Factor VIII chimeric proteins and uses thereof

Abstract

The invention relates to Factor VIII chimeric proteins and uses thereof. The present invention provides a chimeric protein comprising a first polypeptide comprising a FVIII protein and a first Ig constant region or a portion thereof; and a second polypeptide comprising a VWF protein comprising a D'domain and a D3 domain of a VWF, an XTEN sequence having less than 288 amino acids in length, and a second Ig constant region or a portion thereof wherein the first polypeptide and the second polypeptide are associated with each other. The invention also includes nucleotides, vectors, host cells, methods of using the chimeric proteins.

Description

[0001] This application is a divisional application of the invention application with an application date of January 9, 2015, a Chinese application number of 201580009942.9, and an invention title of "Factor VIII Chimeric Proteins and Their Uses".

[0002] Reference to the electronically submitted sequence listing

[0003] The content of the sequence listing submitted electronically in the form of an ASCII text file (name: 2159_441PC02_SequenceListing_ST25.txt; size: 823,500 bytes; and creation date: January 9, 2015) is incorporated herein by reference in its entirety. Background of the Invention

[0004] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding coagulation factor VIII (FVIII) and affects one in ten thousand to two in ten thousand male births. Graw et al., Nat. Rev. Genet. 6(6):488-501 (2005). Patients affected by hemophilia A can be treated by infusion of purified or recombinantly produced FVIII. However, the half-life of all commercially available FVIII products is known to be approximately 8-12 hours, thus requiring frequent intravenous administration to patients. See Weiner M.A. and Cairo, M.S., Pediatric Hematology Secrets, Lee, M.T., 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, many methods have been tried to extend the FVIII half-life. For example, methods under development to extend the half-life of clotting factors include polyethylene glycolylation, glyco-polyethylene glycolylation, and conjugation to albumin. See Dumont et al., Blood. 119(13):3024-3030 (published online January 13, 2012). However, regardless of the protein engineering used, currently developed long-acting FVIII products are reported to have limited half-lives - only about 1.5 to 2 hours in preclinical animal models. See ibid. Consistent results have been demonstrated in humans, for example, it has been reported that compared to The half-life of rFVIIIFc in patients with hemophilia A is improved by up to about 1.7-fold. See ibid. Thus, despite minor improvements, the increased half-life may indicate the presence of other T1 / 2 limiting factors. See Liu, T. et al., 2007 ISTH Congress, abstract #P-M-035; Henrik, A. et al., 2011 ISTH Congress, abstract #P=MO-181; Liu, T. et al., 2011 ISTH Congress abstract #P-WE-131.

[0005] The half-life of plasma von Willebrand Factor (VWF) is about 16 hours (in the range of 13 to 18 hours). Goudemand J et al J Thromb Haemost 2005; 3:2219–27. The VWF half-life can be affected by many factors: glycosylation pattern, ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs-13), and various mutations in VWF.

[0006] 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 maintaining appropriate plasma levels of FVIII in vivo. Lenting et al., Blood. 92(11):3983-96 (1998); Lenting et al., J. Thromb. Haemost. 5(7):1353-60 (2007). Full-length wild-type FVIII exists mainly as a heterodimer with a heavy chain (MW 200kD) and a light chain (MW 73kD). When FVIII is activated by proteolysis at positions 372 and 740 in the heavy chain and at position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII. Activated FVIII together with activated factor IX, calcium, and phospholipids (the "tenase complex") induces the activation of factor X, resulting in the generation of a large amount of thrombin. Thrombin in turn 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 is used to crosslink and stabilize the soluble fibrin polymers, thereby forming cross-linked (insoluble) fibrin. Activated FVIII is rapidly cleared from the circulation by proteolysis.

[0007] Due to the frequent administration and the inconvenience caused by the dosing schedule, there is still a need to develop FVIII products that require less frequent administration, i.e., FVIII products with a half-life longer than 1.5 to 2 times the half-life limit. SUMMARY OF THE INVENTION

[0008] The present invention provides a chimeric protein comprising (i) a first polypeptide comprising a factor VIII ("FVIII") protein fused to a first immunoglobulin ("Ig") constant region or a portion thereof, and (ii) a second polypeptide comprising a von Willebrand factor ("VWF") protein containing the D' domain and the D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via an XTEN sequence therebetween, wherein the XTEN sequence contains less than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide. Certain embodiments include a chimeric protein 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.

[0009] Also disclosed is a chimeric protein as described herein, wherein the chimeric protein exhibits a longer half-life compared to a corresponding fusion protein comprising the first polypeptide and the second polypeptide, wherein the second polypeptide of the fusion protein comprises an XTEN sequence containing at least 288 amino acids. Some embodiments include the XTEN sequence AE288 containing at least 288 amino acids. In some embodiments, AE288 is SEQ ID NO:8.

[0010] Also disclosed is a chimeric protein 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.

[0011] Some embodiments include a chimeric protein as described herein, 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: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.

[0012] In certain embodiments, the first polypeptide further comprises a second XTEN sequence that links the FVIII protein to the first Ig constant region or a portion thereof. Also disclosed are chimeric proteins as described herein, wherein the first polypeptide comprises a third XTEN sequence inserted at one or more insertion sites within the FVIII protein. In some embodiments, the first polypeptide further comprises a second XTEN sequence inserted at one or more insertion sites within the FVIII protein. In certain embodiments, the first polypeptide comprises a third XTEN sequence that links the FVIII protein to the first Ig constant region or a portion thereof.

[0013] Also disclosed are chimeric proteins as described herein, wherein the second XTEN sequence, the third XTEN sequence, or the second XTEN sequence and the third XTEN sequence 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 XTEN sequence and the third XTEN sequence 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 XTEN sequence and the third XTEN sequence 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 via a linker. In certain embodiments, the linker is a cleavable linker.

[0014] Some embodiments include chimeric proteins 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).

[0015] Also disclosed are chimeric proteins as described herein, wherein the linker comprises one or more cleavage sites comprising an amino acid sequence 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 (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 some embodiments, the linker comprises TLDPRSFLLRNPNDKYEPFWED EEK (SEQ ID NO:146). In certain embodiments, the cleavage site comprises the amino acid sequence LVPRG (SEQ ID NO:6). In other embodiments, the cleavage site comprises the amino acid sequence IEPRSFS (SEQ ID NO:194). In other embodiments, the cleavage site comprises the amino acid sequence IEPRSFS (SEQ ID NO:194), wherein 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 IEPR (SEQ ID NO:200).In other embodiments, the cleavage site comprises a fragment of the a2 region of FVIII comprising at least the sequence IEPR (SEQ ID NO:200), wherein 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 known in the art.

[0016] Some embodiments include a chimeric protein as described herein, 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. In some embodiments, 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. In some embodiments, the first polypeptide and the second polypeptide are fused via a linker. In certain embodiments, the first polypeptide and the second polypeptide are fused via a cleavable linker. In some embodiments, the first Ig constant region or a portion thereof associates with the second Ig constant region or a portion thereof. In certain embodiments, the first Ig constant region or a portion thereof associates with the second Ig constant region or a portion thereof via a covalent bond. In some embodiments, the covalent bond is a disulfide bond.

[0017] Also disclosed are chimeric proteins comprising each of the following formulas (a)-(hh):

[0018] (a) FVIII-F1:F2-L2-X-L1-V;

[0019] (b) FVIII-F1:V-L1-X-L2-F2;

[0020] (c) F1-FVIII:F2-L2-X-L1-V;

[0021] (d) F1-FVIII:V-L1-X-L2-F2;

[0022] (e) FVIII-X2-F1:F2-L2-X1-L1-V;

[0023] (f) FVIII-X2-F1:V-L1-X1-L2-F2;

[0024] (g) FVIII(X2)-F1:F2-L2-X1-L1-V;

[0025] (h) FVIII(X2)-F1:V-L1-X1-L2-F2;

[0026] (i) F1-X2-F1:F2-L2-X1-L1-V;

[0027] (j) F1-X2-F1:V-L1-X1-L2-F2;

[0028] (k) V - L1 - X - L2 - F2 - L3 - FVIII - L4 - F1;

[0029] (l) V - L1 - X - L2 - F2 - L3 - F1 - L4 - FVIII;

[0030] (m) F1 - L4 - FVIII - L3 - F2 - L2 - X - L1 - V;

[0031] (n) FVIII - L4 - F1 - L3 - F2 - L2 - X - L1 - V;

[0032] (o) FVIII - L4 - F1 - L3 - V - L1 - X - L2 - F2;

[0033] (p) FVIII - L4 - F1 - L3 - F2 - L2 - X - L1 - V;

[0034] (q) F2 - L2 - X - L1 - V - L3 - F1 - L4 - FVIII;

[0035] (r) F2 - L2 - X - L1 - V - L3 - FVIII - L4 - F1;

[0036] (s) V - L1 - X1 - L2 - F2 - L3 - FVIII(X2) - L4 - F1;

[0037] (t) V - L1 - X1 - L2 - F2 - L3 - F1 - L4 - FVIII(X2);

[0038] (u) F1 - L4 - FVIII(X2) - L3 - F2 - L2 - X1 - L1 - V;

[0039] (v) F - L4 - FVIII(X2) - L3 - V - L1 - X1 - L2 - F2;

[0040] (w) FVIII(X2) - L4 - F1 - L3 - V - L1 - X1 - L2 - F2;

[0041] (x) FVIII(X2) - L4 - F1 - L3 - F2 - L2 - X1 - L1 - V;

[0042] (y) F2 - L2 - X1 - L1 - V - L3 - F1 - L4 - FVIII(X2);

[0043] (z) F2 - L2 - X1 - L1 - V - L3 - FVIII(X2) - L4 - F1;

[0044] (aa) V - L1 - X2 - L2 - F2 - L3 - FVIII - L4 - X2 - L5 - F1;

[0045] (bb) V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII;

[0046] (cc) F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V;

[0047] (dd) F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2;

[0048] (ee) FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1;

[0049] (ff) FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V;

[0050] (gg) F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or

[0051] (hh) F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2;

[0052] Wherein V is a VWF protein, which contains 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 includes an FVIII protein, FVIII(X2) includes an 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; and (:) is a covalent or non-covalent bond.

[0053] Some embodiments include the chimeric proteins as described herein, wherein X or X1 consists of an amino acid sequence having a length between 12 amino acids and 287 amino acids.

[0054] In certain embodiments, compared to the chimeric proteins containing the said formula, with the exception that X or X1 is the corresponding chimeric protein of AE288, the chimeric proteins as described herein exhibit a longer half-life. In some embodiments, AE288 is SEQ ID NO:8.

[0055] Some embodiments include chimeric proteins as described herein, wherein X or X1 contains about 36, about 42, about 72, or about 144 amino acids. In certain embodiments, X or X1 is selected from AE42, AE72, AE144, AG42, AG72, or AG144. In some embodiments, X or X1 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, or 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.

[0056] Chimeric proteins comprising X or X1 and / or X2 as described herein are also disclosed, which exhibit a longer half-life compared to chimeric proteins that do not comprise 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 can be cleaved 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 can be cleaved by factor IIa (thrombin).

[0057] Some embodiments include chimeric proteins as described herein, wherein the linker comprises one or more cleavage sites comprising an amino acid sequence selected from the following: 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), 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 LVPRG (SEQ ID NO:6). In some embodiments, the linker comprises the a1 domain of FVIII, the a2 domain of FVIII, the a3 domain of FVIII, or any combination thereof. In certain embodiments, the linker comprises a fragment of the a2 domain of FVIII. In some cases, the fragment of the a2 domain may comprise the sequence DKNTG DYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO:88). In other embodiments, smaller fragments of the a2 domain of FVIII may be used, including a fragment having the sequence IEPR SFS (SEQ ID NO:194).In a particular embodiment, the linker comprises the amino acid sequence IEPRSFS (SEQ ID NO:194). In another embodiment, the linker comprises the amino acid sequence IEPRSFS (SEQ ID NO:194), wherein the linker is not the full-length a2 region of FVIII.

[0058] Also disclosed are chimeric proteins as described herein, 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) or DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO:88). In some embodiments, the a1 region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95% or 100% identical to ISMKNNEEAEDYDDDLTDSEMDVV RFDDDNSPSFIQIRSV (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 ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSP RSFQ (SEQ ID NO:108). In some embodiments, F1 comprises a first Fc region and / or F2 comprises a second Fc region.

[0059] Some embodiments include chimeric proteins as described herein, wherein the chimeric protein comprising F1 and F2 exhibits a longer half-life compared to a chimeric protein that does not comprise F1 and F2. In certain embodiments, L3 is a cleavable linker. In some embodiments, the VWF protein associates with the FVIII protein by non-covalent bonds. In some embodiments, the half-life of the chimeric protein is extended compared to an FVIII protein without the VWF protein and / or XTEN sequence or compared to wild-type FVIII. In certain embodiments, the half-life of the chimeric protein is extended by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold or at least about 12-fold compared to an FVIII protein without the VWF protein or XTEN sequence or compared to wild-type FVIII.

[0060] Also disclosed are chimeric proteins 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 in HemA mice is about 40 hours. In certain embodiments, the VWF protein does not substantially bind to the VWF clearance receptor. In some embodiments, the VWF protein is capable of protecting the FVIII protein from cleavage by one or more proteases, protecting the FVIII protein from activation, stabilizing the heavy chain and / or light chain of the FVIII protein, or preventing the FVIII protein from being cleared by one or more scavenger receptors.

[0061] Some embodiments include chimeric proteins as described herein, 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. In certain embodiments, the VWF binding site is located in the A3 domain or the C2 domain of the FVIII protein, or both the A3 domain and the C2 domain. In some embodiments, the VWF binding site comprises the amino acid sequences corresponding to amino acids 1669 to 1689 of SEQ ID NO:65 and amino acids 2303 to 2332. In some embodiments, the first Ig constant region or a portion thereof and the second Ig constant region or a 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.

[0062] Also disclosed are chimeric proteins as described herein, wherein the FVIII protein comprises one or more domains of FVIII selected from the group consisting of: A1 domain, a1 acidic region, A2 domain, a2 acidic region, B domain, A3 domain, a3 acidic region, C1 domain, C2 domain, one or more fragments thereof, and any combination thereof.

[0063] Chimeric proteins as described herein are also disclosed, wherein one or more insertion sites in the FVIII protein are within one or more domains of the FVIII protein selected from the group consisting of: A1 domain, a1 acidic region, A2 domain, a2 acidic region, A3 domain, B domain, C1 domain, C2 domain, and any combination thereof, or between one or more domains of the FVIII protein selected from the group consisting of: A1 domain and a1 acidic region, a1 acidic region and A2 domain, A2 domain and a2 acidic region, a2 acidic region and B domain, B domain and A3 domain, A3 domain and C1 domain, C1 domain and C2 domain, and any combination thereof, or between two domains of the FVIII protein selected from the group consisting of: A1 domain and a1 acidic region, a1 acidic region and A2 domain, A2 domain and a2 acidic region, a2 acidic region and B domain, B domain and A3 domain, A3 domain and C1 domain, C1 domain and C2 domain, and any combination thereof. In some embodiments, 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 Tables 7, 8, 9, and 10. In certain embodiments, the insertion site in the FVIII protein is downstream of and adjacent to amino acid 745 corresponding to the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is downstream of and adjacent to residue 1656 and residue 1900 corresponding to the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is downstream of and adjacent to 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 downstream of and adjacent to 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 downstream of and adjacent to residues 745 and 1900 corresponding to the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion site in the FVIII protein is downstream of and adjacent to residues 18 and 745 corresponding to the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the FVIII protein is a double-chain FVIII subtype. In some embodiments, the FVIII protein is a single-chain FVIII subtype. In certain embodiments, the FVIII protein contains the B domain or a portion thereof. In some embodiments, the FVIII protein is an SQ B domain-deleted FVIII.

[0064] Some embodiments include chimeric proteins as described herein, wherein the single-chain FVIII subtype contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645, or both residues of the full-length mature factor VIII polypeptide (SEQ ID NO:65), or residue 754, residue 751, or both residues 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 double-chain FVIII subtype comprises a first chain containing the heavy chain of FVIII and a second chain containing the light chain of FVIII, wherein the heavy chain and the light chain are associated with each other by a metal bond. In certain embodiments, 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. 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 to 1240 of SEQ ID NO:21. In certain embodiments, the VWF protein is a monomer.

[0065] Also disclosed are chimeric proteins as described herein that 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 to 1240 of SEQ ID NO:21. In some embodiments, the VWF protein consists essentially of or consists of amino acids 764 to 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 that replaces 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 both the D1 and D2 domains of VWF.

[0066] Some embodiments include chimeric proteins as described herein, 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.

[0067] Chimeric proteins as described herein are also disclosed, 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.

[0068] Some embodiments include chimeric proteins as described herein, wherein the VWF protein further comprises a signal peptide of VWF or FVIII operably linked to the VWF protein.

[0069] Chimeric proteins as described herein are also disclosed, wherein the length of one or more linkers is 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, the length of one or more linkers is from about 1 to about 2000 amino acid residues. In certain embodiments, one or more linkers comprise a gly / ser peptide. In some embodiments, the gly / ser peptide has the formula (Gly4Ser) n (SEQ ID NO:94) or S(Gly4Ser) n (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. In certain embodiments, (Gly4Ser) n the linker is (Gly4Ser)3 (SEQ ID NO:100) or (Gly4Ser)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 SGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGG (SEQ ID NO:166).

[0070] In some embodiments, the chimeric proteins as described herein are polysialylated, polyethylene glycolated, or hydroxyethyl starchylated.

[0071] Chimeric proteins as described herein are also disclosed, wherein the first polypeptide comprises at least about 80%, 90%, 95%, 99% or 100% identical to 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), FVIII268 (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 the second polypeptide comprises at least about 80%, 90%, 95%, 99% or 100% identical to VWF057 (SEQ ID NO:152) or VWF059 (SEQ ID NO:197). 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 VWF059 (SEQ ID NO:197). In 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 in preventing and / or arresting bleeding in a subject in need thereof.

[0072] A polynucleotide or a set of polynucleotides encoding a chimeric protein as described herein is also disclosed. In some embodiments, the polynucleotide as described herein further comprises a polynucleotide chain encoding PC5 or PC7.

[0073] Some embodiments include a vector comprising the polynucleotide as described herein and one or more promoters operably linked to the polynucleotide or the set of polynucleotides.

[0074] In some embodiments, the vector as described herein further comprises an additional vector comprising a polynucleotide chain encoding PC5 or PC7.

[0075] Also disclosed is a host cell comprising a polynucleotide or vector as described herein. In some embodiments, the host cell is a mammalian cell. In certain embodiments, the mammalian cell is selected from the group consisting of HEK293 cells, CHO cells, and BHK cells.

[0076] Also disclosed is a pharmaceutical composition comprising a chimeric protein, polynucleotide, vector, or host cell as described herein and a pharmaceutically acceptable carrier. In some embodiments, the chimeric protein has an extended half-life compared to the wild-type FVIII protein. In certain embodiments, the half-life of the chimeric protein is extended by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to the wild-type FVIII.

[0077] 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 in HemA mice is about 40 hours. In some embodiments, the 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 intravenous or subcutaneous administration.

[0078] In some embodiments, the compositions as described herein are used to treat bleeding disorders or conditions in a subject in need thereof. In certain embodiments, the bleeding disorder or condition is selected from the group consisting of: bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and any combination thereof. In some embodiments, the subject is scheduled to undergo surgery. In certain embodiments, the treatment is prophylactic or on-demand.

[0079] Also disclosed is a method of prolonging or increasing the half-life of a chimeric protein, wherein the method comprises administering to a subject in need thereof an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition as described herein, wherein the VWF protein, XTEN sequence, first Ig constant region or a portion thereof, and second Ig constant region or a portion thereof increase the half-life of the chimeric protein.

[0080] Some embodiments include a method of treating a bleeding disorder or condition in a subject in need thereof, which comprises administering an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition as described herein, wherein the bleeding disorder or condition is selected from the group consisting of: bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. In some embodiments, the subject is an animal. In certain embodiments, the animal is a human. In some embodiments, the subject is suffering from hemophilia A. In certain embodiments, the treatment is prophylactic or on-demand. In some embodiments, the effective amount is from 0.1 μg / kg to 500 mg / kg.

[0081] Also disclosed is a method as described herein, wherein the 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.

[0082] Some embodiments include a method of preparing a chimeric protein, which comprises transfecting one or more host cells with a polynucleotide or vector as described herein, and expressing the chimeric protein in the host cell. In some embodiments, the method as described herein further comprises isolating the chimeric protein. In certain embodiments, the chimeric protein is effective in preventing and / or precluding bleeding in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Schematic diagram showing a chimeric protein comprising a first polypeptide comprising an FVIII protein (A1-A2 - partially or fully B - A3 - C1 - C2) fused to an Fc region, wherein XTEN is inserted at an insertion site within the FVIII protein; and a second polypeptide comprising a VWF protein containing the D’D3 domain, an XTEN having less than 288 amino acids, a thrombin-cleavable linker, and a second Fc region. Insertion of XTEN in the FVIII protein and / or fusion to the VWF protein prolongs the half-life of the chimeric protein by increasing the hydrodynamic radius and by blocking receptor-mediated clearance. The D’D3 domain of VWF blocks the interaction of FVIII with endogenous VWF, stabilizes the FVIII protein, and prolongs the half-life of the chimeric protein. The Fc domain covalently links the D’D3 domain to the FVIII protein and prolongs the half-life of the chimeric protein via the FcRn-mediated recycling pathway. The thrombin-cleavable linker enables release of the D’D3 domain after FVIII activation and ensures proper alignment between the D’D3 domain of FVIII and VWF.

[0084] Figure 2 Three-plasmid expression system showing the FVIII-XTEN-Fc:D’D3-XTEN-Fc heterodimer: The first plasmid contains a nucleotide sequence encoding single-chain FVIII-XTEN-Fc, wherein XTEN is inserted into the B domain; the second plasmid contains a nucleotide sequence encoding D1D2D’D3-XTEN-Fc, wherein the XTEN sequence contains less than 288 amino acids; and the third plasmid contains a nucleotide sequence encoding PACE (a propeptide processing enzyme). When the three polypeptides are expressed from the three plasmids, the D1D2 propeptide domain of VWF can be processed from the D’D3 domain by intracellular processing. The resulting complex contains three products, the first molecule being the FVIII-XTEN / D’D3 heterodimer, the second molecule being a by-product, namely the homodimer of D’D3-XTEN-Fc, and the third molecule being another by-product, namely FVIII(XTEN)-Fc.

[0085] Figure 3Show the additive effect of the XTEN insert on the extended half-life of the heterodimer. FVIII169 contains the B-domain-deleted FVIII protein fused to the Fc region, where the XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to mature full-length FVIII. FVIII205 contains the B-domain-deleted FVIII protein fused to the Fc region, where the 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 contains the D’ and D3 domains of VWF (without XTEN) fused to the Fc region via a thrombin-cleavable linker. VWF034 contains the D’ and D3 domains of VWF fused to AE288 and the Fc region. The half-life of FVIII169 / VWF031 (inverted triangle) in HemA mice is 16.7 hours; the half-life of FVIII205 / VWF031 (circle) in HemA mice is 29.4 hours; and the half-life of FVIII169 / VWF034 (square) in HemA mice is 31.1 hours.

[0086] Figure 4 Show that when inserted between the D’D3 and Fc domains of VWF, the AE144 XTEN confers better half-life extension than the AE288 XTEN. For example, although the half-life of VWF169 / VWF034 (square) in HemA mice is 31.1 hours, the half-life of FVIII169 / VWF057 (circle) in HemA mice is 42 hours. VWF057 contains the D’D3 domains of VWF fused to AE144 and the Fc region.

[0087] Figure 5 Show that the Fc domain is required for the extended half-life of the chimeric protein heterodimer. When comparing the half-life of FVIII205 / VWF031 (circle) in HemA mice with the half-life of FVIII263 / VWF050 (square) that contains a mutation (IHH triple mutant Fc) at the FcRn binding site and thus cannot be recycled via the FcRn pathway, the half-life of FVIII263 / VWF050 (23 hours) is shorter than the half-life of VWF205 / VWF031 (29.4 hours). This indicates that the Fc region is necessary for the extended half-life.

[0088] Figure 6Panel A shows the similar acute efficacy of the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer compared to the B-domain deleted FVIII (SQ BDD FVIII) in the HemA mouse tail clamp model. Mice were dosed at 75 IU / kg, and activity was measured by aPTT assay. SQ BDD FVIII is shown as circles, while FVIII169 / VWF034 is shown as squares, FVIII169 / VWF057 is shown as diamonds, and the vehicle is shown as inverted triangles. Construct details for FVIII169, VWF034, and VWF057 are shown elsewhere in this paper. Figure 6 Panel B shows the comparison of the acute efficacy of FVIII169 / VWF034 and the B-domain deleted FVIII (SQ BDD FVIII) in HemA mice at a dose of 37.5 IU / kg, and activity was measured by aPTT assay. The median blood loss (uL) of mice in each treatment group is indicated by the horizontal line, blood loss (uL) in C57 / BL6 mice is shown as open triangles; blood loss (uL) after administration of rBDD-FVIII at 37.5 IU / kg is shown as open circles; blood loss (uL) after administration of FVIII169 / VWF034 at 37.5 IU / kg is shown as open squares, and blood loss (uL) after vehicle administration is shown as inverted triangles.

[0089] Figure 7 Panels A-B show that the rFVIII169 / VWF057 heterodimer provides longer protection to HemA mice in the tail vein transection bleeding model. Figure 7 Panel A shows rebleeding data in mice that received rFVIII169 / VWF057 (squares) 72 hours before tail injury, SQ BDD-FVIII (diamonds) 48 hours before tail injury, SQ BDDFVIII (inverted triangles) 24 hours before tail injury, and vehicle (circles). Activity was measured by aPTT assay. The x-axis shows time in hours, and the y-axis shows the percentage of non-bleeders. Figure 7 Panel B shows Figure 7 the corresponding survival data in the four types of mice shown in Panel A. Mice that received 12 IU / kg of FVIII169 / VWF057 72 hours before tail injury showed similar protection against rebleeding and survival compared to mice that received SQ BDD FVIII treatment 24 hours before tail injury.

[0090] Figure 8Panel A shows comparable rebleeding data in mice receiving rFVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer 96 hours before injury relative to mice receiving rBDD-FVIII 24 hours before injury. Solid squares show rebleeding data in mice receiving FVIII169 / VWF034 24 hours before injury; open squares show rebleeding data in mice receiving FVIII169 / VWF034 96 hours before injury; solid diamonds show rebleeding data in mice receiving FVIII169 / VWF057 24 hours before injury; open diamonds show rebleeding data in mice receiving FVIII169 / VWF057 96 hours before injury; solid circles show rebleeding data in mice receiving rBDD-FVIII 24 hours before injury; open circles show rebleeding data in mice receiving rBDD-FVIII 48 hours before injury; and solid triangles show rebleeding data in mice receiving vehicle. The x-axis shows time in hours, and the y-axis shows the percentage of non-bleeders.

[0091] Figure 8 Panel B shows survival curves in mice receiving rFVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer 96 hours before injury relative to mice receiving rBDD-FVIII 24 hours before injury. The x-axis shows time in hours, and the y-axis shows the percentage of survival. The symbols are the same as Figure 8 those in Panel A.

[0092] Figure 9Diagram showing representative FVIII-VWF heterodimers and the FVIII169, FVIII286, VWF057, VWF059, and VWF062 constructs. For example, the FVIII169 construct comprises a B-domain-deleted FVIII protein with an R1648A substitution fused to an Fc region, wherein 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, wherein 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 that comprises the D'D3 domain of a VWF protein (with two amino acid substitutions, namely C336A and C379A in the D'D3 domain) linked to an Fc region via a VWF linker comprising an LVPRG thrombin cleavage site (“LVPRG”; SEQ ID NO:6) and a GS linker (“GS”), wherein 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 that comprises the D'D3 domain of a VWF protein (with two amino acid substitutions, namely C336A and C379A in the D'D3 domain) linked to an Fc region via the acidic region 2 (a2) of FVIII as the VWF linker, wherein an XTEN sequence (i.e., AE144) is inserted between the D'D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct that comprises the D'D3 domain of a VWF protein (with two amino acid substitutions, namely C336A and C379A in the D'D3 domain) linked to an Fc region, wherein an XTEN sequence (i.e., AE144) is inserted between the D'D3 domain and the Fc region (D'D3-144XTEN-Fc).

[0093] Figure 10Schematic representation of FVIII / VWF heterodimer constructs such as FVIII169 / VWF057, FVIII169 / VWF059, FVIII169 / VWF059A, and FVIII169 / VWF073. Arrows show the sites where an optional linker was added to introduce a thrombin cleavage site. FVIII169 / VWF057 has a linker containing LVPRG (SEQ ID NO:6). FVIII169 / VWF059 has a linker containing the FVIII a2 region (i.e., ). FVIII169 / VWF059A has a linker containing a truncated FVIII a2 region (i.e., ). FVIII169 / VWF073 has a linker within the VWF073 construct (SEQ ID NO:175) containing a fragment of the FVIII a2 region consisting of IEPRSFS (SEQ ID NO:194).

[0094] Figure 11 Panels A - C show SDS - PAGE images after thrombin digestion of FVIII169 / VWF057 and FVIII - Fc control. Figure 11 Panel A shows staining of the SDS - PAGE gel with anti - D3 antibody (AB 96340). Arrows highlight "LCFc:D'D3 - XTEN - Fc", which is the uncleaved full - length FVIII169 / VWF057; and "D'D3 - 144 XTEN", which is the resulting fragment after cleavage by thrombin. Figure 11 Panel B shows staining of the SDS - PAGE gel with anti - HC antibody (GMA012). Arrows highlight the FVIII heavy chain ("HC") and the FVIII A2 domain. Figure 11 Panel C shows an overlay of Panels A and B. Samples were collected at the time points indicated at the top of each panel. Arrows point to the relevant proteins.

[0095] Figure 12 Panels A - C show SDS - PAGE images after thrombin digestion of FVIII169 / VWF059. Figure 12 Panel A shows staining of the SDS - PAGE gel with anti - D3 antibody (AB 96340). Arrows highlight "LCFc:D'D3 - XTEN - Fc,", which is the uncleaved full - length FVIII169 / VWF059; and "D'D3 - 144 XTEN,", which is the resulting fragment after cleavage by thrombin. Figure 12Panel B shows the staining of an SDS-PAGE gel with anti-HC antibody (GMA012). Arrows highlight the uncleaved full-length FVIII169 / VWF059; D'D3-144XTEN-a3, which is the fragment obtained after cleavage by thrombin; and "A2," which is the A2 domain of FVIII. Figure 12 Panel C shows an overlay of Panels A and B. Samples were collected at the time points indicated at the top of each panel.

[0096] Figure 13 Shows acute efficacy data for HemA mice treated with FVIII169 / VWF059 (circles) compared to HemA mice treated with BDD-FVIII control (squares). Bleeding values were measured after tail clamping. p = 0.9883. DETAILED DESCRIPTION

[0097] The present invention relates to a chimeric protein comprising two polypeptides, a first polypeptide comprising an FVIII protein fused to a first Ig constant region, and a second polypeptide comprising a VWF protein fused to a second Ig constant region or a portion thereof via an XTEN sequence, wherein the XTEN sequence contains less than 288 amino acids.

[0098] I. DEFINITIONS

[0099] It should be noted that the term "a" entity refers to one or more of that entity; for example, "a nucleotide sequence" should be understood to represent one or more nucleotide sequences. Thus, the terms "a," "one or more," and "at least one" are used interchangeably herein.

[0100] In addition, when used herein, "and / or" is to be regarded as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to cover each of the following aspects: 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 (alone); B (alone); and C (alone).

[0101] It should be understood that whenever aspects are described herein in terms of "comprising," additional similar aspects are also provided in terms of "consisting of" and / or "consisting essentially of."

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. By way of 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide one of ordinary skill in the art with a comprehensive dictionary of many of the terms used in this disclosure.

[0103] Units, prefixes, and symbols are expressed in their Système International de Unites (SI) accepted form. Numerical ranges include the recited values defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully elaborated by reference to the specification as a whole.

[0104] The term “about” is used herein to mean approximate, roughly, around, or in the vicinity of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. In general, the term “about” can modify a numerical value above and below the stated value by varying, for example, up or down (increase or decrease) by 10%.

[0105] The terms "polynucleotide" or "nucleotide" are intended to encompass both single nucleic acids and plural nucleic acids, and refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, the polynucleotide contains conventional phosphodiester bonds or non-conventional bonds (such as amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments present in the polynucleotide, such as DNA or RNA fragments. With respect to an "isolated" nucleic acid or polynucleotide, it means a nucleic acid molecule DNA or RNA that has been removed from its natural environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding a factor VIII polypeptide contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell, or recombinant polynucleotides purified (partially or substantially) from other polynucleotides and in solution form. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. The isolated polynucleotides or nucleic acids of the present invention further include those molecules produced synthetically. In addition, the polynucleotide or nucleic acid may include regulatory elements, such as promoters, enhancers, ribosome binding sites or transcription termination signals.

[0106] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons that can be translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are generally not translated into amino acids, they can be considered part of the coding region, however any flanking sequences (such as promoters, ribosome binding sites, transcription terminators, introns, etc.) are not part of the coding region. The boundaries of the coding region are generally determined by a start codon at the 5' end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the carboxyl terminus of the resulting polypeptide). Two or more coding regions of the present invention can be present in a single polynucleotide construct, such as on a single vector, or in separate polynucleotide constructs, such as on separate (different) vectors. It can then be seen that a single vector can contain only a single coding region, or contain two or more coding regions, for example a single vector can separately encode binding domain A and binding domain B as described below. In addition, the vectors, polynucleotides or nucleic acids of the present invention can encode heterologous coding regions that are fused or not fused to nucleic acids encoding the binding domains of the present invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains.

[0107] Certain proteins secreted by mammalian cells associate with a secretion signal peptide that is cleaved from the mature protein once the initiation of translocation of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art will appreciate that signal peptides are typically fused to the N-terminus of a polypeptide and are cleaved from the full or "full-length" polypeptide to yield the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide or a functional derivative of that sequence that retains the ability to direct the secretion of an operably associated polypeptide is used. Alternatively, a heterologous mammalian signal peptide, such as the human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide or a functional derivative thereof, can be used.

[0108] The term "downstream" when referring to a nucleotide sequence means that the nucleic acid or nucleotide sequence is located 3' of a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence refers to the sequence after the transcription start point. For example, the translation initiation codon of a gene is located downstream of the transcription start site. The term "downstream" when referring to a polypeptide sequence means that the amino acid or amino acid insertion site is located C-terminal to a reference amino acid. For example, an insertion site immediately downstream of amino acid 745 corresponding to the mature wild-type FVIII protein means that the insertion site is between amino acid 745 and amino acid 746 corresponding to the mature wild-type FVIII protein.

[0109] The term "upstream" refers to a nucleotide sequence that is located 5' of a reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to the sequence located 5' of the coding region or transcription start point. For example, most promoters are located upstream of the transcription start site.

[0110] As used herein, the term "regulatory region" means a nucleotide sequence that is located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region and that affects the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended to be expressed in a eukaryotic cell, then a polyadenylation signal and transcription termination sequence will typically be located 3' of the coding sequence.

[0111] A polynucleotide encoding a gene product (such as a polypeptide) may include a promoter and / or other transcriptional or translational control elements operably associated with one or more coding regions. In an operable association, the coding region of the gene product (such as a polypeptide) is associated with one or more regulatory regions in such a way as to place the expression of the gene product under the influence or control of the regulatory region. For example, 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 linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or with the ability of the DNA template to be transcribed, then the coding region and the promoter are "operably associated". In addition to promoters, other transcriptional control elements (such as enhancers, operators, repressors, and transcription termination signals) may also be operably associated with the coding region to direct the expression of the gene product.

[0112] A variety of transcriptional control regions are known to those of ordinary skill in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcomavirus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Also suitable transcriptional control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (such as promoters that can be induced by interferons or interleukins).

[0113] Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (especially internal ribosome entry sites or IRESs, also known as CITE sequences).

[0114] As used herein, the term "expression" refers to the process by which a polynucleotide gives rise to a gene product (e.g., RNA or polypeptide). It includes, without limitation, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of the mRNA into a polypeptide. Expression gives rise to a "gene product". As used herein, a gene product can be a nucleic acid (e.g., messenger RNA produced by gene transcription) or a polypeptide translated from a transcript. The gene products described herein further include nucleic acids having post-transcriptional modifications (e.g., polyadenylation or splicing), or polypeptides having post-translational modifications (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage).

[0115] A "vector" refers to any agent used to clone and / or transfer a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be ligated so as to cause the ligated segment to replicate. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous replication unit in vivo, i.e., is capable of replicating under its own control. The term "vector" includes both viral and non-viral agents for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Many 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 to the selected vector having complementary sticky ends.

[0116] A vector can be engineered to encode a selectable marker or reporter that provides for the selection or identification of cells having the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells having and expressing other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes that confer resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, etc.; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. A selectable marker can also be considered a reporter.

[0117] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. The element can be a linear, circular, or supercoiled autonomously replicating sequence, a genomic integration sequence, a phage, or a nucleotide sequence with single-stranded or double-stranded DNA or RNA from any source, many of which have been ligated or recombined into unique constructs capable of introducing a promoter fragment and the DNA sequence of a selected gene product along with an appropriate 3' untranslated sequence into a cell.

[0118] Eukaryotic viral vectors that can be used include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, and poxviral (e.g., vaccinia virus) vectors, baculoviral vectors, or herpesviral vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cell transfection agents), DNA-protein complexes, and biopolymers.

[0119] A "cloning vector" is a nucleic acid of a certain unit length that replicates sequentially and contains a "replicon" with an origin of replication (such as a plasmid, phage, or cosmid) to which another nucleic acid segment can be ligated to cause the ligated segment to replicate. Some cloning vectors are capable of replicating in one cell type (e.g., bacteria) and expressing in another cell type (e.g., eukaryotic cells). Cloning vectors typically contain one or more sequences that can be used to select cells containing the vector and / or one or more multiple cloning sites for inserting target nucleic acid sequences.

[0120] The term "expression vector" refers to a vehicle that is designed to enable the expression of an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is placed in a manner operably linked to a regulatory region as described above.

[0121] 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, liposome transfection (lysosome fusion), use of a gene gun, or DNA vector transporters.

[0122] As used herein, "culturing" means incubating cells under in vitro conditions that permit cell growth or division or maintaining cells in a viable state. As used herein, "cultured cells" means cells propagated in vitro.

[0123] As used herein, the term "polypeptide" is intended to cover both a single "polypeptide" and plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any one or more chains having two or more amino acids, and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to one or more chains having two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" also means a product having a post-expression modification of the polypeptide, the modifications including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a designated nucleic acid sequence. It can be produced in any manner, including by chemical synthesis.

[0124] An "isolated" polypeptide or fragment, variant, or derivative thereof is a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide may be removed only from its native or natural environment. For the purposes of the present invention, a recombinantly produced polypeptide and protein expressed in a host cell are considered to be isolated, and a native or recombinant polypeptide that has been isolated, fractionated, or partially or substantially purified by any suitable technique is also considered to be isolated.

[0125] Also included in the present invention are fragments or variants of polypeptides and any combinations thereof. The terms "fragment" or "variant" when referring to a polypeptide binding domain or binding molecule of the present invention include any polypeptide that retains at least some properties of the reference polypeptide (e.g., the FcRn binding affinity of an FcRn binding domain or Fc variant, the coagulation activity of an FVIII variant, or the FVIII binding activity of a VWF fragment). Except for specific antibody fragments discussed elsewhere herein, fragments of polypeptides also include proteolytic fragments and deletion fragments, but do not include a naturally occurring full-length polypeptide (or mature polypeptide). Variants of the polypeptide binding domains or binding molecules of the present invention include fragments as described above and polypeptides in which the amino acid sequence is altered due to amino acid substitution, deletion, or insertion. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0126] As used herein, the term "VWF protein" or "VWF protein" means any VWF fragment that interacts with FVIII and retains at least one or more of the properties normally provided by full-length VWF to FVIII, such as preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that can lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII rather than VWF-bound FVIII, and / or stabilizing the interaction between the heavy and light chains of FVIII.

[0127] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a substitution is considered conservative if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family. In another embodiment, a stretch of amino acids can be replaced with a structurally similar stretch of conservative substitutions that differ in the order and / or composition of side chain family members.

[0128] As is known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package for Unix, version 8, 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 obtain the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the present invention, the parameters should of course be set such that the percentage identity is calculated over the full length of the reference polypeptide sequence, and allowing gaps in homology to account for up to 5% of the total number of amino acids in the reference sequence.

[0129] As used herein, "amino acids corresponding to..." or "equivalent amino acids" in a VWF sequence or FVIII protein sequence are identified by aligning to maximize identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The numbering used to identify equivalent amino acids in the second VWF or FVIII sequence is based on the numbering used to identify the corresponding amino acids in the first VWF or FVIII sequence.

[0130] As used herein, the term "insertion site" refers to a position upstream of the position immediately adjacent to where a heterologous moiety can be inserted in an FVIII polypeptide or a fragment, variant or derivative thereof. The "insertion site" is designated by a number which is the number of the amino acid in mature native FVIII (SEQ ID NO:65) corresponding to the insertion site, said amino acid being immediately adjacent to the N-terminus of the insertion position. For example, the phrase "a3 contains XTEN at the insertion site corresponding to amino acid 1656 of SEQ ID NO:65" indicates that the heterologous moiety is located between the two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO:65.

[0131] As used herein, the phrase "downstream of an amino acid" refers to the position immediately adjacent to the terminal carboxyl group of said amino acid. Similarly, the phrase "upstream of an amino acid" refers to the position immediately adjacent to the terminal amine group of said amino acid. Thus, as used herein, the phrase "between two amino acids at an insertion site" refers to the position where XTEN or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases "inserted downstream of an amino acid" and "inserted between two amino acids at an insertion site" are used synonymously with "inserted at the insertion site".

[0132] As used herein, the terms "inserted", "being inserted", "inserted into...", or grammatically related terms refer to the position of XTEN in a chimeric polypeptide relative to a similar position in native mature human FVIII. As used herein, the terms refer to a characteristic of a recombinant FVIII polypeptide relative to native mature human FVIII and do not indicate, imply, or mean any method or process by which the chimeric polypeptide is prepared. For example, with respect to the chimeric polypeptides provided herein, the phrase "inserting XTEN downstream of residue 745 of the FVIII polypeptide" means that the chimeric polypeptide contains XTEN downstream of the amino acid immediately corresponding to amino acid 745 in native mature human FVIII, which is defined, for example, by the amino acids corresponding to amino acids 745 and 746 of native mature human FVIII.

[0133] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. Amino acid sequences that are normally present in separate proteins can be brought together in the form of a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be rearranged and placed in a fusion polypeptide, such as the fusion of the factor VIII domain of the present invention with the Ig Fc domain. A fusion protein is produced, for example, by chemical synthesis or by generating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. The chimeric protein may further comprise a second amino acid sequence that associates with the first amino acid sequence by covalent non-peptide bonds or non-covalent bonds.

[0134] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. The half-life can be represented by the time required for half of the amount administered to a subject to be cleared from the circulation and / or other tissues in an animal. When constructing a clearance curve for a given polypeptide over time, the curve is typically biphasic, i.e., a rapid α-phase and a longer β-phase. The α-phase generally represents the equilibration 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 generally represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins containing FVIII are monophasic and thus do not have an α-phase, but only a single β-phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

[0135] As used herein, the term "linked" means that a first amino acid sequence or nucleotide sequence is covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly joined or contiguous to the second amino acid or nucleotide sequence, or intervening sequences can covalently join the first sequence to the second sequence. The term "linked" not only means that the first amino acid sequence is fused to the second amino acid sequence at the C-terminus or N-terminus, but also includes inserting the entire first amino acid sequence (or the second amino acid sequence) between any two amino acids in the second amino acid sequence (or, correspondingly, the first amino acid sequence). 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 any chemical moiety (for both polypeptide chains and polynucleotide chains). The term "linked" is also indicated by a hyphen (-).

[0136] As used herein, the term "associated with" refers to the formation of a covalent or non-covalent bond between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" means a covalent non-peptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond other than a peptide bond. For example, the amino acid cysteine contains a thiol group that can form a disulfide bond or disulfide bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 region and the CL region are associated by a disulfide bond, and the two heavy chains are associated at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, σ bonds, π bonds, δ bonds, glycosidic bonds, agnostic bonds, bent bonds, dipole bonds, π-back bonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Walls forces, London dispersion forces, mechanical bonds, halogen bonds, metallophilicity, insertion, stacking, entropy forces, or chemical polarity.

[0137] The term "monomer-dimer hybrid" as used herein refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain associated with each other by a disulfide bond, wherein the first chain comprises a clotting factor (e.g., factor VIII) and a first Fc region, and the second chain comprises, consists essentially of, or consists of: a second Fc region without a clotting factor. Thus, a monomer-dimer hybrid construct is a hybrid that comprises a monomeric aspect with only one clotting factor and a dimeric aspect with two Fc regions.

[0138] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Certain enzymatic cleavage sites include intracellular processing sites. In one embodiment, a polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during the coagulation cascade such that cleavage of the site occurs at the site of clot formation. Exemplary such sites include, for example, those recognized by thrombin, factor XIa, or factor Xa. Exemplary FXIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO:1) and SVSQTSKLTR (SEQ ID NO:3). Exemplary 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 enzymatic cleavage sites are known in the art and are described elsewhere herein.

[0139] 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 of an enzyme that acts after translation of the polypeptide. In one embodiment, the enzyme acts during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before the proteins are secreted from the cell. Examples of such processing sites include, for example, those targeted by the PACE / furin family of endopeptidases (where PACE is an acronym for Paired basic Amino acid Cleaving Enzyme). These enzymes are localized to the Golgi membrane and cleave proteins on the carboxyl-terminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein, enzymes of the "furin" family include, 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.

[0140] In constructs that include more than one processing or cleavage site, it should be understood that the sites can be the same or different.

[0141] The term "furin" refers to the enzyme corresponding to EC number 3.4.21.75. Furin is a subtilisin-like proprotein convertase, which is also known as PACE (paired basic amino acid cleaving enzyme). Furin cleaves segments from inactive precursor proteins to convert them into bioactive proteins. During its intracellular trafficking, the propeptide of VWF can be cleaved from the mature VWF molecule by furin. In some embodiments, furin cleaves D1D2 from the D'D3 of VWF. In other embodiments, the nucleotide sequence encoding furin can be expressed together with the nucleotide sequence encoding a VWF fragment so that the D1D2 domain can be cleaved and removed intracellularly by furin.

[0142] In constructs that include more than one processing or cleavage site, it should be understood that the sites can be the same or different.

[0143] As used herein, a "processable linker" refers to a linker that contains at least one intracellular processing site described elsewhere herein.

[0144] As used herein, a hemostatic disorder refers to a genetic or acquired condition characterized by a tendency to spontaneous bleeding or bleeding upon trauma due to impaired ability or inability to form a fibrin clot. Examples of such disorders include hemophilia. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease"), and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand disease; factor XI deficiency (PTA deficiency); factor XII deficiency; fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII deficiency or structural abnormalities; Bernard-Soulier syndrome, which is a GPIb deficiency or defect. The receptor for VWF, GPIb, can be defective and result in a lack of primary clot formation (primary hemostasis) and increased bleeding tendency, as well as Glanzman and Naegeli thrombasthenia (Glanzman thrombasthenia). In liver failure (acute and chronic forms), there is a deficiency of clotting factors produced by the liver; this can increase the risk of bleeding.

[0145] The chimeric molecules of the present invention can be used prophylactically. As used herein, the term "prophylactic treatment" refers to the administration of the molecule before the onset of bleeding. In one embodiment, a subject in need of a general hemostatic agent is undergoing or about to undergo surgery. The chimeric proteins of the present invention can be administered prophylactically before or after surgery. The chimeric proteins of the present invention can be administered during or after surgery to control acute bleeding episodes. Surgery can include, but is not limited to, liver transplantation, hepatectomy, dental procedures, or stem cell transplantation.

[0146] The chimeric proteins of the present invention are also used for on - demand treatment. The term "on - demand treatment" refers to the administration of the chimeric molecule in response to the symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, on - demand treatment can be given to a subject at the onset of bleeding (such as after an injury) or when bleeding is expected (such as before surgery). In another aspect, on - demand treatment can be given before an activity that increases the risk of bleeding (such as contact sports).

[0147] As used herein, the term "acute bleeding" refers to the onset of bleeding regardless of the underlying cause. For example, a subject can have trauma, uremia, a hereditary bleeding disorder (such as factor VII deficiency), a platelet disorder, or resistance due to the production of antibodies against coagulation factors.

[0148] As used herein, treatment refers to, for example, reducing the severity of a disease or condition; decreasing the duration of the disease course; ameliorating one or more symptoms associated with the disease or condition; providing a beneficial effect to a subject having the disease or condition without necessarily curing the disease or condition; or preventing or treating one or more symptoms associated with the disease or condition. In one embodiment, the term "treatment" means maintaining the trough level of FVIII in a subject 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 by administering a chimeric protein or VWF fragment of the invention. In another embodiment, treatment means maintaining the trough level of FVIII between about 1 and about 20 IU / dL, about 2 and about 20 IU / dL, about 3 and about 20 IU / dL, about 4 and about 20 IU / dL, about 5 and about 20 IU / dL, about 6 and about 20 IU / dL, about 7 and about 20 IU / dL, about 8 and about 20 IU / dL, about 9 and about 20 IU / dL or about 10 and about 20 IU / dL. Treating a disease or condition may also include maintaining the FVIII activity in a subject at a level of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% similar to the FVIII activity in non-hemophilic subjects. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0149] II. Chimeric Protein

[0150] The present invention relates to using a VWF protein fused to an XTEN sequence to prolong the half-life of a chimeric protein by preventing or inhibiting the association of FVIII half-life limiting factors, i.e., endogenous VWF, with the FVIII protein. Endogenous VWF associates with approximately 95% to approximately 98% of FVIII in a non-covalent complex. Although endogenous VWF is an FVIII half-life limiting factor, it is also known that the binding of endogenous VWF to the FVIII protein protects FVIII in various ways. For example, full-length VWF (in the form of a multimer having a molecular weight of approximately 250 kDa) protects FVIII from proteolytic cleavage and FVIII activation, stabilizes the FVIII heavy chain and / or light chain, and prevents FVIII from being cleared by scavenger receptors. At the same time, however, endogenous VWF limits the FVIII half-life by preventing endocytosis 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 the half-life limiting factor that prevents the half-life of a chimeric protein fused to a half-life extender from being approximately twice as long as the half-life of wild-type FVIII. Accordingly, the present invention relates to using a VWF protein (e.g., a VWF fragment) comprising a D' domain and a D3 domain to prevent or inhibit the interaction between endogenous VWF and the FVIII protein, and at the same time to increase the half-life of the resulting FVIII protein by using a combination of an XTEN sequence and an Ig constant region or a portion thereof. Specifically, the present invention shows that a shorter XTEN sequence (i.e., an XTEN containing less than 288 amino acids in length, i.e., an XTEN shorter than 288 amino acids) is better in prolonging the half-life of the chimeric protein.

[0151] In one embodiment, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing the D' and D3 domains of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via an XTEN sequence therebetween, 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 consists of an amino acid sequence having a length between 12 and 287 amino acids. In other embodiments, the chimeric protein exhibits a longer half-life as 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 (such as AE288, such as SEQ ID NO:8). In 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 less 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.

[0152] The chimeric protein of the present invention may further comprise a second XTEN sequence that links the FVIII protein to the first Ig constant region or a portion thereof.

[0153] In certain embodiments, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing the D' domain and the D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof by a first XTEN sequence therebetween, 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 further comprises a second XTEN sequence inserted at one or more insertion sites within the FVIII protein or fused to the FVIII protein and / or the first Ig constant region or a portion thereof. Thus, in one embodiment, the second XTEN sequence is inserted at one or more insertion sites within the FVIII protein. In another embodiment, the second XTEN sequence is fused to the FVIII protein and / or the first Ig constant region or a portion thereof. In other embodiments, the second XTEN sequence is inserted at one or more insertion sites within the FVIII protein, and a third XTEN sequence is fused to the FVIII protein and / or the first Ig constant region or a portion thereof.

[0154] The second XTEN sequence and / or the third XTEN sequence can be XTEN amino acids of any length. For example, the second XTEN sequence and / or the third XTEN sequence are disclosed elsewhere herein, such as 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, SEQID NO:11, SEQ ID NO:17; SEQ ID NO:54; SEQ ID NO:19; SEQ ID NO:16; SEQ ID NO:18; SEQID NO:15; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQID 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 and / or the third XTEN sequence is AE288 or AG288, such as SEQ ID NO:8 or 19.

[0155] In certain embodiments, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof via an optional linker, wherein an optional XTEN sequence (X2) is inserted at one or more insertion sites within the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing the D' domain and the D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via 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 via a linker, and wherein the first polypeptide and the second polypeptide associate. In some embodiments, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof via an optional linker, wherein an optional XTEN sequence (X2) is inserted at one or more insertion sites within the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing the D' domain and the D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via 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 second Ig constant region or a portion thereof via a linker, and wherein the first polypeptide and the second polypeptide associate. In other embodiments, the linker that fuses 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 shown elsewhere herein. In certain embodiments, the linker is a thrombin-cleavable linker.

[0156] In some embodiments, the chimeric protein is two polypeptide chains, a first chain comprising the first polypeptide as described above and a second chain comprising the second polypeptide as described above. By way of 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 an optional XTEN sequence inserted at one or more insertion sites within 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 via an XTEN sequence (X1) therebetween, wherein the XTEN sequence (X1) contains less than 288 amino acids.

[0157] In certain embodiments, the chimeric protein is two polypeptide chains, with the first chain comprising the heavy chain of the FVIII protein and the second chain comprising, from the N-terminus to the C-terminus, the light chain of the FVIII protein, an optional XTEN sequence inserted at one or more insertion sites within the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof, and the first Ig constant region or a portion thereof, an optional linker (e.g., a cleavable linker), the VWF protein, an XTEN sequence (X1), a second optional linker (e.g., a cleavable linker), and the second Ig constant region or a portion thereof.

[0158] In other embodiments, the chimeric protein is three polypeptide chains, where (i) the first chain comprises the heavy chain of the FVIII protein, (ii) the second chain comprises the light chain of the FVIII protein, the first Ig constant region or a portion thereof, and an optional XTEN sequence inserted at one or more insertion sites within 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) the third chain comprises the VWF protein fused to the second Ig constant region or a portion thereof via an XTEN sequence (X1) therebetween, wherein the first and second chains associate via a non-covalent bond such as a metal bond, and the second and third chains associate via a covalent bond such as a disulfide bond.

[0159] In other embodiments, the chimeric protein is a single chain that comprises, from the N-terminus to the C-terminus, a single-chain FVIII protein, an optional XTEN sequence inserted at one or more insertion sites within the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof, and the first Ig constant region or a portion thereof, an optional linker (e.g., a cleavable linker), the VWF protein, an XTEN sequence (X1), a second optional linker (e.g., a cleavable linker), and the second Ig constant region or a portion thereof.

[0160] In certain embodiments, the chimeric protein comprises one of the following formulas (a)-(hh):

[0161] (a) FVIII - F1:F2 - L2 - X - L1 - V;

[0162] (b) FVIII - F1:V - L1 - X - L2 - F2;

[0163] (c) F1 - FVIII:F2 - L2 - X - L1 - V;

[0164] (d) F1 - FVIII:V - L1 - X - L2 - F2;

[0165] (e) FVIII - X2 - F1:F2 - L2 - X1 - L1 - V;

[0166] (f) FVIII-X2-F1:V-L1-X1-L2-F2;

[0167] (g) FVIII(X2)-F1:F2-L2-X1-L1-V;

[0168] (h) FVIII(X2)-F1:V-L1-X1-L2-F2;

[0169] (i) F1-X2-F1:F2-L2-X1-L1-V;

[0170] (j) F1-X2-F1:V-L1-X1-L2-F2;

[0171] (k) V-L1-X-L2-F2-L3-FVIII-L4-F1;

[0172] (l) V-L1-X-L2-F2-L3-F1-L4-FVIII;

[0173] (m) F1-L4-FVIII-L3-F2-L2-X-L1-V;

[0174] (n) FVIII-L4-F1-L3-F2-L2-X-L1-V;

[0175] (o) FVIII-L4-F1-L3-V-L1-X-L2-F2;

[0176] (p) FVIII-L4-F1-L3-F2-L2-X-L1-V;

[0177] (q) F2-L2-X-L1-V-L3-F1-L4-FVIII;

[0178] (r) F2-L2-X-L1-V-L3-FVIII-L4-F1;

[0179] (s) V-L1-X1-L2-F2-L3-FVIII(X2)-L4-F1;

[0180] (t) V-L1-X1-L2-F2-L3-F1-L4-FVIII(X2);

[0181] (u) F1-L4-FVIII(X2)-L3-F2-L2-X1-L1-V;

[0182] (v) F-L4-FVIII(X2)-L3-V-L1-X1-L2-F2;

[0183] (w)FVIII(X2)-L4-F1-L3-V-L1-X1-L2-F2;

[0184] (x)FVIII(X2)-L4-F1-L3-F2-L2-X1-L1-V;

[0185] (y)F2-L2-X1-L1-V-L3-F1-L4-FVIII(X2);

[0186] (z)F2-L2-X1-L1-V-L3-FVIII(X2)-L4-F1;

[0187] (aa)V-L1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1;

[0188] (bb)V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII;

[0189] (cc)F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V;

[0190] (dd)F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2;

[0191] (ee)FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1;

[0192] (ff)FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V;

[0193] (gg)F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or

[0194] (hh)F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2;

[0195] wherein V is a VWF protein, which comprises a D' domain and a D3 domain,

[0196] X or X1 is a first XTEN sequence containing less than 288 amino acids,

[0197] X2 is a second XTEN sequence,

[0198] FVIII comprises an FVIII protein,

[0199] FVIII(X2) comprises an FVIII protein having a second XTEN sequence inserted into one or more insertion sites within the FVIII protein,

[0200] F1 is a first Ig constant region or a portion thereof,

[0201] F2 is a second Ig constant region or a portion thereof,

[0202] L1, L2, L3, L4 or L5 is an optional linker,

[0203] (-) is a peptide bond; and

[0204] (:) is a covalent or non-covalent bond.

[0205] 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 a longer half-life as compared to a corresponding fusion protein of the formula in which X or X1 is AE288 (e.g., SEQ ID NO:8).

[0206] 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 less 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 IDNO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ IDNO:62; or SEQ ID NO:63.

[0207] In other embodiments, X2 comprises an amino acid sequence that is 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 in length, such as AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144, such as 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, such as SEQ ID NO:8 or 19.

[0208] In certain embodiments, the chimeric protein comprising X or X1 and / or X2 has an extended half-life compared to a chimeric protein without X or X1 and / or X2. In other embodiments, L1 and / or L2 is a cleavable linker. In other embodiments, L4 and / or L5 is a cleavable linker.

[0209] II.A. von Willebrand Factor (VWF) Protein

[0210] VWF (also known as F8VWF) is a large multimeric glycoprotein present in plasma and constitutively produced in endothelial cells (in Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a protein of 2,813 amino acids. Each monomer contains a number of specific domains with specific functions, namely the D' / D3 domain (which binds factor VIII), the A1 domain (which binds platelet GPIb receptor, heparin, and / or possibly collagen), the A3 domain (which binds collagen), the C1 domain (where the RGD domain binds platelet integrin αIIbβ3 when this domain is activated), and the "cysteine knot" domain at the C-terminus of the protein (VWF shares this domain with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ), and β-human chorionic gonadotropin (βHCG)).

[0211] In one embodiment, the VWF protein is a VWF fragment. As used herein, the term "VWF fragment" includes, but is not limited to, functional VWF fragments containing the D' domain and the D3 domain 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 between the FVIII protein and endogenous VWF. VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0212] The 2813 monomer amino acid sequence of human VWF is reported in Genbank under accession number NP_000543.2. The nucleotide sequence encoding human VWF is reported in Genbank under 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.

[0213] Table 1. VWF Sequences

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] As used herein, the VWF protein can be a VWF fragment comprising the D' domain and the D3 domain of VWF, wherein the VWF fragment binds to factor VIII (FVIII) and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment comprising the D' domain and the D3 domain can further comprise a VWF domain selected from the group consisting of: A1 domain, A2 domain, A3 domain, D1 domain, D2 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, 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 a fragment thereof; (2) the D1, D', and D3 domains of VWF or a fragment thereof; (3) the D2, D', and D3 domains of VWF or a fragment thereof; (4) the D1, D2, D', and D3 domains of VWF or a fragment thereof; or (5) the D1, D2, D', D3, and A1 domains of VWF or a fragment thereof. The VWF fragment described herein does not contain a site that binds to the VWF clearance receptor. In another embodiment, 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 comprise any other sequence linked to or fused to the VWF fragment. For example, the VWF fragment described herein can further comprise a signal peptide.

[0223] In one embodiment, the VWF fragment comprising the D' domain and the D3 domain binds to or associates with the FVIII protein. By binding to or associating with the FVIII protein, the VWF fragment of the present invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy and light chains of FVIII, and prevents FVIII from being cleared by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with the FVIII protein and blocks or prevents the FVIII protein from binding to phospholipids and activated protein C. By preventing or inhibiting the binding of the FVIII protein to endogenous full-length VWF, the VWF fragment of the present invention reduces the clearance of FVIII by the VWF clearance receptor and thus prolongs the half-life of the chimeric protein. Thus, the prolongation of the half-life of the chimeric protein is due to the binding of the VWF fragment lacking the VWF clearance receptor binding site to the FVIII protein and the shielding or protection of the FVIII protein from endogenous VWF containing the VWF clearance receptor binding site by the VWF fragment. The FVIII protein bound to or protected by the VWF fragment also allows for the 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, thereby further prolonging the FVIII half-life.

[0224] In one embodiment, the VWF protein suitable for the present invention comprises the D' domain and the D3 domain of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 866 of SEQ ID NO:21, and wherein the VWF protein prevents or inhibits the binding of endogenous VWF to FVIII. In another embodiment, the VWF protein comprises the D' domain and the D3 domain of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 867 to 1240 of SEQ ID NO:21, and wherein the VWF protein prevents or inhibits the binding of endogenous VWF to FVIII. In some embodiments, the VWF protein described herein comprises, consists essentially of, or consists of: the D' domain and the D3 domain of VWF, the domains being at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 1240 of SEQ ID NO:21, and wherein the VWF protein prevents or inhibits the binding of endogenous VWF to FVIII. In other embodiments, the VWF protein comprises, consists essentially of, or consists of: the D1, D2, D', and D3 domains, the domains being at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 23 to 1240 of SEQ ID NO:21, and wherein the VWF protein prevents or inhibits the binding of endogenous VWF to FVIII. In other embodiments, the VWF protein further comprises a signal peptide operably linked thereto.

[0225] In some embodiments, the VWF protein suitable for the present invention consists essentially of or consists of: (1) a D'D3 domain, a D1D'D3 domain, a D2D'D3 domain, or a D1D2D'D3 domain and (2) an additional VWF sequence of up to about 10 amino acids (e.g., any sequence from amino acid 764 to 1240 of SEQ ID NO: 21 to amino acid 764 to 1250 of SEQ ID NO: 21), up to about 15 amino acids (e.g., any sequence from amino acid 764 to 1240 of SEQ ID NO: 21 to amino acid 764 to 1255 of SEQ ID NO: 21), up to about 20 amino acids (e.g., any sequence from amino acid 764 to 1240 of SEQ ID NO: 21 to amino acid 764 to 1260 of SEQ ID NO: 21), up to about 25 amino acids (e.g., any sequence from amino acid 764 to 1240 of SEQ ID NO: 21 to amino acid 764 to 1265 of SEQ ID NO: 21), or up to about 30 amino acids (e.g., any sequence from amino acid 764 to 1240 of SEQ ID NO: 21 to amino acid 764 to 1260 of SEQ ID NO: 21). In certain embodiments, a VWF protein comprising a D' domain and a D3 domain or consisting essentially of a D' domain and a D3 domain is neither amino acids 764 to 1274 of SEQ ID NO: 21 nor 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.

[0226] In other embodiments, a VWF protein comprising a D'D3 domain linked to a D1D2 domain further comprises an intracellular cleavage site (e.g., a cleavage site for PACE (furin) or PC5), thereby allowing cleavage of the D1D2 domain from the D'D3 domain after expression. Non-limiting examples of intracellular cleavage sites are disclosed elsewhere herein.

[0227] In other embodiments, the VWF protein comprises the D' domain and the D3 domain, but does not comprise an amino acid sequence selected from the group consisting of: (1) amino acids 1241 to 2813 corresponding to SEQ ID NO: 21, (2) amino acids 1270 to amino acid 2813 corresponding to SEQ ID NO: 21, (3) amino acids 1271 to amino acid 2813 corresponding to SEQ ID NO: 21, (4) amino acids 1272 to amino acid 2813 corresponding to SEQ ID NO: 21, (5) amino acids 1273 to amino acid 2813 corresponding to SEQ ID NO: 21, (6) amino acids 1274 to amino acid 2813 corresponding to SEQ ID NO: 21, and any combination thereof.

[0228] In other embodiments, the VWF protein of the invention comprises, consists essentially of, or consists of: an amino acid sequence corresponding to the D' domain, the D3 domain, and the A1 domain, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1479 of SEQ ID NO: 21, and wherein the VWF protein prevents endogenous VWF from binding FVIII. In certain embodiments, the VWF protein is not amino acids 764 to 1274 of SEQ ID NO: 21.

[0229] In some embodiments, the VWF protein of the invention comprises a D' domain and a D3 domain, but does not comprise at least one VWF domain selected from the group consisting of: (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, B3 domain, (14) CK domain, C2 domain, C1 domain, B3 domain, 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, B2 domain, B1 domain and D4 domain, (17) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain and A3 domain, (18) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain and A2 domain, (19) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain, A2 domain and A1 domain, and (20) any combination thereof.

[0230] In other embodiments, the VWF protein comprises a D'D3 domain and one or more domains or modules. Examples of such domains or modules include, but are not limited to, those disclosed in Zhou et al., Blood, published online April 6, 2012: DOI 10.1182 / blood-2012-01-405134, which is incorporated herein by reference in its entirety. For example, the VWF protein may comprise a D'D3 domain and one or more domains or modules selected from the group consisting of: A1 domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL-4 module, C1 module, C2 module, C3 module, C4 module, C5 module, C5 module, C6 module, and any combination thereof.

[0231] In other embodiments, the VWF protein is linked to a heterologous moiety, wherein the heterologous moiety is linked to the N-terminus or C-terminus of the VWF protein or inserted 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 may be in the D' domain, the D3 domain, or both. The heterologous moiety may be a half-life extender.

[0232] In certain embodiments, the VWF proteins suitable for the present invention form multimers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers or higher order multimers. In other embodiments, the VWF protein is a monomer having only one VWF protein. In some embodiments, the VWF proteins of the present invention may have one or more amino acid substitutions, deletions, additions or modifications. In one embodiment, the VWF protein may include amino acid substitutions, deletions, additions or modifications such that the VWF protein cannot form disulfide bonds or cannot form dimers or multimers. In another embodiment, the amino acid substitutions are within the D' domain and the D3 domain. In certain embodiments, the VWF proteins suitable for the present invention contain at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residue 1099 and 1142 of SEQ ID NO:21. The at least one amino acid substitution can be any amino acid not naturally occurring in wild-type VWF. By way of 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 formation of multimers of the VWF protein.

[0233] In certain embodiments, the VWF proteins suitable for use herein may be further modified to improve its interaction with FVIII, such as improving the binding affinity for FVIII. As a non-limiting example, the VWF protein contains a serine residue at a residue corresponding to amino acid 764 of SEQID NO:21 and a lysine residue at a residue corresponding to amino acid 773 of SEQ ID NO:21. Residues 764 and / or 773 may contribute to the binding affinity of the VWF protein for FVIII. In other embodiments, the VWF proteins suitable for the present invention may have other modifications, such as the protein may be pegylated, glycosylated, hydroxyethylstarchylated or polysialylated.

[0234] II.B. XTEN Sequence

[0235] As used herein, an "XTEN sequence" refers to an extended-length polypeptide having a non-naturally occurring, substantially non-repetitive sequence consisting primarily of small hydrophilic amino acids, wherein the sequence has a low degree or no secondary or tertiary structure under physiological conditions. As a chimeric protein partner, XTEN can act as a carrier, thereby conferring certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties, for example, when linked to the VWF protein or FVIII sequence of the present invention to produce a chimeric protein. The desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as single-chain antibodies or Fc fragments of light or heavy chains.

[0236] The present invention provides a shorter XTEN sequence that, when the XTEN sequence is fused to a VWF protein and / or a second Ig constant region or a portion thereof, provides improved half-life extension properties compared to a longer XTEN sequence. 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., 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, or at least about 144 amino acids, but less than 288 amino acids. In other embodiments, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or a portion thereof is selected from AE36, AG36, AE42, AG42, AE72, AG72, AE144, or AG144. In one embodiment, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or a 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 without an XTEN sequence.

[0237] The chimeric proteins of the present invention may further comprise additional (second, third, or more) XTEN sequences. The additional XTEN sequences may 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, an additional XTEN sequence fused to the FVIII protein or the first Ig constant region or a portion thereof is a peptide or polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, the additional XTEN sequence is a peptide or polypeptide having greater than about 20 to about 3000 amino acid residues, greater than about 30 to about 2500 residues, greater than about 40 to about 2000 residues, greater than about 50 to about 1500 residues, greater than about 60 to about 1000 residues, greater than about 70 to about 900 residues, greater than about 80 to about 800 residues, greater than about 90 to about 700 residues, greater than about 100 to about 600 residues, greater than about 110 to about 500 residues, or greater than about 120 to about 400 residues.

[0238] The XTEN sequence (i.e., the XTEN sequence fused to the VWF protein and / or the second Ig constant region or a portion thereof or the XTEN sequence fused to the FVIII protein and / or the first Ig constant region or a portion thereof or inserted at one or more insertion sites within the FVIII protein) may comprise one or more sequence motifs having 9 to 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 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P). See US2010-0239554 A1.

[0239] In some embodiments, the XTEN sequence comprises non-overlapping sequence motifs, wherein 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 is composed of multiple non-overlapping sequence units selected from a single motif family selected from Table 2A, thereby generating a family sequence. As used herein, "family" means that the XTEN has motifs only selected from a single motif type from 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 the family motifs are selected to achieve desired properties, such as allowing incorporation of restriction sites by coding nucleotides, incorporation of cleavage sequences, or achieving better linkage to FVIII or VWF. In some embodiments of the XTEN family, the XTEN sequence comprises multiple non-overlapping sequence motif units of 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, such that the resulting XTEN exhibits the above homology ranges. In other embodiments, the XTEN comprises multiple motif sequence units from two or more motif families from Table 2A. These sequences can be selected to achieve the desired physical / chemical characteristics conferred by the amino acid composition of the motifs, including properties such as net charge, hydrophilicity, lack of secondary structure or lack of repeatability, as described more fully below. In the embodiments described in this paragraph above, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN having from about 36 to about 3000 amino acid residues.

[0240] Table 2A. XTEN sequence motifs and motif families having 12 amino acids

[0241] Motif family Motif sequence AD GESPGGSSGSES (SEQ ID NO:24) AD GSEGSSGPGESS (SEQ ID NO:25) AD GSSESGSSEGGPP (SEQ ID NO:26) AD GSGGEPSESGSS (SEQ ID NO:27) AE, AM GSPAGSPTSTEE (SEQ ID NO:28) AE, AM, AQ GSEPATSGSETP (SEQ ID NO:29) AE, AM, AQ GTSESATPESGP (SEQ ID NO:30) AE, AM, AQ GTSTEPSEGSAP (SEQ ID NO:31) AF, AM GSTSESPSGTAP (SEQ ID NO:32)

[0242] Motif family Motif sequence AF, AM GTSTPESGSASP (SEQ ID NO:33) AF, AM GTSPSGESSTAP (SEQ ID NO:34) AF, AM GSTSSTAESPGP (SEQ ID NO:35) AG, AM GTPGSGTASSSP (SEQ ID NO:36) AG, AM GSSTPSGATGSP (SEQ ID NO:37) AG, AM GSSPSASTGTGP (SEQ ID NO:38) AG, AM GASPGTSSTGSP (SEQ ID NO:39) AQ GEPAGSPTSTSE (SEQ ID NO:40) AQ GTGEPSSTPASE (SEQ ID NO:41) AQ GSGPSTESAPTE (SEQ ID NO:42) AQ GSETPSGPSETA (SEQ ID NO:43) AQ GPSETSTSEPGA (SEQ ID NO:44) AQ GSPSEPTEGTSA (SEQ ID NO:45) BC GSGASEPTSTEP (SEQ ID NO:46) BC GSEPATSGTEPS (SEQ ID NO:47) BC GTSEPSTSEPGA (SEQ ID NO:48) BC GTSTEPSEPGSA (SEQ ID NO:49) BD GSTAGSETSTEA (SEQ ID NO:50) BD GSETATSGSETA (SEQ ID NO:51) BD GTSESATSESGA (SEQ ID NO:52) BD GTSTEASEGSAS (SEQ ID NO:53)

[0243] ● denotes individual motif sequences that generate a "family sequence" when used together in various arrangements

[0244] In some embodiments, the XTEN sequences used in the present invention are at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of: AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908 and AG2004. See US2010-0239554 A1.

[0245] In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: AE42 (SEQ ID NO: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), 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 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_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 particular embodiment, the XTEN sequence is AE288. The amino acid sequences of certain XTEN sequences of the present invention are shown in Table 2B.

[0246] Table 2B. XTEN sequences

[0247]

[0248]

[0249]

[0250] In those embodiments in which the XTEN moiety has less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), or less than 100% of the sequence consists of sequence motifs from Table 3 or XTEN sequences from Tables 4 and 13 - 17, the other amino acid residues of XTEN are selected from any of the other 14 natural L - amino acids, but preferably from hydrophilic amino acids, such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) are dispersed throughout the XTEN sequence, within or between sequence motifs, or concentrated in one or more short segments of the XTEN sequence, for example to create a linker between the XTEN moiety and the FVIII or VWF moiety. In the cases where the XTEN moiety contains 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 is generally devoid of secondary structure, for example having no more than 2% α - helix or 2% β - sheet, as determined by the methods disclosed herein. Hydrophobic residues that are less favorable in constructing XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, the XTEN sequence 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 no amino acids of the following: cysteine (to avoid formation of disulfide bonds and oxidation), methionine (to avoid oxidation), asparagine, and glutamine (to avoid deamidation). Thus, in some embodiments, the XTEN moiety that also contains other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) has a sequence with less than 5% (as measured by the Chou - Fasman algorithm) of residues that contribute to α - helix and β - sheet formation, and has at least 90% or at least about 95% or more random coil formation, as measured by the GOR algorithm.

[0251] In other embodiments, the XTEN sequences used in the present invention affect the physical or chemical properties of the chimeric proteins of the present invention, such as pharmacokinetics. The XTEN sequences used in the present invention may exhibit one or more of the following advantageous properties: conformational flexibility, enhanced water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius. In certain embodiments, the XTEN sequence linked to the FVIII protein in the present invention increases pharmacokinetic properties, such as a longer terminal half-life or increased area under the curve (AUC), such that the chimeric proteins described herein have an increased in vivo residence time compared to wild-type FVIII. In other embodiments, the XTEN sequences used in the present invention increase pharmacokinetic properties, such as a longer terminal half-life or increased area under the curve (AUC), such that the FVIII protein has an increased in vivo residence time compared to wild-type FVIII.

[0252] One embodiment of the invention is an FVIII / VWF fusion protein that comprises an FVIII portion fused to an Fc region and a VWF portion fused to the Fc region, wherein an XTEN sequence (e.g., AE288) is inserted within the FVIII portion, and wherein an XTEN sequence having less than 288 amino acids (e.g., AE144) is inserted between the VWF portion and the Fc portion. As described in the examples, inserting 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 inserting an XTEN having 288 amino acids between the VWF portion and the Fc portion. For example, inserting an XTEN sequence having less than 288 amino acids between the VWF portion and the Fc portion in an FVIII / VWF fusion protein increases 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% relative to inserting an XTEN sequence having 288 amino acids. In one particular embodiment, the terminal half-life is increased by at least about 35% relative to inserting an XTEN having 288 amino acids. Inserting an XTEN sequence having less than 288 amino acids also increases the AUC value of the chimeric protein. In some embodiments, the AUC is increased by at least about 50%, at least about 100%, or at least about 200% relative to inserting an XTEN having 288 amino acids. In one particular embodiment, the AUC is increased by about two-fold. Inserting an XTEN sequence having less than 288 amino acids also decreases the clearance rate of the chimeric protein. For example, the clearance rate can be decreased 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% relative to inserting an XTEN sequence having 288 amino acids. Inserting an XTEN sequence having less than 288 amino acids increases the mean residence time (MRT) and / or decreases the steady-state apparent volume of distribution (Vss) relative to inserting an XTEN sequence having 288 amino acids.

[0253] A variety of methods and assays can be used to determine the physical / chemical properties of proteins containing XTEN sequences. The methods include, but are not limited to, analytical ultracentrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, reverse-phase HPLC, 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. Other methods are disclosed in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

[0254] Other examples of XTEN sequences that can be used in accordance with the present invention are disclosed in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1 or 2011 / 0172146A1 or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228A1, WO 2011028229A1, WO 2011028344A2 or WO 20130122617A1.

[0255] II.C. Factor VIII (FVIII) Protein

[0256] Unless otherwise specified, "FVIII protein" as used herein means a functional FVIII polypeptide that plays its normal role in blood 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" can be used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII function include, but are not limited to, the ability to activate blood coagulation, the ability to act as a cofactor for factor IX, or the ability to form a factor X activating enzyme complex with factor IX in the presence of Ca 2+ and phospholipids, which then converts factor X into its activated form Xa. The FVIII protein can be a human, porcine, canine, rat or murine FVIII protein. In addition, comparison between FVIII from humans and other species has identified conserved residues that may be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

[0257] Many tests can be used to evaluate the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (if the patient's plasma is mixed with normal plasma, is the abnormality corrected), coagulation factor assay, antiphospholipid antibody, D-dimer, genetic test (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry ( For example ) or euglobulin lysis time (ELT).

[0258] The aPTT test is a performance indicator that measures the efficacy of both the "intrinsic" coagulation pathway (also known as the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors (such as FVIII or FIX). It is used in conjunction with the prothrombin time (PT) that measures the extrinsic pathway.

[0259] ROTEM analysis provides information on the following complete hemostatic kinetics: clotting time, clot formation, clot stability, and lysis. Different parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many factors that affect these interactions. This assay can provide a complete overview of secondary hemostasis.

[0260] FVIII polypeptide and polynucleotide sequences are known, and many functional fragments, mutants, and modified forms are also known. The following shows an example of the human FVIII sequence (full length).

[0261] Table 3. Amino acid sequence of full-length factor VIII

[0262] (Full-length FVIII (FVIII signal peptide underlined; FVIII heavy chain double underlined; B domain in italics; and FVIII light chain in plain text)

[0263]

[0264] Table 4. Nucleotide sequence encoding full-length FVIII (SEQ ID NO: 66)*

[0265]

[0266]

[0267]

[0268] *The underlined nucleic acid encodes the signal peptide.

[0269] FVIII polypeptides include full-length FVIII, full-length FVIII minus the N-terminal Met, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and / or FVIII with complete or partial deletion of the B domain. In certain embodiments, FVIII variants include B domain deletions, whether partial or complete.

[0270] The sequence of native mature human FVIII is presented as SEQ ID NO:65. The native FVIII protein has the 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 respect to the positions of the primary amino acid sequence in SEQ ID NO:65, the A1 domain of human FVIII extends from Ala1 to approximately Arg336, the a1 spacer region extends from approximately Met337 to approximately Val374, the A2 domain extends from approximately Ala375 to approximately Tyr719, the a2 spacer region extends from approximately Glu720 to approximately Arg740, the B domain extends from approximately Ser741 to approximately Arg1648, the a3 spacer region extends from approximately Glu1649 to approximately Arg1689, the A3 domain extends from approximately Ser1690 to approximately Leu2025, the C1 domain extends from approximately Gly2026 to approximately Asn2072, and the C2 domain extends from approximately Ser2073 to Tyr2332. Except for specific proteolytic cleavage sites, the designation of the positions of the boundaries between the domains and regions of FVIII can vary in different references. Accordingly, the boundaries indicated herein are designated as approximate boundaries by use of the term "about".

[0271] The human FVIII gene was isolated and expressed in mammalian cells (Toole, J.J. et al., Nature 312:342-347 (1984); Gitschier, J. et al., Nature 312:326-330 (1984); Wood, W.I. et al., Nature 312:330-337 (1984); Vehar, G.A. et al., Nature 312:337-342 (1984); WO 87 / 04187; WO 88 / 08035; WO 88 / 03558; and U.S. Patent No. 4,757,006). The FVIII amino acid sequence was deduced from cDNA as shown in U.S. Patent No. 4,965,199. In addition, FVIII with partial or complete deletion of the B domain is shown in U.S. Patent Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B domain is replaced with the human factor V B domain as shown in U.S. Patent No. 5,004,803. The cDNA sequence and amino acid sequence encoding human factor VIII are shown as SEQ ID NOs: 1 and 2, respectively, of U.S. Application Publication No. 2005 / 0100990.

[0272] The porcine FVIII sequence was published in Toole, J. J. et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). In addition, the complete porcine cDNA sequence obtained by PCR amplification of the FVIII sequence from a porcine spleen cDNA library has been reported in Healey, J. F. et al., Blood 88:4209-4214 (1996). A hybrid human / porcine FVIII with all domains, all subunits, and substitutions in the specific amino acid sequence was disclosed in U.S. Patent No. 5,364,771 to Lollar and Runge and in WO 93 / 20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII and chimeric FVIII with porcine A1 and / or A2 domains replacing the corresponding human domains were reported in WO 94 / 11503. U.S. Patent No. 5,859,204 to Lollar, J. S. also discloses porcine cDNA and deduced amino acid sequences. U.S. Patent No. 6,458,563 discloses a B domain-deleted porcine FVIII.

[0273] U.S. Patent No. 5,859,204 to Lollar, J. S. reports FVIII functional mutants with reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463 to Lollar, J. S. also reports FVIII mutants with reduced immunoreactivity. Published U.S. Application No. 2005 / 0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

[0274] In one embodiment, FVIII (or the FVIII portion of a chimeric protein) can 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 a signal sequence) or the FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO:64 and amino acids 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 a signal sequence), wherein the FVIII has clotting activity, such as as a cofactor to activate factor IX to convert factor X into activated factor X. FVIII (or the FVIII portion of a chimeric protein) can be 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 a signal sequence). FVIII can further comprise a signal sequence.

[0275] As used herein, the "B domain" of FVIII is the same as that known in the art and defined by internal amino acid sequence identity and proteolytic cleavage sites, for example, residues Ser741 - Arg1648 of full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1 - Arg372; A2, residues Ser373 - Arg740; A3, residues Ser1690 - Asn2019; C1, residues Lys2020 - Asn2172; C2, residues Ser2173 - Tyr2332. The A3 - C1 - C2 sequence includes residues Ser1690 - Tyr2332. The remaining sequence residues Glu1649 - Arg1689 are commonly referred to as the a3 acidic region. The boundary positions of all domains including the B domain of porcine, murine, 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 BDD FVIII is (recombinant BDD FVIII), which has the same sequence as the factor VIII moiety having the sequence in Table 5. (The BDD FVIII heavy chain is double - underlined; the B domain is italicized; and the BDD FVIII light chain is in plain text). The nucleotide sequence encoding Table 6 (SEQ ID NO:68) is shown in Table 6.

[0276] Table 5. Amino acid sequence of B domain - deleted factor VIII (BDD FVIII)

[0277]

[0278] Table 6. Nucleotide sequence encoding BDD FVIII (SEQ ID NO:68)*

[0279]

[0280]

[0281] * The underlined nucleic acid encodes the signal peptide.

[0282] "B-domain deleted FVIII" may have a complete or partial deletion as disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In some embodiments, the B-domain deleted FVIII sequence of the present invention comprises any deletion disclosed in column 4, line 4 to column 5, line 28 and Examples 1-5 of U.S. Patent No. 6,316,226 (also in US 6,346,513). In another embodiment, the B-domain deleted factor VIII is S743 / Q1638 B-domain deleted factor VIII (SQ BDD FVIII) (e.g., factor VIII has a deletion from amino acid 744 to amino acid 1637, e.g., factor VIII has amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO:65, i.e., SEQ ID NO:67). In some embodiments, the B-domain deleted FVIII of the present invention has a deletion disclosed in column 2, lines 26-51 and Examples 5-8 of U.S. Patent No. 5,789,203 (as well as 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 described in the following: column 1, line 25 to column 2, line 40 of U.S. Patent No. 5,972,885; column 6, lines 1-22 and Example 1 of U.S. Patent No. 6,048,720; column 2, lines 17-46 of U.S. Patent No. 5,543,502; column 4, line 22 to column 5, line 36 of U.S. Patent No. 5,171,844; column 2, lines 55-68 of U.S. Patent No. 5,112,950, Figure 2and Example 1; columns 2 line 2 to column 19 line 21 and Table 2 of U.S. Patent No. 4,868,112; columns 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 columns 11 line 5 to column 13 line 39 of U.S. Patent No. 7,041,635; or columns 4 lines 25 - 53 of U.S. Patent No. 6,458,563. In some embodiments, the B domain-deleted FVIII lacks most of the B domain but still contains the B domain amino-terminal sequence necessary for proteolytic processing of the primary translation product into two polypeptide chains in vivo, as disclosed in WO 91 / 09122. In some embodiments, the B domain-deleted FVIII is constructed with the deletion of amino acids 747 - 1638, i.e., substantially complete deletion of the B domain. Hoeben R.C. et al. J. Biol. Chem. 265(13):7318 - 7323(1990). The 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 that are part of the present invention include the following: deletion of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939 - 5942)), 797 to 1562 (Eaton et al. Biochemistry (1986) 25:8343 - 8347)), 741 to 1646 (Kaufman (PCT published application No. WO 87 / 04187)), 747 - 1560 (Sarver et al., DNA (1987) 6:553 - 564)), 741 to 1648 (Pasek (PCT application No. 88 / 00831)), or 816 to 1598 or 741 to 1648 (Lagner (Behring Inst. Mitt. (1988) No. 82:16 - 25); EP 295597). In other embodiments, the BDD FVIII comprises an FVIII polypeptide containing a fragment of the B domain that retains one or more N-linked glycosylation sites, such as residues 757, 784, 828, 900, 963 or optionally 943 of the amino acid sequence corresponding to the full-length FVIII sequence.Examples of B domain fragments include the 226 - amino acid or 163 - amino acid B domains (i.e., retaining the first 226 or 163 amino acids of the B domain) disclosed in, for example, Miao, H.Z. et al., Blood 103(a):3412 - 3419 (2004); Kasuda, A et al., J. Thromb. Haemost. 6:1352 - 1359 (2008); and Pipe, S.W. et al., J. Thromb. Haemost. 9:2235 - 2242 (2011). In other embodiments, BDD FVIII further contains a point mutation at residue 309 (from Phe to Ser) to improve the expression of the BDD FVIII protein. See Miao, H.Z. et al., Blood 103(a):3412 - 3419 (2004). In other embodiments, BDDFVIII includes an 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, S.W. et al., J. Thromb. Haemost. 9:2235 - 2242 (2011). Each of the foregoing deletions can be made in any FVIII sequence.

[0283] In some embodiments, FVIII has a partial B domain. In some embodiments, the FVIII protein having a partial B domain is FVIII198. FVIII198 is a single - chain FVIII Fc molecule - 226N6 that contains a partial B domain. The number 226 represents the 226 N - terminal amino acids of the FVIII B domain, and N6 represents six N - glycosylation sites in the B domain.

[0284] In one embodiment, FVIII is cleaved after the arginine or corresponding arginine residue (in other variants) at amino acid 1648 (in full - length factor VIII or SEQ ID NO:65), amino acid 754 (in factor VIII with S743 / Q1638 B - domain deletion or SEQ ID NO:67), thereby generating a heavy chain and a light chain. In another embodiment, FVIII comprises a heavy chain and a light chain that are linked or associated by metal - ion - mediated non - covalent bonds.

[0285] In other embodiments, FVIII is single-chain FVIII and has not been cleaved after the arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO:65), amino acid 754 (in FVIII lacking the S743 / Q1638 B domain or SEQ ID NO:67), or the corresponding arginine residue (in other variants). The single-chain FVIII may comprise one or more amino acid substitutions. In one embodiment, the amino acid substitutions are at the residue corresponding to residue 1648, residue 1645, or both of the full-length mature factor VIII polypeptide (SEQ ID NO:65) or at the residue corresponding to residue 754, residue 751, or both of the SQ BDD factor VIII (SEQ ID NO:67). The amino acid substitution can be any amino acid other than arginine, such as 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.

[0286] FVIII can be further cleaved by thrombin and then activated to FVIIIa, thereby acting as a cofactor for activated factor IX (FIXa). The activated FIX then forms the tenase complex together with activated FVIII and converts factor X to activated factor X (FXa). For activation, FVIII is cleaved by thrombin after three arginine residues at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B domain-deleted FVIII sequence), and the cleavage produces FVIIIa with 50 kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chains. In one embodiment, the FVIII protein suitable for the present invention is inactive FVIII. In another embodiment, the FVIII protein is activated FVIII.

[0287] A protein having an FVIII polypeptide linked or associated with a VWF protein may 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 FVIII clotting activity, such as acting as a cofactor for activated factor IX to convert factor X to activated factor X (FXa).

[0288] As used herein, "hybrid" or "chimeric" polypeptides and proteins include a combination of a first polypeptide chain (e.g., a VWF protein fused to an XTEN sequence having less than 288 amino acids and a first Ig constant region or a portion thereof) and a second polypeptide chain (e.g., an FVIII protein fused to a second Ig constant region or a portion thereof), thereby forming a heterodimer. In one embodiment, the first polypeptide and the second polypeptide in the hybrid associate with each other through protein-protein interactions such as charge-charge or hydrophobic interactions. In another embodiment, the first polypeptide comprises a VWF protein-XTEN-Fc fusion protein and the second polypeptide comprises an FVIII-Fc fusion protein such that the hybrid is a heterodimer, wherein 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 an FVIII(X)-Fc fusion protein such that the hybrid is a heterodimer, wherein XTEN contains less than 288 amino acids. The first polypeptide and the second polypeptide may associate through a covalent bond (e.g., a disulfide bond) between the first Fc region and the second Fc region. The first polypeptide and the second polypeptide may further associate with each other through the binding between the VWF fragment and the FVIII protein.

[0289] The FVIII proteins suitable for use in the present invention may include FVIII having one or more additional XTEN sequences that do not affect the FVIII clotting activity. The XTEN sequence may be fused to the C-terminus or N-terminus of the FVIII protein or inserted between one or more sets of two amino acid residues in the FVIII protein, with the insertion not affecting the FVIII clotting activity or FVIII function. In one embodiment, the insertion improves the pharmacokinetic properties (e.g., half-life) of the FVIII protein. In another embodiment, the insertion may be multiple insertions, such as greater than two, three, four, five, six, seven, eight, nine, or ten 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 any combination thereof.

[0290] The FVIII protein linked to one or more XTEN sequences may be designated as FVIII(X2) or FVIII (a→b) -X-FVIII (c→d) , wherein FVIII (a→b) comprises, consists essentially of, or consists of: a first portion of the 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, and FVIII (c→d) comprises, consists essentially of, or consists of: a second portion of the FVIII protein from amino acid residue "c" to amino acid residue "d";

[0291] a is the N-terminal amino acid residue of the first part of the FVIII protein,

[0292] b is the C-terminal amino acid residue of the first part of the FVIII protein, but also the N-terminal amino acid residue of two amino acids at the insertion site of the XTEN sequence therein,

[0293] c is the N-terminal amino acid residue of the second part of the FVIII protein, but also the C-terminal amino acid residue of two amino acids at the insertion site of the XTEN sequence therein, and

[0294] d is the C-terminal amino acid residue of the FVIII protein, and

[0295] wherein the first part of the FVIII protein is different from the second part of the FVIII protein and together have a length sufficient to confer FVIII clotting activity on the FVIII protein.

[0296] In one embodiment, the first part of the FVIII protein and the second part of the FVIII protein are fragments of SEQ ID NO:65 [Full-length mature FVIII sequence] or SEQ ID NO:67 [FVIII with B domain deleted], for example, the N-terminal part and the C-terminal part, respectively. In certain embodiments, the first part of the FVIII protein comprises the A1 domain and the A2 domain of the FVIII protein. The second part of the FVIII protein comprises the A3 domain, the C1 domain and optionally the C2 domain. In other embodiments, the first part of the FVIII protein comprises the A1 domain and the A2 domain, and the second part of the FVIII protein comprises a part of the B domain, the A3 domain, the C1 domain and optionally the C2 domain. In other embodiments, the first part of the FVIII protein comprises the A1 domain, the A2 domain and a part of the B domain of the FVIII protein, and the second part of the FVIII protein comprises the A3 domain, the C1 domain and optionally the C2 domain. In other embodiments, the first part of the FVIII protein comprises the A1 domain, the A2 domain and the first part of the B domain of the FVIII protein. The second part of the FVIII protein comprises the second part of the B domain, the A3 domain, the C1 domain and optionally the C2 domain. In some embodiments, 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 upstream of the site where one or more XTEN sequences are inserted or linked, and "c" can be the amino acid residue 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 [FVIII amino acid sequence with B domain deleted] or SEQ ID NO:65 [Full-length FVIII], and 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.

[0297] In some aspects, the insertion site in the FVIII protein is located in one or more domains of the FVIII protein (which are the N-terminus, A1 domain, A2 domain, A3 domain, B domain, C1 domain, C2 domain, C-terminus or a combination of two or more thereof) or between two domains of the FVIII protein (which are the A1 domain and the a1 acidic region, and the a1 acidic region and the A2 domain, A2 domain and a2 acidic region, a2 acidic region and B domain, B domain and A3 domain, and A3 domain and C1 domain, C1 domain and C2 domain or any combination thereof). For example, the insertion sites where the XTE N sequence can be inserted are selected from the group consisting of: N-terminus and A1 domain, N-terminus and A2 domain, N-terminus and A3 domain, N-terminus and B domain, N-terminus and C1 domain, N-terminus and C2 domain, N-terminus and C-terminus, A1 domain and A2 domain, A1 domain and A3 domain, A1 domain and B domain, A1 domain and C1 domain, A1 domain and C2 domain, A1 domain and C-terminus, A2 domain and A3 domain, A2 domain and B domain, A2 domain and C1 domain, A2 domain and C2 domain, A2 domain and C-terminus, A3 domain and B domain, A3 domain and C1 domain, A3 domain and C2 domain, A3 domain and C-terminus, B domain and C1 domain, B domain and C2 domain, B domain and C-terminus, C1 domain and C2 domain, C1 domain and C-terminus, C2 domain and C-terminus and a combination of two or more thereof. Non-limiting examples of the insertion sites are listed in Tables 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0298] The FVIII protein with an XTEN sequence inserted immediately downstream of one or more amino acids in the FVIII protein (such as one or more XTEN insertion sites) or linked to the C-terminus or N-terminus retains FVIII activity after being linked to the XTEN sequence or after the XTEN sequence is inserted. The XTEN sequence can be inserted into the FVIII protein once or more than once, twice, three times, four times, five times or six times so that the insertion does not affect FVIII activity (i.e., the FVIII protein still retains its blood coagulation properties).

[0299] The FVIII protein applicable to the present invention can be linked to one or more XTEN polypeptides at the N-terminus or C-terminus of the FVIII protein through an optional linker, or inserted downstream of one or more amino acids (such as one or more XTEN insertion sites) in the FVIII protein through one or more optional linkers. In one embodiment, two amino acid residues at which the XTEN sequence is inserted or the amino acid residues to which the XTEN sequence is linked correspond to two or one amino acid residues selected from the group consisting of the residues in Tables 7, 8, 9, and 10 and any combination thereof of SEQ ID NO:65 [full-length mature FVIII].

[0300] In other embodiments, at least one XTEN sequence is inserted into any one or more of the XTEN insertion sites disclosed herein or any combination thereof. In one aspect, at least one XTEN sequence is inserted into one or more XTEN insertion sites disclosed in one or more amino acids in Table 7.

[0301] Table 7: Exemplary XTEN insertion sites

[0302]

[0303]

[0304]

[0305]

[0306] * Indicates the insertion point of XTEN based on the amino acid numbering of mature full-length human FVIII, where the insertion can be on the N-terminal side or C-terminal side of the indicated amino acid.

[0307] In some embodiments, one or more XTEN sequences are inserted within about six amino acids upstream or downstream of amino acids 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905, or 1910 of SEQ ID NO:65 or any combination thereof.

[0308] Table 8. Exemplary XTEN insertion ranges

[0309]

[0310] * The distance from the inserted residue refers to the relative number of amino acids from the N-terminus (negative number) or C-terminus (positive number) of the designated insertion residue (residue "0") at which the insertion can occur. The label "-x" refers to the insertion site being x amino acids from the N-terminal side of the designated insertion residue. Similarly, the label "+x" refers to the insertion site being x amino acids from the C-terminal side of the designated insertion residue.

[0311] For example, “-1, +2” indicates an insertion at the N-terminus or C-terminus of an amino acid residue represented as -1, 0, +1, or +2.

[0312] In other embodiments, one or more XTEN sequences are inserted downstream of one or more amino acids corresponding to one or more insertion sites selected from the group consisting of the one or more insertion sites in Table 9 that correspond to full-length mature human FVIII.

[0313] Table 9. Exemplary XTEN Insertion Sites or Ranges

[0314]

[0315]

[0316] * Indicates the range of insertion sites numbered relative to the amino acid numbering of mature human FVIII

[0317] In other embodiments, one or more XTENs are inserted into the B domain of FVIII. In one instance, the XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 740 and 1640 is optionally absent. In another instance, the XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 740 and 1690 is optionally absent. In other instances, the XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 741 and 1648 is optionally absent. In other instances, the XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 743 and 1638 is optionally absent. In other instances, the XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 745 and 1656 is optionally absent. In some instances, the XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 745 and 1657 is optionally absent. In certain instances, the XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 745 and 1667 is optionally absent. In other instances, the XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 745 and 1686 is optionally absent. In some other instances, the XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 747 and 1642 is optionally absent. In other instances, the XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO:65, wherein the FVIII sequence between amino acids 751 and 1667 is optionally absent.

[0318] In some embodiments, one or more XTENs are inserted into one or more amino acids immediately downstream of an amino acid at an insertion site selected from the group consisting of the amino acid residues in Table 10.

[0319] Table 10: FVIII XTEN Insertion Sites and Construct Labels

[0320]

[0321]

[0322] *Indicates the amino acid number of the mature FVIII protein

[0323] In one embodiment, one or more XTEN insertion sites are located within one or more surface-exposed flexible loop structures (such as permissive loops) of the FVIII protein. For example, at least one XTEN sequence can be inserted into each FVIII "A" domain containing at least two "permissive loops" in 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 vivo or in vitro in host cells. A permissive loop is a region that allows the insertion of at least one XTEN sequence and that also has high surface or solvent exposure and high conformational flexibility among other properties. The A1 domain contains the permissive loop-1 (A1-1) region and the permissive loop-2 (A1-2) region, the A2 domain contains the permissive loop-1 (A2-1) region and the permissive loop-2 (A2-2) region, and the A3 domain contains the permissive loop-1 (A3-1) region and the permissive loop-2 (A3-2) region.

[0324] In one aspect, the first permissive loop (A1-1) in the FVIII A1 domain is located between β-strand 1 and β-strand 2, and the second permissive loop (A1-2) in the FVIII A2 domain is located between β-strand 11 and β-strand 12. The first permissive loop (A2-1) in the FVIII A2 domain is located between β-strand 22 and β-strand 23, and the second permissive loop (A2-2) in the FVIII A2 domain is located between β-strand 32 and β-strand 33. The first permissive loop (A3-1) in the FVIII A3 domain is located between β-strand 38 and β-strand 39, and the second permissive loop (A3-2) in the FVIII A3 is located between β-strand 45 and β-strand 46. In certain aspects, the surface-exposed flexible loop structure containing A1-1 corresponds to the region from about amino acid 15 to about amino acid 45 of SEQ ID NO:65 in native mature human FVIII, such as the region from about amino acid 18 to about amino acid 41 of SEQ ID NO:65. In other aspects, the surface-exposed flexible loop structure containing A1-2 corresponds to the region from about amino acid 201 to about amino acid 232 of SEQ ID NO:65 in native mature human FVIII, such as the region from about amino acid 218 to about amino acid 229 of SEQ ID NO:65. In other aspects, the surface-exposed flexible loop structure containing A2-1 corresponds to the region from about amino acid 395 to about amino acid 421 of SEQ ID NO:65 in native mature human FVIII, such as the region from about amino acid 397 to about amino acid 418 of SEQ ID NO:65. In other embodiments, the surface-exposed flexible loop structure containing A2-2 corresponds to the region from about amino acid 577 to about amino acid 635 of SEQ ID NO:65 in native mature human FVIII, such as the region from about amino acid 595 to about amino acid 607 of SEQ ID NO:65. In certain aspects, the surface-exposed flexible loop structure containing A3-1 corresponds to the region from about amino acid 1705 to about amino acid 1732 of SEQ ID NO:65 in native mature human FVIII, such as the region from about amino acid 1711 to about amino acid 1725 of SEQ ID NO:65. In other aspects, the surface-exposed flexible loop structure containing A3-2 corresponds to the region from about amino acid 1884 to about amino acid 1917 of SEQ ID NO:65 in native mature human FVIII, such as the region from about amino acid 1899 to about amino acid 1911 of SEQ ID NO:65.

[0325] In another embodiment, one or more amino acids into which at least one XTEN sequence is inserted are located in the a3 domain, such as within amino acids 1649 to 1689 corresponding to the full-length mature FVIII polypeptide. In a particular embodiment, the XTEN sequence is inserted between amino acids 1656 and 1657 of SEQ ID NO:65 (full-length mature FVIII). In a particular embodiment, the FVIII protein comprising the XTEN sequence inserted immediately downstream of the amino acid corresponding to amino acid 1656 of SEQ ID NO:65 further comprises a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO:65.

[0326] In some embodiments, one or more insertion sites for one or more XTEN insertions are immediately downstream of one or more amino acids corresponding to mature full-length FVIII selected from the group consisting of:

[0327] (1) Amino acid 3, (2) amino acid 18, (3) amino acid 22,

[0328] (4) Amino acid 26, (5) amino acid 32, (6) amino acid 40,

[0329] (7) Amino acid 60, (8) amino acid 65, (9) amino acid 81,

[0330] (10) Amino acid 116, (11) amino acid 119, (12) amino acid 130,

[0331] (13) Amino acid 188, (14) amino acid 211, (15) amino acid 216,

[0332] (16) Amino acid 220, (17) amino acid 224, (18) amino acid 230,

[0333] (19) Amino acid 333, (20) amino acid 336, (21) amino acid 339,

[0334] (22) Amino acid 375, (23) amino acid 399, (24) amino acid 403,

[0335] (25) Amino acid 409, (26) amino acid 416, (26) amino acid 442,

[0336] (28) Amino acid 487, (29) amino acid 490, (30) amino acid 494,

[0337] (31) Amino acid 500, (32) amino acid 518, (33) amino acid 599,

[0338] (34) Amino acid 603, (35) Amino acid 713, (36) Amino acid 745,

[0339] (37) Amino acid 1656, (38) Amino acid 1711, (39) Amino acid 1720,

[0340] (40) Amino acid 1725, (41) Amino acid 1749, (42) Amino acid 1796,

[0341] (43) Amino acid 1802, (44) Amino acid 1827, (45) Amino acid 1861,

[0342] (46) Amino acid 1896, (47) Amino acid 1900, (48) Amino acid 1904,

[0343] (49) Amino acid 1905, (50) Amino acid 1910, (51) Amino acid 1937,

[0344] (52) Amino acid 2019, (53) Amino acid 2068, (54) Amino acid 2111,

[0345] (55) Amino acid 2120, (56) Amino acid 2171, (57) Amino acid 2188,

[0346] (58) Amino acid 2227, (59) Amino acid 2277, and

[0347] (60) Combinations of two or more thereof.

[0348] In one embodiment, the FVIII protein suitable for the present invention comprises two XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site and a second XTEN inserted into a second XTEN insertion site. Non-limiting examples of the first XTEN insertion site and the second XTEN insertion site are listed in Table 11.

[0349] Table 11. Exemplary insertion sites for two XTENs

[0350]

[0351]

[0352]

[0353] The two XTENs inserted or linked to the FVIII protein can be the same or different. In some embodiments, the FVIII protein suitable for the present invention comprises two XTEN sequences inserted into the FVIII protein, 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 (C-terminus) corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 18, 26, 40, 1656 or 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 18, 26 or 40 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 599 corresponding to SEQ ID NO: 65. In 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 acids 18, 26, 40, 399, 403, 1725, 1720, 1900, 1905 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 acids 18, 26 or 40 corresponding to SEQ ID NO: 65. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 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, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acids 18, 26 or 40 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 18 corresponding 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 (e.g., R1648A) and amino acid 1680 corresponding to SEQ ID NO:65 (e.g., Y1680F). In certain embodiments, the FVIII protein comprises two XTEN sequences, a first XTEN 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 of 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 1656.

[0354] In some embodiments, the FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN sequence, and a third XTEN sequence inserted into a third XTEN insertion site. The first, second, or third XTEN sequence may be the same or different. The first, second, and third insertion sites may be selected from the group of any of the insertion sites disclosed herein. In some embodiments, the FVIII protein comprising three XTEN sequences may further comprise a mutation or substitution, such as amino acid 1648 corresponding to SEQ ID NO:65, e.g., R1648A. By way of example, non-limiting examples of the first, second, and third XTEN insertion sites are listed in Table 12.

[0355] Table 12. Exemplary Insertion Sites for Three XTENs

[0356]

[0357]

[0358] 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 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 other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 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 other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 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 other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905 or 1910 corresponding to SEQ ID NO:65, the second XTEN sequence is inserted downstream of amino acid 745 corresponding to SEQ ID NO:65, and the third XTEN sequence is inserted downstream of amino acid 2332 corresponding to SEQ ID NO:65.

[0359] In other embodiments, the FVIII protein of the present invention comprises four XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third insertion site, and a fourth XTEN sequence inserted into a fourth insertion site. The first, second, third, and fourth XTEN sequences may be the same, different, or in a combination thereof. In some embodiments, the FVIII protein comprising four XTEN sequences may further comprise a mutation or substitution, such as amino acid 1648 corresponding to SEQ ID NO: 65, such as R1648A. Non-limiting examples of the first, second, third, and fourth XTEN insertion sites are listed in Table 13.

[0360] Table 13. Exemplary insertion sites for four XTENs

[0361]

[0362]

[0363]

[0364] In some embodiments, the FVIII protein comprises five XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, and a fifth XTEN sequence inserted into a fifth XTEN insertion site. The first, second, third, fourth, or fifth XTEN sequences may be the same, different, or in a combination thereof. Non-limiting examples of the first, second, third, fourth, and fifth insertion sites are listed in Table 14.

[0365] Table 14. Exemplary insertion sites for five XTENs

[0366]

[0367] In certain embodiments, the FVIII protein comprises six XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, a fifth XTEN sequence inserted into a fifth XTEN insertion site, and a sixth XTEN sequence inserted into a sixth XTEN insertion site. The first, second, third, fourth, fifth, or sixth XTEN sequences may be the same, different, or in a combination thereof. Examples of the six XTEN insertion sites include, but are not limited to, the insertion sites listed in Table 15.

[0368] Table 15. Exemplary XTEN Insertion Sites for Six XTENs

[0369]

[0370] In one particular instance, the first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, and the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65 (full-length mature FVIII). In another instance, the first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO:65, and the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In some instances, the first XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, and the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In other instances, the first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, and the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In other embodiments, the first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO:65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, and the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In other embodiments, the first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO:65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, and the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65. In certain embodiments, the first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65, and the third XTEN is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO:65. In some embodiments, the first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO:65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO:65, the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:65, and the fourth XTEN is inserted between 1900 and 1901 corresponding to SEQ ID NO:65.

[0371] 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 of B-domain-deleted Factor VIII.

[0372] In some embodiments, the chimeric proteins of the invention comprise two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence that is at least about 80%, 90%, 95% or 100% identical to a sequence selected from 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-267 (SEQ ID NO:81), FVIII-268 (SEQ ID NO:82), FVIII-269 (SEQ ID NO:83), FVIII-271 (SEQID NO:84) or FVIII-272 (SEQ ID NO:85), 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.

[0373] II.D. Ig constant region or a portion thereof

[0374] The chimeric proteins of the invention also include two Ig constant regions or portions thereof, fused to the first Ig constant region or a portion thereof of the FVIII protein via an optional linker, and fused to the second Ig constant region or a portion thereof of the VWF protein via an XTEN sequence having less than 288 amino acids. The Ig constant region or a portion thereof can be combined with the XTEN sequence and the VWF protein to improve the pharmacokinetic or pharmacodynamic properties of the chimeric protein. In certain embodiments, the Ig constant region or a portion thereof extends the half-life of the molecule fused to the Ig constant region or a portion thereof.

[0375] The Ig constant region contains domains designated as CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may contain three or four CH domains. The constant region of some isotypes (e.g., IgG) also contains a hinge region. See Janeway et al 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

[0376] The Ig constant region or a portion thereof used to generate the chimeric proteins of the invention can be obtained from many different sources. In some embodiments, the Ig constant region or a portion thereof is derived from human Ig. However, it should be understood that the Ig constant region or a portion thereof can be derived from the Ig of another mammalian species, including, for example, rodents (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. In addition, the Ig constant region or a portion thereof can be derived from any Ig class (including IgM, IgG, IgD, IgA, and IgE) and any Ig isotype (including IgG1, IgG2, IgG3, and IgG4). In one embodiment, human isotype IgG1 is used.

[0377] Multiple Ig constant region gene sequences (e.g., human constant region gene sequences) can be obtained in the form of publicly available deposits. Constant region domain sequences with specific effector functions (or lacking specific effector functions) or with specific modifications to reduce immunogenicity can be selected. Many antibodies and antibody-encoding gene sequences have been published, and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences or portions thereof) can be obtained from these sequences using techniques recognized in the art. The genetic material obtained using any of the foregoing methods can then be altered or synthesized to obtain the polypeptides of the invention. It should be further understood that the scope of the invention encompasses alleles, variants, and mutations of the constant region DNA sequences.

[0378] The sequence of an Ig constant region or a portion thereof can be cloned, for example, using polymerase chain reaction and primers selected to amplify the target domain. To clone the sequence of an Ig constant region or a portion thereof from an antibody, mRNA can be isolated from a hybridoma, spleen, or lymphocyte, reverse transcribed into DNA, and the antibody gene amplified by PCR. PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, "PCR Protocols: A Guide to Methods and Applications" edited by Innis et al., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR can be primed by consensus constant region primers or by more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, libraries can be screened with consensus primers or larger homology probes (such as murine constant region probes). Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of a purified antibody (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 U.S. Patent No. 5,658,570, filed January 25, 1995, which is incorporated herein by reference.

[0379] The Ig constant region used herein can include all domains and hinge regions or portions thereof. In one embodiment, the Ig constant region or a portion thereof comprises the CH2 domain, the CH3 domain, and the hinge region, i.e., the Fc region or the FcRn binding partner.

[0380] As used herein, the term "Fc region" is defined as the portion of a polypeptide corresponding to the Fc region of a native Ig, i.e., as formed by the dimeric association of the corresponding Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, the term "genetically fused Fc region" or "single-chain Fc region" (scFc region) as used herein refers to a synthetic dimeric Fc region comprising Fc domains genetically linked within a single polypeptide chain (i.e., encoded in a single continuous genetic sequence).

[0381] In one embodiment, the "Fc region" refers to a portion of a single Ig heavy chain that begins in the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, considering the first residue of the heavy chain constant region as 114) and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge domain, CH2 domain, and CH3 domain.

[0382] Depending on the Ig isotype, the Fc region of the Ig constant region may include CH2, CH3, and CH4 domains as well as the hinge region. Chimeric proteins containing the Fc region of an Ig confer several desirable properties on the chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03 / 077834; US2003-0235536A1), the disclosures of which are incorporated herein by reference in their entireties.

[0383] The Ig constant region or a portion thereof can be an FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the intestinal lumen, lung airways, nasal surface, vaginal surface, colon, and rectal surface (U.S. Patent No. 6,485,726). An FcRn binding partner is the portion of an Ig that binds FcRn.

[0384] FcRn receptors have 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). The FcRn receptor binds IgG (but not other Ig classes such as IgA, IgM, IgD, and IgE) at a relatively low pH and actively transports IgG across cells in the luminal to serosal direction, and then releases IgG at the relatively high pH found in interstitial fluid. It is expressed in adult epithelial tissues (U.S. Patent Nos. 6,485,726, 6,030,613, 6,086,875;; WO 03 / 077834; US2003-0235536A1), including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358), and nasal epithelium; vaginal surface; and biliary surface.

[0385] FcRn-binding ligands applicable to the present invention encompass molecules that can be specifically bound by the FcRn receptor, including intact IgG, the Fc fragment of IgG, and other fragments including the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds the FcRn receptor has been described based on X-ray crystallography (Burmeister et al 1994, Nature 372:379). The major contact region of Fc with FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. FcRn-binding ligands include intact IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. Major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References to amino acid numbering of Ig or Ig fragments are based on Kabat et al 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[0386] The Fc region or FcRn-binding ligand bound to FcRn can effectively shuttle across the epithelial barrier via FcRn, thereby providing a non-invasive means of systemically administering a desired therapeutic molecule. Additionally, fusion proteins containing an Fc region or FcRn-binding ligand are endocytosed by cells expressing FcRn. However, instead of being marked for degradation, these fusion proteins are recycled back into the circulation, thereby increasing the in vivo half-life of these proteins. In certain embodiments, a portion of the Ig constant region is an Fc region or FcRn-binding ligand that typically associates with another Fc region or another FcRn-binding ligand via disulfide bonds and other non-specific interactions to form dimers and higher-order multimers.

[0387] Two FcRn receptors can bind a single Fc molecule. Crystallographic data indicate that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking an FcRn-binding ligand (such as the Fc fragment of IgG) to a bioactive molecule provides a means of delivering the bioactive molecule orally, buccally, sublingually, rectally, vaginally, in the form of an aerosol administered via the nasal or pulmonary route, or via the ocular route. In another embodiment, the chimeric protein can be administered invasively, such as subcutaneously or intravenously.

[0388] The FcRn-binding moiety is a molecule or a portion thereof that can be specifically bound by the FcRn receptor and subsequently actively transported by the FcRn receptor of the Fc region. Specific binding means that two molecules form a relatively stable complex under physiological conditions. Specific binding is characterized by a relatively high affinity and a low to medium avidity, as distinguished from non-specific binding, which typically has a relatively low affinity and a medium to high avidity. Generally, when the association constant KA is higher than 10 6 M -1 or higher than 10 8 M -1 , the binding is considered to be specific. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting specific binding. Appropriate binding conditions such as molecular concentration, ionic strength of the solution, temperature, allowable binding time, blocker (e.g., serum albumin, casein) concentration, etc. can be optimized by a person skilled in the art using conventional techniques.

[0389] In certain embodiments, the chimeric proteins of the invention comprise one or more truncated Fc regions, which nonetheless are sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the FcRn-binding portion of the Fc region (i.e., the FcRn-binding moiety) comprises approximately amino acids 282-438 (EU numbering) of IgG1, wherein the major contact sites are 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 can comprise or consist of the FcRn-binding moiety. The FcRn-binding moiety can be derived from the heavy chain of any isotype including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the FcRn-binding moiety from an antibody having the human isotype IgG1 is used. In another embodiment, the FcRn-binding moiety from an antibody having the human isotype IgG4 is used.

[0390] In another embodiment, the "Fc region" includes the amino acid sequence of the Fc domain or an amino acid sequence derived from the Fc domain. In certain embodiments, the Fc region comprises at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain (about amino acids 216 - 230 of the antibody Fc region, according to EU numbering), a CH2 domain (about amino acids 231 - 340 of the antibody Fc region, according to EU numbering), a CH3 domain (about amino acids 341 - 438 of the antibody Fc region, according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, the Fc region comprises the complete Fc domain (i.e., the hinge domain, CH2 domain, and CH3 domain). In some embodiments, the Fc region comprises, consists essentially of, or consists of: a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In 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 the amino acids corresponding to EU numbers 221 to 447.

[0391] The Fc region, denoted herein as F, F1, or F2, can be obtained from many different sources. In one embodiment, the Fc region of the polypeptide is derived from human Ig. However, it should be understood that the Fc region can be derived from the Ig of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, or guinea pig) or non - human primate (e.g., chimpanzee, macaque) species. In addition, the polypeptide of the Fc domain or a portion thereof can be derived from any Ig class (including IgM, IgG, IgD, IgA, and IgE) and any Ig isotype (including IgG1, IgG2, IgG3, and IgG4). In another embodiment, human isotype IgG1 is used.

[0392] In certain embodiments, the Fc variant confers a change in at least one effector function conferred by the Fc region comprising the wild - type Fc domain (e.g., the ability of the Fc region to bind to Fc receptors (e.g., FcγRI, FcγRII, or FcγRIII) or complement proteins (e.g., C1q), or to trigger antibody - dependent cell cytotoxicity (ADCC), phagocytosis, or complement - dependent cell cytotoxicity (CDCC) is improved or reduced). In other embodiments, the Fc variant provides engineered cysteine residues.

[0393] The Fc region of the present invention can employ Fc variants known in the art that confer effector functions and / or changes in FcR or FcRn binding (e.g., enhancement or reduction). Specifically, the binding molecules of the present invention can include, for example, changes (e.g., substitutions) at one or more amino acid positions disclosed in: International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2; US Patent Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or US Patents 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091, each of which is incorporated herein by reference. In one embodiment, specific changes (e.g., specific substitutions of one or more amino acids disclosed in the art) can be made at one or more of the disclosed amino acid positions. In another embodiment, different changes (e.g., different substitutions of one or more of the amino acid positions disclosed in the art) can be made at one or more of the disclosed amino acid positions.

[0394] The Fc region of IgG or an FcRn binding partner can be modified according to well-recognized procedures such as site-directed mutagenesis to produce a modified IgG or its Fc fragment or portion that will be bound by FcRn. Such modifications include modifications distal to the FcRn contact site that maintain or even enhance binding to FcRn and modifications within the contact site. For example, the following single amino acid residues in human IgG1 Fc (Fcγ1) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A and K447A, where, for example, P238A represents substitution of the wild-type proline at position number 238 with alanine. For example, one particular embodiment has an N297A mutation, thereby removing a highly conserved N-glycosylation site. In addition to alanine, other amino acids can substitute for the wild-type amino acid at the positions specified above. Mutations can be introduced singly into the Fc, resulting in more than a hundred Fc regions that are different from the native Fc. Additionally, combinations of two, three, or more of these individual mutations can be introduced together, resulting in hundreds more Fc regions.In addition, one Fc region of the construct of the present invention can be mutated, and the other Fc region of the construct is not mutated at all, or both of them can be mutated, but the mutations are different.

[0395] Certain of the above mutations can confer new functionality to the Fc region or the FcRn binding partner. For example, one embodiment has N297A, thereby removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing the circulating half-life of the Fc region, and rendering the Fc region unable to bind FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA without compromising the affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As another example of new functionality resulting from the above mutations, the affinity for FcRn can in some cases be increased beyond the affinity of the wild type for FcRn. This increase in affinity can reflect an increase in the "association" rate, a decrease in the "dissociation" rate, or both an increase in the "association" rate and a decrease in the "dissociation" rate. Examples of mutations believed to confer an increase in affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0396] In addition, at least three human Fcγ receptors appear to recognize binding sites on IgG in the lower hinge region, typically amino acids 234-237. Thus, another example of reduced new functionality and potential immunogenicity can be generated by mutations in this region, such as, for example, by substituting the amino acids 233-236 "ELLG" of human IgG1 with the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that when the mutation is introduced, FcγRI, FcγRII, and FcγRIII, which mediate various effector functions, will not bind IgG1. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0397] In one embodiment, the Ig constant region or a portion thereof (e.g., the Fc region) is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO:89 or SEQ ID NO:3 of U.S. Patent No. 5,739,277), and optionally further comprises 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 U.S. Patent No. 5,739,277).

[0398] In another embodiment, the immunoglobulin constant region or a portion thereof comprises an amino acid sequence in the hinge region or a portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or a portion thereof. The disulfide bonds formed by the immunoglobulin constant region or a portion thereof place 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 FVIII. Thus, the disulfide bonds between the first immunoglobulin constant region or a portion thereof and the second immunoglobulin constant region or a portion thereof prevent the interaction between endogenous VWF and the FVIII protein. This inhibition of the interaction between VWF and the FVIII protein allows the half-life of the chimeric protein to exceed twice the limit. The hinge region or a portion thereof may further be linked to one or more of the following domains: CH1, CH2, CH3, fragments thereof, and any combination thereof. In a particular embodiment, the immunoglobulin constant region or a portion thereof is the hinge region and CH2.

[0399] In certain embodiments, the Ig constant region or a portion thereof is hypogalactosylated. For example, a chimeric protein comprising two Fc regions or FcRn binding partners may contain a first glycosylated Fc region (e.g., a glycosylated CH2 region) or FcRn binding partner and a second non-glycosylated Fc region (e.g., a non-glycosylated CH2 region) or FcRn binding partner. In one embodiment, a linker may be inserted between the glycosylated Fc region and the non-glycosylated Fc region. In another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all Fc regions are glycosylated. In other embodiments, the Fc region may be non-glycosylated, i.e., no Fc moiety is glycosylated.

[0400] In certain embodiments, the chimeric proteins of the invention comprise amino acid substitutions in the Ig constant region or a portion thereof (e.g., Fc variants) that alter the antigen-independent effector functions of the Ig constant region, particularly the circulating half-life of the protein.

[0401] When compared to proteins lacking such substitutions, the proteins exhibit increased or decreased binding to FcRn and, accordingly, increased or decreased half-lives in serum. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and the molecules have applicable uses in methods of treating mammals where a long half-life of the administered polypeptide is desired, for example, for the treatment of chronic diseases or conditions (see, e.g., U.S. Patents 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and the molecules are also suitable for administration to mammals, for example, when a shortened circulation time may be advantageous, such as for in vivo diagnostic imaging or in cases where the starting polypeptide has toxic side effects when present in the circulation for an extended period. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are thus also suitable for treating diseases or conditions in pregnant women. In addition, other applications where a reduced FcRn binding affinity may be desired include those where targeting to the brain, kidney, and / or liver is required. In one exemplary embodiment, the chimeric proteins of the invention exhibit reduced transport across the epithelium of the glomerulus from the vascular structure. In another embodiment, the chimeric proteins of the invention exhibit reduced transport from the brain across the blood-brain barrier (BBB) into the vascular compartment. 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 within the "FcRn binding loop" of the Ig constant region. The FcRn binding loop comprises amino acid residues 280-299 of the wild-type full-length Fc region (according to EU numbering). In other embodiments, the Ig constant region or a portion thereof having an altered FcRn binding affinity in the chimeric proteins of the invention comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions within the FcRn "contact region". As used herein, the term The FcRn "contact region" includes residues at the following positions in the 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 a portion thereof of the invention having an altered FcRn binding affinity includes at least one Fc region or FcRn binding partner having one or more amino acid substitutions at an amino acid position corresponding to any 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 PCT Publication No. WO05 / 047327, which is incorporated herein by reference.

[0402] The Fc region or FcRn binding partner used in the present invention may also comprise amino acid substitutions that are recognized in the art to alter the glycosylation of the chimeric protein. For example, the Fc region or FcRn binding partner of the chimeric protein linked to the VWF fragment or FVIII protein may comprise an Fc region having a mutation that results in reduced glycosylation (e.g., N-linked or O-linked glycosylation), or may comprise a wild-type Fc portion with an altered glycoform (e.g., low fucose or afucosylated glycan).

[0403] In one embodiment, the unprocessed chimeric protein of the invention may comprise a genetically fused Fc region (i.e., scFc region), and two or more of the constituent Ig constant regions or portions thereof of the Fc region are independently selected from the Ig constant regions or portions thereof described herein. In one embodiment, the Fc regions of the dimeric Fc region are the same. In another embodiment, at least two Fc regions are different. For example, the Fc regions or FcRn binding partners of the proteins of the invention comprise the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In other embodiments, the Fc regions or FcRn binding partners of the proteins of the invention may differ in sequence at one or more amino acid positions. For example, at least two Fc regions or FcRn binding partners may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions.

[0404] II.E. Linker

[0405] 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 blood coagulation. In one embodiment, the cleavable linker allows cleavage from the XTEN sequence at the site of the blood coagulation cascade, and thus cleavage of a portion of the chimeric protein (such as the VWF protein), thereby enabling activated FVIII (FVIIIa) to have its FVIIIa activity. Another type of linker is a processable linker, which contains an intracellular cleavage site and can thus be cleaved by intracellular processing enzymes in the host cell, allowing proper expression of the polypeptide and formation of the chimeric protein.

[0406] One or more linkers can be present between any two proteins in the chimeric protein. In one embodiment, the chimeric protein comprises a first polypeptide that comprises (i) an FVIII protein and (ii) a first Ig constant region or a portion thereof; and a second polypeptide that comprises (iii) a VWF protein, (iv) a linker (such as a cleavable linker), (v) an XTEN sequence, and (vi) a second Ig constant region or a portion thereof. In another embodiment, the chimeric protein comprises a first polypeptide that comprises (i) an FVIII protein and (ii) a first Ig constant region or a portion thereof; and a second polypeptide that comprises (iii) a VWF protein, (iv) an XTEN sequence, (v) a linker (such as a cleavable linker), and (vi) a second Ig constant region or a portion thereof. In other embodiments, the chimeric protein comprises a first polypeptide that comprises (i) an FVIII protein and (ii) a first Ig constant region or a portion thereof; and a second polypeptide that comprises (iii) a VWF protein, (iv) a first linker (such as a cleavable linker), (v) an XTEN sequence, (vi) a second linker (such as a cleavable linker), and (vii) a second Ig constant region or a portion thereof. In some embodiments, the first polypeptide further comprises a linker, such as a cleavable linker, between the FVIII protein and the first Ig constant region.

[0407] In certain embodiments, the chimeric protein comprises a single chain that includes (i) an FVIII protein, (ii) a first Ig constant region or a portion thereof, (iii) a linker (e.g., a cleavable linker), (iv) a VWF protein, (v) an XTEN sequence, and (vi) a second Ig constant region or a portion thereof. In other embodiments, the chimeric protein comprises a single chain that includes (i) an FVIII protein, (ii) a first Ig constant region 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 second Ig constant region or a portion thereof. After the chimeric protein is expressed in a host cell, the cleavable linker can be cleaved; thus, the chimeric protein produced in the host cell can be in a final form that includes two or three polypeptide chains.

[0408] Linkers suitable for use 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 comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids. In one embodiment, the linker includes an XTEN sequence. Other examples of XTEN can be used in accordance with the present invention and are disclosed in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1 or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO2010144508 A1, WO 2011028228A1, WO 2011028229A1, or WO 2011028344A2. In another embodiment, the linker is a PAS sequence.

[0409] 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 comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids or 900-1000 amino acids.

[0410] Examples of linkers are well known in the art. In one embodiment, the linker comprises the sequence G n . The linker can comprise the sequence (GA) n . The linker can comprise the sequence (GGS) n . In other embodiments, the linker comprises (GGGS) n (SEQ ID NO:101). In other embodiments, the linker comprises the sequence (GGS) n (GGGGS) n (SEQ ID NO:95). In these cases, n can be an integer from 1-100. In other cases, n can be an integer from 1-20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Examples of linkers include but are not limited to GGG, SGGSGGS (SEQ ID NO:96), GGSGGSGGSGGSGGG (SEQ ID NO:97), GGSGGSGGGGSGGGGS (SEQ ID NO:98), GGSGGSGGSGGSGGSGGS (SEQ ID NO:99) or GGGGSGGGGSGGGGS (SEQ ID NO:100). The linker does not eliminate or attenuate the VWF protein activity or the coagulation activity of factor VIII. Optionally, the linker enhances the VWF protein activity or the coagulation activity of the factor VIII protein, for example by further reducing the steric hindrance effect and making the VWF protein or factor VIII moiety more accessible to its target binding site.

[0411] In one embodiment, the length of the linker suitable for the chimeric protein is 15 - 25 amino acids. In another embodiment, the length of the linker suitable for the chimeric protein is 15 - 20 amino acids. In some embodiments, the length of the linker for the chimeric protein is 10 - 25 amino acids. In other embodiments, the length of the linker for the chimeric protein is 15 amino acids. In other embodiments, the linker for the chimeric protein is (GGGGS) n (SEQ ID NO:94), where G represents glycine, S represents serine, and n is an integer from 1 - 20.

[0412] II.F. Cleavage Sites

[0413] A cleavable linker can incorporate moieties that can be cleaved chemically (e.g., hydrolyzing an ester bond), enzymatically (i.e., incorporating 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.

[0414] In one embodiment, the cleavable linker contains 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 contains a heterologous amino acid linker sequence or polymer as described herein and one or more cleavable sites.

[0415] In certain embodiments, the cleavable linker contains one or more cleavage sites that can be cleaved in a host cell (i.e., intracellular processing sites). Non - limiting examples of cleavage sites include RRRR (SEQ ID NO:102), RKRRKR (SEQ ID NO:103), and RRRRS (SEQ ID NO:104).

[0416] In some embodiments, the cleavable linker comprises the a1 domain from FVIII, the a2 domain from FVIII, the a3 domain from FVIII, a thrombin-cleavable site comprising X-V-P-R (SEQ ID NO:105) and a PAR1 extracellular binding site interacting motif (wherein X is an aliphatic amino acid), or any combination thereof. The a2 domain is included, which comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to that corresponding to Glu720 to Arg740 of full-length FVIII, wherein the a2 domain is capable of being cleaved by thrombin. In certain embodiments, the cleavable linker suitable for the present invention comprises an a2 domain that comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO:106). In other embodiments, the cleavable linker for the present invention comprises an a1 domain that comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to that corresponding to Met337 to Arg372 of full-length FVIII, wherein the a1 domain is capable of being cleaved by thrombin. In certain embodiments, the a1 domain comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO:107). In some embodiments, the cleavable linker of the present invention comprises an a3 domain that comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to that corresponding to Glu1649 to Arg1689 of full-length FVIII, wherein the a3 domain is capable of being cleaved by thrombin. In certain embodiments, the cleavable linker suitable for the present invention comprises an a3 domain that comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO:108).

[0417] In other embodiments, the cleavable linker comprises a thrombin cleavage site that comprises X-V-P-R (SEQ ID NO: 105) and a PAR1 exosite interaction motif, and wherein the PAR1 exosite interaction motif comprises S-F-L-L-R-N (SEQ ID NO: 109). The PAR1 exosite interaction motif may further comprise an amino acid sequence selected from: P, P-N, P-N-D, P-N-D-K (SEQ ID NO: 110), P-N-D-K-Y (SEQ ID NO: 111), P-N-D-K-Y-E (SEQ ID NO: 112), P-N-D-K-Y-E-P (SEQ ID NO: 113), P-N-D-K-Y-E-P-F (SEQ ID NO: 114), P-N-D-K-Y-E-P-F-W (SEQ ID NO: 115), P-N-D-K-Y-E-P-F-W-E (SEQ ID NO: 116), P-N-D-K-Y-E-P-F-W-E-D (SEQ ID NO: 117), P-N-D-K-Y-E-P-F-W-E-D-E (SEQ ID NO: 118), P-N-D-K-Y-E-P-F-W-E-D-E-E (SEQ ID NO: 119), P-N-D-K-Y-E-P-F-W-E-D-E-E-S (SEQ ID NO: 120), or any combination thereof. In some embodiments, the aliphatic amino acids are selected from glycine, alanine, valine, leucine, or isoleucine.

[0418] In other embodiments, the cleavable linker comprises one or more cleavage sites that are cleaved by a protease after administration of the chimeric protein comprising the cleavable linker to a subject. In one embodiment, the cleavage site is cleaved by a protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, MMP-12, MMP-13, MMP-17, and MMP-20. In another embodiment, the cleavage site is selected from the group consisting of: FXIa cleavage site (e.g., KLTR↓AET (SEQ ID NO:121)), FXIa cleavage site (e.g., DF TR↓VVG (SEQ ID NO:122)), FXIIa cleavage site (e.g., TMTR↓IVGG (SEQ ID NO:123)), kallikrein cleavage site (e.g., SPFR↓STGG (SEQ ID NO:124)), FVIIa cleavage site (e.g., LQVR↓IVGG (SEQ ID NO:125)), FIXa cleavage site (e.g., PLGR↓IVGG (SEQ ID NO:126)), FXa cleavage site (e.g., IEGR↓TVGG (SEQ ID NO:127)), FIIa (thrombin) cleavage site (e.g., LTPR↓SLLV (SEQ ID NO:128)), elastase-2 cleavage site (e.g., LGPV↓SGVP (SEQ ID NO:129)), granzyme-B cleavage (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)), TE V cleavage site (e.g., ENLYFQ↓G (SEQ IDNO:135)), enterokinase cleavage site (e.g., DDDK↓IVGG (SEQ ID NO:136)), protease 3C (PRESCISSION TM)Cleavage sites (e.g., LEVLFQ↓GP (SEQ ID NO:137)) and sortase A cleavage sites (e.g., LPKT↓GSES) (SEQ ID NO:138). In certain embodiments, the FXIa cleavage sites include, but are not limited to, for example, TQSFNDFTR (SEQ ID NO:1) and SVSQTSKLTR (SEQ ID NO:3). Non-limiting exemplary 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 ALRPR (SEQ ID NO:7), consisting essentially of ALRPR (SEQ ID NO:7), or consisting of ALRPR (SEQ ID NO:7) (e.g., ALRPRVVGGA (SEQ ID NO:145)).

[0419] In certain embodiments, 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 a particular embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 194). In another embodiment, the cleavage site comprises EPRSFS (SEQ ID NO: 195), wherein the cleavage site is not the full-length a2 region of FVIII. In another embodiment, the cleavage site comprises IEPR (SEQ ID NO: 200). In another embodiment, the cleavage site comprises IEPR (SEQ ID NO: 200), wherein 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 site comprises 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), 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), AYLLSKNNAIEPRFS (SEQ ID NO:185), YLLSKNNAIEPRSFS (SEQ ID NO:186), LLSKNNAIEPRSFS (SEQ ID NO:187), LSKNNAIEPRSFS (SEQ ID NO:188), SKNNAIEPRSFS (SEQ ID NO:189), KNNAIEPRFS (SEQ ID NO:190), NNAIEPRSFS (SEQ ID NO:191), NAIEPRFS (SEQ ID NO:192), AIEPRSFS (SEQ ID NO:193), or IEPRFS (SEQ ID NO:194).In other embodiments, the cleavage site comprises 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), 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 FVIII a2 region.In certain embodiments, in a thrombin cleavage assay as provided herein or known in the art, the cleavable linker is cleavable.

[0420] III. Polynucleotides, Vectors, and Host Cells

[0421] The present invention also provides a polynucleotide encoding the chimeric protein of the present invention. In one embodiment, the first polypeptide chain and the second polypeptide chain may be encoded by a single polynucleotide chain. In another embodiment, the first polypeptide chain and the second polypeptide chain are encoded by two different polynucleotides (i.e., a first nucleotide sequence and a second nucleotide sequence). In another embodiment, the first nucleotide sequence and the second nucleotide sequence are on two different polynucleotides (such as different vectors).

[0422] The present invention includes a polynucleotide encoding a single polypeptide chain (such as FVIII(X2)-F1-L3-F2-L2-X1-L1-V), wherein FVIII(X2) comprises an FVIII protein with an XTEN sequence inserted at one or more insertion sites, F1 comprises a first Ig constant region or a portion thereof, such as a first Fc region, L1 comprises a first linker, V comprises a VWF protein, X1 comprises an XTEN sequence having less than 288 amino acids in length, L2 comprises a second linker, L3 comprises a third linker, and F2 comprises a second Ig constant region or a portion thereof, such as a second Fc region. The present invention also includes two polynucleotides, a first polynucleotide sequence encoding a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof, and a second polynucleotide sequence encoding a second polypeptide comprising a VWF protein, an XTEN sequence having less than 288 amino acids in length, and a second Ig constant region or a portion thereof. In some embodiments, a chimeric protein comprising two or three polypeptide chains may be encoded by a single polynucleotide chain and then processed into two or three (or more) polypeptide chains. In other embodiments, a chimeric protein comprising these polypeptide chains may be encoded by two or three polynucleotide chains.

[0423] In other embodiments, the polynucleotide set further comprises an additional nucleotide chain encoding a protein convertase (e.g., a second nucleotide chain when the chimeric polypeptide is encoded by a single polynucleotide chain or a third nucleotide chain when the chimeric protein is encoded by two polynucleotide chains). The protein convertase is optionally 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 certain embodiments, the protein convertase is PC5 or PC7. See International Application No. PCT / US2011 / 043568.

[0424] As used herein, an expression vector refers to any nucleic acid construct containing the elements necessary for the transcription and translation of an inserted coding sequence when introduced into a suitable host cell, or in the case of an RNA viral vector, the elements necessary for replication and translation. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0425] The expression vectors of the present invention will include a polynucleotide encoding the chimeric protein described herein. In one embodiment, the coding sequence of one or more of: a first polypeptide comprising an FVIII protein and a first Ig constant region; a second polypeptide comprising a VWF protein, an XTEN sequence having less than 288 amino acids, and a second Ig constant region or a portion thereof; or both, is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to permit each of the constituent nucleic acid sequences to retain its functionality. When a coding sequence and a gene expression control sequence are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence, they are said to be operably linked. Two DNA sequences are said to be operably linked if a promoter in a 5' gene expression sequence causes transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in a frameshift mutation, (2) interfere with the ability of the promoter region to direct 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 will be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of the coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0426] As used herein, a gene expression control sequence is any regulatory nucleotide sequence that facilitates the efficient transcription and translation of an encoding nucleic acid to which it is operably linked, such as a promoter sequence or a promoter-enhancer combination. Gene expression control sequences can be, for example, mammalian or viral promoters, such as constitutive or inducible promoters. Constitutive mammalian promoters include, but are not limited to, the promoters of the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, those from cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeat (LTR) of Moloney leukemia virus, and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. Promoters suitable 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 ordinary skill in the art.

[0427] Generally, gene expression control sequences will, when necessary, include 5' non-transcribed and 5' non-translated sequences that are respectively involved in the initiation of transcription and translation, such as TATA box, capping sequence, CAAT sequence, etc. In particular, the 5' non-transcribed sequence will include a promoter region that includes a promoter sequence for transcriptional control of an operably joined encoding nucleic acid. Gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.

[0428] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and RNA viruses such as retroviruses. Other vectors well 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 by a target gene. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic 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 suitable are those retroviruses that are replication-defective (i.e., capable of directing the synthesis of the desired protein but unable to produce infectious particles). The genetically altered retroviral expression vectors have general utility for high-efficiency transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retroviruses from the packaging cell line, collecting virus particles from tissue culture medium, and infecting target cells with the virus 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, Volume 7, Humana Press, Inc., Clifton, N.J. (1991).

[0429] In one embodiment, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-defective and are capable of infecting a wide range of cell types and species. It further has advantages such as heat and lipid solvent stability; high transduction frequencies in cells of different lineages, including hematopoietic cells; and lack of superinfection inhibition, thus allowing for multilevel transduction. Adeno-associated viruses are reported to integrate into human cell DNA in a site-specific manner, thereby minimizing the likelihood of insertional mutagenesis and variability of the inserted gene expression profile associated with retroviral infection. In addition, wild-type adeno-associated virus infections have been carried out in tissue culture for more than 100 passages in the absence of selection pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also act in an episomal manner.

[0430] Other vectors include plasmid vectors. Plasmid vectors have been widely described 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 the past several years, plasmid vectors have been found to be particularly useful for in vivo gene delivery to cells because they cannot replicate within the host genome and 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 from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Other 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, CA.). Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific DNA fragments.

[0431] In one insect expression system that can be used to produce the proteins of the present invention, the Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an ACNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and the production of non-occluded recombinant virus (i.e., virus lacking the protein coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (See, for example, Smith et al. (1983) J Virol 46:584; U.S. Patent No. 4,215,051). Other 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.

[0432] Another system that can be used to express the proteins of the present invention is the glutamine synthetase gene expression system, also known as the "GS expression system" (Lonza Biologics PLC, Berkshire UK). This expression system is described in detail in U.S. Patent No. 5,981,216.

[0433] In mammalian host cells, many virus-based expression systems can be utilized. In cases where adenovirus is used as an expression vector, the coding sequence can be ligated to an adenovirus transcriptional / translational control complex, such as a late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable in the infected host and capable of expressing the peptide. See, for example, Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5K promoter can be used. See, for example, Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415; Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.

[0434] To increase production efficiency, the polynucleotide can be designed to encode multiple units of the proteins of the present invention separated by an enzyme cleavage site. 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 the polypeptide driven by a single promoter. When used in an appropriate viral expression system, the translation of each polypeptide encoded by the mRNA is directed internally within the transcript; for example, directed by an internal ribosome entry site IRES. Thus, a polycistronic construct directs the transcription of a single large polycistronic mRNA, which in turn directs the translation of multiple individual polypeptides. This method eliminates the production and enzymatic processing of polyproteins and can significantly increase the yield of polypeptides driven by a single promoter.

[0435] The vectors used in transformation will usually contain selectable markers for the identification of transformants. In bacterial systems, this may include antibiotic resistance genes, such as ampicillin or kanamycin. Selectable markers for mammalian cells in culture include genes that confer resistance to drugs such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. The selection of selectable markers is reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable marker is entirely within the level of ordinary skill in the art.

[0436] The selectable marker may be introduced into the cell simultaneously with the target gene on a separate plasmid, or they may be introduced on the same plasmid. If on the same plasmid, then the selectable marker and the target gene may be under the control of different promoters or the same promoter, and the latter arrangement will produce a dicistronic message. This type of construct is known in the art (e.g., U.S. Patent No. 4,713,339).

[0437] Expression vectors may encode tags that allow for the easy purification of the recombinantly produced protein. 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 can be ligated in-frame with the lacZ coding region into the vector to produce a tagged fusion protein; the pGEX vectors can be used to express the proteins of the present invention with glutathione S-transferase (GST) tags. These proteins are generally soluble and can be easily purified from the cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. The vectors include cleavage sites (thrombin or factor Xa protease or PRESCISSION PROTEASE TM (Pharmacia, Peapack, N.J.)) for the easy removal of the tag after purification.

[0438] One or more expression vectors are then transfected or co-transfected into suitable target cells that will express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host expression vector systems can be used to express the proteins described herein, including both prokaryotic and eukaryotic cells. These systems include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli) transformed with recombinant phage DNA or plasmid DNA expression vectors containing the appropriate coding sequences; 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); 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 containing the appropriate coding sequences (e.g., Ti plasmid); or animal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

[0439] 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 defined nucleus. Eukaryotic cells of animals include cells of vertebrates (e.g., mammals) and invertebrates (e.g., insects). Eukaryotic cells of plants can specifically include, but are not limited to, yeast cells. Eukaryotic cells are different from prokaryotic cells such as bacteria, for example.

[0440] In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell line. HEK 293 cells can be obtained from the 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 cell, which is a human cell line derived from the retina. The cells can be obtained from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells. The CHO cells can be obtained from the American Type Culture Collection (Manassas, VA.) (e.g., CHO-K1; CCL-61). In other embodiments, the mammalian cells are baby hamster kidney (BHK) cells. The BHK cells can be obtained from the American Type Culture Collection (Manassas, Va.) (e.g., CRL-1632). In some embodiments, the mammalian cells are HKB11 cells, which are a hybrid cell line of HEK293 cells and a human B cell line. Mei et al., Mol. Biotechnol. 34(2):165-78 (2006).

[0441] In one embodiment, a plasmid comprising the FVIII(X2)-Fc fusion encoding sequence, the VWF protein-L1-X1-L2-Fc encoding sequence, or both and a selectable marker (e.g., zeocin resistance) is transfected into HEK 293 cells to produce a chimeric protein.

[0442] In another embodiment, a plasmid comprising the FVIII-Fc fusion encoding sequence, the VWF protein-L1-X-L2-Fc encoding sequence, or both and a selectable marker (e.g., zeocin resistance) is transfected into HEK 293 cells to produce a chimeric protein.

[0443] In some embodiments, a first plasmid comprising the FVIII(X2)-Fc fusion encoding sequence and a first selectable marker (e.g., the bleomycin resistance gene), a second plasmid comprising the VWF protein-L1-X1-L2-Fc encoding sequence and a second selectable marker (e.g., the neomycin resistance gene), and a third plasmid comprising the protein convertase encoding sequence and a third selectable marker (e.g., the hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., a 1:1 molar ratio), or they can be introduced in unequal amounts.

[0444] In other embodiments, a first plasmid comprising the FVIII-Fc fusion encoding sequence and a first selectable marker (e.g., the bleomycin resistance gene), a second plasmid comprising the VWF protein-L1-X-L2-Fc encoding sequence and a second selectable marker (e.g., the neomycin resistance gene), and a third plasmid comprising the protein convertase encoding sequence and a third selectable marker (e.g., the hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., a 1:1 molar ratio), or they can be introduced in unequal amounts.

[0445] In other embodiments, a first plasmid comprising an FVIII(X2)-Fc fusion encoding sequence and a first selectable marker (e.g., a bleomycin resistance gene), a second plasmid comprising a VWF protein-L1-X1-L2-Fc fusion encoding sequence and a second selectable marker (e.g., a neomycin resistance gene), and a third plasmid comprising a protein convertase encoding sequence and a third selectable marker (e.g., a hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The first and second plasmids may be introduced in equal amounts (i.e., a 1:1 molar ratio), or they may be introduced in unequal amounts.

[0446] In certain embodiments, a first plasmid comprising a chimeric protein encoding FVIII (with or without XTEN)-F1-L3-F2-L2-X-L1-V encoding sequence and a first selectable marker (e.g., a bleomycin resistance gene) and a second plasmid comprising a protein convertase encoding sequence and a second selectable marker (e.g., a hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The promoter of the FVIII(X)-F1 encoding sequence and the promoter of the V-L2-X-L1-F2 encoding sequence may be different, or they may be the same.

[0447] In other embodiments, the transfected cells are stably transfected. Using conventional techniques known to those skilled in the art, these cells can be selected and maintained as a stable cell line.

[0448] Host cells containing a DNA construct encoding a protein are grown in a suitable growth medium. As used herein, the term "suitable growth medium" means a medium containing nutrients required for cell growth. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the medium may contain calf serum or fetal bovine serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium will typically select for cells containing the DNA construct, for example, by drug selection or lack of an essential nutrient, which is supplemented by a selectable marker on or co-transfected with the DNA construct. Cultured mammalian cells are typically grown in a commercially available serum-containing or serum-free medium (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbad, CA.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, CA.). The selection of a medium suitable for the particular cell line used is within the level of ordinary skill in the art.

[0449] To co-express two polypeptide chains of a chimeric protein, a host cell is cultured under conditions that permit expression of the two chains. As used herein, culturing refers to maintaining living cells in vitro for at least a certain period of time. Maintenance may, but need not, include an increase in the living cell population. For example, cells maintained in culture may be static in terms of population but still be viable and capable of producing the desired product, such as a recombinant protein or a recombinant fusion protein. Conditions suitable for culturing eukaryotic cells are well known in the art and include appropriate selection of medium, medium supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing may include the use of any of a variety of scale-up systems, including shake flasks, roller bottles, hollow fiber bioreactors, stirred tank bioreactors, air lift bioreactors, Wave bioreactors, and other systems.

[0450] Cell culture conditions are also selected to permit association of the VWF fragment with the FVIII protein. Conditions that permit expression of the VWF fragment and / or the FVIII protein may include the presence of a vitamin K source. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 medium (Invitrogen, Carlsbad, CA) or OptiCHO medium (Invitrogen, Carlsbad, CA) supplemented with 4 mM glutamine.

[0451] In one aspect, the invention relates to a method of expressing, preparing, or producing a chimeric protein of the invention, which comprises a) transfecting a host cell comprising a polynucleotide encoding the chimeric protein and b) culturing the host cell in a medium under conditions suitable for expression of the chimeric protein, wherein the chimeric protein is expressed.

[0452] In other embodiments, a protein product comprising an FVIII protein linked to a first Ig constant region or a portion thereof and / or a VWF protein fused via an XTEN sequence to a second Ig constant region or a portion thereof is secreted into the medium. The medium is separated from the cells, concentrated, filtered, and then passed through two or three affinity columns, such as a Protein A column and one or two anion exchange columns.

[0453] In certain aspects, the invention relates to a chimeric protein produced by the methods described herein.

[0454] In vitro production allows for scale-up to generate large quantities of the desired altered polypeptides of the invention. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or in a continuously stirred reactor, or, for example, fixed or encapsulated cell culture in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. When 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 over DEAE-cellulose or affinity chromatography.

[0455] IV. Pharmaceutical Compositions

[0456] Compositions containing the chimeric proteins of the invention may contain a pharmaceutically acceptable carrier suitable therefor. By way of example, they may contain excipients and / or adjuvants that assist in processing the active compound into a formulation designed for delivery to the site of action.

[0457] The pharmaceutical compositions can be formulated for parenteral administration (i.e., intravenous, subcutaneous or intramuscular) by bolus injection. Injectable formulations can be provided, for example, in unit dosage forms in ampoules or in multi-dose containers with added preservatives. The compositions can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle (e.g., pyrogen-free water).

[0458] Preparations suitable for parenteral administration also include aqueous solutions of the active compound in water-soluble form (e.g., water-soluble salts). In addition, suspensions of the active compound in the form of suitable oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and dextran. Optionally, the suspension can also contain stabilizers. Liposomes can also be used to encapsulate the molecules of the invention for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. In some embodiments, the composition contains an isotonic agent, such as a sugar, a polyol (such as mannitol, sorbitol) or sodium chloride. In other embodiments, the composition contains a pharmaceutically acceptable substance that enhances the shelf life or effectiveness of the active ingredient, such as a wetting agent or minor auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffering agents.

[0459] The compositions of the present invention can be in a variety of forms, including, for example, liquids (such as injectable and infusible solutions), dispersions, suspensions, semi-solids, and solid dosage forms. The preferred form depends on the mode of administration and the therapeutic application.

[0460] The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amounts into a suitable solvent with one or a combination of the ingredients listed above as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional required ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by using coating agents such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (such as monostearates and gelatin) in the compositions.

[0461] The active ingredient can be formulated with controlled-release formulations or devices. Examples of such formulations and devices include implants, transdermal patches, and microencapsulation delivery systems. Biodegradable 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. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

[0462] Injectable depot formulations can be prepared by forming a microencapsulation matrix of the drug in a biodegradable polymer such as poly(lactide-co-glycolide). The rate of drug release can be controlled depending on the ratio of the drug to the polymer and the nature of the polymer used. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions.

[0463] 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 its variant, fragment, analogue or derivative. For example, coagulation factors include but are 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 the activated form of any of the foregoing. The coagulation factors of the hemostatic agent may also include antifibrinolytic drugs such as ε-aminocaproic acid, tranexamic acid.

[0464] The dosage regimen can be adjusted to provide the optimal desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally decreased or increased as indicated by the exigencies of the treatment situation. Advantageously, parenteral compositions are formulated in dosage unit form for ease of administration and to achieve uniformity of dosage. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0465] In addition to the active compound, liquid dosage forms may also contain inert ingredients such as water, ethanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan.

[0466] Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by E.W. Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition may also contain pH buffering agents and wetting or emulsifying agents.

[0467] For oral administration, the pharmaceutical composition can be in the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid, such as a syrup or suspension. Liquids may include suspending agents (such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (such as almond oil, oily esters, ethanol or fractionated vegetable oils) and preservatives (such as methylparaben or propylparaben or sorbic acid). The formulation may also include flavoring agents, coloring agents and sweetening agents. Alternatively, the composition can be provided in the form of a dry product for reconstitution with water or another suitable vehicle.

[0468] For buccal administration, the composition can be in the form of tablets or lozenges according to conventional formulations.

[0469] For administration by inhalation, the compounds useful in the present invention are preferably delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or a nebulizer with an optional propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gases. 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, such as of gelatin, containing a powder mixture of the compound and a suitable powder matrix such as lactose or starch can be formulated for use in an inhaler or insufflator.

[0470] The pharmaceutical composition can also be formulated for rectal administration in the form of suppositories or retention enemas, for example containing conventional suppository bases such as cocoa butter or other glycerides.

[0471] 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 pharmaceutically acceptable carrier. The FVIII protein in the chimeric protein has an extended half-life compared to the wild-type FVIII protein or the corresponding FVIII protein without the VWF fragment. In one embodiment, the half-life of the chimeric protein is extended by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to the 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.

[0472] 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.

[0473] In other embodiments, the composition is used to treat bleeding disorders or conditions in a subject in need thereof. The bleeding disorder or condition is selected from the group consisting of: bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and any combination thereof. In other embodiments, the subject is scheduled to undergo surgery. In other embodiments, the treatment is prophylactic or on-demand.

[0474] V. Gene Therapy

[0475] The chimeric proteins of the invention can be produced in vivo in a mammal, such as a human patient, and it would be therapeutically beneficial to use gene therapy methods for treating bleeding disorders or conditions selected from the group consisting of: bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. In one embodiment, the bleeding disorder or condition is hemophilia. In another embodiment, the bleeding disorder or condition is hemophilia A. This involves administering a nucleic acid encoding a chimeric protein operably linked to a suitable expression control sequence. In certain embodiments, these sequences are incorporated into a viral vector. Viral vectors suitable for such gene therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein-Barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, and adeno-associated virus (AAV) vectors. The viral vector can be a replication-defective viral vector. In other embodiments, the adenoviral vector has a deletion in its E1 gene or E3 gene. When using an adenoviral vector, the mammal may not be exposed to a nucleic acid encoding a selectable marker gene. In other embodiments, the sequences are incorporated into non-viral vectors known to those skilled in the art.

[0476] VI. Methods of Using Chimeric Proteins

[0477] The invention relates to a method of using a chimeric protein as described herein to prevent or inhibit the binding of endogenous VWF to FVIII protein. The invention also relates to a method of using a chimeric protein having an FVIII protein linked to XTEN and an Ig constant region or a portion thereof.

[0478] One aspect of the present invention relates to preventing or inhibiting the interaction of FVIII with endogenous VWF by blocking or masking the VWF binding site on FVIII from endogenous VWF, and simultaneously using an XTEN sequence in combination with an Ig constant region or a portion thereof, which can also be a half-life extender, to extend the half-life of the chimeric protein. In one embodiment, the present invention relates to a method of constructing an FVIII protein having a longer half-life than wild-type FVIII. The chimeric proteins suitable for use in the method include any one or more of the chimeric proteins described herein.

[0479] Another aspect of the present invention includes a method of administering to a subject in need thereof a chimeric protein comprising an FVIII protein having a longer half-life than wild-type FVIII, wherein the method comprises administering to the subject the chimeric protein described herein.

[0480] In one embodiment, the present invention relates to a method of using an XTEN sequence and an Ig constant region or a portion thereof to improve the half-life of a chimeric protein comprising an FVIII protein and a VWF protein, which prevents or inhibits the interaction of endogenous VWF with the FVIII protein. The FVIII protein linked to the XTEN sequence (e.g., FVIII(X)), and then bound or associated with the VWF protein fused to the XTEN and the Ig constant region or a portion thereof, is shielded or protected from the clearance pathway of VWF, and thus has a reduced clearance rate compared to the FVIII protein not bound to the VWF protein. Accordingly, the half-life of the shielded FVIII protein is maximally extended compared to the FVIII protein not bound or associated with the XTEN sequence and the VWF protein. In certain embodiments, the FVIII protein associated with or protected by the VWF protein and linked to the XTEN sequence is not cleared by the VWF clearance receptor. In other embodiments, the FVIII protein associated with or protected by the VWF protein and linked to the XTEN sequence is cleared from the system more slowly than the FVIII protein not associated with or protected by the VWF protein and linked to the XTEN sequence.

[0481] In one aspect, the clearance rate of a chimeric protein comprising an FVIII protein linked to an XTEN sequence or an FVIII protein bound or associated with a VWF protein linked to XTEN from the circulation is reduced because the VWF protein does not contain a VWF clearance receptor binding site. The VWF protein prevents or inhibits the clearance of the FVIII bound or associated with the VWF protein from the system through the VWF clearance pathway. The VWF protein suitable for use in the present invention can also provide at least one or more VWF-like FVIII protective properties provided by endogenous VWF. In certain embodiments, the VWF protein or the XTEN sequence can also mask one or more FVIII clearance receptor binding sites, thereby preventing the clearance of FVIII through its own clearance pathway.

[0482] In some embodiments, prevention or inhibition of FVIII protein binding to endogenous VWF by VWF protein or XTEN sequence can occur in vitro or in vivo.

[0483] Also provided is a method of increasing the half-life of a chimeric protein, which comprises administering to a subject in need thereof the chimeric protein described herein. The half-life of inactive FVIII that binds or associates with full-length VWF in plasma is about 12 to 14 hours. In type 3 VWD, where there is little VWF in the circulation, the half-life of FVIII is only about six hours, resulting in mild to moderate hemophilia A symptoms in such patients due to reduced FVIII concentration. The half-life of the chimeric protein of the present invention linked or associated with a VWF fragment or XTEN sequence can be increased by at least about 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold or 4.0-fold compared to the half-life of inactive FVIII that binds or associates with full-length VWF.

[0484] In one embodiment, a chimeric protein comprising a first polypeptide (which comprises an FVIII protein and a first Ig constant region or a portion thereof) and a second polypeptide (which comprises a VWF protein, an XTEN having less than 288 amino acids, and an Ig constant region or a portion thereof) exhibits a half-life that is at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 7-fold, 8-fold, 9-fold or 10-fold higher than that of a corresponding chimeric protein comprising the same first polypeptide and a second polypeptide without the XTEN sequence or wild-type FVIII. In another embodiment, a chimeric protein comprising a first polypeptide (which comprises an FVIII protein and a first Ig constant region or a portion thereof) and a second polypeptide (which comprises a VWF protein, an XTEN having less than 288 amino acids, and an Ig constant region or a portion thereof) exhibits a half-life that is about 2 to about 5-fold, about 3 to about 10-fold, about 5 to about 15-fold, about 10 to about 20-fold, about 15 to about 25-fold, about 20 to about 30-fold, about 25 to about 35-fold, about 30 to about 40-fold, about 35 to about 45-fold higher than that of a corresponding chimeric protein comprising the same first polypeptide and a second polypeptide without the XTEN sequence or wild-type FVIII. In a particular embodiment, in FVIII- and VWF-double knockout mice, the half-life of the chimeric protein of the present invention is increased by at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40-fold compared to the half-life of wild-type FVIII.

[0485] In certain embodiments, the chimeric protein exhibits a half-life of approximately 40 hours in mice.

[0486] In some embodiments, the half-life of the chimeric protein is longer than the half-life of FVIII associated with endogenous VWF. In other embodiments, the half-life of the chimeric protein is at least about 1.5-fold, 2-fold, 2.5-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.5-fold, or 5.0-fold the half-life of wild-type FVIII or FVIII protein associated with endogenous VWF.

[0487] In some embodiments, as a result of the present invention, the half-life of the chimeric protein is extended compared to FVIII protein without VWF protein or wild-type FVIII. The half-life of the chimeric protein of the present invention is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold longer than the half-life of the chimeric protein without VWF protein or wild-type FVIII. In one embodiment, the half-life of FVIII is about 1.5-fold to about 20-fold, about 1.5-fold to about 15-fold, or about 1.5-fold to about 10-fold longer than the half-life of wild-type FVIII. In another embodiment, the half-life of FVIII is extended by about 2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2-fold to about 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-fold to about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to about 10-fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-fold to about 5-fold, about 3-fold to about 4-fold, about 4-fold to about 6-fold, about 5-fold to about 7-fold, or about 6-fold to about 8-fold compared to wild-type FVIII or FVIII protein without VWF protein. In other embodiments, the half-life of the chimeric protein of the present invention is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 40 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 other embodiments, the half-life of the chimeric protein of the present invention is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours to about one week, about 24 hours to about one week, about 36 hours to about one week, about 48 hours to about one week, about 60 hours to about one week, about 24 hours to about six days, about 24 hours to about five days, about 24 hours to about four days, about 24 hours to about three days, or about 24 hours to about two days.

[0488] In some embodiments, the chimeric proteins of the invention have an average half-life in a subject of about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about six days, about seven days (one week), about eight days, about nine days, about 10 days, about 11 days, about 12 days, about 13 days or about 14 days.

[0489] In addition, the invention provides a method of treating or preventing a bleeding disease or disorder, which comprises administering an effective amount of the chimeric protein. In one embodiment, the bleeding disease or disorder is selected from the group consisting of: bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space and bleeding in the iliopsoas sheath. In a particular embodiment, the bleeding disease or disorder is hemophilia A.

[0490] Chimeric proteins prepared by the invention comprising an XTEN sequence and an Ig constant region or a portion thereof in combination with a VWF protein (which prevents or inhibits the interaction of FVIII protein with endogenous VWF) as described herein have many uses that will be recognized by those skilled in the art, including but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent. In one embodiment, the invention relates to a method of treating a subject having a hemostatic disorder, which comprises administering a therapeutically effective amount of the chimeric protein.

[0491] The FVIII protein portion in the chimeric protein treats or prevents hemostatic disorders by acting as a cofactor for factor IX on negatively charged phospholipid surfaces, thereby forming the X enzyme complex. Activation of the coagulation factors binds to the phospholipid surface and localizes this process to the site of vascular damage. On the phospholipid surface, factor VIIIa increases the maximum rate of factor X activation achieved by factor IXa by about 200,000-fold, resulting in a large, instantaneous burst of thrombin production.

[0492] The chimeric proteins of the present invention can be used to treat any hemostatic disorder. Hemostatic disorders that can be treated by administering the chimeric proteins of the present invention include, but are not limited to, hemophilia A and deficiencies or structural abnormalities associated with factor VIII. In one embodiment, the hemostatic disorder is hemophilia A.

[0493] The chimeric proteins of the present invention can be prophylactically used to treat a subject having a hemostatic disorder. The chimeric proteins of the present invention can be used to treat an acute bleeding episode in a subject having a hemostatic disorder. In another embodiment, the hemostatic disorder can be the result of a defective coagulation factor, such as von Willebrand factor. In one embodiment, the hemostatic disorder is a genetic disorder. In another embodiment, the hemostatic disorder is an acquired disorder. The acquired disorder can be caused by underlying secondary diseases or conditions. The underlying conditions can be, for example but not limited to, cancer, autoimmune diseases or pregnancy. The acquired disorder can be caused by aging or medical treatment for underlying secondary conditions (such as cancer chemotherapy).

[0494] The present invention also relates to a method of treating a subject who does not have a congenital hemostatic disorder but has an underlying secondary disease or condition that results in an acquired hemostatic disorder (such as due to the production of anti-FVIII antibodies or surgery). Thus, the present invention relates to a method of treating a subject in need of a general hemostatic agent, which comprises administering a therapeutically effective amount of a chimeric protein prepared by the method of the present invention.

[0495] The present invention also relates to a method of reducing the immunogenicity of FVIII or inducing a lesser immunogenicity against FVIII, which comprises administering an effective amount of the chimeric protein described herein or its encoding polynucleotide.

[0496] In one embodiment, the subject in need of a general hemostatic agent is undergoing or about to undergo surgery. The chimeric proteins of the present invention can be administered as a prophylactic regimen before, during or after surgery. The chimeric proteins of the present invention can be administered before, during or after surgery to control an acute bleeding episode.

[0497] The chimeric proteins of the present invention can be used to treat a subject having an acute bleeding episode and not having a hemostatic disorder. The acute bleeding episode can be caused by severe trauma, such as surgery, car accident, wound, gunshot laceration or any other traumatic event that results in uncontrolled bleeding. Non-limiting examples of bleeding episodes include bleeding coagulopathy, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and any combination thereof.

[0498] In prophylactic applications, one or more compositions comprising the chimeric proteins of the invention or mixtures thereof are administered to a patient not yet in a diseased state to enhance the resistance of said patient or to alleviate symptoms associated with a disease or disorder. Such an amount is defined as a "prophylactically effective dose". In therapeutic applications, relatively high doses at relatively short intervals are sometimes required (e.g., about 1 to 400 mg / kg of polypeptide per dose, where doses of 5 to 25 mg are more commonly used for radioimmunoconjugates and higher doses for polypeptide modified with cytotoxic drugs) until disease progression is reduced or terminated and until the patient shows partial or complete improvement of disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient.

[0499] In some embodiments, the chimeric proteins or compositions of the invention are used for on-demand treatment, which includes treating bleeding episodes, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, head trauma (head injury), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space or bleeding in the iliopsoas sheath. The subject may require surgical prophylaxis, perioperative management or surgical treatment. The surgery includes, for example, minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteorrhaphy, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery or joint replacement surgery.

[0500] In one embodiment, the chimeric proteins of the invention are administered intravenously, subcutaneously, intramuscularly or by any mucosal surface, such as orally, sublingually, buccally, nasally, rectally, vaginally or by the pulmonary route. The chimeric proteins of the invention comprising VWF fragments and FVIII proteins can be implanted in a biopolymer solid carrier that allows slow release of the chimeric protein to the bleeding site or conjugated to the biopolymer solid carrier, or implanted in a bandage / dressing. The dose of the chimeric protein will vary depending on the subject and the particular route of administration used. The dose can range from 0.1 to 100,000 μg / kg body weight. In one embodiment, the dose range is 0.1 - 1,000 μg / kg. In another embodiment, the dose range is 0.1 - 500 μg / kg. The protein can be administered continuously or at specific time intervals. In vitro assays can be employed to determine the optimal dose range and / or schedule of administration. In vitro assays for measuring coagulation factor activity are known in the art, such as the STA-CLOT VIIa-rTF coagulation assay or the ROTEM coagulation assay. Additionally, the effective dose can be extrapolated from dose-response curves obtained from animal models (e.g., hemophilic dogs) (Mount et al 2002, Blood 99(8):2670).

[0501] The present invention has been described in detail. The present invention will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the present invention. All patents, publications, and articles mentioned herein are expressly and specifically incorporated herein by reference.

[0502] Example

[0503] Unless otherwise stated, the following materials and methods were used throughout the examples.

[0504] Materials and Methods

[0505] Generally, unless otherwise indicated, the practice of the present invention employs conventional chemical techniques, biophysical techniques, molecular biology techniques, recombinant DNA techniques, immunological techniques (especially, for example, antibody techniques), and standard electrophoresis techniques. See, for example, Sambrook, Fritsch, and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow, et al., CS.H.L.Press, Pub. (1999); and Current Protocols in Molecular Biology, Ausubel, et al., eds., John Wiley & Sons (1992).

[0506] Example 1: FVIII-XTEN-Fc / D’D3-XTEN-Fc Heterodimer

[0507] The present invention relates to the generation of a chimeric FVIII molecule that is coupled to the D’D3 domain of von Willebrand factor (VWF) protein via the Fc domain of IgG. The linked D’D3 domain prevents FVIII from interacting with endogenous VWF multimers. This molecule serves as a platform for incorporating other half-life extension technologies to improve the pharmacokinetics of the chimeric protein. XTEN sequences are incorporated into the FVIII B domain and between the D’D3 and the Fc region to increase the half-life of the FVIII / VWF heterodimer.

[0508] The thrombin cleavage site between D’D3 and Fc allows the release of the D’D3 domain after activation of the FVIII molecule by thrombin.

[0509] Example 2: Plasmid construction of FVIII-XTEN-Fc / D’D3-Fc heterodimer

[0510] Clone VWF050 - VWF031 containing the IHH triple mutation

[0511] The IHH triple mutation in Fc prevents interaction with FcRn, so there is no recycling of Fc-containing molecules through the FcRn pathway. The three mutations in Fc are I253A, H310A, and H435A.

[0512] VWF050 was generated by exchanging the Fc region of the VWF031 plasmid with an Fc fragment containing the IHH triple mutation between the RsRII restriction site and the Not 1 restriction site.

[0513] Clone VWF057 - Clone VWF-Fc with 144AE XTEN + 35aa thrombin-cleavable linker

[0514] Oligomer

[0515] ESC 155 - Oligomer - reverse for 144AE XTEN in VWF034

[0516] CCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGAACCTCCACCGCCGCTCGAGGCACCTTCTTCAGTGCTGGTGGGCGAGCCCGCTGGTGACCCTTCCTC

[0517] ESC 156 - Oligomer - reverse for 144AE XTEN-GS linker in VWF034

[0518] GGGGAAGAGGAAGACTGACGGTCCGCCCAGGAGTTCTGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGCCTCCGCTGCCCCGGGGGACCAGGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCC

[0519] ESC 157 - Oligomer - forward for 144AE XTEN in VWF031

[0520] GTGAAGCCTGCCAGGAGCCGATATCGGGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGC

[0521] Two PCRs were performed to obtain a 35aa GS linker of 144AE-XTEN+ with a thrombin cleavage site.

[0522] The first PCR reaction was carried out using the 144-AE XTEN-encoding DNA as a template and the ESC 157 / ESC 155 primer pair. The PCR product approximately 550bp in length obtained from this reaction was used as a template for the second PCR reaction and amplified using the ESC 157 / 156 primer pair. This reaction produced a product approximately 700bp in length. Then this 700bp PCR product and the VWF034 plasmid were digested with EcoRV-HF and RsRII. The plasmid backbone was digested.

[0523] Then the 700bp PCR product was ligated using VWF034.

[0524] Clone the IHH triple mutation in VWF058-VWF034

[0525] The IHH triple mutation in Fc prevents interaction with FcRn, so there is no recycling of Fc-containing molecules through the FcRn pathway. The 3 mutations in Fc are I253A, H310A, H435A.

[0526] VWF058 was generated by exchanging the Fc region of the VWF034 plasmid with an Fc fragment containing the IHH triple mutation between the RsRII restriction site and the Not 1 restriction site.

[0527] Clone FVIII-263 - FVIII 205 with the IHH triple mutation

[0528] The IHH triple mutation in Fc prevents interaction with FcRn, so there is no recycling of Fc-containing molecules through the FcRn pathway. The 3 mutations in Fc are I253A, H310A, H435A.

[0529] FVIII-263 was generated by exchanging the Fc region of the FVIII 205 plasmid with an Fc fragment containing the IHH triple mutation between the RsRII restriction site and the Not 1 restriction site.

[0530] Clone FVIII-282 - FVIII-Fc with 144AE XTEN in the B domain

[0531] ESC 158 -Oligomer of 144AE XTEN in the B domain - Forward

[0532] AAGAAGCTTCTCTCAAAACGGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGCCACATCTGGGTCGGAAACGCCAGGC

[0533] ESC 159 -Oligomer of 144AE XTEN in the B domain - Reverse

[0534] GGTATCATCATAATCGATTTCCTCTTGATCTGACTGAAGAGTAGTACGAGTTATTTCAGCTTGATGGCGTTTCAAGACTGGTGGGCTCGAGGCACCTTCTTCAGTGCTGGTGGGCGAGCCCGCTGGTGACCCTTCCTCAGTGGACGTAGG

[0535] The first PCR reaction was performed using 144 - AE XTEN - encoding DNA as a template and the ESC 158 / ESC 159 primer pair. Subsequently, the approximately 550bp - long PCR product obtained from this reaction and the FVIII 169 plasmid were digested with AscI and Clal. The plasmid backbone obtained from the digested FVIII 169 was then used to ligate the 550bp PCR product to obtain FVIII 282.

[0536] Clone FVIII - 283 - FVIII 169 with IHH triple mutation

[0537] The IHH triple mutation in Fc prevents interaction with FcRn, so there is no recycling of Fc - containing molecules through the FcRn pathway. The three mutations in Fc are I253A, H310A, and H435A.

[0538] FVIII - 283 was generated by exchanging the Fc region of the FVIII 169 plasmid with an Fc fragment containing the IHH triple mutation between the RsRII restriction site and the Not 1 restriction site.

[0539] Example 3: Generation of FVIII - XTEN - Fc / D’D3 - XTEN - Fc in HEK293 cells

[0540] Figure 2Schematic showing the expression of the FVIII-XTEN-Fc / D’D3-XTEN-Fc construct. Triplex plasmid co-transfection was performed in HEK293 cells using polyethylenimine (PEI). The first plasmid obtained the expression of FVIII-XTEN-Fc, the second plasmid expressed D1D2D’D3-XTEN-Fc, and the third plasmid expressed PACE / furin protease, which was required for the enzymatic removal of the propeptide (i.e., the D1D2 domain) from D1D2D’D3-XTEN-Fc. The products of this triplex plasmid expression system included the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer, the D’D3-XTEN-Fc homodimer, and trace amounts of FVIII-XTEN-Fc hemizygous-like substances.

[0541] Example 4: Purification of the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer

[0542] For the purification of the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer, a tangential flow filtration (TFF) step was used to first concentrate the conditioned medium 10-fold. Subsequently, affinity chromatography and a desalting column were used in sequence to further purify the product in the filtrate. According to HPLC-SEC, the purity of the molecule was acceptable and was further confirmed by Western blot. The specific activity of the molecule was similar to that of B-domain-deleted FVIII, as measured by FVIII activity assay (Example 5) and OD280 measurement.

[0543] Example 5: Specific activity of the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer

[0544] The activity of the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer was measured by FVIII chromogenic assay and activated partial thromboplastin time (aPTT) assay. The specific chromogenic activity and specific aPTT activity of SQ BDD-FVIII, rFVIII169 / VWF034, and rFVIII169 / VWF057 are listed in Table 16. Compared with SQ BDD-FVIII, for rFVIII169 / VWF034 and rFVIII169 / VWF057, we have observed similar specific chromogenic activity and a 60% decrease in specific aPTT activity.

[0545] Table 16: Specific activities of heterodimer variants

[0546]

[0547] FVIII chromogenic assay

[0548] FVIII activity was measured using the COATEST SP FVIII kit from DiaPharma (product number: K824086), and all incubations were performed on a 37 °C plate heater with shaking.

[0549] The WHO International Standard for Factor VIII:C, 8th International Standard (concentrate, code 07 / 350) was used as the assay standard, and the range of the standard was from 100 mIU / mL to 0.78 mIU / mL. Confluent normal human plasma assay controls and test samples (diluted with 1X Coatest buffer) were added in duplicate to an Immulon 2HB 96-well plate (25 μL / well). Freshly prepared IXa / FX / phospholipid mixture (50 μL), 25 μL of 25 mM CaCl2, and 50 μL of FXa substrate were added sequentially to each well, with a 5-minute incubation between each addition. After incubation with the substrate, 25 μL of 20% acetic acid was added to terminate the chromogenic reaction, and the absorbance at OD405 was measured using a SpectraMAX plus (Molecular Devices) instrument. Data were analyzed using SoftMax Pro software (version 5.2). The lowest limit of quantification (LLOQ) was 7.8 mIU / mL.

[0550] FVIII aPTT assay

[0551] The FVIII aPTT assay was performed on a Sysmex CA-1500 coagulation analyzer as follows: First, 50 uL of aPTT buffer (50 mM Tris, 100 mM NaCl, 1% HSA, pH 7.4) containing manually diluted samples, standards, and controls was added to the reaction cup by the instrument, followed by 50 uL of FVIII-deficient plasma (George King Bio-Medical, product number: 0800). After incubation at 37 °C for 1 minute, 50 uL of aPTT reagent ( FSL-activated brain hyaluronate reagent - Dade Behring, reference number B4219 - 2) was added to the reaction mixture and incubated at 37 °C for 4 minutes. Subsequently, 50 ul of 20 mM CaCl2 (Dade Behring, reference number ORFO37) was added and the reaction cuvette was immediately transferred to one of four spectrophotometer channel positions to measure the amount of refracted light in the mixture, which was converted to the occurrence of clotting by the software algorithm of the instrument. The reported clotting time is the duration from the addition of CaCl2 until clot formation occurs. The assay standard was generated by diluting the WHO 8th International FVIII Standard in aPTT buffer in the range from 100 mIU / ml to 0.78 mIU / ml. The standard curve was plotted in MS Excel with the clotting time (in seconds) as the Y-axis versus the logarithm (base 10) of the FVIII activity (mIU / mL) as the X-axis, and the activity of individual samples was calculated using the formula of the linear regression line for this standard curve. Based on the assay performance, the lower limit of quantification (LLOQ) was 7.8 mIU / mL.

[0552] Example 6: Additive effect of XTEN insert on the half-life extension of heterodimer

[0553] The XTEN insert was incorporated into the heterodimer to achieve half-life extension. Insertion of a single 288 amino acid (aa) AE-XTEN at the FVIII B domain resulted in a half-life of 16.7 hours for the heterodimer in HemA mice, as Figure 3 demonstrated by rFVIII169 / VWF031. To further improve the half-life of the heterodimer, a second XTEN insert with a length of 144 aa or 288 aa was incorporated into FVIII169 / VWF031 either in the FVIII A1 domain or downstream of the D’D3 fragment, and the heterodimer variants were named FVIII205 / VWF031 and FVIII169 / VWF034.

[0554] The half-lives of rFVIII169 / VWF031, rFVIII205 / VWF031, and rFVIII169 / VWF034 were evaluated in FVIII-deficient (HemA) mice by single intravenous administration of the test molecules at a dose of 200 IU / kg. Plasma samples were collected at the designated time points as Figure 3 indicated, the FVIII activity of the samples was determined by FVIII chromogenic assay, and the PK parameters were calculated using the WinNonlin-Phoenix program and listed in Table 17.

[0555] As Figure 3As shown in Table 17, adding a second XTEN insert at the A1 domain of FVIII or downstream of D’D3 further improved the half-life of the heterodimer to 29.45 or 31.10, respectively. In addition, a greater than 2-fold improvement in clearance and AUC was also observed due to the two XTEN inserts.

[0556] Table 17: PK parameters of heterodimers in HemA mice

[0557]

[0558] Example 7: When inserted between D’D3 and the Fc domain, 144aa AE-XTEN confers a better half-life benefit than 288aa AE-XTEN.

[0559] Another heterodimer - FVIII169 / VWF057 was constructed to attempt to identify the optimal length of the XTEN insert within the D’D3-XTEN-Fc chain, where the length of the XTEN insert was reduced from 288aa to 144aa. As Figure 4 shown, the half-life of rFVIII169 / VWF057 increased from 31 hours to 42 hours compared to rFVIII169 / VWF034. Improvements in clearance and AUC of rFVIII169 / VWF057 were also observed, and the data are listed in Table 18. Thus, when inserted between D’D3 and the Fc domain of the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer, the 144aa AE-XTEN insert is superior to AE-288aa XTEN.

[0560] Table 18: PK parameters of rFVIII169 / VWF034 and rFVIII169 / VWF057 in HemA mice

[0561]

[0562]

[0563] Example 8: The Fc domain prolongs the half-life of the heterodimer

[0564] The Fc domain prolongs the half-life of its fusion protein via the FcRn-mediated recycling pathway. To confirm the necessity of the Fc domain for the half-life extension of the heterodimer, the wild-type Fc domain in rFVIII205 / VWF031 was replaced with a triple mutant (I253A / H310A / H435A; IHH) to form rFVIII263 / VWF050, and it was confirmed by surface plasmon resonance (Biacore) that the FcRn binding of rFVIII263 / VWF050 was completely abolished. The half-life of FVIII263 / VWF050 was evaluated in HemA mice compared to rFVIII205 / VWF031. An increased clearance rate and decreased half-life and AUC of rFVIII263 / VWF050 were observed, as Figure 5 shown in and Table 19. This result demonstrates that in addition to ensuring the covalent binding of FVIII and D’D3, the Fc domain is also necessary for the half-life improvement of the heterodimer.

[0565] Table 19: PK parameters of rFVIII205 / VWF031 and rFVIII263 / VWF040 in HemA mice

[0566]

[0567] Example 9: Acute efficacy of the FVIII-XTEN-Fc / D’D3-XTEN-Fc heterodimer in the HemA mouse tail clamp bleeding model

[0568] The acute efficacy of lead heterodimer candidates was evaluated using the HemA mouse tail clamp bleeding model.

[0569] Male HemA mice, 8 - 12 weeks old, were randomly assigned to 4 treatment groups and treated with a single intravenous administration of SQBDD-FVIII, rFVIII169 / VWF034, rFVIII169 / VWF057, or vehicle solution, respectively. To mimic episodic treatment of FVIII (to restore 50 - 100% of normal FVIII plasma levels), the selected FVIII treatment dose was 75 IU / kg as measured by FVIII aPTT activity. At this dose level, all tested FVIII variants would restore approximately 70% of normal murine plasma FVIII activity 5 minutes after dosing.

[0570] The blood loss volume of each individual animal in the study was plotted on Figure 6Among the animals treated with the vehicle, a significant reduction in the blood loss volume was observed in all FVIII treatment groups. No statistically significant differences in blood loss reduction were found among the three FVIII treatment groups, indicating that the heterodimeric molecule may potentially be as effective as SQ BDD-FVIII for on-demand treatment.

[0571] The blood loss volume of each individual animal in the study was plotted in Figure 6 Among the animals treated with the vehicle, a significant reduction in the blood loss volume was observed in all FVIII treatment groups. No statistically significant differences in blood loss reduction were found among the three FVIII treatment groups, indicating that the heterodimeric molecule may potentially be as effective as SQ BDD-FVIII for on-demand treatment.

[0572] In addition, HemA mice were treated with lower doses (37.5 IU / kg) of rBDD-FVIII or rFVIII169 / VWF 034, and the results are shown in Figure 6 Panel B. Similar to the 75 IU / kg dose, after tail clamp injury, rFVIII169 / VWF034 provided protection similar to BDD-FVIII to HemA mice, indicating that the molecule is still effective in treating severe bleeding episodes in HemA mice at approximately 35% of normal murine circulating FVIII levels.

[0573] The tail clamp procedure was performed as follows. Briefly, mice were anesthetized with a mixture of 50 mg / kg ketamine / 0.5 mg / kg dexmedetomidine before tail injury and placed on a 37°C heating plate to help maintain body temperature. Then, the tail of the mouse was immersed in 37°C saline for 10 minutes to dilate the lateral veins. After vein dilation, the FVIII variant or vehicle solution was injected via the tail vein, and then the distal 5 mm of the tail was cut...

Claims

1. A chimeric protein comprising (i) a first polypeptide comprising a factor VIII ("FVIII") protein fused to a first immunoglobulin ("Ig") constant region or a portion thereof, and (ii) a second polypeptide comprising a von Willebrand factor ("VWF") protein containing the D' domain and the D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via an XTEN sequence therebetween, wherein the XTEN sequence contains less than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide.

2. The chimeric protein according to claim 1, wherein the XTEN sequence in the second polypeptide consists of an amino acid sequence having a length between 12 and 287 amino acids.

3. The chimeric protein according to claim 1 or 2, wherein the chimeric protein exhibits a longer half-life as compared to a corresponding fusion protein comprising the first polypeptide and the second polypeptide, wherein the second polypeptide of the fusion protein comprises an XTEN sequence containing at least 288 amino acids.

4. The chimeric protein according to claim 3, wherein the XTEN sequence containing at least 288 amino acids is AE288.

5. The chimeric protein according to claim 4, wherein AE288 is SEQ ID NO:

8.

6. The chimeric protein according to any one of claims 1 to 5, wherein the XTEN sequence of the second polypeptide contains about 36, about 42, about 72 or about 144 amino acids.

7. The chimeric protein according to claim 6, wherein the XTEN sequence of the second polypeptide is selected from AE42, AE72, AE144, AG42, AG72 or AG144.

8. The chimeric protein according to claim 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.

9. The chimeric protein according to any one of claims 1 to 8, wherein the first polypeptide further comprises a second XTEN sequence that links the FVIII protein to the first Ig constant region or a portion thereof.

10. The chimeric protein according to claim 9, wherein the first polypeptide comprises a third XTEN sequence inserted at one or more insertion sites within the FVIII protein.

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