of chimeric proteins.

[0099] In one embodiment, the present invention is directed to a chimeric protein comprising (i) a first polypeptide comprising a FVIII protein fused to a first Ig constant region or portion thereof, and (ii) a second polypeptide comprising a VWF protein comprising the D' and D3 domains of VWF fused to a second Ig constant region or portion thereof by an intermediate XTEN sequence, wherein the XTEN sequence contains less than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide. In another embodiment, the XTEN sequence in the second polypeptide comprises an amino acid sequence having a length between 12 amino acids and 287 amino acids. In another embodiment, the chimeric protein exhibits an increased half-life compared to a corresponding fusion protein comprising the first and second polypeptides, wherein the second polypeptide comprises an XTEN sequence containing at least 288 amino acids, e.g., AE288, e.g., SEQ ID NO:8. In yet other embodiments, the XTEN sequence in the second polypeptide contains at least about 36, at least about 42, at least about 72, or at least about 144 amino acids, but fewer than 288 amino acids, such as AE42, AE72, AE144 (AE144, AE144_2A, AE144_3B, AE144_4A, AE144_5A, AE144_6B), AG42, AG72, or AG144 (AG144, AG144_A, AG144_B, AG144_C, AG144_F), e.g., SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63.

[0100] The chimeric protein of the present invention can further comprise a second XTEN sequence linking the FVIII protein to the first Ig constant region or portion thereof.

[0101] In a particular embodiment, the present invention is directed to a chimeric protein comprising: (i) a first polypeptide comprising a FVIII protein fused to a first Ig constant region or portion thereof; and (ii) a second polypeptide comprising a VWF protein comprising the D' and D3 domains of VWF fused to a second Ig constant region or portion thereof by an intermediate first XTEN sequence, wherein the XTEN sequence contains less than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide, and wherein the first polypeptide is further inserted at one or more insertion sites within the FVIII protein or is an intermediate between the FVIII protein and the second polypeptide. and / or a first Ig constant region or portion thereof. Thus, in one embodiment, the second XTEN sequence is inserted at one or more insertion sites in the FVIII protein. In another embodiment, the second XTEN sequence is fused to the FVIII protein or an Ig constant region or portion thereof. In other embodiments, the second XTEN sequence is inserted at one or more insertion sites in the FVIII protein and a third XTEN sequence is fused to the FVIII protein and / or the first Ig constant region or portion thereof.

[0102] The second and / or third XTEN sequence can be any length of XTEN amino acid. For example, the second and / or third XTEN sequence is disclosed elsewhere herein, for example, AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144, such as SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17; SEQ ID NO:54; SEQ ID NO:19; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:15; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the second and / or third XTEN sequence is AE288 or AG288, for example, SEQ ID NO:8 or 19.

[0103] In a specific embodiment, the present invention is directed to a chimeric protein comprising: (i) a first polypeptide comprising a FVIII protein fused to a first Ig constant region or a portion thereof by an optional linker, wherein an optional XTEN sequence (X2) is inserted at one or more insertion sites in the FVIII protein or fused to the FVIII protein or a first Ig constant region or a portion thereof; and (ii) a second polypeptide comprising a VWF protein comprising the D' and D3 domains of VWF fused to a second Ig constant region or a portion thereof by an XTEN sequence (X1) between the VWF protein and the second Ig constant region or a portion thereof, wherein the XTEN sequence (X1) contains less than 288 amino acid residues and is fused to the VWF protein by a linker, and wherein the first and second polypeptides are associated. In some embodiments, the present invention is directed to a chimeric protein comprising: (i) a first polypeptide comprising a FVIII protein fused to a first Ig constant region or a portion thereof by an optional linker, in which an optional XTEN sequence (X2) is inserted at one or more insertion sites in the FVIII protein or fused to the FVIII protein or a first Ig constant region or a portion thereof; and (ii) a second polypeptide comprising a VWF protein comprising a D' domain and a D3 domain of VWF fused to a second Ig constant region or a portion thereof by an XTEN sequence (X1) between the VWF protein and the second Ig constant region or a portion thereof, in which the XTEN sequence (X1) contains less than 288 amino acid residues, is fused to the second Ig constant region or a portion thereof by a linker, and the first polypeptide and the second polypeptide are associated. In other embodiments, the linker fusing the XTEN sequence (X1) to the VWF protein or the second Ig constant region or a portion thereof is a cleavable linker. Non-limiting examples of cleavable linkers are provided elsewhere herein. In certain embodiments, the linker is a thrombin cleavable linker.

[0104] In some embodiments, the chimeric protein is two polypeptide chains, the first chain comprising the first polypeptide described above, and the second chain comprising the second polypeptide described above. For example, the two polypeptide chains comprise (i) a first chain comprising a single chain FVIII protein, a first Ig constant region or a portion thereof, and any XTEN sequence inserted at one or more insertion sites in the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof, and (ii) a second chain comprising a VWF protein fused to a second Ig constant region or a portion thereof by an intermediate XTEN sequence (X1), where the XTEN sequence (X1) contains less than 288 amino acids.

[0105] In certain embodiments, the chimeric protein is two polypeptide chains, the first chain being F The second chain comprises, from N-terminus to C-terminus, the light chain of the FVIII protein, an optional XTEN sequence inserted into one or more insertion sites in the FVIII protein or fused to the FVIII protein or the first constant region or portion thereof, a first Ig constant region or portion thereof, an optional linker (e.g., a processable linker), a VWF protein, an XTEN sequence (X1), a second optional linker (e.g., a cleavable linker), and a second Ig constant region or portion thereof.

[0106] In other embodiments, the chimeric protein has three polypeptide chains: (i) a first chain comprising a heavy chain of a FVIII protein; (ii) a second chain comprising a light chain of a FVIII protein and a first Ig constant region or a portion thereof and an optional XTEN sequence inserted at one or more insertion sites in the heavy or light chain of the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof; and (iii) a third chain comprising a VWF protein fused to a second Ig constant region or a portion thereof by an intermediate XTEN sequence (X1), wherein the first chain and the second chain are associated by a non-covalent bond, e.g., a metal bond, and the second chain and the third chain are associated by a covalent bond, e.g., a disulfide bond.

[0107] In yet other embodiments, the chimeric protein is a single chain comprising, from N-terminus to C-terminus, a single chain FVIII protein, an optional XTEN sequence inserted at one or more insertion sites in the FVIII protein or fused to the FVIII protein or a first Ig constant region or portion thereof, a first Ig constant region or portion thereof, an optional linker (e.g., a processable linker), a VWF protein, an XTEN sequence (X1), a second optional linker (e.g., a cleavable linker), and a second Ig constant region or portion thereof.

[0108] In certain embodiments, the chimeric protein has the following formulas (a)-(hh): (a) FVIII-F1:F2-L2-X-L1-V; (b) FVIII-F1: V-L1-X-L2-F2; (c) F1-FVIII:F2-L2-X-L1-V; (d) F1-FVIII: V-L1-X-L2-F2; (e) FVIII-X2-F1:F2-L2-X1-L1-V; (f) FVIII-X2-F1: V-L1-X1-L2-F2; (g)FVIII(X2)-F1:F2-L2-X1-L1-V; (h)FVIII(X2)-F1:V-L1-X1-L2-F2; (i) F1-X2-F1:F2-L2-X1-L1-V; (j) F1-X2-F1: V-L1-X1-L2-F2; (k)V-L1-X-L2-F2-L3-FVIII-L4-F1; (l)V-L1-X-L2-F2-L3-F1-L4-FVIII; (m)F1-L4-FVIII-L3-F2-L2-X-L1-V; (n)FVIII-L4-F1-L3-F2-L2-X-L1-V; (o)FVIII-L4-F1-L3-V-L1-X-L2-F2; (p) FVIII-L4-F1-L3-F2-L2-X-L1-V; (q) F2-L2-X-L1-V-L3-F1-L4-FVIII; (r) F2-L2-X-L1-V-L3-FVIII-L4-F1; (s) V-L1-X1-L2-F2-L3-FVIII(X2)-L4-F1; (t) V-L1-X1-L2-F2-L3-F1-L4-FVIII(X2); (u) F1-L4-FVIII(X2)-L3-F2-L2-X1-L1-V; (v) F-L4-FVIII(X2)-L3-V-L1-X1-L2-F2; (w) FVIII(X2)-L4-F1-L3-V-L1-X1-L2-F2; (x) FVIII(X2)-L4-F1-L3-F2-L2-X1-L1-V; (y) F2-L2-X1-L1-V-L3-F1-L4-FVIII(X2); (z) F2-L2-X1-L1-V-L3-FVIII(X2)-L4-F1; (aa) V-L1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1; (bb) V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII; (cc) F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V; (dd) F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2; (ee) FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1; (ff) FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V; (gg) F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or (hh) F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2; and includes one of wherein V is a VWF protein comprising the D' and D3 domains, X or X1 is a first XTEN sequence containing less than 288 amino acids, X2 is a second XTEN sequence, and FVIII comprises a FVIII protein; FVIII(X2) comprises a FVIII protein having a second XTEN sequence inserted at one or more insertion sites within the FVIII protein; F1 is a first Ig constant region or a portion thereof; F2 is a second Ig constant region or a portion thereof, L1, L2, L3, L4 or L5 is an optional linker; (-) is a peptide bond, (:) is a covalent or non-covalent bond.

[0109] In one embodiment, X or X1 consists of an amino acid sequence having a length between 12 amino acids and 287 amino acids. In another embodiment, the chimeric protein exhibits an increased half-life compared to a corresponding fusion protein comprising the formula, wherein X or X1 is AE288, e.g., SEQ ID NO:8.

[0110] In other embodiments, X or X1 in the formula contains at least about 36, at least about 42, at least about 72, or at least about 144 amino acids, but fewer than 288 amino acids, such as AE42, AE72, AE144 (AE144, AE144_2A, AE144_3B, AE144_4A, AE144_5A, AE144_6B), AG42, AG72, or AG144 (AG144, AG144_A, AG144_B, AG144_C, AG144_F), such as SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; or SEQ ID NO:63.

[0111] In still other embodiments, X2 comprises an amino acid sequence having a length of at least about 36 amino acids, at least 42 amino acids, at least 144 amino acids, at least 288 amino acids, at least 576 amino acids, or at least 864 amino acids, e.g., AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144, e.g., SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:8; SEQ ID NO:11; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:14. In certain embodiments, X2 is AE288 or AG288, e.g., SEQ ID NO:8 or 19.

[0112] In certain embodiments, a chimeric protein comprising X or X1 and / or X2 has an increased half-life compared to a chimeric protein not comprising X or X1 and / or X2. In other embodiments, L1 and / or L2 are cleavable linkers. In yet other embodiments, L4 and / or L5 are cleavable linkers.

[0113] II.A. von Willebrand factor (VWF) protein VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and constitutively produced by endothelium (in Weibel-Palade bodies), megakaryocytes (α-granules of platelets) and subendothelial connective tissue. The basic VWF monomer is a protein of 2813 amino acids. Each monomer contains a number of specific domains with specific functions: the D' / D3 domain (which binds factor VIII), the A1 domain (which binds platelet GPIb receptors, heparin and / or possibly collagen), the A3 domain (which binds collagen), the C1 domain (which, upon activation, binds the RGD domain to platelet integrin αIIbβ3), and a "cysteine ​​knot" domain at the C-terminus of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ) and β-human chorionic gonadotropin (βHCG)).

[0114] In one embodiment, the VWF protein is a VWF fragment. The term "VWF fragment" as used herein includes, but is not limited to, functional VWF fragments containing the D' and D3 domains that are capable of inhibiting the 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 the interaction of the FVIII protein with endogenous VWF. VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0115] The 2813 monomer amino acid sequence for human VWF is reported in Genbank under the accession number _NP_000543.2_. The nucleotide sequence encoding human VWF is reported in Genbank under the accession number _NM_000552.3_. The nucleotide sequence of human VWF is designated as SEQ ID NO: 20. SEQ ID NO: 21 is the amino acid sequence of full-length VWF. The domains of VWF are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0116] A VWF protein, as used herein, can be a VWF fragment comprising the D' and D3 domains of VWF, wherein the VWF fragment binds to factor VIII (FVIII) and inhibits binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment comprising the D' and D3 domains can further comprise a VWF domain selected from the group consisting of the A1 domain, the A2 domain, the A3 domain, the D1 domain, the D2 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, and any combination thereof. In one embodiment, the VWF fragment comprises, consists essentially of, or consists of (1) the D' and D3 domains of VWF or fragments thereof, (2) the D1, D' and D3 domains of VWF or fragments thereof, (3) the D2, D' and D3 domains of VWF or fragments thereof, (4) the D1, D2, D' and D3 domains of VWF or fragments thereof, or (5) the D1, D2, D', D3 and A1 domains of VWF or fragments thereof. The VWF fragments described herein do not contain a site that binds to the VWF clearance receptor. Alternative embodiments In some embodiments, the VWF fragment described herein is not amino acids 764 to 1274 of SEQ ID NO: 21. The VWF fragment of the present invention can include any other sequence linked or fused to the VWF fragment. For example, the VWF fragment described herein can further include a signal peptide.

[0117] In one embodiment, the VWF fragment comprising the D' and D3 domains binds to or associates with the FVIII protein. By binding to or associating with the FVIII protein, the VWF fragment of the invention protects FVIII from protease cleavage and activation of FVIII, stabilizes the heavy and light chains of FVIII, and prevents clearance of FVIII by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with the FVIII protein and prevents or prevents binding of the FVIII protein to phospholipids and activated protein C. By preventing or inhibiting binding of the FVIII protein by endogenous full-length VWF, the VWF fragment of the invention reduces clearance of FVIII by the VWF clearance receptor, thereby extending the half-life of the chimeric protein. Thus, the increased half-life of the chimeric protein is due to the binding or association of the FVIII protein with the VWF fragment lacking the binding site of the VWF clearance receptor, and the shielding or protection of the FVIII protein by the VWF fragment from endogenous VWF that contains the binding site of the VWF clearance receptor. The FVIII protein bound to or protected by the VWF fragment can also allow recycling of the FVIII protein. By eliminating the VWF clearance pathway receptor binding site contained in the full-length VWF molecule, the FVIII / VWF heterodimer of the present invention is shielded from the VWF clearance pathway, further increasing the half-life of FVIII.

[0118] In one embodiment, a VWF protein useful in the invention comprises the D' and D3 domains 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 764-866 of SEQ ID NO: 21, and wherein the VWF protein prevents or inhibits endogenous VWF binding to FVIII. In another embodiment, a VWF protein comprises the D' and D3 domains 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 867-1240 of SEQ ID NO: 21, and wherein the VWF protein prevents or inhibits endogenous VWF binding to FVIII. In some embodiments, the VWF proteins described herein comprise, consist essentially of, or consist of the D' and D3 domains of VWF that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764-1240 of SEQ ID NO: 21, wherein the VWF protein prevents or inhibits endogenous VWF binding to FVIII. In other embodiments, the VWF protein comprises, consist essentially of, or consists of the D1, D2, D' and D3 domains that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 23-1240 of SEQ ID NO: 21, wherein the VWF protein prevents or inhibits endogenous VWF binding to FVIII. In yet other embodiments, the VWF protein further comprises a signal peptide operably linked thereto.

[0119] In some embodiments, a VWF protein useful in the invention comprises (1) a D'D3 domain, a D1D'D3 domain, a D2D'D3 domain or a D1D2D'D3 domain, and (2) up to about 10 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1250 of SEQ ID NO:21), up to about 15 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1255 of SEQ ID NO:21), up to about 20 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1255 of SEQ ID NO:21), 21), up to about 25 amino acids (e.g., from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1260 of SEQ ID NO:21), or up to about 30 amino acids (e.g., from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1260 of SEQ ID NO:21). In certain embodiments, the VWF protein comprising or consisting essentially of the D' and D3 domains is not amino acids 764-1274 of SEQ ID NO:21 or is not 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.

[0120] In other embodiments, the VWF protein comprising the D'D3 domain linked to the D1D2 domain further comprises an intracellular cleavage site, e.g., a PACE (furin) or PC5 cleavage site, which upon expression allows cleavage of the D1D2 domain from the D'D3 domain. Non-limiting examples of intracellular cleavage sites are disclosed elsewhere herein.

[0121] In yet another embodiment, the VWF protein contains the D' domain and the D3 domain but does not contain an amino acid sequence selected from the group consisting of: (1) amino acids 1241 to 2813 corresponding to SEQ ID NO: 21, (2) amino acids 1270 to 2813 corresponding to SEQ ID NO: 21, (3) amino acids 1271 to 2813 corresponding to SEQ ID NO: 21, (4) amino acids 1272 to 2813 corresponding to SEQ ID NO: 21, (5) amino acids 1273 to 2813 corresponding to SEQ ID NO: 21, (6) amino acids 1274 to 2813 corresponding to SEQ ID NO: 21, and any combination thereof.

[0122] In yet other embodiments, the VWF protein of the invention comprises, consists essentially of, or consists of an amino acid sequence corresponding to the D', D3, and A1 domains, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1479 of SEQ ID NO: 21, and wherein the VWF protein prevents binding of endogenous VWF to FVIII. In certain embodiments, the VWF protein is not amino acids 764-1274 of SEQ ID NO: 21.

[0123] In some embodiments, the VWF protein of the present invention comprises the D' domain and the D3 domain, but may also comprise any one of the following: (1) A1 domain, (2) A2 domain, (3) A3 domain, (4) D4 domain, (5) B1 domain, (6) B2 domain, (7) B3 domain, (8) C1 domain, (9) C2 domain, (10) CK domain, (11) CK domain and C2 domain, (12) CK domain, C2 domain, and C1 domain, (13) CK domain, C2 domain, C1 domain, and B3 domain, (14) CK domain, C2 domain, C1 domain, B3 domain, and B2 domain, (15) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, and B1 domain, (16) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (17) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (18) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (19) CK domain, C2 domain, C1 domain, B3 domain, and B2 domain, (20) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (21) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (22) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (23) CK domain, C2 domain, C1 domain, B3 domain, and B1 domain, (24) CK domain, C2 domain, C1 domain, B3 domain, and B2 domain, (25) CK domain, C2 domain, B1 domain, B2 domain, and B1 domain, (26) CK domain, C2 domain, B1 domain, B1 domain, and B2 domain, (27) CK domain, C2 domain, B1 domain, B1 domain, and B2 domain, (2 (6) does not contain at least one VWF domain selected from the group consisting of a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, and a D4 domain; (17) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, and a A3 domain; (18) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, a A3 domain, and a A2 domain; (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, a A3 domain, a A2 domain, and a A1 domain; and (20) a combination thereof.

[0124] In yet another embodiment, the VWF protein comprises a D'D3 domain and one or more other domains or modules. Examples of such domains or modules include those described herein in their entirety. For example, the VWF protein may include a D'D3 domain and one or more domains or modules selected from the group consisting of an A1 domain, an A2 domain, an A3 domain, a D4N module, a VWD4 module, a C8-4 module, a TIL-4 module, a C1 module, a C2 module, a C3 module, a C4 module, a C5 module, a C5 module, a C6 module, and combinations thereof.

[0125] In yet another embodiment, the VWF protein is linked to a heterologous moiety, where the heterologous moiety is linked to the N-terminus or C-terminus of the VWF protein, or is inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the VWF protein. For example, the insertion site of the heterologous moiety in the VWF protein can be the D' domain, the D3 domain, or both. The heterologous moiety can be a half-life extender.

[0126] In certain embodiments, the VWF proteins useful in the present invention form multimers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, or higher multimers. In other embodiments, the VWF protein is a monomer with only one VWF protein. In some embodiments, the VWF proteins of the present invention can have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF protein can contain amino acid substitutions, deletions, additions, or modifications that prevent the VWF protein from forming disulfide bonds or from forming dimers or multimers. In another embodiment, the amino acid substitutions are within the D' and D3 domains. In certain embodiments, the VWF proteins useful in the present invention contain at least one amino acid substitution at residues corresponding to residue 1099, residue 1142, or both residues 1099 and 1142, corresponding to SEQ ID NO: 21. The at least one amino acid substitution can be any amino acid that does not naturally occur in wild-type VWF. For example, the amino acid substitution can be any amino acid other than cysteine, such as 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 protein from forming multimers.

[0127] In certain embodiments, the VWF proteins useful herein may be further modified to improve interaction with FVIII, e.g., to improve binding affinity to FVIII. As a non-limiting example, the VWF protein contains a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO:21 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO:21. Residues 764 and / or 773 contribute to the binding affinity of the VWF protein to FVIII. In other embodiments, the VWF proteins useful in the invention may have other modifications, e.g., the protein may be PEGylated, glycosylated, HESylated or polysialylated.

[0128] II.B. XTEN Sequences As used herein, "XTEN sequence" refers to an extended length polypeptide having a non-naturally occurring, substantially non-repetitive sequence composed primarily of small hydrophilic amino acids, which has low or no secondary or tertiary structure under physiological conditions. As a chimeric protein partner, XTEN can serve as a carrier and, when linked to a VWF protein or FVIII sequence of the invention to create a chimeric protein, impart certain desirable pharmacokinetic, physicochemical and pharmaceutical properties. Such desirable properties include high pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as single chain antibodies or Fc fragments of light or heavy chains.

[0129] The present invention provides short XTEN sequences that provide improved half-life extension properties compared to longer XTEN sequences when the XTEN sequences are fused to a VWF protein and / or a second Ig constant region or a portion thereof. Thus, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or a portion thereof contains less than 288 amino acids in length, i.e., is shorter than 288 amino acids. In one embodiment, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or a portion thereof consists of an amino acid sequence having a length between 12 amino acids and 287 amino acids. In another embodiment, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or a portion thereof comprises at least about 36 amino acids, at least about 42 amino acids, at least about 72 amino acids, at least about 144 amino acids, but less than 288 amino acids. In other embodiments, the XTEN sequence fused to the VWF protein and / or second Ig constant region or portion thereof is selected from AE36, AG36, AE42, AG42, AE72, AG72, AE144, or AG144. In one embodiment, the XTEN sequence fused to the VWF protein and / or second Ig constant region or portion thereof is an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:14, wherein the chimeric protein exhibits improved half-life compared to a chimeric protein that does not include the XTEN sequence.

[0130] The chimeric protein of the present invention can further comprise additional (second, third or more) XTEN sequences. The additional XTEN sequences can be further fused to the FVIII protein or the first Ig constant region or a portion thereof. The additional XTEN sequences can be of any length. For example, the additional XTEN sequences fused to the FVIII protein or the first Ig constant region or a portion thereof are peptides or polypeptides having more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acid residues. In certain embodiments, the additional XTEN sequence is a peptide or polypeptide having from about 20 to about 3000 amino acid residues, from about 30 to about 2500 residues, from about 40 to about 2000 residues, from about 50 to about 1500 residues, from about 60 to about 1000 residues, from about 70 to about 900 residues, from about 80 to about 800 residues, from about 90 to about 700 residues, from about 100 to about 600 residues, from about 110 to about 500 residues, or from about 120 to about 400 residues.

[0131] The XTEN sequence (i.e., an XTEN sequence fused to a VWF protein and / or a second Ig constant region or portion thereof, or an XTEN sequence fused to a FVIII protein and / or a first Ig constant region or portion thereof or inserted into one or more insertion sites within a FVIII protein) can comprise one or more sequence motifs of 9-14 amino acid residues or an amino acid sequence that is 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 4-6 amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P). See US2010-0239554A1.

[0132] In some embodiments, the XTEN sequences comprise non-overlapping sequence motifs, with at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% of the sequence being composed of multiple units of non-overlapping sequence selected from a single motif family selected from Table 2A, resulting in a family sequence. As used herein, "family" means that the XTEN has a motif selected from only a single motif category found in Table 2A, i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and any other amino acids in the XTEN that are not from a family motif are selected to achieve properties necessary, for example, to incorporate a restriction site by the nucleotides encoded, to incorporate a cleavage sequence, or to achieve good binding to FVIII or VWF. In some embodiments of the XTEN family, the XTEN sequence comprises multiple units of non-overlapping sequence motifs from the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, and the resulting XTEN exhibits 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 are selected to achieve desired physical / chemical characteristics, including properties such as charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness conferred by the amino acid composition of the motif, as described more fully below. In the embodiments described above in this paragraph, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve XTENs of between about 36 and about 3000 amino acid residues. [Table 2A-1] [Table 2A-2]

[0133] In some embodiments, the XTEN sequences used in the present invention are 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, AF57 6, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE8 28, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044 , AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG104 4, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US2010-0239554A1.

[0134] In one embodiment, the XTEN sequence is AE42 (SEQ ID NO: 9), AE72 (SEQ ID NO: 10), AE144_2A (SEQ ID NO: 55), AE144_3B (SEQ ID NO: 56), AE144_4A (SEQ ID NO: 57), AE144_5A (SEQ ID NO: 58), AE144_6B (SEQ ID NO: 59), AG144_A (SEQ ID NO: 60), AG144_B (SEQ ID NO: 61), AG144_C (SEQ ID NO: 62), AG144_F (SEQ ID NO: 63), AE864 (SEQ ID NO: 15 ...6B (SEQ ID NO: 64), AG144_7A (SEQ ID NO: 65), AG144_8A (SEQ ID NO: 66), AG144_9A (SEQ ID NO: 67), AG144_10A (SEQ ID NO: 68), AG144_11A (SEQ ID NO: 69), AG144_12A (SEQ ID NO: 70), AG144_13A (SEQ ID NO: 71), AG144_14B (SEQ ID NO: 72), AG144_15C (SEQ ID NO: ), AE576 (SEQ ID NO: 16), AE288 (SEQ ID NO: 8), AE288_2 (SEQ ID NO: 54), AE144 (SEQ ID NO: 11), AG864 (SEQ ID NO: 17), AG576 (SEQ ID NO: 18), AG288 (SEQ ID NO: 19), AG144 (SEQ ID NO: 14), and any combination thereof. In another 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: 9), AE72 (SEQ ID NO: 10), AE144_2A (SEQ ID NO: 55), AE144_3B (SEQ ID NO: 56), AE144_4A (SEQ ID NO: 57), AE144_5B (SEQ ID NO: 58), AE144_6B (SEQ ID NO: 59), AE144_7B (SEQ ID NO: 60), AE144_8B (SEQ ID NO: 61), AE144_9B (SEQ ID NO: 62), AE144_10B (SEQ ID NO: 63), AE144_11B (SEQ ID NO: 64), AE144_12B (SEQ ID NO: 65), AE144_13B (SEQ ID NO: 66), AE144_14B (SEQ ID NO: 67), AE144_15B (SEQ ID NO: 68), AE144_16B (SEQ ID NO: 69), AE144_17B (SEQ ID ), AE144_4A (SEQ ID NO:57), AE144_5A (SEQ ID NO:58), AE144_6B (SEQ ID NO:59), AG144_A (SEQ ID NO:60), AG144_B (SEQ ID NO:61), AG144_C (SEQ ID NO:62), AG144_F (SEQ ID NO:63), AE864 (SEQ ID NO:15), AE576 (SEQ ID NO:16), AE288 (SEQ ID NO:8), AE288_2 (SEQ ID NO:54), AE144 (SEQ ID NO:11), AG864 (SEQ ID NO:17), AG576 (SEQ ID NO:18), AG288 (SEQ ID NO:19), AG144 (SEQ ID NO:14), and any combination thereof. In a specific embodiment, the XTEN sequence is AE288. The amino acid sequences of certain XTEN sequences of the invention are shown in Table 2B. [Table 2B-1] [Table 2B-2] [Table 2B-3]

Table 2B-4

Table 2B-5

[0135] In those embodiments in which the XTEN component has less than 100% of amino acids consisting of 4, 5 or 6 of the amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P), or has less than 100% of a sequence consisting of a sequence motif of Table 3 or the XTEN sequences of Tables 4 and 13-17, the other amino acid residues of the XTEN are selected from any of the other 14 naturally occurring L-amino acids, but are 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. XTEN amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) are interspersed throughout the XTEN sequence, located within or between sequence motifs, or concentrated in one or more short stretches of the XTEN sequence, for example, to create a linker between the XTEN and FVIII or VWF. In those cases where the XTEN component includes amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids are hydrophobic residues, such that the resulting sequence generally lacks secondary structure, e.g., has no more than 2% alpha helices or more than 2% beta sheets as measured by the methods disclosed herein. Hydrophobic residues that are less preferred in XTEN construction include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine and methionine. Additionally, the XTEN sequences can be designed to 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: cysteine ​​(to avoid disulfide formation and oxidation), methionine (to avoid oxidation), arginine and glutamine (to avoid deamidation).Thus, in some embodiments, XTEN components containing amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) have sequences with less than 5% of the residues contributing to alpha helices and beta sheets as determined by the Chou-Fasman algorithm, and at least 90% or at least about 95% or more random coil formation as determined by the GOR algorithm.

[0136] In further embodiments, the XTEN sequence used in the present invention affects the physical or chemical properties, such as 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: structural flexibility, high water solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or large hydrodynamic (or Stokes) radius. In a specific embodiment, the XTEN sequence linked to the FVIII protein of the present invention enhances pharmacokinetic properties, such as, for example, a long terminal half-life or a large area under the curve (AUC), so that the chimeric protein disclosed herein persists in vivo for a longer period of time compared to wild-type FVIII. In further embodiments, the XTEN sequence used in the present invention enhances pharmacokinetic properties, such as, for example, a long terminal half-life or a large area under the curve (AUC), so that the FVIII protein persists in vivo for a longer period of time compared to wild-type FVIII.

[0137] One embodiment of the present invention is a FVIII / VWF fusion protein comprising a FVIII protein fused to an Fc region and a VWF protein fused to an Fc region, wherein an XTEN sequence (e.g., AE288) is inserted within the FVIII portion and has an amino acid sequence less than 288. An XTEN sequence having an amino acid sequence (e.g., AE144) is inserted between the VWF portion and the Fc portion. As described in the Examples, the insertion of an XTEN having less than 288 amino acids between the VWF portion and the Fc portion has a greater effect on the pharmacokinetics of the chimeric protein than the insertion of an XTEN having 288 amino acids between the VWF portion and the Fc portion. For example, the insertion of an XTEN sequence having less than 288 amino acids between the VWF portion and the Fc portion of a FVIII / VWF fusion protein can increase the terminal half-life of the chimeric protein compared to an XTEN having 288 amino acids. In some embodiments, the terminal half-life is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% compared to the insertion of an XTEN sequence having 288 amino acids. In a particular embodiment, the terminal half-life is increased by at least about 35% compared to the insertion of an XTEN sequence having 288 amino acids. Insertion of an XTEN sequence having less than 288 amino acids can also increase the AUC value of the chimeric protein. In some embodiments, the AUC value is increased by at least about 50%, at least about 100%, or at least about 200% compared to insertion of an XTEN having 288 amino acids. In one particular embodiment, the AUC is increased by about 2-fold. Insertion of an XTEN sequence having less than 288 amino acids can also reduce the clearance of the chimeric protein. For example, the clearance can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% compared to insertion of an XTEN sequence having 288 amino acids. Insertion of an XTEN sequence having less than 288 amino acids can increase the mean residence time (MRT) and / or reduce the apparent volume of distribution at steady state (Vss) compared to insertion of an XTEN having 288 amino acids.

[0138] A variety of methods and assays can be used to measure the physical / chemical properties of proteins containing XTEN sequences. Such methods include, but are not limited to, analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV / visible spectroscopy. Additional methods are disclosed in Amau et al., Prot. Expr. and Purif. 48, 1-13 (2006).

[0139] Additional examples of XTEN sequences that can be used in accordance with the present invention are disclosed in US2010 / 0239554A1, US2010 / 0323956A1, US2011 / 0046060A1, US2011 / 0046061A1, US2011 / 0077199A1, or US2011 / 0172146A1, or WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, WO2011028344A2, or WO20130122617A1.

[0140] II.C. Factor VIII (FVIII) Protein "FVIII protein" as used herein, unless specified, means a FVIII polypeptide functional in its normal role in coagulation. The term FVIII protein includes functional fragments, variants, analogs or derivatives thereof that retain the function of full-length wild-type factor VIII in the coagulation pathway. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include the ability to activate coagulation, act as a cofactor for factor IX, or inhibit Ca. 2+These include, but are not limited to, the ability to form a tenase complex with factor IX in the presence of phospholipids and then convert factor X to the activated form Xa. The FVIII protein can be human, porcine, canine, rat or mouse FVIII protein. In addition, comparisons of human-derived FVIII with FVIII from other species are also possible. have identified conserved residues that may be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US Pat. No. 6,251,632).

[0141] Numerous tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM assay, prothrombin time (PT) test (also used to measure INR), fibrinogen test (often by Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected 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), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g. ROTEM®) or euglobulin lysis time (ELT).

[0142] The aPTT test is a performance indicator that measures the effectiveness of both the "intrinsic" (also called the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors, e.g., FVIII or FIX. It is used in conjunction with the prothrombin test (PT), which measures the extrinsic pathway.

[0143] ROTEM analysis provides information on the global dynamics of hemostasis: clotting time, clot formation, clot stability and lysis. The various parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis or on the numerous factors that affect their interactions. This assay can provide a complete picture of secondary hemostasis.

[0144] The polypeptide and polynucleotide sequences of FVIII are known, as are a number of functional fragments, variants and modifications. An example of a human FVIII sequence (full length) is shown below. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 4-3]

[0145] FVIII polypeptides include full-length FVIII, full-length FVIII with Met removed at the N-terminus, mature FVIII (without the signal peptide), mature FVIII with an additional Met at the N-terminus, and / or FVIII with a complete or partial deletion of the B domain, in certain embodiments, the FVIII variants include a deletion of the B domain, either a partial deletion or a complete deletion.

[0146] The sequence of native human mature FVIII is presented as SEQ ID NO: 65. The native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2 and A3 are structurally related "A domains", B is the "B domain", C1 and C2 are structurally related "C domains", and a1, a2 and a3 are acidic spacer regions. With reference to the primary amino acid sequence positions in SEQ ID NO:65, the A1 domain of human FVIII extends from Ala1 to about Arg336, the a1 spacer region extends from about Met337 to about Val374, the A2 domain extends from about Ala375 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg1648, and the a3 spacer region extends from about Glu1649 to about Arg1689. The A3 domain extends from about Ser1690 to about Leu2025, the C1 domain extends from about Gly2026 to about Asn2072, and the C2 domain extends from about Ser2073 to about Tyr2332. Other than specific proteolytic cleavage sites, the designation of the locations of boundaries between domains and regions of FVIII can vary in various literature references. Thus, the boundaries referred to herein are designated as approximations by use of the term "approximately".

[0147] The human FVIII gene has been isolated and expressed in mammalian cells (Toole, JJ et al., Nature, 312:342-347 (1984); Gitschier, J. et al., Nature, 312:326-330 (1984); Wood, WI et al., Nature, 312:330-337 (1984); Vehar, GA et al., Nature, 312:337-342 (1984); WO87 / 04187; WO88 / 08035; WO88 / 03558; and US4,757,006). The amino acid sequence of FVIII has been deduced from cDNA as shown in US4,965,199. In addition, partially or completely B-domain deleted FVIIIs are shown in US4,994,371 and US4,868,112. In some embodiments, the B domain of human FVIII is replaced with the B domain of human factor V as set forth in US 5,004,803. The cDNA and amino acid sequences encoding human factor VIII are set forth in SEQ ID NOs: 1 and 2, respectively, of US 2005 / 0100990.

[0148] The porcine FVIII sequence has been published in Toole, JJ et al., Proc. Natl. Acad. Sci. USA, 83:5939-5942 (1986). Furthermore, the complete porcine cDNA sequence obtained by PCR amplification of FVIII sequence from a porcine spleen cDNA library has been reported in Healey, JF et al., Blood, 88:4209-4214 (1996). Hybrid human / porcine FVIII with all domains, all subunits and specific amino acid sequence replacements have been disclosed in US 5,364,771 by Lollar and Runge and in WO 93 / 20093. More recently, the nucleotide sequence and corresponding amino acid sequence of the A1 and A2 domains of porcine FVIII, as well as chimeric FVIII with the porcine A1 and / or A2 domains replaced for the corresponding human domains, have been reported in WO 94 / 11503. US 5,859,204, Lollar, JS also discloses a porcine cDNA and deduced amino acid sequence. US 6,458,563 discloses a B domain deleted porcine FVIII.

[0149] US 5,859,204, Lollar, JS, reports functional mutants of FVIII with reduced antigenicity and reduced immunoreactivity. US 6,376,463, Lollar, JS, also reports mutants of FVIII with reduced immunoreactivity. Saenko et al., US 2005 / 0100990, reports functional mutations in the A2 domain of FVIII.

[0150] In one embodiment, FVIII (or the FVIII portion of the chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 67 or amino acids 1 to 2332 of SEQ ID NO: 65 (without the signal sequence) or the FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 64 and 1 to 1438 of SEQ ID NO: 67 or amino acids 1 to 19 of SEQ ID NO: 64 and amino acids 1 to 2332 of SEQ ID NO: 65 (with the signal sequence), where FVIII has coagulation activity, e.g., activates factor IX as a cofactor and converts factor X to activated factor X. FVIII (or the FVIII portion of the chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 67 or amino acids 1 to 2332 of SEQ ID NO: 65 (without the signal sequence). The FVIII may further comprise a signal sequence.

[0151] The "B domain" of FVIII, as used herein, is identical to the B domain known in the art, defined by the internal amino acid sequence identity and site of proteolytic cleavage of human full-length FVIII, e.g., residues Ser741 to Arg1648. The other domains of human FVIII are defined by the following amino acid residues: A1, residues Ala1 to Arg372; A2, residues Ser373 to Arg740; A3, residues Ser1690 to Asn2019; C1, residues Lys2020 to Asn2172; C2, residues Ser2173 to Tyr2332. The A3-C1-C2 sequence includes residues Ser1690 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is commonly referred to as the a3 acidic region. The locations of all the domain boundaries, including the B domain, for porcine, mouse and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B domain deleted factor VIII" or "BDD-FVIII"). An example of a BDD-FVIII is REFACTO® (recombinant BDD-FVIII), which has the same sequence as the factor VIII portion of the sequence in Table 5 (BDD-FVIII heavy chain double underlined, B domain italicized, BDD-FVIII light chain in regular text). The nucleotide sequence encoding Table 6 (SEQ ID NO:68) is shown in Table 6. [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2]

[0152] A "B domain deleted FVIII" may have complete or partial deletions as disclosed in US6,316,226, US6,346,513, US7,041,635, US5,789,203, US6,060,447, US5,595,886, US6,228,620, US5,972,885, US6,048,720, US5,543,502, US5,610,278, US5,171,844, US5,112,950, US4,868,112, and US6,458,563. In some embodiments, the sequence of a B domain deleted FVIII of the invention comprises any one of the deletions disclosed in US 6,316,226 (also US 6,346,513) at column 4, line 4 to column 5, line 28 and Examples 1 to 5. In another embodiment, the B domain deleted factor VIII is S743 / Q1638 B domain deleted factor VIII (SQ BDD FVIII) (e.g., a factor VIII having a deletion of amino acids 744 to 1637, e.g., a factor VIII having amino acids 1 to 743 and amino acids 1638 to 2332 of SEQ ID NO: 65, i.e., having SEQ ID NO: 67). In some embodiments, the B domain deleted FVIII of the present invention has a deletion as disclosed in column 2, lines 26-51 and Examples 5-8 of US 5,789,203 (also US 6,060,447, US 5,595,886 and US 6,228,620). In some embodiments, the B domain deleted factor VIII has a deletion as disclosed in column 1, line 25 to column 2, line 40 of US 5,972,885; column 6, lines 1-22 and Example 1 of US 6,048,720; column 2, lines 17-46 of US 5,543,502; column 4, lines 22 to column 5, line 36 of US 5,171,844; column 2, lines 55-68 of US 5,112,950. lines, FIG. 2 and Example 1; column 2, line 2 to column 19, line 21 and Table 2 of US 4,868,112; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of US 7,041,635; or column 4, lines 25 to 53 of US 6,458,563.In some embodiments, the B domain deleted FVIII has most of the B domain deleted, but still contains sequences at the amino terminus of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains, as disclosed in WO 91 / 09122. In an embodiment, a B domain deleted FVIII is constructed by deleting amino acids 747-1638, i.e., by practically complete deletion of the B domain. Hoeben, RC et al. J. Biol. Chem. 265(13):7318-7323 (1990). B domain deleted factor VIII may also contain deletions 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 which are part of the present invention include deletions of amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986), 83, 5939-5942), 797-1562 (Eaton et al., Biochemistry, (1986), 25:8343-8347), 741-1646 (Kaufman (WO 87 / 04187)), 747-1560 (Sarver et al., DNA, (1987), 6:553-564)), 741-1648 (Pasek (PCT 88 / 00831)), or 816-1598 or 741-1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597). In other embodiments, BDD-FVIII includes a fragment of the B domain that retains one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or optionally 943, corresponding to the amino acid sequence of the full-length FVIII sequence. Examples of fragments of the B domain include 226 or 163 amino acids of the B domain (i.e., the first 226 or 163 amino acids of the B domain are retained) as disclosed in Miao, HZ et al., Blood, 103(a):3412-3419 (2004), Kasuda, A et al., J. Thromb. Haemost. 6:1352-1359 (2008), and Pipe, SW et al., J. Thromb. Haemost. 9:2235-2242 (2011). In yet other embodiments, the BDD-FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve expression of the BDD-FVIII protein.See Miao, HZ et al., Blood, 103(a):3412-3419 (2004). In yet other embodiments, BDD-FVIII includes a FVIII polypeptide that contains a portion of the B domain but does not contain one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, SW et al., J. Thromb. Haemost. 9:2235-2242 (2011). Each of the foregoing deletions may be made in any FVIII sequence.

[0153] In some embodiments, FVIII has a partial B domain. In some embodiments, the FVIII protein with a partial B domain is FVIII198. FVIII198 is a single chain FVIIIFc molecule containing a partial B domain-226N6. The number 226 represents the N-terminal 226 amino acids of the B domain of FVIII, and N6 represents the six N-glycosylation sites in the B domain.

[0154] In one embodiment, FVIII is cleaved immediately after the arginine at amino acid 1648 (in full length factor VIII or SEQ ID NO: 65), the arginine at amino acid 754 (in S743 / Q1638B domain deleted factor VIII or SEQ ID NO: 67), or the corresponding arginine residue (in other variants), thereby generating a heavy chain and a light chain. In another embodiment, FVIII comprises heavy and light chains that are linked or associated by a non-covalent bond mediated by a metal ion.

[0155] In other embodiments, the FVIII is a single-chain FVIII that is not truncated immediately after the arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 65), the arginine at amino acid 754 (in S743 / Q1638B domain deleted FVIII or SEQ ID NO: 67), or the corresponding arginine residue (in other variants). The single-chain FVIII may contain one or more amino acid substitutions. In one embodiment, The amino acid substitution is at residue 1648, residue 1645, or both, of the full-length mature factor VIII polypeptide (SEQ ID NO:65), or at residue 754, residue 751, or both, of the SQ BDD factor VIII (SEQ ID NO:67). The amino acid substitution can be an amino acid other than arginine, for example, 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.

[0156] FVIII can be cleaved by thrombin and then activated as FVIIIa, which can serve as a cofactor for activated factor IX (FIXa). Activated FIX then forms an Xase complex with activated FVIII, converting 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), and cleavage produces FVIIIa with a 50 kDa A1 chain, a 43 kDa A2 chain and a 73 kDa A3-C1-C2 chain. In one embodiment, the FVIII protein useful in the present invention is an inactive FVIII. In another embodiment, the FVIII protein is an active FVIII.

[0157] A protein having a FVIII polypeptide linked or associated with a VWF protein can comprise a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65 or 67, wherein the sequence has the clotting activity of FVIII, e.g., the activity of activating factor IX as a cofactor and converting factor X to activated factor X (FXa).

[0158] "Hybrid" or "chimeric" polypeptides and proteins as used herein include a combination of a first polypeptide chain, e.g., a VWF protein fused to a first Ig constant region or portion thereof with an XTEN sequence having less than 288 amino acids, and a second polypeptide chain, e.g., a FVIII protein fused to a second Ig constant region or portion thereof, thereby forming a heterodimer. In one embodiment, the first and second polypeptides in the hybrid associate with each other via protein / protein interactions, e.g., charge / charge or hydrophobic interactions. In another embodiment, the first polypeptide includes a VWF protein / XTEN-Fc fusion protein and the second polypeptide includes a FVIII-Fc fusion protein, making the hybrid a heterodimer, where the XTEN contains less than 288 amino acids. In other embodiments, the first polypeptide comprises a VWF protein / XTEN-Fc fusion protein and the second polypeptide comprises a FVIII(X)-Fc fusion protein, making the hybrid a heterodimer, where the XTEN contains less than 288 amino acids. The first and second polypeptides can be associated via a covalent bond, such as, for example, a disulfide bond between the first and second Fc regions. The first and second polypeptides can further be associated with each other by a bond between the VWF fragment and the FVIII protein.

[0159] The FVIII protein useful in the present invention can include FVIII with one or more additional XTEN sequences that do not affect the coagulation activity of FVIII. Such XTEN sequences can be fused to the C-terminus or N-terminus of the FVIII protein, or can be inserted between one or more two amino acid residues in the FVIII protein, where the insertion does not affect the coagulation activity of FVIII or the function of FVIII. In one embodiment, The insertion improves the pharmacokinetic properties (e.g., half-life) of the FVIII protein. In another embodiment, the insertion can be multiple insertions, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions. Examples of insertion sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, 15, or combinations thereof.

[0160] The FVIII protein linked to one or more XTEN sequences may be FVIII(X2) or FVIII (a→b) -X-FVIII (c→d) where FVIII is (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"; X2 comprises, consists essentially of, or consists of one or more XTEN sequences; FVIII (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", where "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 two amino acids from the insertion site where 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 two amino acids from the insertion site where the XTEN sequence is inserted; and "d" is the C-terminal amino acid residue of the FVIII protein, and wherein the first portion of the FVIII protein and the second portion of the FVIII protein are not identical to each other and are both of sufficient length such that the FVIII protein has the clotting activity of FVIII.

[0161] In one embodiment, the first portion of the FVIII protein and the second portion of the FVIII protein are fragments of SEQ ID NO: 65 (full-length mature FVIII sequence) or SEQ ID NO: 67 (FVIII with the B domain deleted), e.g., the N-terminal portion and the C-terminal portion, respectively. In a particular embodiment, the first portion of the FVIII protein comprises the A1 and A2 domains of the FVIII protein. The second portion of the FVIII protein comprises the A3 and C1 domains, and optionally the C2 domain. In yet another embodiment, the first portion of the FVIII protein comprises the A1 and A2 domains of the FVIII protein, and the second portion of the FVIII protein comprises a portion of the B domain, the A3 and C1 domains, and optionally the C2 domain. In yet another embodiment, the first portion of the FVIII protein comprises the A1 and A2 domains of the FVIII protein, and a portion of the B domain of the FVIII domain, and the second portion of the FVIII protein comprises the A3 and C1 domains, and optionally the C2 domain. In yet another embodiment, the first portion of the FVIII protein comprises the A1 domain, the A2 domain, and a first portion of the B domain of the FVIII protein. The second portion of the FVIII protein comprises a second portion of the B domain, the A3 domain, the C1 domain, and optionally the C2 domain. In some embodiments, the two amino acids ("b" and "c") can be any one or more of the amino acid residue 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 where one or more XTEN sequences are inserted or linked, and "c" can be the amino acid residue immediately downstream of the site where one or more XTEN sequences are inserted or linked. In some embodiments, "a" is the first mature amino acid sequence of the FVIII protein, and "d" is the last amino acid sequence of the FVIII protein. For example, FVIII (a→b) can be an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 745 of SEQ ID NO: 67 (the amino acid sequence of B-domain deleted FVIII) or SEQ ID NO: 65 (full-length FVIII),(c→d) can be amino acids 746 to 1438 of SEQ ID NO: 67 or amino acids 1641 to 2332 of SEQ ID NO: 65, respectively.

[0162] In some aspects, the insertion site of the FVIII protein is located in one or more domains of the FVIII protein that are the N-terminus, the A1 domain, the A2 domain, the A3 domain, the B domain, the C1 domain, the C2 domain, the C-terminus, or a combination of two or more thereof, or between two domains of the FVIII protein that are the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, and the A3 domain and the C1 domain, the C1 domain and the C2 domain, or any combination thereof. For example, insertion sites into which an XTEN sequence may be inserted are selected from the group consisting of the N-terminus and the A1 domain, the N-terminus and the A2 domain, the N-terminus and the A3 domain, the N-terminus and the B domain, the N-terminus and the C1 domain, the N-terminus and the C2 domain, the N-terminus and the C-terminus, the A1 and the A2 domains, the A1 and the A3 domains, the A1 and the B domains, the A1 and the C1 domains, the A1 and the C2 domains, the A1 domain and the C-terminus, the A2 and the A3 domains, the A2 and the B domains, the A2 and the C1 domains, the A2 and the C2 domains, the A2 domain and the C-terminus, the A3 and the B domains, the A3 and the C1 domains, the A3 and the C2 domains, the A3 domain and the C-terminus, the B and the C1 domains, the B and the C2 domains, the B domain and the C-terminus, the C1 and the C2 domains, the C1 and the C-terminus, the C2 domain, and the C-terminus, and combinations of two or more thereof. Non-limiting examples of insertion sites are listed in Tables 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0163] FVIII proteins in which an XTEN sequence is inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites) or linked to the C-terminus or N-terminus retain FVIII activity after linkage to or insertion by the XTEN sequence. The XTEN sequence can be inserted once, more than once, two, three, four, five or six times into the FVIII protein such that the insertion does not affect FVIII activity (i.e., the FVIII protein still retains clotting properties).

[0164] The FVIII proteins 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 any linker, or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein by any linker. In one embodiment, the two amino acid residues at which the XTEN sequence is inserted or the amino acid residues at which the XTEN sequence is linked correspond to two or one amino acid residues of SEQ ID NO:65 (full-length mature FVIII) selected from the group consisting of residues in Table 7, Table 8, Table 9, and Table 10, and combinations thereof.

[0165] In other embodiments, at least one XTEN sequence is inserted into one or more of the XTEN insertion sites disclosed herein or a combination thereof. In one aspect, at least one XTEN sequence is inserted into one or more of the XTEN insertion sites disclosed at one or more amino acids disclosed in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

[0166] In some embodiments, one or more XTEN sequences are inserted within about 6 amino acids above or below amino acid 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905, or 1910, or a combination thereof, corresponding to SEQ ID NO:65. [Table 8]

[0167] In other embodiments, the one or more XTEN sequences are inserted immediately downstream of one or more amino acids corresponding to human full-length mature FVIII selected from the group consisting of one or more insertion sites in Table 9. [Table 9]

[0168] In yet another embodiment, one or more XTENs are inserted into the B domain of FVIII. In one example, XTENs are inserted between amino acids 740 and 1640 corresponding to SEQ ID NO:65, where the FVIII sequence between amino acids 740 and 1640 is optionally absent. In another example, XTENs are inserted between amino acids 741 and 1690 corresponding to SEQ ID NO:65, where the FVIII sequence between amino acids 740 and 1690 is optionally absent. In another example, XTENs are inserted between amino acids 741 and 1648 corresponding to SEQ ID NO:65, where the FVIII sequence between amino acids 741 and 1648 is optionally absent. In yet another example, XTENs are inserted between amino acids 743 and 1638 corresponding to SEQ ID NO:65, where the FVIII sequence between amino acids 743 and 1638 is optionally absent. In yet other examples, XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO: 65, where the FVIII sequence between amino acids 745 and 1656 is optionally absent. In some examples, XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO: 65, where the FVIII sequence between amino acids 745 and 1657 is optionally absent. In certain examples, XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO: 65, where the FVIII sequence between amino acids 745 and 1667 is optionally absent. In yet other examples, XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO: 65, where the FVIII sequence between amino acids 745 and 1686 is optionally absent. In some other examples, XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO: 65, where the FVIII sequence between amino acids 747 and 1642 is optionally absent. In yet another example, XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO:65, where the FVIII sequence between amino acids 751 and 1667 is optionally absent.

[0169] In some embodiments, the one or more XTENs are inserted at one or more amino acids immediately downstream of an amino acid at the insertion site selected from the group consisting of the amino acid residues in Table 10. [Table 10-1] [Table 10-2]

[0170] In one embodiment, one or more XTEN insertion sites are located within one or more surface-exposed flexible loop structures (e.g., permissive loops) of the FVIII protein. For example, at least one XTEN sequence can be inserted into each FVIII "A" domain, which contains at least two "permissive loops" into which at least one XTEN polypeptide can be inserted without eliminating the procoagulant activity of the recombinant protein or the ability of the recombinant protein to be expressed in a host cell in vivo or in vitro. Permissive loops are regions that allow, among other attributes, the insertion of at least one XTEN sequence with high surface or solvent exposure and conformational flexibility. The A1 domain is referred to as permissive loop-1 (A1- The A1 domain contains a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region, and the A3 domain contains a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region.

[0171] In one embodiment, the first permissive loop (A1-1) in the A1 domain of FVIII is located between beta strand 1 and beta strand 2, and the second permissive loop (A1-2) in the A1 domain of FVIII is located between beta strand 11 and beta strand 12. The first permissive loop (A2-1) in the A2 domain of FVIII is located between beta strand 22 and beta strand 23, and the second permissive loop (A2-2) in the A2 domain of FVIII is located between beta strand 32 and beta strand 33. The first permissive loop (A3-1) in the A3 domain of FVIII is located between beta strand 38 and beta strand 39, and the second permissive loop (A3-2) in the A3 domain of FVIII is located between beta strand 45 and beta strand 46. In certain embodiments, the surface-exposed flexible loop structure comprising A1-1 corresponds to a region of human native mature FVIII from about amino acid 15 to about amino acid 45 of SEQ ID NO:65, e.g., from about amino acid 18 to about amino acid 41 of SEQ ID NO:65. In other embodiments, the surface-exposed flexible loop structure comprising A1-2 corresponds to a region of human native mature FVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO:65, e.g., from about amino acid 218 to about amino acid 229 of SEQ ID NO:65. In yet other embodiments, the surface-exposed flexible loop structure comprising A2-1 corresponds to a region of human native mature FVIII from about amino acid 395 to about amino acid 421 of SEQ ID NO:65, e.g., from about amino acid 397 to about amino acid 418 of SEQ ID NO:65. In yet other embodiments, the surface-exposed flexible loop structure comprising A2-2 corresponds to a region of human native mature FVIII from about amino acid 577 to about amino acid 635 of SEQ ID NO:65, e.g., from about amino acid 595 to about amino acid 607 of SEQ ID NO:65. In certain aspects, the surface-exposed flexible loop structure comprising A3-1 corresponds to a region of human native mature FVIII from about amino acid 1705 to about amino acid 1732 of SEQ ID NO:65, e.g., from about amino acid 1711 to about amino acid 1725 of SEQ ID NO:65.In yet another embodiment, the surface-exposed flexible loop structure comprising A3-2 corresponds to a region of human native mature FVIII from about amino acid 1884 to about amino acid 1917 of SEQ ID NO:65, e.g., from about amino acid 1899 to about amino acid 1911 of SEQ ID NO:65.

[0172] In another embodiment, one or more amino acids into which at least one XTEN sequence is inserted are located within the a3 domain, e.g., within the range of amino acids 1649 to 1689, corresponding to a full-length mature FVIII polypeptide. In a particular embodiment, the XTEN sequence is inserted between amino acid 1656 and amino acid 1657 of SEQ ID NO:65 (full-length mature FVIII). In a particular embodiment, the FVIII protein comprising an XTEN sequence inserted immediately downstream of amino acid 1656, corresponding to SEQ ID NO:65, further comprises a deletion from amino acid 745 to amino acid 1656, corresponding to SEQ ID NO:65.

[0173] In some embodiments, the one or more insertion sites for the one or more XTEN insertions are: (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 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 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) immediately downstream of one or more amino acids corresponding to mature full-length FVIII selected from the group consisting of a combination of two or more thereof.

[0174] In one embodiment, a FVIII protein useful in the invention comprises two XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site and a second XTEN sequence inserted into a second XTEN insertion site. Non-limiting examples of first and second XTEN insertion sites are listed in Table 11. [Table 11-1] [Table 11-2]

[0175] The two XTEN inserted or linked into the FVIII protein are identical or different. In some embodiments, a FVIII protein useful in the invention comprises a first XTEN sequence inserted just downstream of amino acid 745 corresponding to SEQ ID NO:65 and a second XTEN sequence inserted just downstream of amino acid 2332 (C-terminus) corresponding to SEQ ID NO:65. In other embodiments, the first XTEN sequence is inserted just downstream of amino acid 18, 26, 40, 1656 or 1720 corresponding to SEQ ID NO:65 and the second XTEN sequence is inserted just downstream of amino acid 403 corresponding to SEQ ID NO:65. In yet other embodiments, the first XTEN sequence is inserted just downstream of amino acid 18, 26 or 40 corresponding to SEQ ID NO:65 and the second XTEN sequence is inserted just downstream of amino acid 599 corresponding to SEQ ID NO:65. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO:65 and the second XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1725, 1720, 1900 or 2332 corresponding to SEQ ID NO:65. In certain embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO:65 and the second XTEN sequence is inserted immediately downstream of amino acid 18, 26 or 40 corresponding to SEQ ID NO:65. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26 or 40 corresponding to SEQ ID NO:65 and the second XTEN sequence is inserted immediately downstream of amino acid 399 corresponding to SEQ ID NO:65. In other embodiments, a first XTEN sequence is inserted just downstream of amino acid 1720, which corresponds to SEQ ID NO:65, and a second XTEN sequence is inserted just downstream of amino acid 18, 26, or 40, which corresponds to SEQ ID NO:65. In yet other embodiments, a first XTEN sequence is inserted just downstream of amino acid 1720, which corresponds to SEQ ID NO:65, and a second XTEN sequence is inserted just downstream of amino acid 18, which corresponds to SEQ ID NO:65.In certain embodiments, the FVIII protein comprises two XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO:65 and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO:65, wherein the FVIII protein further has a deletion from amino acid 745 corresponding to SEQ ID NO:65 to amino acid 1685 corresponding to SEQ ID NO:65; a mutation or substitution at amino acid 1680 corresponding to SEQ ID NO:65, e.g., Y1680F; a mutation or substitution at amino acid 1648 corresponding to SEQ ID NO:65, e.g., R1648A; or at least two mutations or substitutions at amino acid 1648 corresponding to SEQ ID NO:65 and amino acid 1680 corresponding to SEQ ID NO:65, e.g., R1648A and Y1680F. In a specific embodiment, the FVIII protein comprises two XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO:65 and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO:65, wherein the FVIII protein further has a deletion of amino acids 745 through 1656 corresponding to SEQ ID NO:65.

[0176] In certain embodiments, the FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted into the first XTEN insertion site, a second XTEN sequence inserted into the second XTEN sequence insertion site, and a third XTEN sequence inserted into the third XTEN insertion site. The first XTEN sequence, the second XTEN sequence, and the third XTEN sequence can be the same or different. The first, second, and third insertion sites can be selected from any one of the groups of insertion sites disclosed herein. In some embodiments, the FVIII protein comprising three XTEN sequences can further comprise a mutation or substitution at amino acid 1648, e.g., R1648A, e.g., corresponding to SEQ ID NO:65. For example, non-limiting examples of the first, second, and third XTEN insertion sites are listed in Table 12. [Table 12]

[0177] In some embodiments, the FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO:65, a second XTEN sequence inserted downstream of amino acid 403 corresponding to SEQ ID NO:65, and a third XTEN sequence inserted downstream of amino acid 1656, 1720, or 1900 corresponding to SEQ ID NO:65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO:65, the second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO:65, and the third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO:65. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO:65, the second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO:65, and the third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO:65. In yet other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO:65, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO:65, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO:65. In other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 corresponding to SEQ ID NO:65, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO:65, and a third XTEN sequence is inserted downstream of amino acid ... a second XTEN sequence is inserted downstream of amino acid 745, which corresponds to SEQ ID NO:65; and a third XTEN sequence is inserted downstream of amino acid 2332, which corresponds to SEQ ID NO:65.

[0178] In other embodiments, the FVIII protein of the present invention comprises four XTEN sequences, a first XTEN sequence inserted at the first insertion site, a second XTEN sequence inserted at the second insertion site, a third XTEN sequence inserted at the third insertion site, and a fourth XTEN sequence inserted at the fourth insertion site. The first, second, third, and fourth XTEN sequences can be the same, different, or a combination thereof. In some embodiments, the FVIII protein comprising four XTEN sequences can further comprise a mutation or substitution at amino acid 1648, e.g., R1648A, corresponding to, e.g., SEQ ID NO: 65. Non-limiting examples of the first, second, third, and fourth XTEN insertion sites are listed in Table 13. [Table 13-1] [Table 13-2] [Table 13-3]

[0179] In some embodiments, the FVIII protein comprises five XTEN sequences, a first XTEN sequence inserted at the first insertion site, a second XTEN sequence inserted at the second insertion site, a third XTEN sequence inserted at the third XTEN insertion site, a fourth XTEN sequence inserted at the fourth XTEN insertion site, and a fifth XTEN sequence inserted at the fifth XTEN insertion site. The first, second, third, fourth, and fifth XTEN sequences can be the same, different, or a combination thereof. Non-limiting examples of first, second, third, fourth, and fifth insertion sites are listed in Table 14. [Table 14]

[0180] In certain embodiments, the FVIII protein comprises six XTEN sequences, a first XTEN sequence inserted into the first XTEN insertion site, a second XTEN sequence inserted into the second XTEN insertion site, a third XTEN sequence inserted into the third XTEN insertion site, a fourth XTEN sequence inserted into the fourth XTEN insertion site, a fifth XTEN sequence inserted into the fifth XTEN insertion site, and a sixth XTEN sequence inserted into the sixth XTEN insertion site. The first, second, third, fourth, fifth, or sixth XTEN sequences can be the same, different, or a combination thereof. Examples of six XTEN insertion sites include, but are not limited to, the insertion sites listed in Table 15. [Table 15]

[0181] In a particular example, a first XTEN sequence is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65 (full-length mature FVIII) and a second XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In another example, a first XTEN sequence is inserted between amino acids 403 and 404 corresponding to SEQ ID NO:65 and a second XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In some examples, a first XTEN sequence is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65 and a second XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In other examples, a first XTEN sequence is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, a second XTEN sequence is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65 and a third XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In yet another embodiment, a first XTEN sequence is inserted between amino acids 403 and 404 corresponding to SEQ ID NO:65, a second XTEN sequence is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, and a third XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In yet another embodiment, a first XTEN sequence is inserted between amino acids 403 and 404 corresponding to SEQ ID NO:65. , the second XTEN sequence is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, and the third XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In certain embodiments, the first XTEN sequence is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, the second XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65, and the third XTEN sequence is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO:65. In some embodiments, the first XTEN sequence is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, the second XTEN sequence is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, the third XTEN sequence is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65, and the fourth XTEN sequence is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO:65.

[0182] In certain embodiments, the XTEN sequence is inserted between amino acids 745 and 746 of full-length factor VIII, or at the corresponding insertion site in B-domain deleted factor VIII.

[0183] In some embodiments, a chimeric protein of the invention comprises two polypeptide sequences: FVIII-161 (SEQ ID NO:69), FVIII-169 (SEQ ID NO:70), FVIII-170 (SEQ ID NO:71), FVIII-173 (SEQ ID NO:72); FVIII-195 (SEQ ID NO:73); FVIII-196 (SEQ ID NO:74), FVIII199 (SEQ ID NO:75), FVIII-201 (SEQ ID NO:76); FVIII-203 (SEQ ID NO:77), FVIII-204 (SEQ ID NO:78), FVIII-205 (SEQ ID NO:79), FVIII-266 (SEQ ID NO:80), FVIII and a second polypeptide sequence comprising an amino acid sequence that is at least about 80%, 90%, 95% or 100% identical to a sequence selected from VWF031 (SEQ ID NO:86), VWF034 (SEQ ID NO:87) or VWF-036.

[0184] II.D. Ig Constant Region or Portion Thereof The chimeric protein of the present invention also comprises two Ig constant regions or portions thereof, a first Ig constant region or portion thereof fused to a FVIII protein by an optional linker, and a second Ig constant region or portion thereof fused to a VWF protein via an XTEN sequence having less than 288 amino acids. The Ig constant region or portion thereof can improve the pharmacokinetic or pharmacodynamic properties of the chimeric protein in combination with the XTEN sequence and the VWF protein. In certain embodiments, the Ig constant region or portion thereof extends the half-life of the molecule fused to the Ig constant region or portion thereof.

[0185] The Ig constant region is composed of domains designated CH (constant heavy chain) (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may be composed of 3 or 4 CH domains. The constant regions of some isotypes (e.g., IgG) also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, NY, NY.

[0186] The Ig constant region or portion thereof for making the chimeric proteins of the present invention may be obtained from a number of different sources. In some embodiments, the Ig constant region or portion thereof is derived from human Ig. However, the Ig constant region or portion thereof may also be derived from the Ig of another mammal, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, monkey) species. Additionally, the Ig constant region may be derived from a human Ig. Or portions thereof may be derived from Ig classes including IgM, IgG, IgD, IgA and IgE, and Ig isotypes including IgG1, IgG2, IgG3, and IgG4, in one embodiment, human isotype IgG1 is used.

[0187] Various Ig constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domain sequences can be selected that have specific effector functions (or lack of effector functions) or have specific modifications that reduce immunogenicity. Numerous sequences of antibodies or 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 the above methods may then be altered or synthesized to obtain the polypeptides of the invention. It will be further appreciated that the scope of the present invention encompasses alleles, variants and mutations of the constant region DNA sequences.

[0188] For example, the Ig constant region or a portion thereof can be cloned using polymerase chain reaction and primers selected to amplify the domain. To clone the Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen or lymphoid cells, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. PCR amplification methods are described in detail in US 4,683,195; US 4,683,202; US 4,800,159; US 4,965,188; and for example, in "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 published heavy and light chain DNA and amino acid sequences. As mentioned above, PCR may also be used to isolate DNA clones encoding the light and heavy chains of the antibody. In this case, libraries may be screened with consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on purified antibody N-terminal sequences (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)). Cloning of antibody sequences is further described in US 5,658,570 to Newman et al., filed January 25, 1995, which is incorporated herein by reference.

[0189] As used herein, an Ig constant region can include all domains and a hinge region or a portion thereof, hi one embodiment, an Ig constant region or a portion thereof includes a CH2 domain, a CH3 domain, and a hinge region, i.e., the Fc region or the binding partner of FcRn.

[0190] As used herein, "Fc region" is defined as that portion of a polypeptide that corresponds to the Fc region of a native Ig, i.e., as formed by the dimeric association of the 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) refers to a synthetic dimeric Fc region composed of genetically linked (i.e., a single contiguous genetic sequence) Fc domains in a single polypeptide chain.

[0191] In one embodiment, "Fc region" refers to that portion of a single Ig heavy chain starting at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, considering the first residue of the heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge domain, CH2 domain, and CH3 domain.

[0192] The Fc region of the Ig constant region, depending on the Ig isotype, can include CH2, CH3, CH4 domains as well as the hinge region. Chimeric proteins containing the Ig Fc region confer several desirable properties to the chimeric protein, including high stability, increased serum half-life (Capon et al., 1989, Nature, 337:525), as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (US 6,086,875; US 6,485,726; US 6,030,613; WO 03 / 077834; US 2003-0235536 A1), which are incorporated by reference in their entirety.

[0193] An Ig constant region or a portion thereof can be a binding partner of FcRn. FcRn is active in epithelial tissues in adults and is expressed in the lumen of the intestinal tract, the pulmonary airways, the nasal surface, the vaginal surface, and the surface of the colon and rectum (US 6,485,726). A binding partner of FcRn is a portion of an Ig that binds to FcRn.

[0194] FcRn has been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). FcRn binds IgG at a relatively low pH (but does not bind other Ig classes, e.g., IgA, IgM, IgD and IgE), actively transports IgG across the cell in the direction from the lumen to the serosal membrane, and then releases IgG at the relatively high pH found in the interstitial fluid. It is expressed in adult epithelial tissues 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 surface, and biliary surface (US 6,485,726; US 6,030,613; US 6,086,875; WO 03 / 077834; US 2003-0235536 A1).

[0195] Binding partners of FcRn useful in the present invention include molecules to which the FcRn receptor can specifically bind, including whole IgG, Fc fragments of IgG, and other fragments that contain 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 main contact area of ​​Fc with FcRn is near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within a single Ig heavy chain. Binding partners of FcRn include whole IgG, Fc fragments of IgG, and other fragments of IgG that contain the complete binding region of the FcRn receptor. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All references to amino acid numbering of Igs or Ig fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, USDepartment of Public Health, Bethesda, Md.

[0196] Fc regions or binding partners of FcRn that bind to FcRn can be effectively trafficked across epithelial barriers by FcRn, providing a non-invasive means of systemically administering desired therapeutic molecules. Furthermore, fusion proteins containing Fc regions or binding partners of FcRn can be taken up into cells by cells expressing FcRn. However, instead of being marked for degradation, these fusion proteins are recycled back into the circulation, which may be beneficial. In certain embodiments, a portion of the Ig constant region is an Fc region or another FcRn binding partner that associates with another Fc region or another FcRn binding partner via disulfide bonds or other non-specific interactions to form dimers and higher order multimers.

[0197] Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggests that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking a binding partner of FcRn, e.g., an Fc fragment of IgG, to a biologically active molecule provides a means to deliver the biologically active molecule orally, bucally, 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.

[0198] A binding partner region of FcRn is a molecule or portion thereof to which the FcRn receptor can specifically bind, resulting in active transport of the Fc region by the FcRn receptor. Specific binding refers to two molecules forming a complex that is relatively stable under physiological conditions. Specific binding is usually characterized by high affinity and moderate to low avidity, as distinguished from nonspecific binding, which usually has low affinity with moderate to high avidity. Specific binding is usually characterized by an affinity constant KA of 10 6 M -1 Higher, or 10 8 M -1 If the binding is higher, the binding is considered specific. If necessary, the binding conditions can be varied to reduce non-specific binding without substantially affecting the specific binding. Suitable binding conditions, such as, for example, the concentration of the molecule, the ionic strength of the solution, the temperature, the time allowed for binding, the concentration of the blocking agent (e.g., serum albumin, milk casein), and the like, can be optimized by a skilled artisan using routine techniques.

[0199] In certain embodiments, the chimeric proteins of the invention comprise one or more truncated Fc regions that are sufficient to still confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of the Fc region that binds to FcRn (i.e., the FcRn binding portion) comprises approximately amino acids 282-438 of IgG1, EU numbering (with 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, the Fc region of the invention may comprise or consist of an FcRn binding portion. The FcRn binding portion may be derived from a heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn binding portion derived from an antibody of human isotype IgG1 is used. In another embodiment, an FcRn binding portion derived from an antibody of human isotype IgG4 is used.

[0200] In another embodiment, an "Fc region" includes an amino acid sequence of an Fc domain or an amino acid sequence derived from an Fc domain. In certain embodiments, an Fc region includes at least one of a hinge (e.g., upper, middle and / or lower hinge region) domain (approximately amino acids 216-230 of an antibody Fc region according to EU numbering), a CH2 domain (approximately amino acids 231-340 of an antibody Fc region according to EU numbering), a CH3 domain (approximately amino acids 341-438 of an antibody Fc region according to EU numbering), a CH4 domain, or variants, portions or fragments thereof. In other embodiments, an Fc domain includes a complete Fc region (i.e., a hinge domain, a CH2 domain and a CH3 domain). In some embodiments, an Fc region includes 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 hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, the Fc region comprises, consists essentially of, or consists of a CH2 domain (or a portion thereof) fused to both the Fc region and the Fc region (e.g., a portion of the CH2 domain). In yet other embodiments, the Fc region lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). In certain embodiments, the Fc region comprises or consists of amino acids corresponding to EU numbers 221-447.

[0201] The Fc region, designated herein as F, F1 or F2, may be obtained from a number of different sources. In one embodiment, the Fc region of the polypeptide may be derived from human Ig. However, the Fc region may also be derived from Ig of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit or guinea pig) or non-human primate (e.g., chimpanzee, monkey) species. Furthermore, the Fc domain or a portion thereof polypeptide may be derived from Ig classes including IgM, IgG, IgD, IgA and IgE, and Ig isotypes including IgG1, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgG1 is used.

[0202] In certain embodiments, the Fc variants confer an alteration in at least one effector function conferred by the Fc region comprising the wild-type Fc domain (e.g., an improved or reduced ability of the Fc region to bind an Fc receptor (e.g., FcγRI, FcγRII or FcγRIII) or a complement protein (e.g., C1q), or to induce antibody-dependent cellular cytotoxicity (ADCC), phagocytosis or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variants provide an engineered cysteine ​​residue.

[0203] The Fc regions of the present invention may employ art-recognized Fc variants known to confer altered (e.g., improved or decreased) effector function and / or FcR or FcRn binding. In particular, the binding molecules of the present invention may employ Fc variants described in, for example, International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO00 / 09560 ... 3 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 0 40217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350 A2 and WO06 / 085967A2; U.S. Patent Publications US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or US5,648,260; US5,739,277; US5,834,250; US5,869 ,046; US 6,096,871; US ​​6,121,022; US 6,194,551; US ​​6,242,195; US 6,277,375; US 6,528,624; US 6,538,124; US 6,737,056; US 6,821,505; US 6,998,253; US 7,083,784; US 7,404,956, and US 7,317,091. In one embodiment, a specific change (e.g., a 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 alternative embodiments, a different change at one or more of the disclosed amino acid positions (eg, a different substitution at one or more of the disclosed amino acid positions) may be made.

[0204] For example, the Fc region of IgG may be engineered according to well-recognized procedures such as site-directed mutagenesis. The domains or binding partners of FcRn can be modified to obtain modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications within the contact sites that protect or even enhance binding to FcRn as well as modifications away from the contact sites of FcRn. For example, the following single amino acid residues in the Fc of human IgG1 (Fcγ1): 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 can be substituted without significant loss of Fc binding affinity to FcRn, where P238A represents the wild type proline substituted by alanine at position 238. As an example, a specific embodiment incorporates the N297A mutation, which removes 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 identified above.Mutations may be introduced into the Fc one at a time to produce over 100 Fc regions that are distinct from the native Fc. Furthermore, combinations of two, three or more of these individual mutations may be introduced together to produce hundreds or more Fc regions. Furthermore, one of the Fc regions of the construct of the invention may be mutated, the other Fc region of the construct may not be mutated at all, or both may be mutated, but with different mutations.

[0205] Certain of the above mutations may confer new functionality to the Fc region or to the binding partner of FcRn. For example, one embodiment incorporates N297A, which removes a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby improving the circulating half-life of the Fc region, and to render the Fc region unable to bind to FcγRI, FcγRIIA, FcγRIIB, and FcγIIIA, without compromising affinity to FcRn. (Routledge et al. 1995, Transplantation, 60:847; Friend et al. 1999, Transplantation, 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality resulting from the above mutations, in some cases, affinity to FcRn may be increased beyond that of the wild type. 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 that may confer increased affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0206] Furthermore, at least three human Fcγ receptors appear to recognize a binding site on IgG within the lower hinge region, generally within amino acids 234 to 237. Thus, another example of novel functionality and potentially reduced immunogenicity would be, for example, replacing amino acids 233-236 "ELLG" of human IgG1 with the corresponding amino acids "PVA" from IgG2 (one amino acid deletion). This may result from mutations in this region by altering the FcγRI domain, resulting in a loss of IgG1 binding. It has been shown that, with such mutations, FcγRII and FcγRIII, which mediate various effector functions, will not bind IgG1. Ward and Ghetie, 1995, Therapeutic Immunology, 2:77; Armour et al., 1999, Eur. J. Immunol. 29:2613.

[0207] In one embodiment, the Ig constant region or portion thereof, e.g., the Fc region, is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO:89 or SEQ ID NO:3 of US 5,739,277), and optionally further comprising a sequence selected from HQSLGTQ (SEQ ID NO:90), HQNLSDGK (SEQ ID NO:91), HQNISDGK (SEQ ID NO:92) or VISSHLGQ (SEQ ID NO:93) (or SEQ ID NOs:11, 1, 2 and 31, respectively, of US 5,739,277).

[0208] In another embodiment, the constant region or portion of the immunoglobulin comprises an amino acid sequence in the hinge region or portion thereof that forms one or more disulfide bonds with the constant region or portion of another immunoglobulin. The disulfide bond by the constant region or portion of the immunoglobulin positions the first polypeptide comprising FVIII and the second polypeptide comprising the VWF fragment together such that endogenous VWF does not displace the VWF fragment and does not bind to FVIII. Thus, the disulfide bond between the constant region or portion of the first immunoglobulin and the constant region or portion of the second immunoglobulin prevents the interaction between endogenous VWF and FVIII protein. Inhibition of the interaction between VWF and FVIII protein allows the half-life of the chimeric protein to exceed the two-fold limit. The hinge region or portion thereof can be further linked to one or more domains of CH1, CH2, CH3, fragments thereof, and any combination thereof. In a particular embodiment, the constant region or portion of the immunoglobulin is the hinge region and CH2.

[0209] In certain embodiments, the constant region of an Ig or a portion thereof is hemiglycosylated. For example, a chimeric protein comprising two Fc regions or binding partners of FcRn contains a first glycosylated Fc region (e.g., a glycosylated CH2 region) or binding partner of FcRn and a second non-glycosylated Fc region (e.g., a non-glycosylated CH2 region) or binding partner of FcRn. In one embodiment, a linker may be interposed between the glycosylated Fc region and the non-glycosylated Fc region. In another embodiment, the Fc region or binding partner of FcRn is fully glycosylated, i.e., all of the Fc region is glycosylated. In other embodiments, the Fc region is not glycosylated, i.e., none of the Fc molecules are glycosylated.

[0210] In certain embodiments, the chimeric proteins of the present invention contain amino acid substitutions to an Ig constant region or a portion thereof (e.g., Fc variants) that alter the antigen-independent effector functions of the Ig constant region, in particular the circulating half-life of the protein.

[0211] Such proteins will exhibit increased or decreased binding to FcRn when compared to proteins lacking these substitutions, and therefore will exhibit increased or decreased half-lives, respectively, in serum. Fc variants with improved affinity for FcRn are expected to have extended serum half-lives, and such molecules will have useful applications in methods of treating mammals, for example, when extended half-life of a polypeptide administered to treat a chronic disease or disorder is desired (see, e.g., US 7,348,004, US 7,404,956, and US 7,862,820). In contrast, Fc variants with reduced binding affinity for FcRn are expected to have a short half-life, and such molecules will also be useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, for example, for in vivo diagnostic imaging, or where the starting polypeptide may not exhibit toxic side effects if present in the circulation for an extended period of time. The chimeric proteins of the invention are useful in situations where the FcRn binding is impaired. Fc variants with reduced FcRn binding affinity are also useful in treating diseases or disorders in pregnant women, as they are less likely to cross the placenta. In addition, other applications in which reduced FcRn binding affinity may be desirable include applications in which localization to the brain, kidney and / or liver is desirable. In an exemplary embodiment, the chimeric proteins of the invention reduce transport across the epithelium of the kidney glomerulus from the vasculature. In another embodiment, the chimeric proteins of the invention reduce transport across the blood-brain barrier (BBB) ​​from the brain to the vascular space. In one embodiment, the 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 in the "FcRn binding loop" of the Ig constant region. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering) of the wild-type full-length Fc region. In other embodiments, the Ig constant region or portion thereof in the chimeric protein of the present invention with altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions within the 15 Å FcRn "contact zone." As used herein, the 15 Å FcRn "contact zone" includes residues at the following positions of a wild-type full-length Fc portion: 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, an Ig constant region or portion thereof of the invention with altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner with 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 that alter FcRn binding activity are disclosed in International Publication No. WO 05 / 047327, which is incorporated herein by reference.

[0212] The Fc region or FcRn binding partner used in the present invention may also contain art-recognized amino acid substitutions that alter the glycosylation of the chimeric protein. For example, the Fc region or FcRn binding partner of a chimeric protein linked to a VWF fragment or FVIII protein may contain an Fc region with a mutation that results in reduced glycosylation (e.g., N- or O-linked glycosylation) or may contain an altered glycoform of the wild-type Fc moiety (e.g., a glycan with less or no fucose).

[0213] In one embodiment, the unprocessed chimeric protein of the invention may comprise a genetically fused Fc region (i.e., scFc region) with two or more of its constituent constant regions or portions thereof independently selected from the constant regions or portions of an Ig described herein. In one embodiment, the Fc regions of the dimeric Fc region are identical. In another embodiment, at least two of the Fc regions are different. For example, the Fc regions of the protein of the invention or the binding partner of FcRn may comprise the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., by approximately 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), approximately 10 residues, approximately 15 residues, approximately 20 residues, approximately 30 residues, approximately 40 residues, or approximately 50 residues). In yet other embodiments, the Fc regions of the protein of the invention or the binding partner of FcRn may differ at one or more amino acid positions. For example, at least two of the Fc regions or FcRn binding partners may differ at approximately 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), approximately 10 positions, approximately 15 positions, approximately 20 positions, approximately 30 positions, approximately 40 positions, or approximately 50 positions).

[0214] II.E. Linker The chimeric protein of the present invention further comprises one or more linkers. One type of linker is a cleavable linker, which can be cleaved by various proteases when administered to a subject in vivo, for example, at the site of coagulation. In one embodiment, the cleavable linker allows the cleavage of a moiety, for example, from the XTEN sequence of the VWF protein, thereby allowing cleavage from the chimeric protein at the site of the coagulation cascade, thereby allowing the activated FVIII (FVIIIa) to have its FVIIIa activity. Another type of linker is a processable linker, which contains an intracellular cleavage site and can be cleaved by intracellular processing enzymes in the host cell, allowing convenient expression of the polypeptide and formation of the chimeric protein.

[0215] There can be one or more linkers between the two proteins in the chimeric protein. In one embodiment, the chimeric protein comprises a first polypeptide comprising (i) a FVIII protein and (ii) a constant region of a first Ig or a portion thereof, and a second polypeptide comprising (iii) a VWF protein and (iv) a linker (e.g., a cleavable linker), (v) an XTEN sequence and (vi) a constant region of a second Ig or a portion thereof. In another embodiment, the chimeric protein comprises a first polypeptide comprising (i) a FVIII protein and (ii) a constant region of a first Ig or a portion thereof, and a second polypeptide comprising (iii) a VWF protein and (iv) an XTEN sequence and (v) a linker (e.g., a cleavable linker) and (vi) a constant region of a second Ig or a portion thereof. In other embodiments, the chimeric protein comprises (i) a first polypeptide comprising a FVIII protein and (ii) a constant region of a first Ig or a portion thereof, and (iii) a second polypeptide comprising a VWF protein, (iv) a first linker (e.g., a cleavable linker), (v) an XTEN sequence, (vi) a second linker (e.g., a cleavable linker), and (vii) a constant region of a second Ig or a portion thereof. In some embodiments, the first polypeptide further comprises a linker, e.g., a cleavable linker, between the FVIII protein and the first Ig constant region.

[0216] In certain embodiments, the chimeric protein comprises a single chain comprising (i) a FVIII protein, (ii) a first polypeptide comprising a constant region of a first Ig or a portion thereof, (iii) a linker (e.g., a cleavable linker), (iv) a VWF protein, (v) an XTEN sequence, and (vi) a constant region of a second Ig or a portion thereof. In other embodiments, the chimeric protein comprises a single chain comprising (i) a FVIII protein, (ii) a first polypeptide comprising a constant region of a first Ig or a portion thereof, (iii) a first linker (e.g., a cleavable linker), (iv) a VWF protein, (v) a second linker (e.g., a cleavable linker), (vi) an XTEN sequence, and (vii) a constant region of a second Ig or a portion thereof. The processable linker can be processed after the chimeric protein is expressed in a host cell; thus, the chimeric protein produced in the host cell can be in a final form comprising two or three polypeptide chains.

[0217] Linkers useful in the present invention can include any organic molecule. In one embodiment, the linker includes a polymer, such as polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker includes an amino acid sequence. The linker can include 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 may be 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids. In one embodiment, the linker comprises an XTEN sequence. Additional examples of XTENs that can be used in accordance with the present invention are disclosed in US2010 / 0239554A1, US2010 / 0323956A1, US2011 / 0046060A1, US2011 / 0046061A1, US2011 / 0077199A1, or US2011 / 0172146A1, or WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, or WO2011028344A2. In another embodiment, the linker is a PAS sequence.

[0218] In one embodiment, the linker is a polymer, such as polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker is an amino acid sequence. The linker can include 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 contain 1 to 5 amino acids, 1 to 10 amino acids, 1 to 20 amino acids, 10 to 50 amino acids, 50 to 100 amino acids, 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids.

[0219] Examples of linkers are well known in the art. In one embodiment, the linker has the sequence G n The linker comprises the sequence (GA) n The linker may include the sequence (GGS). n In other embodiments, the linker can include the sequence (GGGS). n (SEQ ID NO: 101). In yet another embodiment, the linker comprises the sequence (GGS)n (GGGGS) n (SEQ ID NO: 95). In these examples, n can be an integer from 1 to 100. In other examples, n can be an integer from 1 to 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: 96), GGSGGSGGSGGSGGG (SEQ ID NO: 97), GGSGGSGGGGSGGGGS (SEQ ID NO: 98), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 99), or GGGSGGGGSGGGGGS (SEQ ID NO: 100). The linker does not eliminate or reduce VWF protein activity or the clotting activity of factor VIII. Optionally, the linker improves VWF protein activity or factor VIII clotting activity, for example by reducing the effects of steric hindrance and making the VWF protein or factor VIII protein more accessible to its target binding site.

[0220] In one embodiment, linkers useful for chimeric proteins are 15-25 amino acids in length. In another embodiment, linkers useful for chimeric proteins are 15-20 amino acids in length. In some embodiments, linkers for chimeric proteins are 10-25 amino acids in length. In other embodiments, linkers for chimeric proteins are 15 amino acids in length. In yet other embodiments, linkers for chimeric proteins are (GGGGS) n (SEQ ID NO: 94), where G represents glycine, S represents serine, and n is an integer from 1 to 20.

[0221] II.F. Cutting site Cleavable linkers can incorporate a moiety that can be cleaved chemically (e.g., hydrolysis of an ester bond), enzymatically (e.g., incorporation of a protease cleavage sequence), or photolytically (e.g., a chromophore such as 3-amino-3-(2-nitrophenyl)propionic acid (ANP)) to release one molecule from another.

[0222] In one embodiment, the cleavable linker comprises 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 a heterologous amino acid linker sequence as described herein, or a polymer and one or more cleavable sites.

[0223] In certain embodiments, the cleavable linker comprises one or more cleavage sites (i.e., intracellular processing sites) that can be cleaved in a host cell. Non-limiting examples of cleavage sites include RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: 103), and RRRRS (SEQ ID NO: 104).

[0224] In some embodiments, the cleavable linker comprises an a1 region derived from FVIII, an a2 region derived from FVIII, an a3 region derived from FVIII, a thrombin cleavable site comprising XVPR (SEQ ID NO: 105), and a PAR1 non-active site interacting motif, where X is an aliphatic amino acid or any combination thereof. The a2 region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95% or 100% identical to Glu720 to Arg740 corresponding to full-length FVIII, where the a2 region is capable of being cleaved by thrombin. In certain embodiments, the cleavable linker of the invention comprises an a2 region comprising ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106). In other embodiments, cleavable linkers useful in the invention include an a1 region that includes an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95% or 100% identical to Met337 to Arg372 corresponding to full-length FVIII, where the a1 region is capable of being cleaved by thrombin. In certain embodiments, the a1 region includes ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107). In some embodiments, cleavable linkers of the invention include an a3 region that includes an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95% or 100% identical to Glu1649 to Arg1689 corresponding to full-length FVIII, where the a3 region is capable of being cleaved by thrombin. In a specific embodiment, a cleavable linker of the invention comprises an a3 region, wherein the a3 region comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 108).

[0225] In other embodiments, the cleavable linker comprises a thrombin cleavable site comprising XVPR (SEQ ID NO: 105) and a PAR1 non-active site interaction motif, wherein the PAR1 non-active site interaction motif comprises SFLLRN (SEQ ID NO: 109). The PAR1 non-active site interaction motif can further comprise an amino acid sequence selected from P, PN, PND, PNDK (SEQ ID NO: 110), PNDKY (SEQ ID NO: 111), PNDKYE (SEQ ID NO: 112), PNDKYEP (SEQ ID NO: 113), PNDKYEPF (SEQ ID NO: 114), PNDKYEPFW (SEQ ID NO: 115), PNDKYEPFWE (SEQ ID NO: 116), PNDKYEPFWED (SEQ ID NO: 117), PNDKYEPFWEDE (SEQ ID NO: 118), PNDKYEPFWEDEE (SEQ ID NO: 119), PNDKYEPFWEDEES (SEQ ID NO: 120), or a combination thereof. In some embodiments, the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine.

[0226] In another embodiment, the cleavable linker is a chimeric protein comprising a cleavable linker. The protein comprises one or more cleavage sites that are cleaved by a protease after administration of the protein to a subject, hi one embodiment, the cleavage sites are cleaved by a protease selected from the group consisting of Factor XIa, Factor XIIa, kallikrein, Factor VIIa, Factor IXa, Factor Xa, Factor IIa (thrombin), elastase-2, MMP-12, MMP-13, MMP-17, and MMP-20. In another embodiment, the cleavage site is a FXIa cleavage site (e.g., KLTR↓AET (SEQ ID NO: 121)), a FXIa cleavage site (e.g., DFTR↓VVG (SEQ ID NO: 122)), a FXIIa cleavage site (e.g., TMTR↓IVGG (SEQ ID NO: 123)), a kallikrein cleavage site (e.g., SPFR↓STGG (SEQ ID NO: 124)), a FVIIa cleavage site (e.g., LQVR↓IVGG (SEQ ID NO: 125)), a FIXa cleavage site (e.g., PLGR↓IVGG (SEQ ID NO: 126)), a FXa cleavage site (e.g., IEGR↓TVGG (SEQ ID NO: 127)), a FIIa (thrombin) cleavage site (e.g., LTPR↓SLLV (SEQ ID NO: 128)), an elastase-2 cleavage site (e.g., LGPV↓SGVP (SEQ ID NO: 129)), a granzyme-B ...30)), a granzyme-B cleavage site (e.g., LGPV↓SGVP (SEQ ID NO: 131)), a granzyme-B cleavage site (e.g., LGPV↓SGVP (SEQ ID NO: 132)), a granzyme-B cleavage site (e (e.g., VAGD↓SLEE (SEQ ID NO: 130)), MMP-12 cleavage site (e.g., GPAG↓LGGA (SEQ ID NO: 131)), MMP-13 cleavage site (e.g., GPAG↓LRGA (SEQ ID NO: 132)), MMP-17 cleavage site (e.g., APLG↓LRLR (SEQ ID NO: 133)), MMP-20 cleavage site (e.g., PALP↓LVAQ (SEQ ID NO: 134)), TEV cleavage site (e.g., ENLYFQ↓G (SEQ ID NO: 135)), enterokinase cleavage site (e.g., DDDK↓IVGG (SEQ ID NO: 136)), protease 3C (PRESCISSION™) cleavage site (e.g., LEVLFQ↓GP (SEQ ID NO: 137)), and sortase A cleavage site (e.g., LPKT↓GSES) (SEQ ID NO: 138). In certain embodiments, FXIa cleavage sites include, but are not limited to, for example, TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3).Non-limiting examples of thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88) or IEPRSFS (SEQ ID NO: 194), and sequences comprising, consisting essentially of, or consisting of ALRPR (SEQ ID NO: 7) (e.g., ALRPRVVGGA (SEQ ID NO: 145)).

[0227] In a particular embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). In another embodiment, the cleavage site comprises DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88) or a fragment thereof. In one particular embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 194). In another embodiment, the cleavage site comprises EPRSFS (SEQ ID NO: 195), where the cleavage site is not the full-length a2 region of FVIII. In yet another embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 200). In another embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 200), where the cleavage site is not the full-length a2 region of FVIII or does not comprise the full-length a2 region of FVIII. In other embodiments, the cleavage sites are DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), KNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 139), NTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 140), TGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 141), GDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 142), DYYEDSYEDISAYLLSKNNA IEPRSFS (SEQ ID NO: 143), YYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 144), YEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 176), EDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 177), DSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 178), SYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 179), YEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 180), EDISAYLLSKNNAIEPR SFS (SEQ ID NO: 181), DISAYLLSKNNAIEPRSFS (SEQ ID NO: 182), ISAYLLSKNNAIEPRSFS (SEQ ID NO: 183), SAYLLSKNNAIEPRSFS (SEQ ID NO: 184), AYLLSKNNAIEPRSFS (SEQ ID NO: 185), YLLSKNNAIEPRSFS (SEQ ID NO: 186), LLSKNNAIEPRSFS (SEQ ID NO: 187), LSKNNAIEPRSFS (SEQ ID NO: 188), SKNNAIEPRSFS (SEQ ID NO: 189), KNNAIEPRSFS (SEQ ID NO: 190), NNAIEPRSFS (SEQ ID NO: 191), NAIEPRSFS (SEQ ID NO: 192), AIEPRSFS (SEQ ID NO: 193), or IEPRSFS (SEQ ID NO: 194).In other embodiments, the cleavage site is DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), KNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 139), NTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 140), TGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 141), GDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 142), DYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 143), YYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 144), YEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 176), EDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 177), DSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 178), SYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 179), RSFS (SEQ ID NO: 179), YEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 180), EDISAYLLSKNNAIEPRSFS (SEQ ID NO: 181), DISAYLLSKNNAIEPRSFS (SEQ ID NO: 182), ISAYLLSKNNAIEPRSFS (SEQ ID NO: 183), SAYLLSKNNAIEPRSFS (SEQ ID NO: 184), AYLLSKNNAIEPRSFS (SEQ ID NO: 185), YLLSKNNAIEPRSFS (SEQ ID NO: 186), LLSKNNAIEPRSFS (SEQ ID NO: 187), LSKNNAIEPRSFS (SEQ ID NO: 188), SKNNAIEPRSFS (SEQ ID NO: 189), KNNAIEPRSFS (SEQ ID NO: 190), NNAIEPRSFS (SEQ ID NO: 191), NAIEPRSFS (SEQ ID NO: 192), AIEPRSFS (SEQ ID NO: 193), or IEPRSFS (SEQ ID NO: 194), wherein the cleavage site is not the full length FVIIIa2 region. In certain embodiments, the cleavable linker is cleavable in a thrombin cleavage assay as provided herein or as known in the art.

[0228] III. Polynucleotides, Vectors and Host Cells Also provided herein are polynucleotides encoding the chimeric proteins of the invention. In one embodiment, the first and second polypeptide chains can be encoded by a single polynucleotide chain. In another embodiment, the first and second polypeptide chains are encoded by two different polynucleotides, i.e., a first nucleotide sequence and a second nucleotide sequence. In another embodiment, the first and second nucleotide sequences are on two different polynucleotides (e.g., different vectors).

[0229] The invention includes a polynucleotide encoding a single polypeptide chain (e.g., FVIII(X2)-F1-L3-F2-L2-X1-L1-V), where FVIII(X2) has an XTEN sequence inserted into one or more insertion sites, F1 comprises a first Ig constant region or portion thereof, e.g., a first Fc region, L1 comprises a first linker, V comprises a VWF protein, X1 comprises an XTEN sequence having a length of less than 288 amino acids, L2 comprises a second linker, L3 comprises a third linker, and F2 comprises a second Ig constant region or portion thereof, e.g., a second Fc region. The invention also includes a polynucleotide encoding a first polypeptide comprising a FVIII protein fused to a first Ig constant region or portion thereof, e.g., a second Fc region, The chimeric protein comprises a VWF protein and an XTEN sequence having a length of less than 288 amino acids, and a second polypeptide comprising a second Ig constant region or a portion thereof. In some embodiments, chimeric proteins comprising two 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, chimeric proteins comprising these polypeptide chains can be encoded by two or three polynucleotide chains.

[0230] In other embodiments, the set of polynucleotides further comprises an additional nucleotide strand encoding a protein convertase (e.g., a second nucleotide strand where the chimeric polypeptide is encoded by a single polynucleotide strand, or a third nucleotide strand where the chimeric protein is encoded by two polynucleotide strands). 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), yeast Kex2, proprotein convertase subtilisin / kexin type 3 (PACE or PCSK3), and combinations of two or more thereof. In some embodiments, the protein convertase is PACE, PC5, or PC7. In specific embodiments, the protein convertase is PC5 or PC7. See PCT / US2011 / 043568.

[0231] As used herein, an expression vector refers to a nucleic acid construct that contains the necessary elements for transcription and translation, or, in the case of RNA viral vectors, replication and translation, of an inserted coding sequence when introduced into an appropriate host cell. Expression vectors include plasmids, phagemids, viruses and their derivatives.

[0232] The expression vector of the present invention will comprise a polynucleotide encoding the chimeric protein described herein. In one embodiment, one or more coding sequences for a first polypeptide comprising a FVIII protein and a first Ig constant region, a second polypeptide comprising a VWF protein and an XTEN sequence having less than 288 amino acids and a second Ig constant region or a portion thereof, 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 a manner that allows 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 a manner that places 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 the introduction of a promoter at 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 frameshift 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 is operably linked to a coding nucleic acid sequence if it is capable of affecting the transcription of the coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0233] A gene expression control sequence, as used herein, is a regulatory nucleotide sequence, such as, for example, a promoter sequence or a promoter / enhancer combination, that promotes the efficient transcription and translation of a coding nucleic acid to which it is operably linked. A gene expression control sequence may be, for example, a mammalian or viral promoter, e.g., a constitutive or inducible promoter. Constitutive mammalian promoters include promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, the promoter of β-actin, and other constitutive Examples of viral promoters that function constitutively in eukaryotic cells include, but are not limited to, promoters from cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, and the herpes simplex virus thymidine kinase promoter. Other constitutive promoters are known to those of skill in the art. Promoters useful as gene expression sequences of the present invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducer. 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 skill in the art.

[0234] In general, gene expression control sequences should include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, as appropriate, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed sequences will include a promoter region that includes a promoter sequence for transcriptional control of an operably linked coding nucleic acid. Gene expression sequences optionally include enhancer sequences or upstream activator sequences, as desired.

[0235] Viral vectors include, but are not limited to, the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40 type viruses; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; polio virus; and nucleic acid sequences derived from RNA viruses such as retroviruses. Other vectors known in the art can be readily employed. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with genes of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of the genome's viral RNA into DNA followed by integration of the provirus into the host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins but unable to manufacture infectious particles). Such genetically altered retroviral expression vectors have general utility for efficient transfer of genes in vivo. Standard protocols for generating replication-defective retroviruses, including incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retrovirus by the packaging cell line, recovery of viral particles from tissue culture medium, and infection of target cells with the viral particles, are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0236] In one embodiment, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication-deficient and can infect a wide range of cell types and species. It has further advantages such as thermostability and lipid solvent stability; high transduction frequency in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition, allowing multiple series of transductions. Adeno-associated virus can reportedly integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in the inserted gene expression characteristics of retroviral infection. In addition, the fact that wild-type adeno-associated virus infection has been sustained for more than 100 passages in tissue culture in the absence of selective pressure indicates that the adeno-associated virus genome is integrated into the cellular DNA. is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0237] Other vectors include plasmid vectors. Plasmid vectors have been described extensively in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they cannot replicate or integrate into the host genome. However, these plasmids with promoters compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids available commercially from vendors include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids will be known to those of skill in the art. Additionally, plasmids may be custom designed to remove and / or add specific segments of DNA using standard molecular biology techniques.

[0238] In one insect expression system that may be used to produce proteins of the invention, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. Coding sequences are cloned into non-essential regions of the virus (e.g., the polyhedron gene) and placed under control of an ACNPV promoter (e.g., the polyhedron promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat encoded therefor by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (see, e.g., Smith et al. (1983), J. Virol. 46:584; US 4,215,051). Further examples of this expression system can be found in Ausubel et al., eds. (1989), Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

[0239] Another system that can be used to express the proteins of the invention is the Glutamine Synthetase Gene Expression System, also called the "GS Expression System" (Lonza Biologics PLC, Berkshire UK). This expression system is described in detail in US 5,981,216.

[0240] In mammalian host cells, a number of viral-based expression systems may be utilized. When adenovirus is used as an expression vector, the coding sequence may be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in non-essential regions of the viral genome (e.g., regions E1 or E3) will yield recombinant viruses capable of viability and expression of the peptide in infected hosts. See, e.g., Logan and Shenk (1984), Proc. Natl. Acad. Sci. USA. 81:3655). Alternatively, the vaccinia 7.5K promoter may be used. See, e.g., T. et al. (1982), Proc. Natl. Acad. Sci. USA. 79:7415; Mackett et al. (1984), J. Virol. 49:857; Panicali et al. (1982), Proc. Natl. Acad. Sci. USA. 79:4927.

[0241] To increase the efficiency of production, the polynucleotide can be designed to encode multiple units of the protein of the invention separated by enzyme cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with an appropriate enzyme) to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in a suitable viral expression system, the translation of each polypeptide encoded by the mRNA is directed internally in the transcript, for example, by an internal ribosome entry site (IRES). Thus, a polycistronic construct directs the transcription of a single large polycistronic mRNA, which then directs the translation of multiple individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and can significantly increase the yield of polypeptides driven by a single promoter.

[0242] Vectors used in transformation will usually contain a selectable marker that is used to identify transformants. In bacterial systems, this can include, for example, antibiotic resistance genes, such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include, for example, genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may also be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen, C. C. et al. (1983), Proc. Natl. Acad. Sci. USA. 80:249 5-9. Selectable markers are reviewed in Thilly, (1986), Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable markers is well within the level of ordinary skill in the art.

[0243] The selectable marker may be introduced into the cell simultaneously as the gene on a separate plasmid, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene may be under the control of different promoters or the same promoter, the latter arrangement resulting in a bicistronic message. Constructs of this type are known in the art (e.g., US 4,713,339).

[0244] Expression vectors can encode tags that allow for easy purification of recombinantly produced proteins. Examples include, but are not limited to, the vector pUR278 (Ruther et al. (1983), EMBO J. 2:1791), in which the coding sequence of the protein to be expressed is ligated into the vector in frame with the lacZ coding region to produce a tagged fusion protein; pGEX vectors may be used to express proteins of the invention with glutathione S-transferase (GST) tags. These proteins are normally soluble and can be easily purified from cells by adsorption to glutathione / agarose beads followed by elution in the presence of free glutathione. The vector contains a cleavage site (for thrombin or factor Xa protease or PRESCISSION PROTEASE™ (Pharmacia, Peapack, NJ)) for easy removal of the tag after purification.

[0245] The expression vector or vectors are then transfected or co-transfected into suitable target cells, which will express the polypeptide. Transfection techniques known in the art include calcium phosphate precipitation (Wigler et al. (1978), Cell, 1999). 14:725), electroporation (Neumann et al. (1982), EMBO J.1:841), and liposome-based reagents. A variety of host expression vector systems, including both prokaryotic and eukaryotic cells, may be utilized to express the proteins described herein. These include, but are not limited to, bacteria (e.g., E. coli) transformed with recombinant bacteriophage or plasmid DNA expression vectors containing the appropriate coding sequences; microorganisms such as yeast or filamentous fungi transformed with recombinant yeast or fungal expression vectors containing the appropriate coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the appropriate coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the appropriate coding sequences; or animal cell systems, including mammalian cells (e.g., HEK293, CHO, Cos, HeLa, HKB11, and BHK cells).

[0246] In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell that has a distinct nucleus. Animal eukaryotic cells include vertebrate, e.g., mammalian, and invertebrate, e.g., insect, cells. Plant eukaryotic cells can include, but are not limited to, yeast cells. Eukaryotic cells are distinct from prokaryotic cells, e.g., bacteria.

[0247] In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK293 cell, which is a human embryonic kidney cell line. HEK293 cells are available from American Type Culture Collection, Manassas, VA as CRL-1533, and from Invitrogen (Carlsbad, Calif.) as 293-H cells, catalog number 11631-017 or 293-F cells, catalog number 11625-019. In some embodiments, the mammalian cell is a PER.C6® cell, a human cell line derived from the retina. ER.C6® cells are available from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from the American Type Culture Collection, Manassas, VA (e.g., CHO-K1; CCL-61). In yet other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from the American Type Culture Collection, Manassas, VA (e.g., CRL-1632). In some embodiments, the mammalian cell is an HKB11 cell, which is a hybrid cell line of HEK293 cells and a human B cell line. Mei et al., Mol. Biotechnol. 34(2):165-78 (2006).

[0248] In one embodiment, a plasmid containing a FVIII(X2)-Fc fusion coding sequence, a VWF protein-L1-X1-L2-Fc coding sequence, or both, and a selectable marker, e.g., Zeocin resistance, is transfected into HEK293 cells to produce the chimeric protein.

[0249] In another embodiment, a plasmid containing a FVIII-Fc fusion coding sequence, a VWF protein-L1-X-L2-Fc coding sequence, or both, and a selectable marker, e.g., Zeocin resistance, is transfected into HEK293 cells to produce the chimeric protein.

[0250] In some embodiments, a first plasmid containing a FVIII(X2)-Fc fusion coding sequence and a first selectable marker, such as a zeocin resistance gene, a second plasmid containing a VWF protein-L1-X1-L2-Fc coding sequence and a second selectable marker, such as a neomycin resistance gene, and a third plasmid containing a protein convertase coding sequence and a third selectable marker, such as a hygromycin resistance gene, are co-transfected into HEK293 cells to produce, for example, a chimeric protein. The first and second plasmids can be introduced in the same amount (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

[0251] In yet another embodiment, a first plasmid containing a FVIII-Fc fusion coding sequence and a first selectable marker, such as a zeocin resistance gene, a second plasmid containing a VWF protein / L1-X-L2-Fc coding sequence and a second selectable marker, such as a neomycin resistance gene, and a third plasmid containing a protein convertase coding sequence and a third selectable marker, such as a hygromycin resistance gene, are co-transfected into HEK293 cells for the production of chimeric proteins. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

[0252] In yet another embodiment, a first plasmid containing FVIII(X2)-Fc fusion coding sequence and a first selectable marker, such as Zeocin resistance gene, a second plasmid containing VWF protein / L1-X-L2-Fc coding sequence and a second selectable marker, such as Neomycin resistance gene, and a third plasmid containing protein convertase coding sequence and a third selectable marker, such as Hygromycin resistance gene, are co-transfected into HEK293 cells for the production of chimeric proteins. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

[0253] In a particular embodiment, a first plasmid containing the FVIII (with or without XTEN)-F1-L3-F2-L2-X-L1-V coding sequence and a first selectable marker, e.g., Zeocin resistance gene, and a second plasmid containing the protein convertase coding sequence and a second selectable marker, e.g., Hygromycin resistance gene, are co-transfected into HEK293 cells for production of chimeric proteins. The promoters for the FVIII(X)-F1 coding sequence and the V-L2-X-L1-F2 coding sequence can be different or they can be the same.

[0254] In yet other embodiments, the transfected cells are stably transfected and can be selected and maintained as stable cell lines using conventional techniques known to those of skill in the art.

[0255] Host cells containing the protein of the DNA construct of the present invention are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" refers to a medium containing nutrients required for cell growth. Nutrients required for cell growth include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium can contain one or more selection factors. Optionally, the medium can contain calf serum or fetal calf serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium will generally select for cells containing the DNA construct, for example, by drug selection or by depletion of an essential nutrient that is complemented by a selectable marker in the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is selected from CD293 (Invitrogen, C In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.). Selection of an appropriate medium for the particular cell line used is within the level of ordinary skill in the art.

[0256] To co-express the two polypeptide chains of the chimeric protein, the host cells are cultured under conditions that allow for the expression of both chains. As used herein, culturing refers to maintaining living cells in a test tube for at least a definite period of time. Maintaining can, but need not, include an increase in the population of living cells. For example, cells maintained in culture can be quiescent in population, but still live and capable of producing a desired product, e.g., a recombinant protein or a recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include the appropriate selection of culture medium, culture supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing can include the use of various types of scale-up systems, including shaker flasks, roller bottles, hollow fiber bioreactors, stirred tank bioreactors, airlift bioreactors, wave bioreactors, and the like.

[0257] Cell culture conditions are selected to allow for the association of the VWF fragment with the FVIII protein. Conditions allowing for expression of the VWF fragment and / or the FVIII protein include the presence of a source of Vitamin K. For example, in one embodiment, stably transfected HEK293 cells are cultured in CD293 medium (Invitrogen, Carlsbad, Calif.) or OptiCHO medium (Invitrogen, Carlsbad, Calif.) supplemented with 4 mM glutamine.

[0258] In one aspect, the invention is directed to a method of expressing, making or producing a chimeric protein of the invention comprising: (a) transfecting a host cell with a polynucleotide encoding the chimeric protein; and (b) culturing the host cell in a culture medium suitable for expressing the chimeric protein under conditions in which the chimeric protein is expressed.

[0259] In a further embodiment, a protein product containing a FVIII protein linked to a first Ig constant region or portion thereof and / or a VWF protein fused to a second Ig constant region or portion thereof by an XTEN sequence is secreted into the medium. The medium is separated from the cells, concentrated, filtered, and then passed over two or three affinity columns, e.g., a Protein A column and one or two anion exchange columns.

[0260] In certain aspects, the invention relates to chimeric proteins produced by the methods described herein.

[0261] In vitro production allows for scale-up to produce large quantities of the desired altered polypeptide of the invention. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogenous suspension culture in airlift or continuous stirred reactors, or immobilized or entrapped cell culture in hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC), chromatography on DEAE cellulose, or affinity chromatography.

[0262] IV. Pharmaceutical Compositions Compositions containing the chimeric proteins of the invention may contain suitable pharma- ceutically acceptable carriers. For example, they may be formulated to deliver active compounds to a site of action. The formulation may also contain excipients and / or adjuvants that facilitate processing.

[0263] The pharmaceutical composition can be formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular) by bolus injection. The formulation for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers with added preservatives. The composition can take such forms as suspensions, solutions, or emulsions in oily or aqueous media, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, pyrogen-free water.

[0264] Suitable formulations for parenteral administration also include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds may be administered as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and dextran. Optionally, the suspension may also contain stabilizers. Liposomes may be used to encapsulate the molecules of the invention for delivery to cells or interstitial spaces. Exemplary pharmacologic carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments, the compositions include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions contain pharma- ceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that enhance the shelf life or effectiveness of the active ingredient.

[0265] The compositions of the present invention may be in a variety of forms, including, for example, liquids (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the method of administration and the therapeutic application.

[0266] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of the above listed ingredients as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active ingredient in a sterile vehicle containing the basic dispersion medium listed above and the other ingredients required. In the case of sterile powders for sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including an agent that delays absorption, for example, monostearate salts and gelatin, in the composition.

[0267] The active ingredient can be formulated with sustained release formulations or devices. Examples of such formulations and devices include implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations and devices are known in the art. For example, see Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, ed. , Inc., New York, 1978.

[0268] Injectable depot formulations can be made by forming microencapsulated matrices of the drug in biodegradable polymers such as, for example, polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the polymer employed, the rate of drug release can be controlled. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions.

[0269] Supplementary active compounds can be incorporated into the composition. In one embodiment, the chimeric protein of the present invention is formulated with another coagulation factor, or a variant, fragment, analog, or derivative thereof. For example, the coagulation factor can include, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF), or an activated form of any of the above. The hemostatic coagulation factor can also include an antifibrinolytic agent, such as ε-aminocaproic acid, tranexamic acid.

[0270] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0271] In addition to the active compound, liquid dosage forms may contain, for example, water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- It may also contain inactive ingredients such as butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.

[0272] Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by EW Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc. The composition can also contain pH buffering agents, and wetting or emulsifying agents.

[0273] For oral administration, the pharmaceutical composition can take the form of a tablet or capsule prepared by conventional means. The composition can also be prepared as a liquid, for example, a syrup or suspension. The liquid can contain suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The formulation can also contain flavoring agents, coloring agents, and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

[0274] For buccal administration, the compositions are formulated in the form of tablets or lozenges according to conventional protocols. You may take this stance.

[0275] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol, with or without excipients, or in the form of an aerosol spray from pressurized packs or nebulizers, optionally with a propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0276] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0277] In one embodiment, the pharmaceutical composition comprises a chimeric protein, a polynucleotide encoding the chimeric protein, a vector comprising the polynucleotide, or a host cell comprising the vector, and a pharma- ceutically acceptable carrier. The FVIII protein in the chimeric protein has an extended half-life compared to wild-type FVIII protein or compared to a corresponding FVIII protein that does not contain a VWF fragment. In one embodiment, the half-life of the chimeric protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild-type FVIII. In another embodiment, the half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0278] In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. Parenteral administration can be intravenous or subcutaneous administration.

[0279] In other embodiments, the composition is used to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of bleeding coagulopathy, hemarthrosis, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and combinations thereof. In yet other embodiments, the subject is scheduled to undergo surgery. In yet other embodiments, the treatment is prophylactic or on-demand.

[0280] V. Gene Therapy The chimeric proteins of the invention can be produced in vivo in a mammalian, e.g., human, subject and can be used to treat hemorrhagic coagulopathy, hemarthrosis, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. It is therapeutically beneficial to use gene therapy approaches for the treatment of bleeding diseases or disorders selected from the group consisting of bleeding associated with hemophilia, hemophilia B, and hemorrhage as...

Claims

[Claim 1] The invention described in the specification.

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