BR112015031194B1 - CHIMERICAL MOLECULE, PHARMACEUTICAL COMPOSITION, AND USE OF THE FOREGOING - Google Patents

Abstract

Chimerical molecule and method for doing it, polynucleotide or a set of polynucleotides, vector or a set of vectors, host cell, pharmaceutical composition, use of the previous ones, and method to increase the activity of FVII of the chimerical molecule. The present invention relates to a chimeric molecule, composed of a VWF protein fused to a heterologous portion through a VWF ligand. The invention provides an efficient VWF linker that can be cleaved in the presence of thrombin. The chimeric molecule may also comprise a polypeptide chain composed of a FVIII protein and a second heterologous portion, wherein the chain comprising the VWF protein and the FVIII protein are associated with each other. The invention also includes nucleotides, vectors, host cells and methods for using the chimeric proteins.

Description

BACKGROUND OF THE INVENTION

[001] Hemophilia A is a bleeding disorder caused by defects in the gene encoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 male births. Graw et al, Nat. Rev. Genet. 6(6): 488501 (2005). Patients affected with hemophilia A can be treated with infusion of purified or recombinantly produced FVIII. All commercially available FVIII products, however, are known to have a half-life of about 8-12 hours, which requires 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). Furthermore, a number of approaches have been tried in order to extend the FVIII half-life. For example, approaches under development to extend the half-life of clotting factors include pegylation, glycopegylation, and albumin conjugation. See Dumont et al., Blood. 119(13): 3024-3030 (published online on Jan. 13, 2012). However, regardless of the protein engineering used, the long-acting FVIII products currently in development have improved half-lives, but the half-lives are reported to be limited - in preclinical animal models, the improvement is only 1. 5 to 2 times, approximately. See Id. Consistent results have been demonstrated in humans, for example, rFVIIIFc has been reported to improve half-life ~1.7-fold compared to ADVATE® in patients with hemophilia A. See Id. Therefore, half-life increases life, despite small improvements, may indicate the presence of other limiting factors t1/2.

[002] Due to frequent dosing and inconvenience caused by the dosing regimen, there is still a need to develop FVIII products that require less frequent administration, i.e., an FVIII product that has a half-life longer than the limitation of 1 .5 to 2 times.

BRIEF SUMMARY OF THE INVENTION

[003] The present invention is directed to a chimeric molecule, composed of a Von Willebrand Factor (VWF) protein, a heterologous portion (H1), an XTEN sequence, and a VWF linker connecting the VWF protein with the heterologous portion, in which the VWF ligand is composed of a polypeptide selected from: (i) an a2 region of Factor VIII (FVIII); (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof, and in which the XTEN sequence is linked to the VWF protein, or the heterologous portion (H1), the VWF ligand or any combination thereof. In one embodiment, the XTEN sequence connects the VWF protein to the VWF ligand or the VWF ligand to the heterologous moiety. In another embodiment, the chimeric molecule also comprises a second polypeptide chain comprising an FVIII protein, in which the first polypeptide chain and the second polypeptide chain are associated with each other. In other embodiments, the FVIII protein in the chimeric molecule comprises an additional XTEN sequence. The additional XTEN sequence can be linked to the N-terminus or the C-terminus of the FVIII protein or inserted between two FVIII amino acids adjacent to each other. In still other embodiments, the second polypeptide chain also comprises a second heterologous portion (H2).

[004] The present description also includes a chimeric molecule composed of a first polypeptide chain comprising a VWF protein, a heterologous portion (H1) and a VWF linker connecting the VWF protein and the heterologous portion (H1), and a second chain of polypeptides composed of a FVIII protein and an XTEN sequence, in which the VWF ligand in the first polypeptide chain comprises: (i) an a2 region of FVIII; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof, and in which the first polypeptide chain and the second polypeptide chain are associated with each other. The additional XTEN sequence is connected to the N-terminus or C-terminus of the FVIII protein or inserted between two FVIII amino acids adjacent to each other. In another embodiment, the chimeric molecule comprises an additional XTEN sequence, which is linked to the VWF protein, the heterologous moiety, the VWF ligand, or any combination thereof. In other embodiments, the chimeric molecule also comprises a second heterologous moiety (H2). In still other embodiments, the second heterologous portion is connected to the FVIII protein, the XTEN sequence, or both.

[005] For the chimeric molecules of the present description, the XTEN sequence, connected to a VWF protein, a VWF ligand, an FVIII protein or any other components in the chimeric molecules, comprises about 42 amino acids, about 72 amino acids , about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids , about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids , about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids , about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. In some embodiments, the XTEN polypeptide is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. In other embodiments, the XTEN polypeptide is selected from SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45; SEQ ID NO: 44; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46; SEQ ID NO: 44; or SEQ ID NO: 42.

[006] In other aspects, the additional XTEN sequence in the chimeric molecules comprises about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. In some embodiments, the XTEN polypeptide is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. In other embodiments, the XTEN polypeptide is selected from SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45; SEQ ID NO: 43; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46; SEQ ID NO: 44; or SEQ ID NO: 42.

[007] In one embodiment, the VWF linker useful for linking a VWF protein and a heterologous portion in the chimeric molecules comprises an a2 region, which is composed of an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu720 to Arg740, corresponding to full-length FVIII, in which the a2 region is capable of being cleaved by thrombin. In a certain embodiment, region a2 comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4). In another embodiment, the VWF linker useful for linking a VWF protein and a heterologous moiety comprises an α1 region that is composed of an amino acid sequence of at least about 80%, about 85%, about 90%, about 95 %, or 100% identical to Met337 to Arg372, corresponding to complete FVIII, in which the a1 region is capable of being cleaved by thrombin. In some embodiments, region a1 includes ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5).

[008] In other embodiments, the VWF linker useful for linking a VWF protein and a heterologous moiety comprises an a3 region that is composed of an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu1649 to Arg1689, corresponding to full-length FVIII, in which the a1 region is capable of being cleaved by thrombin. In a specific embodiment, the a3 region encompasses ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6).

[009] In still other embodiments, the VWF linker useful for linking a VWF protein and a heterologous moiety comprises a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif in the which PAR1 interaction motif motif at an external site is composed of S-F-L-L-R-N (SEQ ID NO: 7). In one embodiment, the external site-interacting PAR1 motif also comprises a sequence selected from P, P-N, N-P-D, P-N-D-K (SEQ ID NO: 8), P-N-D-K-Y (SEQ ID NO: 9), P-N-D-K-Y-E (SEQ ID NO: 10) , P-N-D-K-Y-E-P (SEQ ID NO: 11), P-N- D-K-Y-E-P-F (SEQ ID NO: 12), P-N-D-K-Y-E-P-F-W (SEQ ID NO: 13), P-N-D-K-Y-E-P-F-W-E (SEQ ID NO: 14), P-N-D-K-Y-E-P-F- W-E-D (SEQ ID NO: 20), P-N-D-K-Y-E-P-F-W-E-D-E ( SEQ ID NO: 21), P-N-D- K-Y-E-P-F-W-E-D-E-E (SEQ ID NO: 22), P-N-D-K-Y-E-P-F-W-E-D-E- E-S (SEQ ID NO: 23), or any combination thereof. In another embodiment, the aliphatic amino acid is selected from Glycine, Alanine, Valine, Leucine and Isoleucine. In a certain embodiment, the VWF ligand is composed of GGLVPRSFLLRNPNDKYEPFWEDEES (SEQ ID NO: 24).

[0010] In certain embodiments, thrombin cleaves the VWF ligand faster than thrombin would cleave the thrombin cleavage site, if the thrombin cleavage site were replaced by the VWF ligand in the chimeric molecule. In other embodiments, thrombin cleaves the VWF ligand at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times. times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times faster than thrombin would cleave the thrombin cleavage site, if the thrombin cleavage site was replaced by the VWF ligand in the chimeric molecule.

[0011] In some embodiments, the VWF linker also comprises one or more amino acids with a length of at least about 5, 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, 8 50, 900, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acids. In one example, the single amino acid or more amino acids make up a gly peptide. In another example, the single amino acid or more amino acids make up a GliGli peptide. In other examples, the single amino acid or more amino acids make up a gly/ser peptide. In other examples, the gly/ser peptide has a formula of (Gly4Ser)n or S(Gly4Ser)n, where n is a positive integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100. In certain examples, the ligand (Gly4Ser)n is ( Gli4Ser)3 (SEQ ID NO: 89) or (Gly4Ser) 4 (SEQ ID NO: 90).

[0012] The VWF protein useful for the chimeric molecule of the invention can include the D' domain and the D3 domain of VWF, where the D' domain and the D3 domain are capable of binding to a FVIII protein. In one embodiment, the D' Domain of the VWF protein is composed of an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 866 of the VWF protein. SEQ ID NO: 2. In another embodiment, the D3 Domain of the VWF protein is composed of an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to those amino acids 867 to 1240 of SEQ ID NO: 2. In other 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: 2. In In still other embodiments, in the VWF protein sequence, an amino acid other than cysteine is replaced by a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. In still other embodiments, the protein sequence VWF is composed of amino acids 764 to 1240 of SEQ ID NO: 2. In certain embodiments, the VWF protein also comprises the D1 domain, D2 domain or the D1 and D2 domains of VWF. In some embodiments, the VWF protein further comprises a VWF domain selected from the A1 domain, the A2 domain, the A3 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain , the CK domain, one or more fragments thereof or any combinations thereof. In other embodiments, the VWF protein essentially consists of, or consists of: (1) the VWF D' and D3 domains or fragments thereof; (2) the VWF D1, D' and D3 domains or fragments thereof; (3) the VWF D2, D' and D3 domains or fragments thereof; (4) the VWF D1, D2, D' and D3 domains or fragments thereof; or (5) the VWF D1, D2, D', D3 and A1 domains or fragments thereof. In still other embodiments, the VWF protein also comprises a VWF signal peptide. In still other embodiments, the VWF protein is pegylated, glycosylated, hesylated, or polysialylated. The term "pegylated" refers to having polyethylene glycol (PEG) in the protein; the term "glycosylated" refers to having glycosylation on the protein; the term "hesilated" refers to having hydroxyethyl starch (HES) in the protein; and the term "polysialylated" refers to having polysialic acids (PSA) in the protein. Examples of PEG, HES, and PSA are shown elsewhere in this document.

[0013] In some aspects, the heterologous portion (H1), fused to the VWF protein through a VWF ligand is capable of extending the half-life of the chimeric molecule. In one embodiment, the heterologous portion (H1) comprises an immunoglobulin constant region or a portion thereof, albumin, albumin-binding portion, PAS, HAP, transferrin or a fragment thereof, polyethylene glycol (PEG), hydroxyethyl starch (HES). ), PSA, the C-terminal peptide (CTP) of the human chorionic gonadotropin β subunit, or any combination thereof. In another embodiment, the heterologous portion is composed of an FcRn binding partner. In other embodiments, the heterologous portion comprises an Fc region. In other embodiments, the heterologous portion (H1) includes a clearance receptor, or fragment thereof, in which the clearance receptor blocks the binding of the FVIII protein to the FVIII clearance receptors. In some embodiments, the clearance receptor is a receptor-like low-density lipoprotein 1 (LRP1) or an FVIII binding fragment thereof.

[0014] In some aspects, the second heterologous portion fused to the FVIII protein through an optional FVIII linker comprises an immunoglobulin constant region or a portion thereof, albumin, albumin-binding polypeptide, PAS, the C-terminal peptide (CTP). ) of the β subunit of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), small albumin-binding molecules, or any combination thereof. In one embodiment, the second heterologous portion (H2) is capable of extending the half-life of the FVIII protein. In another embodiment, the second heterologous portion (H2) comprises a polypeptide, a non-polypeptide portion, or both. In another embodiment, the second heterologous portion (H2) comprises an immunoglobulin constant region or a portion thereof. In still other embodiments, the second heterologous portion is composed of an FcRn binding partner. In still other embodiments, the second heterologous portion comprises a second Fc region.

[0015] In some embodiments, the first heterologous portion fused to the VWF protein through a VWF linker and the second heterologous portion fused to the FVIII protein through an optional linker, in which an XTEN sequence is fused to either component, are associated each other. In one embodiment, the association between the first polypeptide chain and the second polypeptide chain is a covalent bond. In another embodiment, the association between the first heterologous moiety and the second heterologous moiety is a disulfide bond. In other embodiments, the first heterologous portion is an FcRn binding partner and the second heterologous portion is an FcRn binding partner. In still other embodiments, the first heterologous portion is an Fc region, and the second heterologous portion is an Fc region.

[0016] In certain embodiments, the FVIII protein is linked to the second heterologous portion by a FVIII linker. In one embodiment, the second linker is a cleavable linker. In another embodiment, the FVIII ligand is identical to the VWF ligand. In other embodiments, the FVIII ligand is different from the VWF ligand.

[0017] In some aspects, a chimeric molecule of the invention comprises a formula selected from: (a) V-L1-X1-H1:H2-L2-X2-C; (b) V-X1-L1-H1:H2-L2-X2-C; (c) V-L1-X1-H1:H2-X2-L2-C; (d) V-X1-L1-H1:H2-X2-L2-C; (e) V-L1-X1-H1: H2-L2-C(X2); (f) V-X1-L1-H1:H2-L2-C(X2); (g) C-X2-L2-H2:H1-X1-L1-V; (h) C-X2-L2-H2:H1-L1-X1-V; (i) C-L2-X2-H2:H1-L1-X1-V; (j) C-L2-X2-H2:H1-L1-X1-V; (k) C(X2)-L2-H2:H1-X1-L1-V; or (l) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF ligand; L2 is an optional FVIII ligand; H1 is a heterologous first moiety; H2 is a second heterologous moiety; X1 is an XTEN sequence; X2 is an optional XTEN sequence; C is an FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, where the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond between H1 and H2.

[0018] In some aspects, a chimeric molecule of the invention comprises a formula selected from: (a) V-L1-X1-H1: H2-L2-X2-C; (b) V-X1-L1-H1: H2-L2-X2-C; (c) V-L1-X1-H1: H2-X2-L2-C; (d) V-X1-L1-H1: H2-X2-L2-C; (e) V-L1-X1-H1: H2-L2-C(X2); (f) V-X1-L1-H1: H2-L2-C(X2); (g) C-X2-L2-H2: H1-X1-L1-V; (h) C-X2-L2-H2: H1-L1-X1-V; (i) C-L2-X2-H2:H1-L1-X1-V; (j) C-L2-X2-H2:H1-L1-X1-V; (k) C(X2)-L2-H2:H1-X1-L1-V; or (l) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF ligand; L2 is an optional FVIII ligand; H1 is a heterologous first moiety; H2 is a second heterologous moiety; X1 is an XTEN sequence; X2 is an optional XTEN sequence; C is an FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, where the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond between H1 and H2.

[0019] In the chimeric molecules of the invention, the VWF protein can inhibit or prevent the binding of endogenous VWF to the FVIII protein.

[0020] In certain aspects, the FVIII protein in chimeric molecules may include a third heterologous portion (H3). The third heterologous portion (H3) may be an XTEN sequence. In certain aspects, the FVIII protein in the chimeric molecules may include a fourth heterologous moiety (H4). The fourth heterologous portion (H4) may be an XTEN sequence. In certain aspects, the FVIII protein in chimeric molecules may include a fifth heterologous portion (H5). The fifth heterologous portion (H5) may be an XTEN sequence. In certain aspects, the FVIII protein in chimeric molecules may include a heterologous sixth moiety (H6). The sixth heterologous portion (H6) may be an XTEN sequence. In certain aspects, one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) is capable of extending the half-life of the chimeric molecule. . In other aspects, the third heterologous portion (H3), the fourth heterologous portion (H4), the fifth heterologous portion (H5), and the sixth heterologous portion (H6) are linked to the C terminus or the N terminus of FVIII or inserted between two amino acids of the FVIII protein. In certain aspects, one or more of the third heterologous portion, the fourth heterologous portion, the fifth heterologous portion, and the sixth heterologous portion span a length selected from one or more of about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. For example, the XTEN sequence of the third heterologous portion, the fourth heterologous portion, the fifth heterologous portion, or the sixth heterologous portion may be selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. More specifically, the sequence XTEN can be selected from SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45; SEQ ID NO: 43; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46; SEQ ID NO: 44; SEQ ID NO: 42.

[0021] In certain embodiments, the half-life of the chimeric molecule is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times or at least about 12 times longer than wild-type FVIII.

[0022] The present description also provides a polynucleotide or a set of polynucleotides encoding a chimeric molecule or a complementary sequence thereof. The polynucleotide or set of polynucleotides may also comprise a polynucleotide chain, which encodes PC5 or PC7.

[0023] Also included is a vector or set of vectors comprising the polynucleotide or set of polynucleotides and one or more promoters operably linked to the polynucleotide or set of polynucleotides. In some embodiments, the vector or set of vectors may also comprise an additional polynucleotide chain encoding PC5 or PC7.

[0024] The present invention also includes a host cell, comprising the polynucleotide or set of polynucleotides or the vector or set of vectors. In one embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is selected from a HEK293 cell, a CHO cell or a BHK cell.

[0025] In some aspects, the invention includes a pharmaceutical composition comprising a chimeric molecule described herein, the polynucleotide or set of polynucleotides encoding the chimeric molecule, the vector or set of vectors comprising the polynucleotide or set of polynucleotides, or the host cell described herein and a pharmaceutically acceptable carrier. In one embodiment, the chimeric molecule in the composition has an extended half-life compared to the wild-type FVIII protein. In another embodiment, the half-life of the chimeric molecule is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times or at least about 12 times more than wild-type FVIII.

[0026] Also included is a method of reducing the frequency or degree of a bleeding episode in an individual by administering an effective amount of a chimeric molecule described herein, the polynucleotide or set of polynucleotides encoding the chimeric molecule, the vector or the set of vectors described in this document, the host cell described in this document, or the composition described in this document. Also included is a method of preventing the occurrence of a bleeding episode in a subject by administering an effective amount of a chimeric molecule described herein, the polynucleotide or set of polynucleotides encoding the chimeric molecule, the vector or set of vectors described herein. herein, the host cell described herein or the composition described herein. In one embodiment, the bleeding episode is caused by a blood clotting disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, trauma capitis, gastrointestinal hemorrhage, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, retropharyngeal space bleeding, retroperitoneal space bleeding, illiopsoas muscle sheath hemorrhage, or any combination thereof. In another embodiment, a chimeric molecule described herein, the polynucleotide or set of polynucleotides encoding the chimeric molecule, the vector or set of vectors described herein, the host cell described herein, or the composition described herein may be administered by a route selected from topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, oral administration or any combinations thereof.

[0027] The present description also includes a method for making a chimeric molecule, comprising transfecting one or more host cells with a polynucleotide described herein or a vector described herein and expressing the chimeric molecule in the host cell. The method also involves isolating the chimeric molecule. In some embodiments, the FVIII activity of the chimeric molecule can be measured by aPTT examination or ROTEM examination.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

[0028] Figure 1 shows an exemplary diagram of a chimeric molecule (FVIII-XTEN/VWF heterodimer), consisting of two polypeptide chains, the first chain composed of a VWF protein (for example, a D' domain and a D3 domain of VWF) fused to an Fc region via a thrombin-cleavable VWF linker and the second chain composed of a FVIII protein fused to a second Fc region via a FVIII linker. The FVIII protein is composed of one or more XTENs in several domains of FVIII.

[0029] Figure 2 shows several VWF constructs, each construct comprising a D' domain and a D3 domain fused to an Fc region through a thrombin-cleavable VWF linker except control (i.e., VWF-052). VWF-031 comprises a 48 amino acid linker, comprising a L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-034 is composed of an XTEN sequence having 288 amino acids and a linker of 35 amino acids, comprising a L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-035 is composed of a 73 amino acid linker, comprising a L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-036 is composed of a 98 amino acid linker, comprising a L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-039 is composed of a 26 amino acid linker, comprising an L-V-P-R thrombin cleavage site (SEQ ID NO: 25) and an external site PAR1 interaction motif. VWF-051 is composed of a 54 amino acid linker, comprising an A-L-R-P-R-V-V thrombin cleavage site (SEQ ID NO: 26). VWF-052 comprises a 48 amino acid linker without any thrombin cleavage site (control). VWF-054 comprises a 40 amino acid VWF linker, which constitutes an α1 region of FVIII. VWF-055 comprises a 34 amino acid VWF linker, which constitutes an a2 region of FVIII. VWF-056 comprises a 46 amino acid VWF linker, which constitutes an a3 region of FVIII.

[0030] Figure 3A shows the rate of thrombin-mediated cleavage in resonance units per second (RU/s) as a function of capture density in RU units for VWF-Fc fusion constructs, i.e., VWF-031, VWF-034, VWF-036, VWF-039, VWF-051 and 052-VWF. Figure 3B shows the rate of thrombin-mediated cleavage in resonance units per second (RU/s) as a function of capture density in RU units for VWF-Fc fusion constructs, i.e., VWF-031, VWF-034 , VWF-036, VWF-039, VWF-051 and 052-VWF. In these experiments, each VWF-Fc fusion construct was captured at various densities and subsequently exposed to a fixed concentration of human alpha-thrombin. The slope of each curve in Figure 3A and Figure 3B directly reflects the susceptibility of each construct to thrombin cleavage.

[0031] Figure 4A shows the rate of thrombin-mediated cleavage in resonance units per second (RU/s) as a function of capture density in RU units for VWF-Fc fusion constructs, i.e., VWF-054, VWF-055, and VWF-056. FIG 4B shows the rate of thrombin-mediated cleavage in resonance units per second (RU/s) as a function of capture density in RU units for fusion constructs, i.e., VWF-031, VWF-039, VWF- 054, VWF-055, and VWF-056. In these experiments, each VWF-Fc fusion construct was captured at various densities and subsequently exposed to a fixed concentration of human alpha-thrombin. The slope of each curve in Figure 4A and Figure 4B directly reflects the susceptibility of each construct to thrombin cleavage.

[0032] Figure 5 shows the results of a linear regression analysis to determine the susceptibility of the various constructs VWF-Fc, VWF-031, VWF-034, VWF-036, VWF-039, VWF-051, VWF-052, VWF -054, VWF-055, and VWF-056 to thrombin-mediated cleavage. Values are expressed in units of inverse seconds and reflect the slopes of the curves presented in Figure 3 and Figure 4. The relative susceptibility of two different constructs is derived from the quotient of their respective slopes. SlopeVWF-039/slopeVWF-031 is 71, indicating that the VWF-Fc fusion construct VWF-039 is 71 times more susceptible to thrombin-mediated cleavage than VWF-031. TiltVWF-055/tiltVWF-031 is 65, and tiltVWF-051/tiltVWF-031 is 1.8.

[0033] Figure 6 shows the clotting time of several chimeric molecules in HemA patient measured by ROTEM whole blood examination. FVII155/VWF-031 is composed of two polypeptide chains, the first chain comprising BDD FVIII fused to an Fc region and the second chain comprising a D'Domain and a VWF D3 domain fused to an Fc region through a site of minimal thrombin cleavage (i.e., L-V-P-R (SEQ ID NO: 25)). FVII155/VWF-039 is composed of two polypeptide chains, the first chain comprising BDD FVIII fused to an Fc region and the second chain comprising a D'Domain and a VWF D3 domain fused to an Fc region through a site thrombin cleavage motif (i.e., L-V-P-R (SEQ ID NO: 25) and an external site-interacting PAR1 motif. FVII155/VWF-055 is composed of two polypeptide chains, the first chain comprising BDD FVIII fused to a region of Fc and the second chain comprising a D'Domain and a D3 domain of VWF fused to an Fc region through a VWF linker composed of an a2 region of FVIII.

[0034] Figure 7 shows a diagram of a representative FVIII-VWF heterodimer and constructs FVIII169, FVIII286, VWF057, VWF059, and VWF062. For example, the FVIII169 construct is composed of a B-domain deleted FVIII protein with R1648A substitution fused to an Fc region, in which an XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to the full length of mature FVIII ( A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-Fc). The FVIIIs86 construct is composed of a B-domain deleted FVIII protein with R1648 substitution fused to an Fc region, in which an XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to the full length of mature FVIII, with region a2 additional between FVIII and Fc (A1-a1-A2-a2- 288XTEN-a3-A3-C1-C2-a2-Fc). VWF057 is a VWF-Fc fusion construct comprising the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A), linked to the Fc region via a VWF linker, which comprises the thrombin site LVPR ("LVPR") and the GS linker ("GS"), in which an XTEN sequence (i.e., 144XTEN) is inserted between the D'D3 domain and the VWF linker (D'D3-144XTEN-GS +LVPR-Fc). VWF059 is a VWF-Fc fusion construct comprising the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A), linked to the Fc region via an acidic region 2 ( a2) as a VWF ligand, in which an XTEN sequence is inserted between the D'D3 domain and the VWF ligand. VWF062 is a VWF-Fc fusion construct comprising the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A), linked to the Fc region, in which an XTEN sequence it is inserted between the D'D3 domain and the Fc region (D'D3-144XTEN-Fc). VWF ligand.

[0035] Figure 8 shows the marked effectiveness of the FVIII-XTEN-Fc/D'D3-Ligante-Fc FVIII-XTEN-Fc/D heterodimers (i.e., FVIII169/VWF034 FVIII169/VWF059 and FVIII169/VWF057), compared to the FVIII domain B deleted ("SQ BDD FVIII" or "BDD-rFVIII") or with vehicle control in the HemA model of rat tail docking. BDD-rFVIII is shown as a circle while FVIII169/VWF034 is shown as a square, FVIII169/VWF059 is shown as a triangle, FVIII169/VWF057 is shown as an empty circle and the vehicle is shown as an inverted triangle. VWF034 is a VWF-Fc fusion construct comprising a VWF D' domain and a D3 domain fused to an Fc region via a VWF linker, which comprises LVPR, in which an XTEN sequence (i.e., 288XTEN) is inserted between the D'D3 domain and the VWF ligand (D'D3-288XTEN-LVPR-Fc). Construct details of FVIII169, VWF059, and VWF057 are shown elsewhere in this document. The average blood loss (uL) of mice after a dose of 75 IU/kg of construct in each of the treatment groups are indicated by horizontal lines.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention is directed to a chimeric molecule, composed of an XTEN sequence and a thrombin cleavable linker connecting a VWF protein or a FVIII protein to a heterologous moiety, for example, a half-life prolongation moiety. The invention also provides a chimeric molecule comprising two polypeptide chains, the first chain comprising a VWF protein fused to a heterologous moiety and a second chain comprising an FVIII protein and a second heterologous moiety, wherein the chimeric molecule is composed of a sequence XTEN in the first or second polypeptide chain and where the VWF protein or the FVIII protein (or both) are fused to a heterologous moiety via a VWF linker or a FVIII linker (or both). The thrombin cleavable ligand (VWF ligand or FVIII ligand) can be efficiently cleaved by thrombin at the site of injury where thrombin is readily available. Exemplary chimeric molecules are illustrated in the present description and in the figures. In some embodiments, the invention relates to chimeric molecules having the structures set forth, for example, in FIGS. 1 to 7. In other embodiments, the invention relates to polynucleotide constructs encoding chimeric molecules described herein.

[0037] In order to provide a clear understanding of the specifications and claims, the following definitions are provided below.

I. Definitions

[0038] It should be noted that the term "one" or "an" entity refers to one or more of that entity; "a nucleotide sequence" is understood, for example, to represent one or more nucleotide sequences. Accordingly, the terms "one", "one or more" and "at least one" may be used interchangeably in this document.

[0039] The term "about" is used here to mean approximately, around, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range, widening the limits above and below established numerical values. In general, the term "about" is used in this document to indicate a 10% variation up or down (greater or smaller) about an indicated numerical value.

[0040] The term "polynucleotide" or "nucleotide" is intended to encompass a single nucleic acid, as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or Plasma DNA (pDNA). In certain embodiments, a polynucleotide comprises a conventional phosphodiester linkage or an unconventional linkage (e.g., an amide linkage, such as those found in peptide nucleic acids (PNA)). The term "nucleic acids" refers to any one or more of the nucleic acid segments, e.g., fragments of DNA or RNA, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is meant a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention also include such synthetically produced molecules. Furthermore, a polynucleotide or nucleic acid may include regulatory elements such as promoters, enhancers, ribosomal binding sites, or transcription termination signals.

[0041] As used herein, a "coding region" or "coding sequence" is a portion of the polynucleotide that consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not normally translated into an amino acid, it can be considered part of a coding region, but any flanking sequences, e.g. promoters, ribosomal binding sites, transcriptional termini, introns and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5'-terminus, encoding the amino terminus of the resulting polypeptide, and translation stop codon at the 3'-terminus of the resulting polypeptide. Two or more coding regions of the present invention may be present in a single polynucleotide construct, e.g., in a single vector, or separate polynucleotide constructs, e.g., in separate (different) vectors. It follows, then, that a single vector may contain only a single coding region, or comprise two or more coding regions, for example, a single vector may separately encode a first polypeptide chain and a second polypeptide chain of a chimeric molecule as described below. Furthermore, a vector, polynucleotide or nucleic acid of the invention may encode heterologous coding regions, fused or unfused to a nucleic acid encoding a chimeric molecule of the invention. Heterologous coding regions include, without limitation, specialized elements or patterns, such as a secretory signal peptide or a heterologous functional domain.

[0042] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein once export of the growing protein chain through the rough endoplasmic reticulum has begun. Those skilled in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide and are cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, for example, an FVIII signal peptide or a VWF signal peptide is used, or a functional derivative of this sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide may be used, for example, a human tissue plasminogen activator (TPA), a rat β-glucuronidase signal peptide or functional derivatives thereof.

[0043] The term "downstream" refers to a nucleotide sequence that is located 3' of a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to sequences that follow the transcription starting point. For example, the translation initiation codon of a gene is located downstream of the transcription starting point.

[0044] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence relates to sequences that are located on the 5' side of a coding region or a transcription starting point. For example, most promoters are located upstream of the transcription start site.

[0045] As used herein, the term "regulatory region" refers to the nucleotide sequence, located upstream (5' non-coding sequence), within or downstream (3' non-coding sequence) of a coding region, and that influences transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadelination and transcription termination signal sequence will generally be located 3' to the coding sequence.

[0046] A polynucleotide encoding a gene product, for example, a polypeptide, may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association, a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions so as to place expression of the gene product under the influence or control of the regulatory region(s). (s). For example, a coding region and a promoter are operably "associated" if induction of promoter function results in transcription of the mRNA encoding the gene product encoded by the coding region, and if the nature of the link between the promoter and the region coding does not interfere with the ability of the promoter to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, in addition to a promoter, for example, enhancers, operators, repressors and transcription termination signals, can also be operably associated with a coding region to direct the expression of gene products.

[0047] One embodiment of transcription control regions is known to those skilled in the art. These include, without limitation, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments of cytomegalovirus (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter) and retroviruses (e.g. Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional appropriate transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., interferon- or interleukin-inducible promoters).

[0048] Likewise, a modality of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and picornavirus-derived elements (particularly an internal ribosomal entry site, or IRES, also known as a CITE sequence).

[0049] The term "expression", as used herein, refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes, without limitation, the transcription of polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product and the translation of an mRNA into a polypeptide. Expression produces a "gene product". As used herein, a gene product can be any nucleic acid, for example, a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. The gene products described herein also include nucleic acids with post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0050] A "vector" refers to any vehicle for cloning and/or transferring a nucleic acid to a host cell. A vector can be a replicon to which another nucleic acid segment can be attached in order to cause replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and non-viral vehicles for introducing nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses or modified bacterial viruses. Insertion of a polynucleotide into an appropriate vector can be accomplished by ligating appropriate fragments of the polynucleotide into a chosen vector that has complementary cohesive termini.

[0051] Vectors can be designed to encode selectable markers or reporters that enable the selection or identification of cells that have incorporated the vector. The expression of selectable markers or reporters allows the identification and/or selection of host cells that incorporate and express other coding regions contained in the vector. Examples of selectable marker genes known and used in the art: genes providing resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase gene and the like. Examples of reporters known and used in the art: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus) and the like. Selectable markers can also be considered reporters.

[0052] The term "plasmid" refers to an extrachromosomal element that often carries a gene that is not part of the cell's central metabolism, and generally in the form of circular, double-stranded DNA molecules. Such elements may be autonomous replication sequences, genome integration sequences, phages, or nucleotide sequences, linear, circular or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which various nucleotide sequences have been joined or recombined into a single construct, which is capable of introducing a promoter fragment and a DNA sequence into a selected gene product together with the appropriate 3' untranslated sequence in a cell.

[0053] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adenovirus-associated vectors, poxviruses, for example, vaccinia virus vectors, baculovirus vectors or herpesvirus vectors. Nonviral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0054] A "cloning vector" refers to a "replicon," which is a unit length of a nucleic acid that replicates sequentially and that comprises an origin of replication, such as a plasmid, phage or cosmid, to which another Nucleic acid segment can be attached in order to cause replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria, and expression in another, e.g., eukaryotic cells. Cloning vectors typically encompass one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

[0055] The term "expression vector" refers to a vehicle designed to allow expression of an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with the regulatory regions as described above.

[0056] Vectors are introduced into host cells by methods well known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), the use of a gene gun, or a DNA vector vehicle.

[0057] "Culture", "cultivate" and "cultivation", as used herein, means means for incubating cells under in vitro conditions that allow the growth or division of cells to maintain the cells in a living state. "Cultivated cells", as used herein, means cells that are propagated in vitro.

[0058] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain", or any other terms used to refer to a chain or chains of two or more amino acids, are included in the definition of "polypeptide", and the The term "polypeptide" may be used instead of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or by modification by amino acids that do not occur naturally. A polypeptide may be derived from a natural biological source or produced with recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any way, including chemical synthesis.

[0059] An "isolated" polypeptide or a fragment, variant or derivative thereof refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced proteins and polypeptides expressed in host cells are considered isolated for the purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated or partially or substantially purified by any suitable technique.

[0060] Fragments or variants of polypeptides and any combinations thereof are also included in the present invention. The term "fragment" or "variant" when referring to polypeptide binding domains or binding molecules of the present invention includes any polypeptides that maintain at least some of the properties (e.g., FcRn binding affinity for an FcRn binding domain or variant Fc, clotting activity for a FVIII variant or FVIII binding activity for the VWF protein) of the reference polypeptide. Polypeptide fragments include proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere in this document, but do not include the naturally occurring complete polypeptide (or mature polypeptide). Variant polypeptide binding domains or binding molecules of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may encompass amino acid substitutions, deletions or additions, conservative or non-conservative.

[0061] The term "VWF fragment" or "VWF fragments" used herein means all VWF fragments that interact with FVIII and retain at least one or more properties that are normally provided to FVIII by the full length of VWF, e.g., preventing premature activation to FVIIIa, prevent premature proteolysis, prevent association with phospholipid membranes that could lead to premature clearance, prevent binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and/or stabilize FVIII heavy chain or light chain interactions. In a certain embodiment, the "VWF fragment" as used herein comprises a D' domain and a D3 domain of the VWF protein, but does not include the A1 domain, the A2 domain, the A3 domain, the D4 domain, the B1, the B2 domain, the B3 domain, the C1 domain, the C2 domain and the CK domain of the VWF protein.

[0062] The term "half-life limiting factor" or "FVIII half-life limiting factor", as used herein, indicates a factor that prevents the half-life of an FVIII protein from being more than 1.5 or 2-fold compared to wild-type FVIII (e.g. ADVATE® or REFACTO®). For example, full-length or mature VWF can act as a limiting factor for FVIII half-life by inducing the FVIII and VWF complex to be cleared from the system by one or more VWF clearance pathways. In one example, endogenous VWF is a limiting factor of FVIII half-life. In another example, a full-length recombinant VWF molecule non-covalently linked to a FVIII protein is a limiting factor for FVIII half-life.

[0063] The term "endogenous VWF", as used here, indicates VWF molecules naturally present in plasma. The endogenous VWF molecule can be multimer, but can be a monomer or a dimer. Endogenous VWF in plasma binds to FVIII and forms a noncovalent complex with FVIII.

[0064] A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. , threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, tryptophan, phenylalanine, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, an amino acid sequence may be conservatively replaced with a structurally similar sequence that differs in the order and/or composition of side chain family members.

[0065] As 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 given polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711 ). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a given sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, so that the percentage of identity is calculated over the total length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0066] As used herein, a "corresponding amino acid" or an "equivalent amino acid" in a VWF sequence or a FVIII protein sequence is identified by alignment to maximize the identity or similarity between a first VWF or FVIII sequence and a second sequence VWF or FVIII. The number used to identify an equivalent amino acid in a second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0067] A "fusion" or "chimeric" molecule comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. Amino acid sequences that normally exist in separate proteins can be brought together into the fusion polypeptide, or amino acid sequences that normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, for example, the fusion of a Factor domain. VIII of the invention with an immunoglobulin Fc domain. A fusion protein is created, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein may also comprise a second amino acid sequence associated with the first amino acid sequence by a covalent bond, non-peptide bond or non-covalent bond.

[0068] As used herein, the term "half-life" refers to a biological half-life of a particular polypeptide in vivo. Half-life can be represented by the time required for half of the amount administered to an individual to be cleared from the circulation and/or other tissues of the animal. When a clearance curve for a given polypeptide is constructed as a function of time, the curve is usually biphasic with a fast α phase and a longer β phase. The α phase typically represents an equilibrium of the administered polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide. The β phase normally represents catabolism of the polypeptide in the intravascular space. In some embodiments, the chimeric molecule of the invention is monophasic, and thus does not have an alpha phase, but only the single beta phase. Therefore, in certain embodiments, the term half-life 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.

[0069] The term "heterologous" as applied to a polynucleotide or a polypeptide means that the polynucleotide or polypeptide is derived from an entity distinct from the entity to which it is being compared. Therefore, a heterologous polypeptide, linked to a VWF protein, means a polypeptide chain that is linked to a VWF protein and is not a natural part of the VWF protein. For example, a heterologous polynucleotide or antigen may be derived from a different species, a different cell type from an individual, or the same or different cell type from different individuals.

[0070] The terms "linked," "fused," or "connected" as used herein refer to a first amino acid sequence or nucleotide sequence joined to a second amino acid sequence or nucleotide sequence (e.g., through of a peptide bond or a phosphodiester bond, respectively). The term "covalently linked" or "covalent bond" refers to a covalent bond, e.g. a disulfide bond, a peptide bond or one or more amino acids, e.g. a linker, between the two moieties that are bonded together. yes. The first amino acid or nucleotide sequence may be joined directly to the second amino acid or nucleotide sequence or alternatively an intervening sequence may join the first sequence to a second sequence. The terms "linked," "fused," or "connected" mean not only the fusion of a first amino acid sequence to a second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first sequence of amino acids (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively). In one embodiment, the first amino acid sequence may be joined to a second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be joined to a second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for polypeptide and polynucleotide chains). The covalent bond is sometimes indicated as (-) or a hyphen.

[0071] In this document the term "associated" refers to a covalent or non-covalent bond formed between a first chain of amino acids and a second chain of amino acids. In one embodiment, the term "associated with" means a non-covalent peptide bond or a non-covalent bond. In some embodiments, this association is indicated by a colon, that is, (:). In another embodiment, it means a covalent bond, other than a peptide bond. In other embodiments, the term "covalently associated", as used herein, means an association between two moieties by a covalent bond, e.g., a disulfide bond, a peptide bond, or one or more amino acids (e.g., a linker). For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond, or bridge, with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by a disulfide bond and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229 , US numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a metallic bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agostic bond, a curved bond, a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibond. Non-limiting examples of non-covalent bonds include an ionic bond (e.g., cation-pi bond or salt bond), a metallic bond, a hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, a low- barrier or a symmetric hydrogen bond), van der Walls force, London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, entropic force, stacking, or chemical polarity.

[0072] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. In one embodiment, a polypeptide has an enzymatic cleavage site cleaved by an enzyme that is activated during the coagulation cascade, such that cleavage of such sites occurs at the site of clot formation. In another embodiment, a FVIII linker connecting a FVIII protein and a second heterologous moiety may include a cleavage site. 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: 27) and SVSQTSKLTR (SEQ ID NO: 28). Exemplary thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 29), TTKIKPR (SEQ ID NO: 30), LVPRG (SEQ ID NO: 31) and ALRPR (amino acids 1 to 5 of SEQ ID NO: 26 ). Other enzymatic cleavage sites are known in the art. A cleavage site that can be cleaved by thrombin is referred to herein as a "thrombin cleavage site."

[0073] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site on a polypeptide that is the target for enzymes that operate after translation of the polypeptide. In one embodiment, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before secretion of the protein from the cell. Examples of such processing sites include, for example, those directed by the PACE/furin family of endopeptidases (PACE is the acronym for Paired basic Aminoacid Cleaving Enzyme). These enzymes localize to the Golgi membrane and cleave proteins on the carboxy-terminal side of the Arg-[any residue]-(Lys or Arg)-ARG motif sequence. As used herein, the "furin" family of enzymes includes, for example, PCSK1 (also known as PC1/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4) or PCSK7 (also known as PC7/LPC, PC8 or SPC7). Other processing locations are known in the art. The term "processable ligand" referred to herein means a ligand comprising an intracellular processing site.

[0074] The term "furin" refers to enzymes corresponding to EC No. 3.4.21.75. Furin is a subtilisin-type proprotein convertase, which is also known as PACE (Paired basic Aminoacid Cleaving Enzyme). Furin deletes sections of inactive precursor proteins to convert them into biologically active proteins. During its intracellular transport, the propeptide is cleaved from the mature VWF molecule by a furin enzyme in the Golgi.

[0075] In constructs that include more than one processing or cleavage site, it will be understood that sites may be the same or different.

[0076] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, spontaneously or as a result of trauma, due to a deficient ability or inability to form a fibrin clot. Examples of such disorders are 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, tendency to mild bleeding). Other hemostatic disorders include, for example, von Willebrand disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, in factor X or factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb, the receptor for VWF, may be defective and lead to lack of preliminary clot formation (primary hemostasis) and increased tendency to bleeding, and to Glanzman and Naegeli's thrombasthenia (Glanzmann's thrombasthenia). In liver functional failures (acute and chronic forms), there is an insufficient production of clotting factors by the liver; this may increase the risk of bleeding.

[0077] The chimeric molecules of the invention can be used prophylactically. As used herein, the term "prophylactic treatment" refers to the administration of a molecule prior to an episode of bleeding. In one embodiment, the individual in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered before or after surgery as a prophylactic. The chimeric protein of the invention can be administered during or after surgery to control an acute bleeding episode. Surgery may include, but is not limited to, liver transplantation, hepatectomy, dental procedures or stem cell transplantation.

[0078] The chimeric molecule of the invention is also used for on-demand (also referred to as "episodic") treatment. The term "on-demand treatment" or "episodic treatment" refers to the administration of a chimeric molecule in response to the symptoms of a bleeding episode or prior to an activity that may cause bleeding. In one aspect, on-demand (episodic) treatment may be given to an individual when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, on-demand treatment can be given before activities that increase the risk of bleeding, such as contact sports.

[0079] In this document the term "acute hemorrhage" refers to an episode of bleeding regardless of the underlying cause. For example, an individual may have trauma, uremia, an inherited bleeding disorder (e.g., factor VII deficiency), a platelet disorder, or resistance due to the development of antibodies against clotting factors.

[0080] Treat, treatment, treating, as used herein refer, for example, to reducing the severity of a disease or condition; reduction of duration of a disease course; the improvement of one or more symptoms associated with a disease or condition; the provision of beneficial effects to an individual with a disease or condition, without necessarily curing the disease or condition, or the prophylaxis of one or more symptoms associated with a disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining a trough FVIII concentration of at least about 1IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7 IU/dL, 8UL/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12 IU/dL, 13 IU/dL, 14 IU/dL, 15 IU/dL, 16 IU /dL, 17 IU/dL, 18 IU/dL, 19 IU/dL or 20 IU/dL in an individual through administration of a chimeric molecule of the invention. In another embodiment, treating or treating means maintaining a minimum FVIII concentration level between about 1 and about 20 IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL, about 7 and about 20 IU/dL, about 8, about 20 IU/dL, about 9 and about 20 IU/dL, or about 10 and 20 IU/dL. Treating or treating a disease or condition may also include maintaining FVIII activity in an individual at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8 %, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of FVIII activity in a non-hemophilic individual. The minimum concentration level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each person.

II. Chimeric Molecules

[0081] The chimeric molecules of the invention are designed to enhance the release of a VWF protein or a FVIII protein from another portion to which the VWF protein or FVIII protein is fused. The invention provides a thrombin-cleavable linker that can be cleaved quickly and efficiently at the site of injury. In one aspect of the invention, the chimeric molecule may comprise a Von Willebrand Factor (VWF) protein, a heterologous portion (H1), an XTEN sequence, and a VWF linker connecting the VWF protein with the heterologous portion, in which the linker VWF is composed of a polypeptide selected from: (i) an a2 region of Factor VIII (FVIII); (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof, and in which the XTEN sequence is linked to the VWF protein, or the heterologous portion (H1), the VWF ligand or any combination thereof. In another aspect of the invention, the chimeric molecule may include a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1) and a VWF linker connecting the VWF protein and the heterologous moiety (H1), and a second polypeptide chain. composed of a FVIII protein and an XTEN sequence, in which the VWF linker in the first polypeptide chain comprises: (i) an a2 region of FVIII; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof, and in which the first polypeptide chain and the second polypeptide chain are associated with each other.

[0082] In other aspects of the invention, a chimeric molecule comprises a polypeptide chain composed of a FVIII protein fused to a heterologous portion through a FVIII linker, where the FVIII linker is composed of: (i) an a2 region of FVIII; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination of these and an external local PAR1 interaction motif, where X is an aliphatic amino acid; or (v) any combination thereof.

I.A. Chimeric Molecules with WF, XTEN or VWF Ligand

[0083] The present invention provides a chimeric molecule, composed of a VWF protein fused to an XTEN sequence through a VWF linker, where the VWF linker is composed of a polypeptide selected from: (i) an a2 region of Factor VIII (FVIII) ; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof.

[0084] In one embodiment, the chimeric molecule is composed of a VWF protein, a heterologous portion (H1), an XTEN sequence, and a VWF linker connecting the VWF protein with the heterologous portion where the XTEN sequence is located between the protein VWF and the VWF ligand and wherein the VWF ligand comprises a polypeptide selected from: (i) an a2 region of Factor VIII (FVIII); (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof. In one embodiment, the chimeric molecule is composed of a VWF protein, a heterologous portion (H1), an XTEN sequence, and a VWF linker connecting the VWF protein with the heterologous portion where the XTEN sequence is located between the VWF protein and the VWF ligand and wherein the VWF ligand comprises a polypeptide selected from (i) an a2 region of Factor VIII (FVIII); (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof.

[0085] In other embodiments, the chimeric molecule also comprises a chain of polypeptides, consisting of a FVIII protein, where the first chain comprising the VWF protein and the second chain comprising the FVIII protein are associated with each other. In one example, the association may be a covalent bond, e.g., disulfide bond or association. In still other embodiments, the polypeptide chain composed of an FVIII protein also comprises an additional XTEN sequence. The additional XTEN sequence can be linked to the N-terminus or the C-terminus of the FVIII protein or inserted between two FVIII amino acids adjacent to each other. In still other embodiments, the chain comprising an FVIII protein also comprises a second heterologous portion (H2). In some embodiments, the FVIII protein is fused to the second heterologous portion via an FVIII linker. In certain embodiments, the FVIII linker is identical to the VWF linker connecting the VWF protein and the heterologous portion. In other embodiments, the FVIII linker is different from the VWF linker connecting the VWF protein and the heterologous portion.

[0086] In certain embodiments, a chimeric molecule comprises a formula selected from: (i) V-L1-X1-H1:H2- L2-X2-C; (ii) V-X1-L1-H1:H2-L2-X2-C; (iii) V-L1-X1-H1:H2-X2-L2-C; (iv) V-X1-L1-H1:H2-X2-L2-C; (v) V-L1-X1-H1:H2-L2-C(X2); (vi) V-X1-L1-H1:H2-L2-C(X2); (vii) C-X2-L2-H2:H1-X1-L1-V; (viii) C-X2-L2-H2:H1-L1-X1-V; (ix) C-L2-X2-H2:H1-L1-X1-V; (x) C-L2-X2-H2:H1-L1-X1-V; (xi) C(X2)-L2-H2:H1-X1-L1-V; or (xii) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF ligand; L2 is an optional FVIII ligand; H1 is a heterologous first moiety; H2 is a second heterologous moiety; X1 is an XTEN sequence; X2 is an optional XTEN sequence; C is an FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, where the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond between H1 and H2.

[0087] In some embodiments, the FVIII protein in the chimeric molecule comprises a third heterologous portion (H3), which may be an XTEN sequence. In other embodiments, the FVIII protein of the chimeric molecule comprises a fourth heterologous portion (H4), which may be an XTEN sequence. In some embodiments, the FVIII protein in the chimeric molecule comprises a fifth heterologous portion (H5), which may be an XTEN sequence. In some embodiments, the FVIII protein in the chimeric molecule comprises a heterologous sixth portion (H6), which may be an XTEN sequence. In certain aspects, one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are capable of extending the half-life of the chimeric molecule. . In some embodiments, the third heterologous portion (H3), the fourth heterologous portion (H4), the fifth heterologous portion (H5), and the sixth heterologous portion (H6) are linked to the C terminus or N terminus of FVIII or inserted between two amino acids of the FVIII protein.

II.B. Chimeric Molecules with FVIII, XTEN, VWF Protein, VWF Ligand

[0088] The present description also includes a chimeric molecule composed of a first polypeptide chain comprising a VWF protein, a heterologous portion (H1) and a VWF linker connecting the VWF protein and the heterologous portion (H1) and a second chain of polypeptides composed of a FVIII protein and an XTEN sequence, where the VWF ligand of the first polypeptide chain comprises: (i) an a2 region of FVIII; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof, and in which the first polypeptide chain and the second polypeptide chain are associated with each other. The additional XTEN sequence can be linked to the N-terminus or the C-terminus of the FVIII protein or inserted between two FVIII amino acids adjacent to each other. In another embodiment, the chimeric molecule also comprises an additional XTEN sequence, which is linked to the VWF protein, the heterologous portion, the VWF ligand or any combination thereof. In other embodiments, the chimeric molecule also comprises a second heterologous moiety (H2). In still other embodiments, the second heterologous portion is linked to the FVIII protein, the XTEN sequence, or both. In still other embodiments, the second heterologous portion is linked to the FVIII protein or the XTEN sequence via a FVIII linker. In still other embodiments, the FVIII ligand is identical to the VWF ligand. In some embodiments, the FVIII ligand is different from the VWF ligand.

[0089] In certain embodiments, a chimeric molecule comprises a formula selected from: (i) V-L1-X1-H1:H2-L2-X2-C; (ii) V-X1-L1-H1:H2-L2-X2-C; (iii) V-L1-X1-H1:H2-X2-L2-C; (iv) V-X1-L1- H1:H2-X2-L2-C; (v) V-L1-X1-H1:H2-L2-C(X2); (vi) V-X1-L1-H1:H2-L2- C(X2); (vii) C-X2-L2-H2:H1-X1-L1-V; (viii) C-X2-L2-H2:H1-L1-X1-V; (ix) C-L2-X2-H2:H1-L1-X1-V; (x) C-L2-X2-H2:H1-L1-X1-V; (xi) C(X2)-L2- H2:H1-X1-L1-V; or (xii) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF ligand; L2 is an optional FVIII ligand; H1 is a heterologous first moiety; H2 is a second heterologous moiety; X1 is an XTEN sequence; 2X is an optional XTEN sequence; C is an FVIII protein; C(x2) is a FVIII protein fused to an XTEN sequence, in which the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond between H1 and H2 o. In one embodiment, the VWF ligand and the FVIII ligand may be the same. In another embodiment, the FVIII ligand is identical to the VWF ligand.

[0090] In some embodiments, the FVIII protein in the chimeric molecule comprises a third heterologous portion (H3), which may be an XTEN sequence. In some embodiments, the FVIII protein in the chimeric molecule comprises a fourth heterologous portion (H4), which may be an XTEN sequence. In some embodiments, the FVIII protein in the chimeric molecule comprises a fifth heterologous portion (H5), which may be an XTEN sequence. In certain aspects, one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are capable of extending the half-life of the chimeric molecule. . In some embodiments, the third heterologous portion (H3), the fourth heterologous portion (H4), the fifth heterologous portion (H5), and the sixth heterologous portion (H6) are attached to the C terminus or N terminus of FVIII or inserted between two amino acids of the FVIII protein.

II.C. Chimeric Molecules with FVIII, XTEN and FVIII Ligand

[0091] A chimeric molecule of the invention may comprise a FVIII protein, an XTEN sequence, and a heterologous portion fused to a FVIII linker, which comprises (i) an a2 region of FVIII; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof. In certain embodiments, a chimeric molecule comprises two polypeptide chains, the first chain comprising a FVIII protein fused to a first Fc region via an FVIII linker, and a second chain comprising a VWF protein (e.g., a D domain and a VWF D3 domain) fused to an Fc region, where the FVIII linker in the first polypeptide chain comprises: (i) an FVIII a2 region; (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof in which the first polypeptide chain and the second polypeptide chain are associated with each other and in which an XTEN sequence is linked to the first polypeptide (e.g., N-terminal or C-terminal of FVIII protein, the linker or the first Fc region or within the FVIII protein), the second polypeptide (e.g., N-terminal or C-terminal of the VWF protein or Fc region or within the FVIII protein), or both. In a specific embodiment the linker of the first polypeptide chain comprises an a2 region of FVIII.

[0092] In certain embodiments, a chimeric molecule comprises a formula selected from: (i) V-L1-X1-H1:H2-L2-X2-C; (ii) V-X1-L1-H1:H2-L2-X2-C; (iii) V-L1-X1-H1:H2-X2-L2-C; (iv) V-X1-L1- H1:H2-X2-L2-C; (v) V-L1-X1-H1: H2-L2-C(X2); (vi) V-X1-L1-H1:H2-L2- C(X2); (vii) C-X2-L2-H2:H1-X1-L1-V; (viii) C-X2-L2-H2:H1-L1-X1-V; (ix) C-L2-X2-H2:H1-L1-X1-V; (x) C-L2-X2-H2:H1-L1-X1-V; (xi) C(X2)-L2- H2:H1-X1-L1-V; or (xii) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is an FVIII ligand; L2 is an optional VWF ligand; H1 is a heterologous first moiety; H2 is a second heterologous moiety; X1 is an XTEN sequence; X2 is an optional XTEN sequence; C is an FVIII protein; C(X1) is a FVIII protein fused to an XTEN sequence, where the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond between H1 and H2 and in which at least one XTEN sequence is present in the chimeric molecule. In another embodiment, the FVIII ligand is identical to the VWF ligand. In another embodiment, the FVIII ligand is identical to the VWF ligand.

II.D. Components of Chimeric Molecules II. C. 1. VWF ligand or FVIII ligand

[0093] The VWF linker or FVIII linker useful for a chimeric molecule of the invention is a thrombin-cleavable linker fusing a VWF protein to a heterologous moiety or a FVIII protein to a heterologous moiety. In one embodiment, the VWF ligand or FVIII ligand comprises an a1 region of FVIII. In another embodiment, the VWF linker or FVIII linker comprises an a2 region of FVIII. In other embodiments, the VWF linker or FVIII linker comprises an a3 region of FVIII. In still other embodiments, the VWF ligand or FVIII ligand comprises a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external PAR1 local interaction motif, where X is an aliphatic amino acid.

[0094] In one embodiment, the VWF ligand or FVIII ligand comprises an a1 region, which is composed of an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, or 100 % identical to Met337 to Arg372, corresponding to complete FVIII, in which the a1 region is capable of being cleaved by thrombin. In one embodiment, the VWF ligand or FVIII ligand comprises an a1 region, which comprises an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, 99% or 100% identical to 337 to 374 corresponding to complete FVIII, in which the a1 region is capable of being cleaved by thrombin. In other embodiments, the VWF ligand or FVIII ligand also comprises additional amino acids, for example, one, two, three, four, five, ten or more. In a certain embodiment, the VWF ligand or FVIII ligand is composed of ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5).

[0095] In some embodiments, the VWF ligand or FVIII ligand comprises a region of a2, which comprises an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96 %, about 97%, about 98%, 99% or 100% identical to Glu720 to Arg740 corresponding to full-length FVIII, in which the a2 region is capable of being cleaved by thrombin. In one embodiment, the VWF ligand or FVIII ligand comprises a region of a1, which comprises an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96%, about of 97%, about 98%, 99% or 100% identical to amino acids 712 to 743 corresponding to complete mature FVIII. In other embodiments, the VWF ligand or FVIII ligand also comprises additional amino acids, for example, one, two, three, four, five, ten or more. In a certain embodiment, region a2 comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4)

[0096] In certain embodiments, the VWF ligand or FVIII ligand comprises a region of a3, which comprises an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96 %, about 97%, about 98%, 99% or 100% identical to Glu1649 to Arg1689 corresponding to the full length of mature FVIII, in which the a3 region is capable of being cleaved by thrombin. In one embodiment, the VWF ligand or FVIII ligand comprises a region of a3, which comprises an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, 99% or 100% identical to 1649 to 1692 corresponding to the full length of mature FVIII, in which the a3 region is capable of being cleaved by thrombin. In other embodiments, the VWF ligand or FVIII ligand also comprises additional amino acids, for example, one, two, three, four, five, ten or more. In a specific embodiment, the VWF ligand or the FVIII ligand comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6)

[0097] In still other embodiments, the VWF linker or FVIII linker comprises a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external local PAR1 interaction motif and in which the external local PAR1 interaction motif is composed of S-F-L-L-R-N (SEQ ID NO: 7). In some embodiments, the external local PAR1 interaction motif further comprises a sequence selected from P, P-N, N-P-D, P-N-D-K (SEQ ID NO: 8), P-N-D-K-Y (SEQ ID NO: 9), P-N-D-K-Y-E (SEQ ID NO: 10), P-N-D-K-Y-E-P (SEQ ID NO: 11) E-D (SEQ ID NO: 20), P-N-D-K-Y-E-P-F-W-E-D-E (SEQ ID NO: 21), P-N-D-K-Y-E-P-F-W-E-D-E-E (SEQ ID NO: 22), -N-D-K-Y-E- P-F-W-E-D-E-E-S (SEQ ID NO: 23), or any combination thereof. In other embodiments, in which the aliphatic amino acid is selected from Glycine, Alanine, Valine, Leucine and Isoleucine. In a specific embodiment, the thrombin cleavage site comprises L-V-P-R. In other embodiments, thrombin cleaves the VWF ligand from FVIII faster than thrombin would cleave the thrombin cleavage site (e.g., L-V-P-R) if the thrombin cleavage site (L-V-P-R) were replaced by the VWF ligand or the FVIII ligand. , respectively (i.e., without the PAR1 external site interaction motif). In other embodiments, thrombin cleaves the VWF ligand or FVIII ligand at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times faster than thrombin would cleave the thrombin cleavage site (e.g., L-V-P-R ) if the thrombin cleavage site was replaced by the VWF ligand or FVIII ligand.

[0098] In some embodiments, a VWF linker or FVIII linker composed of (i) an a1 region, (ii) an a2 region, (iii) an a3 region, or (iv) an X-V-P-R thrombin cleavage site and an PAR1 external interaction site also comprises one or more amino acids with a length of at least 2, 5, 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, 1 000, 1200, 1400, 1600, 1800 or 2000 amino acids. In one embodiment, the single amino acid or more amino acids comprise a gly peptide. In another embodiment, the single amino acid or more amino acids comprise a GliGli peptide. In other embodiments, the single amino acid or more amino acids make up IleSer. In still other embodiments, the single amino acid or more amino acids comprise a gly/ser peptide. In still other embodiments, the single amino acid or amino acids comprising a peptide has a formula of (Gly4Ser)n or S(Gly4Ser)n, wherein n is a positive integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the single amino acid or more amino acids make up (Gly4Ser)3 (SEQ ID NO: 89) or (Gly4Ser)4 (SEQ ID NO: 90).

II. C. 2. VWF protein

[0099] VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and produced constitutively in the endothelium (in Weibel-Palade bodies), megakaryocytes (platelet a-granules) and subendothelial connective tissue. The basic monomer of VWF is a protein of 2813 amino acids. Each monomer contains a number of specific domains, with specific functions, the D'/D3 domain (which binds to Factor VIII), the A1 domain (which binds to GPIb receptor platelets, heparin, or possibly collagen), the A3 domain (which binds collagen), the C1 domain (in which the RGD domain binds to platelet αIIbβ3 integrin when it is activated), and the "cysteine knot" domain at the C-terminal end of the protein (which VWF shares with platelet-derived growth factor (PDGF) transforming into growth factor-β (TGFβ) and β-subunit of human chorionic gonadotropin (βHCG)).

[00100] The term "VWF protein" as used herein includes, but is not limited to, full-length VWF or functional VWF fragments comprising a D' domain or D3 domain, which are capable of inhibiting the binding of endogenous VWF to FVIII. . In one embodiment, a VWF protein binds to FVIII. In another embodiment, the VWF protein blocks the VWF binding site on FVIII, thereby inhibiting the interaction of FVIII with endogenous VWF. In other embodiments, a VWF protein is not cleared by a VWF clearance pathway. VWF proteins include derivatives, variants, mutants, or analogs that retain these VWF activities.

[00101] The 2813 amino acid monomer sequence for human VWF is reported as Accession Number _NP_000543.2__ in Genbank. The nucleotide sequence encoding human VWF is reported as Accession Number __NM_000552.3_ in Genbank. The nucleotide sequence encoding human VWF is designated as SEQ ID NO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. Each VWF domain is listed in Table 1. TABLE 1. VWF Sequences

[00102] The VWF protein, as used herein, may include a D' Domain and a VWF D3 domain, in which the VWF protein binds to FVIII and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF protein comprising the D' Domain and the D3 domain may also include a VWF domain selected from an A1 domain, an A2 domain, an A3 domain, a D1 domain, a D2 domain, a D4 domain, a B1 domain, a B2 domain, a B3 domain, a C1 domain, a C2 domain, a CK domain, one or more fragments thereof, or any combination thereof. In one embodiment, a VWF protein includes, consists essentially of, or consists of: (1) the VWF D' and D3 domains or fragments thereof; (2) the VWF D1, D' and D3 domains or fragments thereof; (3) the VWF D2, D' and D3 domains or fragments thereof; (4) the VWF D1, D2, D' and D3 domains or fragments thereof; or (5) the VWF D1, D2, D', D3 and A1 domains or fragments thereof. The VWF protein described herein does not contain a binding site for clearance VWF receptors. The VWF protein of the present invention may encompass any other sequences linked or fused to the VWF protein. For example, a VWF protein described herein may also comprise a signal peptide.

[00103] In one embodiment, a VWF protein binds to, or is associated with, an FVIII protein. By binding to, or being associated with, a FVIII protein, the VWF protein of the invention can protect FVIII from protease cleavage and FVIII activation, stabilize the FVIII heavy chain and light chain, and prevent FVIII clearance by scavenger receptors. In another embodiment, the VWF protein binds or associates with a FVIII protein and blocks or prevents the binding of the FVIII protein to phospholipids and activated Protein C. By preventing or inhibiting the binding of the FVIII protein with endogenous, full-length VWF, the VWF protein of the invention reduces the clearance of FVIII by endogenous VWF clearance receptors and thereby extends the half-life of the FVIII protein. The half-life extension of a FVIII protein occurs, then, due to the association of a FVIII protein with a VWF protein without a clearance VWF receptor and, thus, shields and/or protects the FVIII protein from the endogenous VWF that contains the binding site for clearance VWF receptors. The FVIII protein bound to or protected by the VWF protein may also allow recycling of a FVIII protein. By eliminating the VWF clearance pathway receptor binding sites on the full length of the VWF molecule, the FVIII/VWF heterodimers of the invention are protected against the VWF clearance pathway, further extending the half-life of FVIII.

[00104] In one embodiment, a VWF protein of the present invention comprises a D' Domain and a D3 domain of VWF, wherein the D' Domain is at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2, in which the VWF protein prevents the binding of endogenous VWF to FVIII. In another embodiment, a VWF protein of the present invention comprises a D' Domain and a D3 domain of VWF, wherein the D' Domain is at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2, in which the VWF protein prevents the binding of endogenous VWF to FVIII. In some embodiments, a VWF protein described herein comprises, essentially consists of or consists of, a VWF D' Domain and a D3 Domain, which are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2, in which the VWF protein prevents the binding of endogenous VWF to FVIII. In other embodiments, a VWF protein comprises, consists essentially of, or consists of, the D1, D2, D', and D3 domains at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99%, or 100% identical to amino acids 23 to 1240 of SEQ ID NO: 2, in which the VWF protein prevents the binding of endogenous VWF to FVIII. In still other embodiments, the VWF protein also comprises a signal peptide, operably linked thereto.

[00105] In some embodiments, a VWF protein of the invention consists essentially of, or consists of (1) the D' D3 domain, the D1D' D3 domain, the D2D' D3 domain, or the D1D2D'D3 domain and (2) an additional VWF sequence of up to about 10 amino acids (e.g., amino acid sequences 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up to about 15 amino acids (e.g. , any sequences between amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about 20 amino acids (for example, any sequences between amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), up to about 25 amino acids (e.g., any sequences between amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1265 of SEQ ID No.: 2), up to about 30 amino acids (e.g., any sequences between amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a certain embodiment, the VWF protein comprising, or consisting essentially of, a D' Domain and a D3 domain is neither amino acids 764 to 1274 of SEQ ID NO: 2 nor the full length of 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.

[00106] In other embodiments, a VWF protein comprising D'D3 domains linked to the D1D2 domains also comprises an intracellular processing site, for example, (a PACE (furin) or PC5 processing site), allowing the cleavage of the D1D2 domains of the D'D3 domains after expression. Non-limiting examples of intracellular processing sites are described elsewhere herein.

[00107] In still other embodiments, a VWF protein comprises a D'Domain and a D3 domain, but does not comprise an amino acid sequence selected from (1) amino acid 1241 to 2813 of SEQ ID NO: 2, (2) amino acid 1270 to amino acid 2813 of SEQ ID NO: 2, (3) amino acid 1271 to amino acid 2813 of SEQ ID NO: 2, (4) amino acid 1272 to amino acid 2813 of SEQ ID NO: 2, (5) amino acid 1273 to amino acid 2813 of SEQ ID NO: 2, (6) amino acid 1274 to amino acid 2813 of SEQ ID NO: 2), or any combination thereof.

[00108] In still other embodiments, a VWF protein of the present invention comprises, essentially consists of or consists of an amino acid sequence corresponding to a D' domain, D3 domain and A1 domain, of which 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: 2, in which the protein VWF prevents the binding of endogenous VWF to FVIII. In a certain embodiment, the VWF protein is not amino acids 764 to 1274 of SEQ ID NO: 2.

[00109] In some embodiments, a VWF protein of the invention comprises a D' Domain and a D3 domain, but does not comprise at least one VWF domain selected from (1) an A1 domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2 domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CK, (11) a CK domain and a C2 domain, (12) a CK domain, a C2 domain, and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, and a B3 domain, (14 ) a CK domain, a C2 domain, a C1 domain, a B3 domain, and a B2 domain, (15) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, and a B1 domain, ( 16) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain and a D4 domain, (17) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain and an A3 domain, (18) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain and an A2 domain, (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, an A2 domain and an A1 domain or (20) any combination thereof.

[00110] In still other embodiments, a VWF protein comprises D'D3 domains and one or more domains or modules. Examples of such domains or modules include, but are not limited to, the domains and modules described in Zhour et al., Blood published online April 6, 2012: DOI 10.1182/blood-2012-01- 405134. For example, the VWF protein may include a D'D3 domain and one or more domains or modules selected from: the A1 domain, the A2, the A3 domain, the D4N module, the VWD4 module, the C8-4 module, the TIL-4 module, the C1 module, the C2 module, the C3 module, the C4 module, the C5 module, the C6 module or any combination thereof.

[00111] In certain embodiments, a VWF protein of the invention forms a multimer, for example, dimer, trimer, tetramer, pentamer, hexamer, heptamer or higher order multimers. In other embodiments, the VWF protein is a monomer having only one VWF protein. In some embodiments, the VWF protein 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 is not capable of forming a disulfide bond or forming a dimer or multimer. In another embodiment, the amino acid substitution is between the D' Domain and the D3 domain. In a certain embodiment, the VWF protein of the invention contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142 or both residues 1099 and 1142 of SEQ ID NO: 2. At least one amino acid substitution may be any amino acid that does not occur naturally in wild-type VWF. For example, the amino acid substitution may be any amino acid except cysteine, e.g., isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine , tyrosine, arginine or histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit VWF proteins from forming multimers.

[00112] In some embodiments, the VWF protein comprises an amino acid substitution from cysteine to alanine at residue 336 corresponding to the D'D3 domain of VWF (residue 1099 of SEQ ID NO: 2) and an amino acid substitution from cysteine to alanine at residue 379 corresponding to the VWF D'D3 domain (residue 1142 of SEQ ID NO: 2), or both.

[00113] In certain embodiments, the VWF protein useful herein can be further modified to improve its interaction with FVIII, for example, to improve its binding affinity to FVIII. As a non-limiting example, the VWF protein is composed of a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO: 2 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO: 2. residues 764 and/or 773 may contribute to the binding affinity of VWF proteins to FVIII. In other embodiments, the VWF protein useful for the invention may have other modifications, for example, the protein may be pegylated, glycosylated, hesylated, or polysialylated.

II.C.3. Heterologous Portion

[00114] A heterologous moiety that can be fused to a VWF protein through a VWF linker or to a FVIII protein through a FVIII linker can be a heterologous polypeptide or a heterologous non-polypeptide moiety. In certain embodiments, the heterologous moiety is a half-life extension molecule, which is known in the art and is composed of a polypeptide, a non-polypeptide moiety, or a combination of both. A heterologous polypeptide moiety may include an FVIII protein, an immunoglobulin constant region or a part thereof, albumin or a fragment thereof, an albumin binding moiety, transferrin or a fragment thereof, a PAS sequence, a HAP sequence, a derivative or variant thereof, the C-terminal peptide (CTP) of human chorionic gonadotropin β subunit, or any combination thereof. In some embodiments, the non-polypeptide linker moiety comprises polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), a derivative, or any combination thereof. In certain embodiments, there may be one, two, three or more heterologous moieties, which may be the same or different molecules.

II.C.3.a. Constant Immunoglobulin Region or Portion Thereof

[00115] A constant immunoglobulin region is composed of domains denoted CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA IgD or IgE), the constant region can be composed of three or four CH domains. The constant regions of some isotypes (e.g. IgG) also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

[00116] A constant immunoglobulin region or part thereof, to produce the chimeric protein of the present invention, can be obtained from several different sources. In some embodiments, an immunoglobulin constant region or a portion thereof is derived from a human immunoglobulin. It is understood, however, that the constant region of the immunoglobulin or a portion thereof may be derived from an immunoglobulin from other mammalian species, including for example, a rodent (mouse, rat, rabbit, guinea pig) or non-human primates (chimpanzee , monkey). Furthermore, the immunoglobulin constant region or a portion thereof may be derived from any class of immunoglobulin, including IgM, IgD, IgG, IgA and IgE and any isotype of immunoglobulin, including IgGl, IgG2, IgG3 and IgG4. In one embodiment, the human IgG1 isotype is used.

[00117] One embodiment of immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domain sequences can be selected having a certain effector function (or without a certain effector function) or with a certain modification to reduce immunogenicity. Many antibody and antibody-encoding gene sequences have been published and Ig constant region sequences (e.g., hinge, CH2 or CH3 sequences, or parts thereof) can be derived from these sequences using known techniques. The genetic material obtained using any of the above methods can then be altered or synthesized to obtain the polypeptide of the present invention. It will further be considered that the scope of the present invention encompasses alleles, variants and mutations of constant region DNA sequences.

[00118] The immunoglobulin constant region sequences or a part thereof can be cloned, for example, using the polymerase chain reaction and primers that are selected to amplify the domain of interest. To clone an immunoglobulin constant region sequence or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and the antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Patent No. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, for example, "PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Enzymol Methods. 217:270).

[00119] An immunoglobulin constant region used herein may include all domains and the hinge region or parts thereof. In one embodiment, the immunoglobulin constant region or a portion thereof comprises the CH2 domain, the CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[00120] As used herein, the term "Fc region" is defined as that portion of a polypeptide that corresponds to the Fc region of the native immunoglobulin, that is, as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region.

[00121] In one embodiment, the "Fc" region refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, the first being heavy chain residue of the constant region 114) and ending at the C-terminus of the antibody. In this sense, a complete Fc domain comprises at least a hinge domain, a CH2 domain and a CH3 domain.

[00122] The Fc region of an immunoglobulin constant region, depending on the immunoglobulin isotype, may include the CH2, CH3 and CH4 domains, as well as the hinge region. Chimeric proteins comprising the Fc region of an immunoglobulin confer several desirable properties to a chimeric protein, including increased stability, increased half-life in serum (see Cardoso et al., 1989, Nature 337:525) as well as 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), which are incorporated in their entirety herein by reference.

[00123] An immunoglobulin constant region or part thereof can be an FcRn binding partner. FcRn is active in adult epithelial tissues and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colonic surfaces, and rectal surfaces (U.S. Patent No. 6,485,726). An FcRn binding partner is a portion of an immunoglobulin that binds to FcRn.

[00124] The FcRn receptor has been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other immunoglobulin classes, such as IgA, IgM, IgD, and IgE) at relatively low f, actively transports IgG transcellularly from the lumen to the serosa, and then releases the IgG at a relatively higher f found in interstitial fluids. It is expressed in adult epithelial tissue (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) proximal tubular epithelium renal (Kobayashi et al. 2002, Am. J. Fysiol. Renal Fysiol. 282:F358) as well as the nasal epithelium, vaginal surfaces, and surfaces of the biliary tree.

[00125] FcRn binding partners useful in the present invention encompass molecules that can specifically be bound by the FcRn receptor including full-length IgG, Fc fragment of IgG, and by other fragments that include the entire binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on x-ray crystallography (Burmeister et al. 1994, Nature 372:379). Most of the area of Fc contact with FcRn is near the junction of the CH2 and CH3 domains. The FC-FcRn contacts are all within a single Ig heavy chain. FcRn binding partners include the entire IgG, IgG Fc fragment, and other IgG fragments that include the entire FcRn binding region. The main contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 385-387, 428 and 433-436 of the CH3 domain . References made to the numbering of amino acids of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[00126] Fc regions or FcRn binding partners bound to FcRn can be effectively transported across epithelial barriers by FcRn, thus providing a non-invasive means to systemically deliver a desired therapeutic molecule. Furthermore, fusion proteins that encompass an Fc region or an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these fusion proteins are recycled into circulation again, thus increasing the in vivo half-life of these proteins. In certain embodiments, portions of the immunoglobulin constant regions are an Fc region or an FcRn binding partner that generally associates, through disulfide bonds and other nonspecific interactions, with another Fc region or another FcRn binding partner to form dimers. and other higher order multimers.

[00127] An FcRn binding partner region is a molecule or a moiety that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the Fc region. "Specifically linked" refers to two molecules forming a complex that is relatively stable under physiological conditions. A specific bond is characterized by a high affinity and a capacity between low and moderate, in contrast to a nonspecific bond which generally has a low affinity with a capacity between low and high. Typically, binding is considered specific when the KA affinity constant is greater than 106 M-1, or greater than 108 M-1. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Appropriate binding conditions, such as the concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, the concentration of a blocking agent (e.g., albumin, dairy casein), etc., can be optimized by a skilled technician using routine techniques.

[00128] Many mutants, fragments, variants and derivatives are described, for example, in PCT Publication Nos. WO 2011/069164 A2, WO 2012/006623 A2, WO 2012/006635 A2, or WO 2012/006633 A2, all of which are incorporated herein in their entireties by reference.

II.C.3.b. Albumin, or fragment, or variant thereof

[00129] In certain embodiments, a heterologous moiety linked to the VWF protein through a VWF linker or linked to a FVIII protein through a FVIII linker is albumin or a functional fragment thereof. In some embodiments, the albumin fused to the VWF protein is covalently associated with an albumin fused to an FVIII protein.

[00130] Human serum albumin (HSA, or HA), a protein of 609 amino acids in its complete form, is responsible for a significant proportion of serum osmotic pressure and also functions as a vehicle for endogenous and exogenous ligands. The term "albumin" as used herein includes total albumin, a fragment, a variant, a derivative, or a functional analogue thereof. Examples of albumin or fragments or variants thereof are described in Patent Publ. Us. 2008/0194481A1, 2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or 2008/0153751 A1 or PCT Order. Publ. Us. 2008/033413 A2, 2009/058322 A1, or 2007/021494 A2, which are incorporated herein in their entirety by reference.

II.C.3.c. Albumin Binding Portion

[00131] In certain embodiments, a heterologous moiety linked to a VWF protein through a VWF linker or to a FVIII protein through a FVIII linker is an albumin-binding moiety, which comprises an albumin-binding peptide, a bacterial albumin binding, an albumin binding antibody fragment or any combination thereof. For example, the albumin-binding protein may be a bacterial albumin-binding protein, an antibody, or an antibody fragment including domain antibodies (see U.S. Patent No. 6,696,245). An albumin-binding protein, for example, may be a bacterial albumin-binding domain, such as that of streptococcal G protein (Konig, T. and Skerra, a. (1998) J. Immunol. Methods 218, 73-83 ). Other examples of albumin-binding peptides that can be used as conjugation partners are, for example, those that have the consensus sequences Cis-Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Cis, where Xaa1 is Asp, Asn, Ser, Tr or Trp; Xaa2 is Asn, Gln, H, Ile, Leu or Lys; Xaa3 is Ala, Asp, Fe, Trp or Tir; and Xaa4 is Asp, Gly, Leu, Fe, Ser or Tr as described in US patent application 2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 3503535043).

II.C.3.d. PAS sequence

[00132] In certain embodiments, a heterologous moiety linked to the VWF protein through a VWF linker or linked to a FVIII protein through a FVIII linker is albumin or a functional fragment thereof. In one embodiment, a chimeric molecule comprises a VWF protein described herein fused to a PAS sequence, via a VWF linker. In another embodiment, a chimeric molecule of the invention comprises a first chain composed of a VWF protein fused to a PAS sequence, via a VWF linker, and a second chain composed of a FVIII protein and an additional optional PAS sequence, in which the PAS sequence blocks or protects the VWF binding site on the FVIII protein, thereby inhibiting or preventing the interaction between the FVIII protein and endogenous VWF. The two PAS sequences can be covalently associated with each other.

[00133] A PAS sequence, as used herein, represents an amino acid sequence composed primarily of alanine and serine residues or composed primarily of alanine, serine and proline residues, the amino acid sequence forming a random coil conformation under physiological conditions. . In this sense, the PAS sequence is a building block, an amino acid polymer or a cassette sequence comprising, essentially consisting of, or consisting of alanine, serine and proline, which can be used as part of the heterologous portion of the chimeric protein. Furthermore, the skilled artisan is aware that an amino acid polymer can also form a random coil conformation when residues other than alanine, serine and proline are added as a minor constituent in the PAS sequence. The term "minor constituent" as used herein means that amino acids other than alanine, serine and proline can be added to the PAS sequence to some extent, for example up to about 12%, i.e. about 12 of 100 amino acids of the PAS sequence, up to about 10%, i.e., about 10 out of 100 amino acids of the PAS sequence, up to about 9%, i.e., about 9 out of 100 amino acids, up to about 8%, i.e., about 8 of 100 amino acids, about 6%, i.e. about 6 of 100 amino acids, about 5%, i.e. about 5 of 100 amino acids, about 4%, i.e. about 4 of 100 amino acids, about 3%, i.e. about 3 of 100 amino acids, about 2%, i.e. about 2 of 100 amino acids, about 1%, i.e. approximately 1 of 100 amino acids. Amino acids other than alanine, serine and proline can be selected from the group consisting of Arg, Asn, Asp, Cis, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Fe, Tr, Trp, Tir and Val.

[00134] Under physiological conditions, the PAS sequence strand forms a random coil conformation and thus can mediate greater in vivo or in vitro stability for the VWF factor or coagulation activity protein. Since the random coil domain does not adopt a stable structure or function on its own, biological activity mediated by the VWF protein or the FVIII protein to which it is fused is essentially preserved. In other embodiments, the PAS sequences that form the random coil domain are biologically inert, especially with respect to blood plasma proteolysis, immunogenicity, electrostatic behavior/isoelectric point, binding to surface receptors, or cell internalization, but are still biodegradable, which provides clear advantages over synthetic polymers such as PEG.

[00135] Non-limiting examples of PAS sequences forming a random coil conformation comprise an amino acid sequence selected from the group consisting of combination thereof. Additional examples of PAS sequences are known from, for example, Patent Publ. No. 2010/0292130 A1 and PCT Publ Order. No. WO 2008/155134 A1.

II.C.3.e. HAP sequence

[00136] In certain embodiments, a heterologous moiety linked to the VWF protein through a VWF linker or linked to a FVIII protein through a FVIII linker is a glycine-rich homo-amino acid polymer (HAP). The HAP sequence may encompass a glycine repetitive sequence, which is at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids or 500 amino acids long. In one embodiment, the HAP sequence is capable of extending the half-life of a moiety fused or linked to the HAP sequence. Non-limiting examples of HAP sequences include (Gli)n, (Gli4Ser)n, or S(Gly4Ser)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200. See, for example, Schlapschy M et al, Protein Eng. Design Selection, 20: 273-284 (2007).

II.C.3.f. Transferrin or fragment thereof

[00137] In certain embodiments, a heterologous moiety linked to the VWF protein through a VWF linker or linked to a FVIII protein through a FVIII linker is transferrin or a fragment thereof. Any transferrin can be used to make the chimeric molecules of the invention. As an example, wild-type human Tf (Tf) is a protein of 679 amino acids, approximately 75 kDa (not accounting for glycosylation), with two main domains, N (about 330 amino acids) and C (about 340 amino acids), that appear originate from gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, Transferrin is composed of two domains, N domain and C domain. The N domain comprises two subdomains, N1 domain and N2 domain, and the C domain comprises two subdomains, C1 domain and C2 domain.

[00138] In one embodiment, the transferrin portion of the chimeric molecule includes a transferrin splice variant. In an example, transferrin splice variant may be human transferrin splice variant, e.g., Genbank Accession AAA61140. In another embodiment, the transferrin portion of the chimeric molecule includes one or more domains of the transferrin sequence, e.g., the N domain, the C domain, the N1 domain, the N2 domain, the C1 domain, the C2 domain, or any combination thereof. same.

II.C.3.g. Polymer, e.g. Polyethylene glycol (PEG)

[00139] In other embodiments, a heterologous moiety attached to a VWF protein through a VWF linker or to a FVIII protein through a FVIII linker is a soluble polymer known in the art, including, but not limited to, polyethylene glycol, ethylene glycol/ethylene glycol copolymers. propylene glycol, carboxymethyl cellulose, dextran or polyvinyl alcohol. The heterologous moiety as a soluble polymer can be attached at any positions within the chimeric molecule.

[00140] In certain embodiments, a chimeric molecule comprises a VWF protein fused to a heterologous moiety (e.g., an Fc region) through a VWF linker, in which the VWF protein is also linked to PEG. In another embodiment, a chimeric molecule comprises a VWF protein fused to an Fc region via a VWF linker and a FVIII protein, which are associated with each other, wherein the FVIII protein is linked to PEG.

[00141] Also provided by the invention are chemically modified derivatives of the chimeric molecule of the invention that may provide additional advantages, such as greater solubility, stability and circulation time of the polypeptide, or lower immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for modification may be selected from water-soluble polymers, including, but not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol. A chimeric molecule may be modified at random positions within the molecule or at the N- or C-terminus or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

[00142] A polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, in one embodiment, the molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing. Other sizes may be used depending on the desired profile (e.g., the duration of sustained release desired, the effects, if any, on biological activity, ease of handling, degree or lack of antigenicity, and other known effects of polyethylene glycol in a protein or analogue). For example, polyethylene glycol may have an average molecular weight of 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17. 000,17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 kDa.

[00143] In some embodiments, polyethylene glycol may have a branched structure. Branched polyethylene glycols glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Cálicoeti et al., Bioconjug. chem. 10:638-646 (1999), each of which is incorporated herein in its entirety by reference.

[00144] The number of polyethylene glycol moieties attached to each chimeric molecule (i.e., the degree of substitution) can also vary. For example, the pegylated proteins of the invention can be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. Similarly, the average degree of substitutions varies between 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11 -13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19 or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

[00145] In other embodiments, a FVIII protein used in the invention is conjugated to one or more polymers. A polymer may be water-soluble and covalently or non-covalently linked to factor VIII or other moieties conjugated to Factor VIII. Non-limiting examples of polymers may be poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline or poly(acryloyl morpholine). Additional types of polymer-conjugated FVIII are described in U.S. Patent No. 7,199,223.

II.C.3.h. Hydroxyethyl starch (HES)

[00146] In certain embodiments, a heterologous moiety linked to the VWF protein through a VWF linker or linked to a FVIII protein through a FVIII linker is a polymer, for example, hydroxyethyl starch or a functional fragment thereof.

[00147] Hydroxyethyl starch (HES) is a naturally occurring derivative of amylopectin and is degraded by alpha-amylase in the body. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch in a concentration of up to 95% by weight. HES has advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in clinics (Sommermeyer et al, Krankenhausfarmazie, 8(8), 271-278 (1987); and Weidler et al., Arzneim. - Forschung/Drug Res., 41, 494-498 (1991)).

[00148] Amylopectin contains glucose moieties, and in the main chain, alpha-1,4-glucosidic bonds are present and alpha-1,6-glucosidic bonds are present in the branching sites. The physicochemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the fragmented alpha-1,4-glycosidic bond, helical structures with about six glucose monomers in each turn are produced. The physicochemical properties as well as the biochemical properties of the polymer can be modified through substitution. The introduction of a hydroxyethyl group can be achieved through alkaline hydroxyethylation. By adapting the reaction conditions it is possible to explore the different reactivities to the hydroxyethylation of the respective hydroxy group in the unsubstituted glucose monomer. Due to this fact, the qualified technician is able to influence the replacement reason to some extent.

[00149] HES is mainly characterized by molecular weight distribution and degree of substitution. The degree of substitution, denoted as DS, relates to molar substitution, and is known among those of skill. See Sommermeyer et al, Krankenhausfarmazie, 8(8), 271-278 (1987), as cited above, in particular p. 273.

[00150] In one embodiment, the hydroxyethyl starch has an average molecular weight (weight average) of 1 to 300 kD, 2 to 200kD, 3 to 100 kD, or 4 to 70kD. Hydroxyethyl starch may also exhibit a molar degree of substitution of 0.1 to 3, preferably 0.1 to 2, more preferably 0.1 to 0.9, preferably 0.1 to 0.8 and a ratio of C2 substitution: C6 in the range of 2 to 20 with regard to hydroxyethyl groups. A non-limiting example of a HES with an average molecular weight of about 130 kD is a HES with a degree of substitution of 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0 .6, 0.7 or 0.8, preferably 0.4 to 0.7 such as 0.4, 0.5, 0.6 or 0.7. In a specific embodiment, HES with an average molecular weight of about 130 kD is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid, used, for example, for volume replacement, used in therapeutic indications for the therapy and prophylaxis of hypovolemia. The characteristics of VOLUVEN® are an average molecular weight of 130,000+/-20,000 D, a molar substitution of 0.4 and a C2:C6 ratio of about 9:1. In other embodiments, the average molecular weight ranges of hydroxyethyl starch are, for example, 4 to 70 kD or 10 to 70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10 to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 kD or 12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD. In still other embodiments, the average molecular weight of the hydroxyethyl starch employed is in the range of more than 4 kD and below 70 kD, such as about 10 kD, or in the range of 9 to 10 kD or 10 to 11 kD or 9 to 11 kD, or about 12 kD, or in the range 11 to 12 kD or 12 to 13 kD or 11 to 13 kD, or about 18 kD, or in the range 17 to 18 kD or 18 to 19 kD or from 17 to 19 kD, or about 30 kD, or in the range of 29 to 30, or from 30 to 31 kD, or about 50 kD, or in the range of 49 to 50 kD or from 50 to 51 kD or from 49 to 51 kD.

[00151] In certain embodiments, the heterologous portion may be mixtures of hydroxyethyl starches with different average molecular weights and/or different degrees of substitution and/or different C2:C6 substitution ratios. Therefore, mixtures of hydroxyethyl starches can be employed having different average molecular weights and different degrees of substitution and different C2:C6 substitution ratios, or having different average molecular weights and different degrees of substitution and the same or almost the same C2 substitution ratios. :C6, having different average molecular weights and the same or almost the same degree of substitution and different substitution ratios C2:C6, or having the same or almost the same average molecular weights and different degrees of substitution and different substitution ratios C2 :C6, or having different average molecular weights and the same or almost the same degree of substitution and the same or almost the same substitution ratios C2:C6, or having the same or almost the same average molecular weights and different degrees of substitution and the same or nearly the same C2:C6 substitution ratios, or having the same or nearly the same average molecular weights and the same or nearly the same degree of substitution and different C2:C6 substitution ratios, or having the same or nearly the same almost the same molecular weight averages and the same or almost the same degree of substitution and the same or almost the same C2:C6 substitution ratios.

II.C.3.i. Polysialic Acids (PSA)

[00152] In certain embodiments, a heterologous peptide moiety linked to the VWF protein through a VWF linker or linked to a FVIII protein through a FVIII linker is a polymer, for example, polysialic acids (PSAs) or a derivative thereof. Polysialic Acids (PSA) are naturally occurring polymers of unbranched sialic acid produced by certain bacterial strains and in certain cells in mammals. J. Roth, et al. (1993) in Polysialic Acid: From Microbes to Man, eds. Roth J., Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp 335-348. They can be produced in varying degrees of polymerization, from n=about 80 or more sialic acid residues to n=2, by limited acid hydrolysis or by digestion with neuraminidases or by fractionation of the natural, bacterially derived polymer forms. The composition of the different polysialic acids also varies such that there are homopolymeric forms, that is, the alpha-2,8-linked polysialic acid comprising the capsular polysaccharide of E. coli strain K1 and group B meningococci, which are also found in the form embryonic neuronal cell adhesion molecule (N-CAM). Heteropolymeric forms also exist — such as the alternative polysialic acids alpha-2,8 and alpha-2,9 from E. coli strain K92 and polysaccharides from group C N. meningitidis. Sialic acid can also be found in alternative copolymers with different monomers of sialic acid, such as the W135 group or the Y group of N. meningitidis. Polysialic acids have important biological functions, including the evasion of the immune and complementary systems by pathogenic bacteria and the regulation of glial adhesiveness of immature neurons during fetal development (in which the polymer has an anti-adhesive function) Cho and Troy, P.N.A.S., USA, 91 (1994) 1142711431, although there are no known polysialic acid receptors in mammals. Alpha-2,8-linked polysialic acid from E. coli strain K1 is also known as 'colominic acid' and is used (in various lengths) to exemplify the present invention. Various methods of attaching or conjugating polysialic acids to a polypeptide have been described (for example, see U.S. Patent No. 5,846,951; WO-A-0187922, and US 2007/0191597 A1, which are incorporated herein in their entireties by reference.

II.C.4. XTEN sequence.

[00153] As used herein, "XTEN sequence" refers to a long-length, non-natural polypeptide, a substantially non-repetitive sequence that is composed primarily of small hydrophilic amino acids, each sequence having a low degree of, or no, secondary structure. or tertiary, under physiological conditions. As a partner of chimeric proteins, XTENs can serve as a carrier, and impart certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a VWF protein or a FVIII protein of the invention to create a chimeric protein. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments such as single chain antibodies or Fc fragments of a light chain or a heavy chain.

[00154] In some embodiments, the XTEN sequence of the invention is a peptide or a polypeptide having more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 , 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or a polypeptide having more than about 20 to about 3000 amino acid residues, more than 30 to about 2500 residues, more than 40 to about 2000 residues, more than 50 to about 1500 residues , more than 60 to about 1000 waste, more than 70 to about 900 waste, more than 80 to about 800 waste, more than 90 to about 700 waste, more than 100 to about 600 waste, more than 110 to about 500 residues, or more than 120 to about 400 residues.

[00155] The XTEN sequence of the invention may encompass one or more sequence patterns of 9 to 14 amino acid residues or an amino acid sequence of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence motif, where the motif is composed of, consists essentially of, or consists of 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A ), serine (S), threonine (T), glutamate (E) and proline (P). See US 2010-0239554 A1.

[00156] In some embodiments, XTEN comprises non-overlapping sequence patterns wherein about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of several non-overlapping sequence units selected from one of the unique pattern families selected from Table 2A, resulting in a familiar sequence. As used herein, "family" means that XTEN has standards selected only from a single standards category selected from Table 2A; that is, AD, AE, AF, AG, AM, AQ, AC, or BD restriction by coding nucleotides, incorporation of a cleavage sequence, or to achieve better binding to FVIII or VWF. In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence patterns from the AD pattern family, or the AE pattern family, or the AF pattern family, or the AG pattern family, or the AM standards, or the AQ family of standards, or the BC family or the BD family, with the resulting XTEN exposing the homology modality described above. In other embodiments, XTEN comprises multiple motif sequence units from two or more of the pattern families of Table 2A. These sequences can be selected to achieve desired physicochemical characteristics, including net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the standards, described in more detail below. In the embodiments described above in this paragraph, XTEN-incorporated standards can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues. Table 2A. XTEN 12-Amino Acid Sequence Patterns and Pattern Families • Denotes individual sequence patterns that, when used together in various permutations, result in a "familiar" sequence

[00157] XTEN can have different lengths for insertion into or binding to FVIII or VWF or any other components of the chimeric molecule. In one embodiment, the length of the XTEN sequence(s) is chosen based on the function or property to be achieved in the fusion protein. Depending on the intended property or function, XTEN can be a short or intermediate sequence or a longer sequence that can serve as a carrier. In certain embodiments, XTEN includes short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to 399 amino acid residues, and longer lengths of about 400 to about 1000 to about 3000 amino acid residues. Thus, XTEN inserted into or linked to FVIII or VWF can have lengths of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288 , about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to 3000 amino acid residues in length. In other embodiments, the XTEN sequences are about 6 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 250, about 250 to about 400, about 400 to about 500, about 500 to about 900, about 900 to about 1500, about 1500 to about 2000, or about 2000 to about 3000 amino acid residues in length. The exact length of an XTEN inserted in. or linked to FVIII or VWF can vary without adversely affecting FVIII or VWF activity. In one embodiment, one or more of the XTEN sequences used herein is 36 amino acids, 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from one or more of the XTEN sequence families; i.e. AD, AE, AF, AG AM, AQ, BC or BD.

[00158] In some embodiments, the XTEN sequence used in the invention is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% identical to a sequence selected from the group consisting of AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF5 76, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD8 64, AE948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948,AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620,AG171 6, AG1812, AG1908 and AG2004. See US 2010-0239554 A1.

[00159] 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, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144 and any combination thereof. In another embodiment, the XTEN sequence is selected from the group consisting of AE42, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144 and any combination thereof. In a specific embodiment, the XTEN sequence is AE288. Amino acid sequences for certain XTEN sequences of the invention are shown in Table 2B. TABLE 2B. XTEN sequences.

[00160] In those embodiments where the XTEN component used has less than 100% of its amino acids consisting of 4, 5 or 6 types of amino acid selected from glycine (G), alanine (A), serine (S), threonine (T) , glutamate (E) and proline (P) or less than 100% of the sequence consisting of the sequence patterns of Table 2A or XTEN sequences of Table 2B, the other amino acid residues of XTEN are selected from any of the other 14 L- natural amino acids, but are preferably selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least minus about 99% hydrophilic amino acids. XTEN amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) are interspersed throughout the XTEN sequence, or are located within or between the sequence patterns or focus on one or more short stretches of the XTEN sequence, for example, to create a linker between XTEN and other components; for example, the VWF protein. In such cases where the XTEN component is composed of amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is preferable that less than ca. 2% or less than about 1% of the amino acids are hydrophobic residues, such that the resulting sequences generally have no secondary structure, for example, having no more than 2% alpha-helices or 2% beta-sheets, as determined by the methods described in this document. Hydrophobic residues that are less favored in the construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design XTEN sequences to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: cysteine (to prevent the formation of disulfide and oxidation), methionine (to prevent oxidation), asparagine and glutamine (to prevent deamidation). Thus, in some embodiments, XTEN comprising amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) have a sequence with less than 5% of the residues contributing to alpha-helices and beta-sheets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.

[00161] In additional embodiments, the XTEN sequence used in the invention affects the physical or chemical property, for example, pharmacokinetics, of the chimeric protein of the present invention. The XTEN sequence used in the present invention may exhibit one or more of the following advantageous properties: conformational flexibility, improved water solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radials. ). In a specific embodiment, the XTEN sequence linked to an FVIII protein in this invention enhances pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that the chimeric proteins described herein remain in vivo for a period of longer time compared to wild type FVIII. In additional embodiments, the XTEN sequence used in the present invention enhances pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that the FVIII protein remains in vivo for a longer period of time relative to FVIII. wild type.

[00162] A variety of methods and tests can be employed to determine the physicochemical properties of proteins that comprise the XTEN sequence. Such methods include, but are not limited to, analytical centrifugation, EPR, Ion Exchange HPLC, size exclusion HPLC, reverse phase HPLC, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, iv, NMR , Raman spectroscopy, refractometry and UV/Visible spectroscopy. Additional methods are described in Amau et al, Prot Expr and Purif 48, 1-13 (2006).

[00163] Other examples of XTEN sequences that can be used in accordance with the present invention are described in US Patent Publication Nos. 2010/0239554 A1, A1 2010/0323956, 2011/0046060 A1, 2011/0046061 A1, A1 2011/0077199 or A1 2011/0172146, or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2, or WO2013123457 A1 International Application Nos. PCT/US2013/049989. II.C.5. FVIII protein

[00164] A "FVIII protein", as used herein, means a FVIII polypeptide functional in its normal coagulation role, unless otherwise specified. The term an FVIII protein includes a functional fragment, variant, analogue, or derivatives thereof that retain the function of complete wild-type Factor VIII in the coagulation pathway. A "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of the functions of FVIII include, but are not limited to, the ability to activate coagulation, the ability to act as a cofactor for factor IX, or the ability to form a complex with factor IX in the presence of Ca2+ and phospholipids, which then converts the factor X to the activated form Xa. The FVIII protein can be human, porcine, canine, rat or murine FVIII protein. Furthermore, comparisons between FVIII from humans and other species have identified conserved residues that may be necessary for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

[00165] A number of tests are available to assess the function of the coagulation system: activated partial thromboplastin time (APTT), chromogenic test, ROTEM test, prothrombin time (PT) test (also used to determine INR), fibrinogen (often by Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (if an abnormality is corrected if the patient's plasma is mixed with normal plasma), clotting factor tests, antiphospholipid antibody test, D-dimer test, genetic tests (e.g., Factor V Leiden test, G20210A prothrombin mutation test), Diluted Russell's Viper Venom Test (dRVVT) , various platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g. ROTEM®), or euglobulin lysis time (ELT).

[00166] The aPTT test is a performance indicator that measures the effectiveness of both the "intrinsic" coagulation pathway (also referred to as the contact activation pathway) and the common coagulation pathways. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors, for example, FVIII or FIX. It is used in conjunction with the prothrombin time (PT) test, which measures the extrinsic pathway.

[00167] The ROTEM test provides information about the entire kinetics of hemostasis: clotting time, clot formation, clot stability and lysis. The different parameters in thromboelastometry are dependent on the activity of the plasma coagulation system, platelet function, fibrinolysis or many factors that influence these interactions. This test can provide a complete picture of secondary hemostasis.

[00168] FVIII polynucleotide sequences and polynucleotides are known, as are many functional fragments, mutants and modified versions. Examples of human FVIII sequences (complete) are shown as subsequences in SEQ ID NO: 16 or 18. Table 3. Full length of FVIII (FVIII signal peptide underlined; FVIII heavy chain is double underlined; B domain is italicized ; and the FVIII light chain is in plain text) VWF signal peptide (SEQ ID NO: 15) Table 4. Nucleotide Sequence Encoding Complete FVIII (SEQ ID NO: 17)* * Underlined nucleic acids encode a signal peptide.

[00169] The FVIII polypeptide includes full-length FVIII, full-length FVIII minus Met at the N-terminus, mature FVIII (minus the signal sequence), mature FVIII with additional Met at the N-terminus, and/or FVIII with a full deletion or partial domain B. In certain embodiments, FVIII variants include partial or complete domain B deletions.

[00170] 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. Furthermore, the partially or fully deleted FVIII B domain is shown in U.S. Pat. 4,994,371 and 4,868,112. In some embodiments, the human FVIII domain B is replaced with the human factor V domain B as shown in U.S. Patent No. 5,004,803. The cDNA sequence encoding human factor VIII and the coding amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively, of U.S. Patent No. 7,211,559.

[00171] The porcine FVIII sequence is published in Toole, J. J., et al., Proc. Natl. academic Sci. USA 83:5939-5942 (1986). Furthermore, the complete porcine cDNA sequence obtained by PCR amplification of FVIII sequences from a porcine spleen cDNA library has been reported in Healey, J. F., et al., Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII with substitutions of all domains, all subunits and specific amino acid sequences have been described in U.S. Patent No. 5,364,771 by Lollar and Runge and in WO 93/20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII and a chimeric FVIII with porcine A1 and/or A2 domains replaced by the corresponding human domains were reported in WO 94/11503. U.S. Patent No. 5,859,204, Lollar, J. S., also discloses porcine cDNA and deduced amino acid sequences. U.S. Patent No. 6,458,563 discloses a porcine FVIII with deleted B domain.

[00172] U.S. Patent No. 5,859,204 to Lollar, J. S. reports functional mutants of FVIII having reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463 to Lollar, J. S. also reports FVIII mutants having reduced antigenicity and reduced immunoreactivity. Public Request US NO. 2005/0100990 to Saenko et al reports functional mutations in the A2 domain of FVIII.

[00173] In one embodiment, the FVIII protein (or the FVIII portion of a chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% identical to a FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 18 or amino acids 1 to 2332 of SEQ ID NO: 16 (without a signal sequence), wherein FVIII it has a clotting activity, for example, it activates Factor IX as a cofactor to convert factor X into activated factor X. FVIII (or FVIII part of a chimeric protein) may be identical to an FVIII amino acid sequence of amino acids 1 of 1438 of SEQ ID NO: 18 or amino acids 1 to 2332 of SEQ ID NO: 16 (without a signal sequence ). The FVIII protein may further include a signal sequence.

[00174] The "B Domain" of FVIII, as used herein, is the same B Domain known in the art that is defined by the internal identity of the amino acid sequence and proteolytic cleavage sites, e.g., residues Ser741-Arg1648 of human FVIII complete. The other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373- Arg740; A3, residues Ser1690-Asn2019; C1, residues Lis2020-Asn2172; C2, residues Ser2173-Tyr2332. Sequence A3-C1-C2 includes residues Ser1690-Tir2332. The remaining sequence, residues Glu1649-Arg1689, is generally referred to as the a3 acidic region. The locations of the boundaries of all domains, including Domain B, for porcine, rat and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("Factor-VIII-B-domain-deleted" or "BDD FVIII"). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequences as the Factor VIII portion of the sequence in Table 5. (The BDD FVIII heavy chain is double underlined; the B domain is italicized; and the light chain BDD FVIII is in plain text). Table 5. BDD FVIII (SEQ ID NO: 18) Table 6. Nucleotide Sequence Encoding Complete FVIII (SEQ ID NO: 19)* * Underlined nucleic acids encode a signal peptide.

[00175] A "FVIII B-deleted domain" may have the complete or partial deletions described 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, a FVIII B-domain deleted sequence of the present invention comprises any of the deletions described in col. 4, row 4 to col. 5, line 28 and examples 1-5 of U.S. Patent No. 6,316,226 (also in U.S. 6,346,513). In another embodiment, a Factor VIII Domain B deleted is Factor VIII Domain B deleted S743/Q1638 (BDD SQ FVIII) (e.g., Factor VIII that has a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII that has the amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 16, i.e., SEQ ID NO: 18). In some embodiments, a B-deleted FVIII Domain of the present invention has the deletion described in col. 2, lines 26-51 and examples 5-8 of U.S. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886 and US 6,228,620). In some embodiments, a B-domain deleted FVIII has the deletion described in col. 1, line 25 to col. 2, line 40 in U.S. Patent no. ° 5,972,885; col. 6, lines 1-22 and examples 1 of U.S. Patent No. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent No. 5,171,844; col. 2, lines 55-68, figure 2, and example 1 of U.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and example 2 of U.S. Patent No. 4,868,112; col. 2, line 1 to col. 3 line 19, col. 3, line 40 to col. 4 line 67, col. 7, line 43 to col. 8, line 26 et al. 11, line 5 to col. 13, line 39 of U.S. Patent No. 7,041,635; or col. 4, lines 25-53 of U.S. Patent No. 6,458,563.

[00176] In some embodiments, a B-domain deleted FVIII has most of the B domain deleted, but still contains amino-terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary two-chain translation product. polypeptide, as described in WO 91/09122. In some embodiments, a B-domain deleted FVIII is constructed with the deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R.C., et al. J. Biol. chem. 265 (13): 7318-7323 (1990). An FVIII B-deleted domain may also contain the deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng 2.4: 301-6 (1988). Additional B domain deletions forming part of the invention include: 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., 6:553 of DNA (1987)-564)), 1648 to 741 (Pasek (PCT application No.88/00831)), or 816 1598 or 741 to 1648 (Lagner (Inst. Behring Mitt. (1988) No. 82:16- EP 25, 295597)). In other embodiments, BDD FVIII includes an FVIII polypeptide containing Domain B fragments that maintain one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or, optionally, 943, that correspond to the sequence of amino acids of the complete FVIII sequence. Examples of B domain fragments include 226 amino acids or 163 amino acids of the B Domain as described in Miao Miao, H.Z., et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, S. W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011) (i.e., the first 226 amino acids or 163 amino acids of the B domain are retained). In some embodiments, FVIII with a partial B domain is FVIII198. FVIII198 is a partial B domain containing a single-chain FVIIIFc molecule-226N6. 226 represents the N-terminal amino acid 226 of FVIII domain B and N6 represents six N-glycosylation sites in domain B. In still other embodiments, BDD FVIII further comprises a point mutation at residue 309 (from Fe to Ser) to improve expression of the BDD FVIII protein. See Miao, H.Z., et al., Blood 103(a): 3412-3419 (2004). In still other embodiments, BDD FVIII includes a FVIII polypeptide containing a portion of the B domain, but not containing one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, S. W., et al., J. Thromb. Haemost. 9:2235-2242 (2011). Each of the above-mentioned deletions can be made in any FVIII sequence.

[00177] A FVIII protein useful in the present invention may include FVIII having one or more additional heterologous sequences or chemical or physical modifications, which do not affect FVIII coagulation activity. Such heterologous sequences or chemical or physical modifications may be fused to the C-terminus or N-terminus of the FVIII protein or inserted between one or more of the two amino acid residues in the FVIII protein. Such insertions in the FVIII protein do not affect FVIII coagulation activity or FVIII function. In one embodiment, the insertions improve the pharmacokinetic properties of the FVIII protein (e.g., half-life). In another embodiment, the insertions may be more than two, three, four, five or six locations.

[00178] In one embodiment, FVIII is cleaved just after Arginine at amino acid 1648 (in full-length Factor VIII or SEQ ID NO: 16), amino acid 754 (in Factor VIII B-deleted domain S743/Q1638 or SEQ ID NO : 16), or the corresponding Arginine residue (in other variants), thus resulting in a heavy chain and a light chain. In another embodiment, FVIII is composed of a heavy chain and a light chain, which are linked or associated by an ion-mediated non-covalent metallic bond.

[00179] In other embodiments, FVIII is a single chain that has not been cleaved just after Arginine amino acid 1648 FVIII (in FVIII full length or SEQ ID NO: 16), amino acid 754 (Factor VIII domain B-deleted S743/Q1638 or SEQ ID N°: 18), or the corresponding Arginine residue (in other variants). A single FVIII chain may include one or more amino acid substitutions. In one embodiment, the amino acid substitution is at a residue corresponding to residue 1648, residue 1645, or both, of the full-length mature Factor VIII polypeptide (SEQ ID NO: 16) or residue 754, residue 751, or both of Factor VIII SQ BDD (SEQ ID NO: 18). The replacement amino acid can be any amino acid other than Arginine, for example, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine , serine, tyrosine, histidine, ornithine, pyrrolysine or taurine.

[00180] FVIII can further be cleaved by thrombin and then activated as FVIIIa, serving as a cofactor for activated Factor IX (FIXa). And activated FIX together with activated FVIII forms a Xase complex and converts 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 of the Factor VIII domain B-deleted sequence), the cleavage generating FVIIIa having the 50kDa A1, 43kDa A2 and 73kDa A3-C1-C2 chains. In one embodiment, the FVIII protein useful for the present invention is inactive FVIII. In another embodiment, the FVIII protein is an activated FVIII.

[00181] The FVIII protein having polypeptide linked or associated with the VWF protein may include a sequence of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16 or 18, in which the sequence has FVIII coagulation activity, for example, activating Factor IX as a cofactor to convert Factor X to activated Factor X (FXa).

[00182] In some embodiments, the FVIII protein further comprises one or more heterologous portions that are fused to the C-terminal or N-terminal of the FVIII protein or that are inserted between two adjacent amino acids of the FVIII protein. In other embodiments, the heterologous portions comprise an amino acid sequence of at least about 50 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, at least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 550 amino acids, at least about 600 amino acids, at least about of 650 amino acids, at least about 700 amino acids, at least about 750 amino acids, at least about 800 amino acids, at least about 850 amino acids, at least about 900 amino acids, at least about 950 amino acids, or at least about 1000 amino acids. In some embodiments, the half-life of the chimeric molecule is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times. times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times or at least about 12 times longer than wild-type FVIII.

[00183] Other exemplary FVIII variants are also described in U.S. Publication no. US2013/0017997, published on January 17, 2013, International Publication No. 2013/122617, published on January 22, 2013, International Publication No. 2014/011819, published on January 16, 2014, Publication No. International WO2013123457 A1, or International Application No. PCT/US2013/049989. III. Polynucleotides, Vectors, Host Cells and Methods of Making

[00184] Also provided by the invention is a polynucleotide encoding the chimeric molecule described herein. When a VWF protein is linked to a heterologous moiety via a VWF linker and to a FVIII protein and an XTEN sequence in a chimeric protein as a single polypeptide chain, the invention becomes a single polynucleotide encoding single polypeptide chain. When the chimeric protein comprises a first and a second polypeptide chain, the first polypeptide chain formed by a VWF protein, an XTEN sequence and a first heterologous portion (e.g., a first Fc region) through a VWF linker and the second polypeptide chain composed of an FVIII protein and a second heterologous portion (e.g., a second Fc region), a polynucleotide may comprise the first nucleotide region and the second nucleotide region. In one embodiment, the first nucleotide region and the second nucleotide region are on the same polynucleotide. In another embodiment, the first nucleotide region and the second nucleotide region are on two different polynucleotides. In certain embodiments, the present invention is directed to a set of polynucleotides that includes a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes a VWF protein, an XTEN sequence, a VWF ligand, and a heterologous portion of the chimeric protein and the second nucleotide chain encodes a FVIII protein and a second heterologous portion. In certain embodiments, the present invention is directed to a set of polynucleotides that includes a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes a VWF protein, and a heterologous portion of the chimeric protein and the The second nucleotide chain encodes a FVIII protein linked to a second heterologous portion via a FVIII linker, wherein at least one XTEN sequence is fused to the chimeric molecule. In certain embodiments, the present invention is directed to a set of polynucleotides that includes a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes a VWF protein, a VWF ligand, and a heterologous portion of the protein. chimeric and the second nucleotide chain encodes a FVIII protein, a FVIII linker, and a heterologous portion and a second portion in which at least one XTEN sequence is fused to the chimeric molecule.

[00185] In other embodiments, the set of polynucleotides further comprises an additional nucleotide chain (e.g., a second nucleotide chain when the chimeric polypeptide is encoded by a single polynucleotide chain, or a third nucleotide chain when the chimeric protein is encoded by two polynucleotide chains) that encodes a convertase protein. The protein convertase can be selected from proprotein subtilisin/chexin convertase type 5 (PCSK5 or PC5), proprotein subtilisin/chexin convertase type 7 (PCSK7 or PC5), yeast Kex 2, proprotein subtilisin/chexin type 3 (PACE or PCSK3), or two or more combinations thereof. In some embodiments, the convertase protein is PACE, PC5, or PC7. In a specific embodiment, the convertase protein is PC5 or PC7. See International Order No. PCT/US2011/043568, which is incorporated herein by reference. In another embodiment, the convertase protein is PACE/furin.

[00186] In certain embodiments, the invention includes a set of polynucleotides composed of a first nucleotide sequence encoding a VWF protein comprising a D' Domain and a D3 domain of VWF fused to a first heterologous portion via a VWF linker, a second nucleotide sequence encoding a FVIII protein and a second heterologous portion, and a third nucleotide sequence, encoding a VWF D1 domain and D2 domain and in which an XTEN sequence is present, either in the first or in the second chain. In this modality, the D1 domain and D2 domain are expressed separately (not linked to the D'D3 domain of the VWF protein) for the formation of an appropriate disulfide bond and folding of the D'D3 domains. D1D2 domain expression can be in cis or trans.

[00187] As used herein, an expression vector refers to any nucleic acid construct that contains the elements necessary for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation when introduced into a suitable host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

[00188] Expression vectors of the invention will include polynucleotides encoding the chimeric molecule.

[00189] In one embodiment, a coding sequence for the chimeric molecule is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked so as to allow each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are considered operably linked when they are covalently linked so as to place expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are considered operably linked if the induction of a promoter in the 5' gene expression sequence leads to transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a mutation reading frame, (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 the nucleic acid coding sequence if the gene expression sequence is capable of effecting transcription of that nucleic acid coding sequence such that the resulting transcript is translated into the desired protein or polypeptide. .

[00190] A gene expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or promoter enhancer combination, that facilitates efficient transcription and translation of the nucleic acid encoding to which it is operably connected. The gene expression control sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Mammalian constitutive promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters with constitutive function in eukaryotic cells include, for example, promoters from cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an induction agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other constitutive promoters are known to those skilled in the art.

[00191] In general, the gene expression control sequence should include, as necessary, the 5' untranscribed and 5' untranslated sequences of those involved with the initiation of transcription and translation, respectively, as a TATA box, sequence of capping, CAAT sequence and the like. Especially, such 5' nontranscribed sequences include a promoter region that includes a promoter sequence for transcriptional control of the operably spliced encoding nucleic acid. The gene expression sequences optionally include upstream enhancer sequences or activator sequences, as desired.

[00192] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney's murine leukemia virus, Harvey's murine sarcoma virus, murine mammary tumor virus, and Rous' sarcoma virus ; adenovirus, adeno-associated virus; SV40 type virus; polyomaviruses; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; polio virus; and RNA viruses, such as a retrovirus. Other vectors known in the art can readily be employed. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Noncytopathic viruses include retroviruses, whose life cycle involves the reverse transcription of viral genomic RNA into DNA, with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. More useful are retroviruses that are replication deficient (i.e., capable of directing protein synthesis as desired, but unable to manufacture an infectious particle). Such genetically modified retroviral expression vectors have general utility for high-efficiency transduction of genes in vivo. Motif protocols for producing retroviruses that are replication deficient (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line into a plasmid, producing recombinant retroviruses by the packaging cell line, collecting viral particles from tissue culture media and infection of target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Human Press, Inc., Cliffton, N.J. (1991).

[00193] In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and capable of infecting a wide range of species and cell types. It has even more advantages, such as stability in heat and lipid solvents; high frequencies of transduction in cells of different lineages, including hematopoietic cells; and lack of superinfection inhibition, allowing multiple series of transductions. Reportedly, adeno-associated viruses can integrate into human cellular DNA at a specific location, thus minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. Furthermore, wild-type adeno-associated virus infections have been followed in tissue culture for more than 100 passages in the absence of selective pressure, implying that adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function extrachromosomally.

[00194] Other vectors include plasmid vectors. Plasmid vectors have been widely described and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, it has been found that plasmid vectors are particularly advantageous for delivering genes into cells in vivo by because of its inability to replicate and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well known to those skilled in the art. Additionally, plasmids can be customized using molecular biology motif techniques to remove and/or add specific DNA fragments.

[00195] In an insect expression system that can be used to produce the proteins of the invention, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express the exogenous genes. The virus grows in Spodoptera frugiperda cells. A coding sequence can be cloned into non-essential regions (e.g., the polyhedron gene) of the virus and placed under the control of an ACNPV promoter (e.g., polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of unobstructed recombinant virus (i.e., viruses without the coat protein encoded by the polyhedron gene). Such recombinant viruses are 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). More 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.

[00196] Another system that can be used to express the proteins of the 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.

[00197] In mammalian host cells, several viral-based expression systems can be used. In cases where an adenovirus is used as an expression vector, a coding sequence can be linked to an adenovirus transcription/translation control complex, for example, the late promoter and the tripartite leader sequence. This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the peptide in infected hosts. See, for example, Logan & Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K 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.

[00198] To increase production efficiency, the polynucleotide can be designed to encode several units of the protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with the appropriate enzyme) to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in suitable viral expression systems, translation of each polypeptide encoded by the mRNA is directed internally in transcription; for example, by an internal ribosomal entry site. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA, which in turn directs the translation of several, individual polypeptides. This approach eliminates the enzymatic production and transformation of polyproteins and can significantly increase the yield of polypeptides driven by a single promoter.

[00199] Vectors used in transformation generally contain a selectable marker, used to identify transformants. In bacterial systems, this may include a gene for resistance to antibiotics such as ampicillin or kanamycin. Selectable markers for use in mammalian cell cultures include genes that confer resistance to drugs such as neomycin, hygromycin, and methotrexate. The selectable marker may be an enlargeable selectable marker. An amplifiable selectable marker is the dehydrofolate reductase (DHFR) gene. Simonsen C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. Selectable markers are reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable markers is at the level of skill in the art.

[00200] Selectable markers can be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they can be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest will be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (e.g., U.S. Patent No. 4,713,339).

[00201] Expression vectors can encode tags that allow easy purification of the recombinant proteins produced. Examples include, but are not limited to, the pUR278 vector (Ruther et al. (1983) EMBO J 2:1791), in which sequences encoding the protein to be expressed can be ligated into the vector in frame with the lac z coding region for that a labeled fusion protein is produced; pGEX vectors can be used to express the proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are generally soluble and can be purified easily from cells by absorption from glutathione-agarose beads followed by elution in the presence of free glutathione. Vectors include cleavage sites (thrombin or Factor Xa protease or PRESCISSION PROTEASETM (Farmacia, Peapack and, NJ)) to facilitate removal of the tag after purification.

[00202] The expression vector or vectors are then transfected or cotransfected into an appropriate target cell, which will express the polypeptides. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841) and reagents based on liposomes. One embodiment of vector expression systems in hosts can be used to express the proteins described herein, including prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli) transformed with bacteriophage DNA, or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungal expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a sequence appropriate encoder; or animal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

[00203] 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 definitive nucleus. Eukaryotic animal cells include cells from vertebrates, e.g. mammals, and cells from invertebrates, e.g. insects. Eukaryotic plant cells may specifically include, without limitation, yeast cells. A eukaryotic cell is distinct from a prokaryotic cell, for example, bacteria.

[00204] In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell line. HEK 293 cells are available as CRL-1533 from the American Type Culture Collection, Manassas, VA and as 293-H cells, catalog no. 11631-017 or 293-F cells, catalog no. 11625-019 from Invitrogen (Carlsbad, California). In some embodiments, the mammalian cell is a PER.C6® cell, which is a human cell line derived from the retina. PER.C6® cells are available from Crucell (Leiden, Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from the American Type Culture Collection, Manassas, VA. (e.g., CHO-K1; CCL-61). In still other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from the American Type Culture Collection, Manassas, VA. (e.g. CRL-1632). In some embodiments, the mammalian cell is an HKB11 cell, which is a hybrid cell line of a HEK293 cell and a human B cell line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

[00205] In one embodiment, a plasmid encoding a VWF protein, a VWF ligand, a heterologous moiety, or the chimeric protein of the invention also includes a selectable marker, e.g., zeocin resistance, and is transfected into HEK 293 cells, to the production of the chimeric protein.

[00206] In still other embodiments, transfected cells are stably transfected. These cells can be selected and maintained as a stable cell line using conventional techniques known to those skilled in the art.

[00207] Host cells containing protein DNA constructs are grown in suitable culture medium. In this document, the term "appropriate culture medium" represents a medium that contains the nutrients necessary 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 calf serum (FCS). In one embodiment, the medium substantially does not contain IgG. The culture medium will generally select for cells that contain the DNA construct through, for example, drug selection or deficiency of an essential nutrient, which is complemented by the selectable marker on the DNA construct or co-transfected with the construct. of DNA. Mammalian cells are generally cultured in commercially available serum-containing or serum-free media (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 suitable medium for a given cell line is common among those skilled in the art.

[00208] To co-express two polypeptide chains of the chimeric molecule as described herein, host cells are cultured under conditions that allow expression of both chains. As used here, cultivation refers to keeping cells alive in vitro for at least a defined period. Maintenance may generate, but need not include, an increase in the population of living cells. For example, cells maintained in culture may be static in population, but still viable and capable of producing a desired product, for example, a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include appropriate selection of culture media, media supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, cultivation may include the use of any of several types of scale systems, including shake flasks, spinner flasks, hollow fiber bioreactors, stirred tank bioreactors, airlift-type bioreactors, wave bioreactors, and others.

[00209] Cell culture conditions are also selected to allow association of the first and second chains with the chimeric molecule. Conditions that permit expression of the chimeric molecule may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 (Invitrogen, Carlsbad, CA) or OptiCHO (Invitrogen, Carlsbad, CA) medium. CA) supplemented with 4 mM glutamine.

[00210] In one aspect, the present invention is directed to a method of expressing, making or producing the chimeric protein, comprising a) transfecting the host cell with a polynucleotide encoding the chimeric molecule and b) culturing the host cell in culture medium under a condition appropriate for expression of the chimeric molecule, characterized by the fact that the chimeric molecule is expressed.

[00211] In other embodiments, the protein product containing the chimeric molecule is secreted into the culture medium. The culture medium is separated from the cells, concentrated, filtered, and then passed through two or three affinity columns, for example, a protein A column and one or two anion exchange columns.

[00212] In certain aspects, the present invention relates to the chimeric polypeptide produced by the methods described herein.

[00213] In vitro production allows proportional increase to generate large quantities of the desired altered polypeptides of the invention. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, for example, in an airlift-type reactor or in a perfectly stirred reactor, or immobilized or conveyed cell culture, by example, in hollow fibers, microcapsules, agarose microspheres or ceramic cartridges. If necessary and/or desired, polypeptide solutions can be purified by customary chromatography methods, for example, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC, DEAE-cellulose chromatography or affinity chromatography.

[00214] The invention also includes a method of improving the FVIII activity of a chimeric FVIII protein composed of a VWF protein fused to a first heterologous portion and an XTEN sequence and a FVIII protein fused to a second heterologous portion, the method involving inserting of a VWF linker between the VWF protein and the first heterologous portion, where the VWF linker is composed of a polypeptide selected from: (i) an a2 region of Factor VIII (FVIII); (ii) an a1 region of FVIII; (iii) an a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an external site-interacting PAR1 motif, where X is an aliphatic amino acid; or (v) any combination thereof. In some embodiments, FVIII activity is measured by an aPTT exam or ROTEM exam.

IV. Pharmaceutical Composition

[00215] Compositions containing the chimeric molecule of the present invention may contain a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and/or auxiliaries that facilitate the transformation of active compounds into preparations designed to be released at the site of action.

[00216] The pharmaceutical composition can be formulated for parenteral administration (that is, intravenously, subcutaneously or intramuscularly) by bolus injection. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers with an added preservative. The compositions may take forms such as suspensions, solutions or emulsions in oily or aqueous vehicles and contain formulating agents such as suspenders, stabilizers and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable carrier, such as pyrogen-free water.

[00217] Formulations suitable for parenteral administration also include aqueous solutions of active compounds in water-soluble form, for example, water-soluble salts. Furthermore, suspensions of the active compounds such as suitable oily injection suspensions can be administered. Suitable lipophilic solvents or carriers include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the molecules of the invention for release into cells or interstitial spaces. Pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol and the like. In some embodiments, the composition comprises isotonic agents, for example, sugars, and polyalcohols such as sodium chloride, sorbitol or mannitol. In other embodiments, the compositions comprise pharmaceutically acceptable substances such as wetting agents or small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffer solutions, which reinforce the validity or effectiveness of the active ingredients.

[00218] Compositions of the invention may come in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid pharmaceutical forms. The preferable form depends on the mode of administration and therapeutic application.

[00219] The composition can be formulated as a solution, microemulsion, dispersion, in liposomes or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required quantity in an appropriate solvent with an ingredient or a combination of the ingredients enumerated above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produce a powder of the active ingredient plus any desired additional ingredient from a previously filtered sterile solution. Adequate fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the necessary particle size in case of dispersion, and by the use of surfactants. Prolonged absorption of injectable compositions can be caused by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[00220] The active ingredient can be formulated with a controlled release formulation or device. Implants, transdermal patches, and microencapsulated delivery systems are examples of such formulations and devices. Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparing such formulations and devices are known in the art. See for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[00221] Injectable slow-release formulations can be made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactic-polyglycolic polymers. Depending on the ratio of drug to polymer, and the nature of the polymer used, the rate of drug release can be controlled. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Injectable slow-release formulations can also be prepared by encapsulating the drug in liposomes or microemulsions.

[00222] Complementary active compounds can be incorporated into the compositions. In one embodiment, a chimeric molecule of the invention is formulated with another clotting factor, or a variant, fragment, analogue or derivative thereof. For example, clotting factor includes, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant factor of soluble tissue (rsTF) or activated forms of any of the above. The hemostatic agent clotting factor may also include antifibrinolytic drugs, for example, epsilon-aminocaproic acid and tranexamic acid.

[00223] Dosage regimens can be adjusted to provide the desired optimal response. For example, it may be administered as a single bolus, several divided doses may be administered over time, or the dose may be proportionately reduced or increased as indicated by the demands of the therapeutic situation. It is advantageous to formulate parenteral compositions in unit dosage form due to ease of administration and uniformity of dosage. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[00224] In addition to the active compound, the dosage in liquid form may contain inert ingredients such as water, ethyl alcohol, ethyl carbamate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters.

[00225] 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, skimmed milk powder, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain pH buffering reagents and wetting or emulsifying agents.

[00226] For oral administration, the pharmaceutical composition may take the form of tablets or capsules prepared by conventional means. The composition may also be prepared as a liquid, for example a syrup or a suspension. The liquid may include suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or vegetable oils). fractionated) and preservatives (sorbic acid methyl or propyl-p-hydroxybenzoates). The preparations may also include flavoring agents, coloring agents and sweeteners. Alternatively, the composition may be presented as a dry product for constitution with water or another suitable vehicle.

[00227] For buccal administration, the composition can take the form of tablets or lozenges in accordance with conventional protocols.

[00228] For administration by inhalation, the compounds for use in accordance with the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray in a pressurized package or nebulizer, optionally with a propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to release a metered dose. Capsules and cartridges of (for example) gelatin for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base, such as lactose or starch.

[00229] The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

V. Gene Therapy

[00230] A chimeric molecule of the invention can be produced in vivo in a mammal, for example, a human patient, using a gene therapy approach for the treatment of a bleeding disease or disorder such as blood clotting disorder, hemarthrosis, muscle bleeding , oral bleeding, hemorrhage, muscular hemorrhage, oral hemorrhage, trauma, capitis trauma, gastrointestinal hemorrhage, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space , hemorrhage of the illiopsoas muscle sheath, which would be therapeutically beneficial. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is Hemophilia A. This involves administering an appropriate chimeric molecule that encodes nucleic acids operably linked to suitable expression control sequences. In one embodiment, these sequences are incorporated into a viral vector. Suitable viral vectors for such gene therapy include adenovirus 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 may be a replication-defective viral vector. In other embodiments, an adenoviral vector has a deletion in its E1 gene or E3 gene. When an adenoviral vector is used, the mammal cannot be exposed to a nucleic acid encoding a selectable marker gene. In other embodiments, the sequences are incorporated into a non-viral vector known to those skilled in the art.

SAW. Methods of Using Chimeric Protein

[00231] Further the present invention provides a method for reducing the frequency or degree of a bleeding episode in a patient in need using a chimeric molecule of the invention. An exemplary method comprises administering to the patient a therapeutically effective amount of a chimeric molecule of the invention. In other aspects, the invention includes a method of preventing the occurrence of a bleeding episode in a patient using a chimeric molecule of the invention. In other aspects, a composition comprising a DNA encoding the recombinant protein of the invention can be administered to a patient in need. In certain aspects of the invention, a cell expressing a chimeric molecule of the invention can be administered to a patient in need. In certain aspects of the invention, the pharmaceutical composition comprises (i) a chimeric molecule, (ii) an isolated nucleic acid encoding a chimeric molecule, (iii) a vector comprising a nucleic acid encoding a chimeric molecule, (iv) a cell comprising isolated nucleic acid encoding a chimeric molecule and/or a vector comprising a nucleic acid encoding a chimeric molecule, or (v) a combination thereof, and the pharmaceutical compositions also comprise an acceptable excipient or carrier.

[00232] The bleeding episode may be caused by a blood clotting disorder or derivative thereof. A blood clotting disorder may also be referred to as a coagulopathy. In one example, the blood clotting disorder that can be treated with a pharmaceutical composition of the present disclosure is hemophilia or von Willebrand disease (vWD). In another example, the blood clotting disorder, which can be treated with a pharmaceutical composition of the present disclosure, is hemophilia A.

[00233] In some embodiments, the type of hemorrhage associated with the bleeding condition is selected from hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscular hemorrhage, oral hemorrhage, trauma, trauma capitis, gastrointestinal hemorrhage, intracranial hemorrhage, intracranial hemorrhage, abdominal, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, hemorrhage of the illiopsoas muscle sheath, or any combinations thereof.

[00234] In other embodiments, the patient suffering from bleeding requires treatment for surgery, including, for example, surgical prophylaxis or perioperative management. In one example, the surgery is selected between minor surgery and major surgery. Exemplary surgical procedures include tooth extraction, tonsillectomy, herniotomy, synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraabdominal surgery, intrathoracic surgery, joint replacement surgery (e.g., total knee replacement, hip replacement, and the like), heart surgery and cesarean section.

[00235] In another example, the patient is concomitantly treated with Factor IX. Since the compounds of the invention are capable of activating FIXa, they can be used to pre-activate the FIXa polypeptide before administering FIXa to the patient.

[00236] The methods of the invention can be practiced on a patient who requires prophylaxis or treatment on demand.

[00237] Pharmaceutical compositions comprising a chimeric molecule of the invention can be formulated for any suitable form of administration, including, for example, topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral.

[00238] The term parenteral in this document includes subcutaneous, intradermal, intravascular (e.g., intravenously), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial, and intraperitoneal injection, as well as any injection or infusion technique similar. The composition may also be, for example, a suspension, emulsion, sustained-release formulation, cream, gel or powder. The composition may be formulated as a suppository, with traditional binders and carriers, such as triglycerides.

[00239] Having been described in detail, the present invention will be more clearly understood by reference to the following examples, which are included here for purposes of illustration only and are not intended to be limiting of the invention. All patents and publications referred to herein are expressly incorporated by reference.

Examples

[00240] In all examples, the following materials and methods were used unless otherwise indicated.

Materials and methods

[00241] In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (especially, for example, antibody technology) and motif techniques in electrophoresis . 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, eds. Ausubel et al., John Wiley & Sons (1992).

Example 1. Assessment of D'D3 thrombin-mediated release of various VWF constructs

[00242] This example evaluates the kinetics of D'D3 thrombin-mediated release at 37° C of several VWF constructs, mentioned in Figure 2. Biocore experiments were conducted with VWF-Fc constructs that contain different thrombin-cleavable linkers between the D domain 'D3 of VWF and Fc. The goal is to apply the information gathered from VWF-Fc thrombin digestion to FVIII-VWF heterodimers as described herein. All VWF-D'D3 constructs were passed through the chip to achieve protein capture density ranging between 100-700 RU. After the VWF construct was captured on the chip, 5 U/ml of thrombin was injected onto the surface for 5 minutes. The Fc remains bound to the chip, while the D'D3 in the cleavable constructs is released. The capture rate (RU/s) versus capture density (RU) was plotted as shown in Figures 3 and 4. The cleavage rate is proportional to the initial capture density while the slope provided a measurement of the susceptibility to cleavage by thrombin. of each construct.

[00243] Figure 3 shows that VWF-052 (which does not have a thrombin cleavage site in the linker region), as expected, is not cleaved by thrombin. The rate of VWF-039 (LVPR with PAR1 site) is comparable to the rate of FVIII cleavage (data not shown). Thus, VWF-039 served as a reference for the complete release of D'D3 from Fc. The ratio slopes of various VWF-Fc constructs relative to VWF-039 were used to determine the efficiency of thrombin cleavage. VWF-039 (LVPR with PAR1 site) is cleaved with thrombin approximately 70-80-fold faster than VWF-031 (LVPR). VWF-51 (ALRPRVV) is cleaved 1.8 times faster than VWF-031 (LVPR). VWF-034, which contains 288 XTEN along the LVPR site, exhibited slower cleavage compared with VWF-031.

[00244] VWF-Fc constructs were also produced by introducing different acidic regions (a1, a2 and a3) of the FVIII protein into the linker region. VWF-055, which contains the a2 region between D'D3 and Fc, exhibited thrombin cleavage similar to that of the VWF-039 construct. As shown in Figure 4, VWF-054 (a1 region) and VWF-056 (a3 region) showed ~5-fold lower thrombin cleavage.

[00245] Figure 5 shows the slope values of the thrombin cleavage curves for different VWF constructs. From such results, the acidic region 2 (a2) of FVIII appears to be a highly efficient thrombin cleavage site and was incorporated into the FVIII-VWF heterodimers as described herein.

Example 2. Assessment of the hemostatic potency of FVIII/VWFD'D3 heterodimers with ROTEM test of whole blood from a patient with HemA

[00246] FVIII/VWFD'D3 heterodimers containing different thrombin-cleavable ligands had their hemostatic potency evaluated in a ROTEM test (rotational thromboelastometry) of whole blood from a patient with HemA. A whole blood sample was collected from the donor with the severe bleeding disorder Hemophilia A using sodium citrate as an anticoagulant. 40 minutes after blood sample collection, variants of the FVIII/VWFD'D3 heterodimer containing different thrombin-cleavable linkers - FVIII155/VWF031 (48aa, LVPR site), FVIII155/VWF039 (26aa, LVPR + PAR1 site), FVIII155/VWF055 (34aa, a2 of FVIII) were diluted in the whole blood sample to final concentrations of 100%, 30%, 10% and 3% of normal as measured by the FVIII chromogenic test. Immediately after addition of FVIII/VWFD heterodimers 'D3, the ROTEM reaction was initiated by the addition of CaCl2. The clotting time (time to reach 2mm of amplitude, from the beginning of the test) was recorded by an instrument and plotted against the concentration of FVIII in the samples (Figure 6). It was hypothesized that a more potent FVIII/VWFD'D3 heterodimer would induce a faster clotting process, resulting in a shorter clotting time compared to a less potent FVIII/VWFD'D3 heterodimer. As shown in Figure 6, samples with the addition of the FVIII/VWF039 heterodimer had the shortest clotting time at all concentrations that were tested, and samples with the addition of the FVIII/VWF031 heterodimer had the longest clotting time at all. concentrations. The clotting time for samples with the addition of the FVIII155/VWF055 heterodimer is in the middle. Therefore, the hemostatic potency rank is FVIII155/VWF039 > FVIII155/VWF055 > FVIII155/VWF031. Since the only difference between the three molecules is the thrombin-cleavable linker between the VWF protein and the Fc region, the result indicates that the linker containing the LVPR site and PAR1 interaction motif at the external site and the a2 region of FVIII works better. than the LVPR site alone.

Example 3. Assessment of the activity of FVIII/VWF heterodimers

[00247] FVIII-XTEN/VWF heterodimer constructs were transfected into HEK293F cells using three plasmids: the first expressing FVIII-XTEN-Fc, the second expressing VWF-XTEN-Fc and the third expressing PACE. The polyethyleneimine (PEI) motif protocol was used for transfection and after 5 days of transfection, tissue culture medium was harvested. Various combinations of FVIII-VWF heterodimers were purified from the culture medium. The activity of the purified protein was tested in a chromogenic coagulation test (two stages) and aPTT (one stage), using motif protocols. Introduction of an acidic region 2 (a2) of FVIII between FVIII and Fc or between D'D3 and Fc (as shown in Table 7A and Figure 7) improved the TTPa activity of the FVIII-VWF heterodimer, as shown in Table 7. For example , the FVIII169/VWF059 heterodimer has a2 thrombin cleavage site in the D'D3-Fc linker region and has better aPTT activity than FVIII169/VWF057, which contains the thrombin LVPR site in the D'D3Fc linker, as shown in table 7C

[00248] Similarly, the incorporation of the a2 region between FVIII and Fc increased the coagulation activity of one stage of the heterodimer, as evident by the better ratio between chromogeny and aPTT of FVIII286/VWF059 and FVIII286/VWF062, as shown in table 7B. Table 7A Table 7B Table 7C

Example 4: Marked efficacy of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers in the tail docking model in mice

[00249] The enhanced efficiency of heterodimers containing different thrombin-cleavable ligands was evaluated using a tail bleeding model from mice carrying HemA.

[00250] 8-12 week old male mice carrying HemA were randomly divided into 5 treatment groups and treated with a single intravenous administration of SQ BDD-FVIII, rFVIII169/VWF034, rFVIII169/VWF057, rFVIII169/VWF059 or solution- vehicle, respectively. In order to mimic episodic FVIII treatment (to reconstitute 50-100% of normal plasma FVIII level), the selected FVIII treatment dose is 75 IU/kg, measured by FVIII aPTT activity. At this dose level, all FVIII variants tested will reconstitute ~70% of normal murine plasma FVIII activity 5min after dosing.

[00251] The tail docking procedure was performed as follows. Briefly, mice were anesthetized with a cocktail of 50 mg/kg ketamine/0.5 mg/kg dexmedetomidine prior to tail injury and placed on a 37°C heating pad to help maintain body temperature. The tails of the mice were then immersed in 37°C saline for 10 minutes to dilate the lateral vein. After vein dilation, FVIII variants or carrier solution were injected through the tail vein and 5mm of the tail tip was then cut using a #11 straight blade scalpel 5min after dosing. Spilled blood was collected in 13 ml of saline solution at 37° C for 30 minutes and the volume of blood loss was determined by the change in weight of the blood collection tube: volume of blood loss = (weight of the whole at the end of collection - weight at the beginning + 0.10) ml. Statistical analysis was performed using the t test (Kolmogorov-Smirnov test) and 1-way ANOVA (KRUSKAL-Wallis test, after the test: Dunn multiple comparison test).

[00252] The volume of blood loss from each individual animal in the study was plotted in Figure 8. Significant reduction in the volume of blood loss was observed for all FVIII treatment groups compared to vehicle-treated animals (p < 0.05, table 8). Similar blood loss reduction was observed in all heterodimer treatment groups compared to BDD-FVIII treatment (p > 0.5, Table 8), suggesting that heterodimer molecules could potentially be as effective as SQ BDD-FVIII for demand treatment.Table 8: P-value of the Kolmogorov-Smirnov test

[00253] The above description of specific embodiments will so fully disclose the general nature of the invention that others may, through the application of knowledge within the skill in the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation and without deviate from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and scope of the equivalents of the modalities described, based on the teaching and guidance presented here. It should be understood that the phraseology or terminology in this document is for purposes of description and not prescription, so that the terminology or phraseology of this specification should be interpreted by the skilled artisan in light of the teachings and guidance.

[00254] Other embodiments of the invention will be apparent to those skilled in the art after consideration of the specifications and practice of the invention described in this document. The specifications and examples are intended to be exemplary only, the true scope and spirit of the invention being indicated by the following claims.

[00255] All patents and publications cited in this document are incorporated by reference in this document in their entirety. [00256] This application claims the benefit of the priority of U.S. Patent Application Number 61/840,872, filed on June 28, 2013. The contents of the above application are incorporated herein by reference in their entirety.

Claims (5)

1. Chimeric molecule, characterized by the fact that it comprises: (1) a first polypeptide chain comprising a von Willebrand Factor (VWF) protein, a first XTEN sequence, a VWF ligand and a first Fc, in that specific order from the N-terminal to C-terminal, wherein the VWF protein comprises a D' domain and a D3 domain comprising amino acid residues 1-124 of SEQ ID NO: 82, wherein the D' domain and the D3 domain are capable of binding to a factor VIII (FVIII) protein, wherein the VWF ligand comprises an a2 region of FVIII comprising an amino acid sequence from Glu720 to Arg740 corresponding to SEQ ID NO: 16, wherein the a2 region is capable of being cleaved by thrombin, and wherein the first XTEN sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 and 78; (2) a second polypeptide chain comprising a FVIII protein fused to a second Fc region and a second XTEN sequence inserted into the FVIII protein, wherein the second XTEN sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 and 78; wherein the first polypeptide chain and the second polypeptide chain are associated with each other by a covalent bond. 2. Chimeric molecule according to claim 1, characterized by the fact that the first and second polypeptide chains are associated with each other by a disulfide bond. 3. Pharmaceutical composition, characterized by the fact that it comprises the chimeric molecule as defined in claim 1 or 2, and a pharmaceutically acceptable carrier. 4. Use of the chimeric molecule, as defined in claim 1 or 2, or the pharmaceutical composition, as defined in claim 3, characterized by the fact that it is in the manufacture of a medicament to reduce the frequency or degree of or prevent the occurrence of a episode of bleeding in an individual in need of it. 5. Use, according to claim 4, characterized by the fact that the individual has hemophilia A.