EA041588B1 - THROMBIN CLEAVABLE LINKER CONTAINING XTEN AND ITS USE - Google Patents

Description

State of the art

Hemophilia A is a bleeding disorder caused by defects in the coding of the coagulation factor VIII (FVIII) gene and affects 1-2 in 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients suffering from hemophilia A can be treated with an infusion of purified or recombinantly produced FVIII. However, all commercially available FVIII products are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to patients. See Weiner MA and Cairo, MS, Pediatric Hematology Secrets, Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried to increase the half-life of FVIII. For example, approaches used to increase the half-life of coagulation factors include pegylation, glycopegylation, and albumin conjugation. See Dumont et al., Blood. 119(13): 3024-3030 (online published January 13, 2012). Regardless of the protein engineering used, however, sustained-release FVIII products currently under development have improved half-lives, but half-lives are reported to be limited to only about 1.5- to 2-fold increases in preclinical animal models. See ibid. Consistent results have been demonstrated in humans, for example, rFVIIIFc has been reported to improve half-life by about 1.7-fold compared to ADVATE® in patients with hemophilia A. See ibid. Thus, an increase in the half-life, despite minor improvements, may indicate the existence of other factors that limit t _1/2 .

Due to frequent dosing and the inconvenience caused by the administration schedule, there is a need to develop FVIII products requiring less frequency of administration, ie. an FVIII product having a half-life greater than 1.5-2 times the half-life limit.

The essence of the invention

The present invention relates to a chimeric molecule containing a von Willebrand factor (VWF) protein, a heterologous fragment (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous fragment, wherein the VWF linker contains a polypeptide selected from: (i) the a2 region of factor VIII (FVIII); (ii) regions a1 of FVIII; (iii) a3 regions from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof, wherein the XTEN sequence is linked to a VWF protein, a heterologous fragment (H1), a VWF linker, or any combination thereof. In one embodiment, the XTEN sequence connects a VWF protein to a VWF linker, or a VWF linker to a heterologous fragment. In another embodiment, the chimeric molecule further comprises a second polypeptide chain that contains the FVIII protein, wherein the first polypeptide chain and the second polypeptide chain are associated with each other. In other embodiments, the FVIII protein in the chimeric molecule further comprises an additional XTEN sequence. An additional XTEN sequence may be linked to the N-terminus or C-terminus of the FVIII protein, or inserted between two adjacent FVIII amino acids. In still other embodiments of the invention, the second polypeptide chain further comprises a second heterologous fragment (H2).

The present description also includes a chimeric molecule containing a first polypeptide chain that contains a VWF protein, a heterologous fragment (H1) and a VWF linker connecting the VWF protein and a heterologous fragment (H1), and a second polypeptide chain containing the FVIII protein and the XTEN sequence, while the VWF linker in the first polypeptide chain contains: (i) the a2 region of FVIII; (ii) region a1 from FVIII; (iii) region a3 from FVIII; (iv) a thrombin cleavage site containing XV-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof, wherein the first polypeptide chain and the second polypeptide chain are associated with each other. In one embodiment, the XTEN sequence is fused to the N-terminus or C-terminus of the FVIII protein, or inserted between two adjacent FVIII amino acids. In another embodiment, the chimeric molecule further comprises an additional XTEN sequence that is linked to a VWF protein, a heterologous fragment, a VWF linker, or any combination thereof. In other embodiments of the invention, the chimeric molecule further comprises a second heterologous fragment (H2). In yet other embodiments of the invention, the second heterologous fragment is connected to the FVIII protein, the XTEN sequence, or both.

For chimeric molecules according to the present invention, the XTEN sequence linked to the VWF protein, VWF linker, FVIII protein, or any other components in the chimeric molecules contains about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 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

- 1,041,588 no 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.

In other aspects, the additional XTEN sequence in the chimeric molecules is 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 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 additional XTEN polypeptide is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. In certain embodiments, the additional 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.

In one embodiment of the invention, a VWF linker suitable for connecting a VWF protein and a heterologous fragment in chimeric molecules contains an a2 region that contains an amino acid sequence that is at least about 80, about 85, about 90, about 95, or 100% identical to the region from Glu720 to Arg740 corresponding to full-length FVIII, while the a2 region is capable of being cleaved by thrombin. In a particular embodiment, region a2 contains ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4). In another embodiment, a VWF linker suitable for connecting the VWF protein and the heterologous fragment contains an al region that contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the Met337 to Arg372 region. corresponding to full-length FVIII, while the a1 region is capable of being cleaved by thrombin. In some embodiments, region a1 contains ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5). In other embodiments, a VWF linker suitable for connecting the VWF protein and the heterologous fragment contains an a3 region that contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the Glu1649 to Arg1689 region. corresponding to full-length FVIII, while the a3 region is capable of being cleaved by thrombin. In a specific embodiment, the a3 region contains ISEITRTTLQSDQEEroYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6). In still other embodiments, a VWF linker suitable for connecting the VWF protein and the heterologous fragment comprises a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, wherein the PAR1 interaction exosite motif contains S-F-LL-R-N (SEQ ID NO: 7). In one embodiment, the PAR1 interaction exosite motif further comprises a sequence selected from P, P-N, P-N-D, P-N-D-K (SEQ ID NO: 8), PN-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-EP-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) or any combination thereof. In another embodiment, the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine. In a particular embodiment, the VWF linker contains GGLVPRSFLLRNPNDKYEPFWEDEES (SEQ ID NO: 24). In certain embodiments, thrombin cleaves the VWF linker faster than thrombin would cleave the thrombin cleavage site if the thrombin cleavage site replaced the VWF linker in the chimeric molecule. In other embodiments, thrombin cleaves the VWF linker at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, 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 if the thrombin cleavage site replaced the VWF linker in the chimeric molecule.

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In some embodiments, the VWF linker further comprises one or more amino acids and is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 in length, 150 160 170 180 190 200 210 220 230 240 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000, 1200, 1400, 1600, 1800 or 2000 amino acids. In one example, one or more amino acids comprise a gly peptide. In another example, one or more amino acids contain GlyGly. In other examples, one or more amino acids comprise the gly/ser peptide. In some examples, the gly/ser peptide has the formula (Gly ₄ Ser)n or S(Gly ₄ Ser)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 (Gly4Ser)n linker is (Gly ₄ Ser) ₃ (SEQ ID NO: 89) or (Gly ₄ Ser) ₄ (SEQ ID NO: 90).

A VWF protein suitable for a chimeric molecule of the invention may comprise a D' domain and a VWF D3 domain, wherein the D' domain and D3 domain are capable of binding to the FVIII protein. In one embodiment, the D' domain of the VWF protein contains an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-866 of SEQ ID NO: 2. In another embodiment, the domain D3 of the VWF protein contains an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO: 2. In other embodiments, the VWF protein contains at least one amino acid a substitution at residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. In still other embodiments, in the VWF protein sequence, an amino acid other than cysteine replaces the residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. In other embodiments, the VWF protein sequence contains amino acids 764-1240 of SEQ ID NO: 2. In certain embodiments, the invention ie, the VWF protein additionally contains a D1 domain, a D2 domain, or the D1 and D2 domains of the VWF. In some embodiments, the VWF protein further comprises a VWF domain selected from an A1 domain, an A2 domain, an A3 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 of them. In other embodiments, the VWF protein consists essentially of or consists of: (1) VWF D' and D3 domains or fragments thereof; (2) VWF D1, D' and D3 domains or fragments thereof; (3) VWF D2, D' and D3 domains or fragments thereof; (4) VWF D1, D2, D' and D3 domains or fragments thereof; or (5) VWF D1, D2, D', D3 and A1 domains or fragments thereof. In still other embodiments, the VWF protein further comprises a VWF signal peptide. In other embodiments, the VWF protein is pegylated, glycosylated, gekylated (hydroxyethyl starch conjugated), or polysialylated. The term pegylated refers to the presence of polyethylene glycol (PEG) attached to a protein; the term glycosylated refers to the presence of protein glycosylation; the term gekylated refers to the presence of hydroxyethyl starch (HES) attached to a protein; and the term polysialylated refers to the presence of polysialic acid (PSA) attached to a protein. Examples of PEG, HES and PSA are given elsewhere in this document.

In some aspects, a heterologous fragment (H1) coupled to a VWF protein via a VWF linker is capable of increasing the half-life of the chimeric molecule. In one embodiment, the heterologous fragment (H1) comprises an immunoglobulin constant region or portion thereof, albumin, albumin-binding fragment, PAS, HAP, transferrin or fragment thereof, polyethylene glycol (PEG), hydroxyethyl starch (HES), PSK, C-terminal peptide (STR ) β-subunit of human chorionic gonadotropin, or any combination thereof. In another embodiment, the heterologous fragment contains an FcRn binding partner. In other embodiments of the invention, the heterologous fragment contains an Fc region. In other embodiments of the invention, the heterologous fragment (H1) contains a clearance receptor, or a fragment thereof, and the clearance receptor blocks the binding of the FVIII protein to the clearance FVIII receptors. In some embodiments, the clearance receptor is a low density lipoprotein receptor associated protein 1 (LRP1) or a FVIII binding fragment thereof. In some aspects, the second heterologous fragment linked to the FVIII protein via an optional FVIII linker comprises an immunoglobulin constant region or region, albumin, albumin-binding polypeptide, PAS, human chorionic gonadotropin β-subunit C-terminal peptide (TPP), polyethylene glycol (PEG ), hydroxyethyl starch (HES), albumin-binding small molecules, or any combination thereof. In one embodiment, the second heterologous fragment (H2) is capable of increasing the half-life of the FVIII protein. In another embodiment, the second heterologous fragment (H2) contains a polypeptide, a non-polypeptide fragment, or both. In another embodiment, the second heterologous fragment (H2) contains an immunoglobulin constant region or portion thereof. In still other embodiments of the invention, the second heterologous fragment contains an FcRn binding partner. In other embodiments of the invention, the second heterologous fragment contains a second Fc region. In some embodiments of the invention

- 3 041588 the first heterologous fragment connected to the VWF protein using a VWF linker, and the second heterologous fragment connected to the FVIII protein using an optional linker, in which the XTEN sequence is linked to any of the components, are associated with each other. In one embodiment, the association between the first polypeptide chain and the second polypeptide denotes a covalent bond. In another embodiment of the invention, the association between the first heterologous fragment and the second heterologous fragment is a disulfide bond. In other embodiments, the first heterologous fragment is an FcRn binding partner and the second heterologous fragment is an FcRn binding partner. In still other embodiments, the first heterologous fragment is an Fc region and the second heterologous fragment is an Fc region.

In certain embodiments of the invention, the FVIII protein is connected to the second heterologous fragment using a FVIII linker. In one embodiment of the invention, the second linker is a cleavable linker. In another embodiment, the FVIII linker is identical to the VWF linker. In other embodiments, the FVIII linker is different from the VWF linker. In some aspects, the chimeric molecule of the invention has the 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:H2X2-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:H1X1-L1-V; or (1) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF linker; L2 is an optional FVIII linker; H1 is the first heterologous fragment; H2 denotes the second heterologous fragment; X1 denotes the sequence XTEN; X2 denotes an optional XTEN sequence; C is FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, with the XTEN sequence inserted between two adjacent FVIII amino acids; (-) denotes a peptide bond or one or more amino acids; and (:) denotes a covalent bond between H1 and H2.

In other aspects, the chimeric molecule has the 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-X1H1: 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-L1X1-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 (1) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF linker; L2 is an optional FVIII linker; H1 denotes the first heterologous fragment; H2 denotes the second heterologous fragment; X1 denotes an optional XTEN sequence; X2 denotes the sequence XTEN; C is FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, with the XTEN sequence inserted between two adjacent FVIII amino acids; (-) denotes a peptide bond or one or more amino acids; and (:) denotes a covalent bond between H1 and H2. In the chimeric molecules of the invention, the VWF protein can inhibit or prevent endogenous VWF from binding to the FVIII protein.

In certain aspects, the FVIII protein in the chimeric molecules may contain a third heterologous fragment (H3). The third heterologous fragment (H3) may be the XTEN sequence. In other aspects, the FVIII protein contains a fourth heterologous fragment (H4). The fourth heterologous fragment (H4) may be the XTEN sequence. In some aspects, the FVIII protein contains a fifth heterologous fragment (H5). The fifth heterologous fragment may be the XTEN sequence. In other aspects, the FVIII protein contains a sixth heterologous fragment (H6). The sixth heterologous fragment may be the 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 increasing the half-life of the chimeric molecule. In other aspects, the third heterologous fragment (H3), the fourth heterologous fragment (H4), the fifth heterologous fragment (H5) and the sixth heterologous fragment (H6) are attached to the C-terminus or N-terminus of FVIII or are inserted between two amino acids of the FVIII protein. In other aspects, one or more of the third heterologous fragment, the fourth heterologous fragment, the fifth heterologous fragment, and the sixth heterologous fragment contain a segment 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 sequence

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The XTEN of the third heterologous moiety, the fourth heterologous moiety, the fifth heterologous moiety, or the sixth heterologous moiety may be selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. In particular, 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, or SEQ ID NO: 42. In certain embodiments, the half-life of the chimeric molecule is increased by at least about 1.5-fold, at least about 2-fold, 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 compared to wild-type FVIII.

The present disclosure also provides for a polynucleotide or set of polynucleotides encoding a chimeric molecule or its complementary sequence. The polynucleotide or set of polynucleotides may further comprise a polynucleotide chain that encodes for PC5 or PC7.

Also included is a vector or set of vectors containing a polynucleotide or set of polynucleotides and one or more promoters operably linked to the polynucleotide or set of polynucleotides. In some embodiments of the invention, the vector or set of vectors may additionally contain an additional polynucleotide chain encoding PC5 or PC7.

The present invention also includes a host cell containing a polynucleotide or set of polynucleotides or a 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.

In some aspects, the invention includes a pharmaceutical composition comprising a chimeric molecule as disclosed herein, a polynucleotide or set of polynucleotides encoding a chimeric molecule, a vector or set of vectors containing a polynucleotide or set of polynucleotides, or a host cell as disclosed herein, and a pharmaceutically acceptable carrier. In one embodiment, the chimeric molecule in the composition has an increased half-life compared to the wild-type FVIII protein. In another embodiment, the half-life of the chimeric molecule in the composition is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least 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, compared to wild-type FVIII.

Also included is a method for reducing the frequency or severity of a bleeding episode in a subject in need thereof, comprising administering an effective amount of a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding a chimeric molecule, a vector or set of vectors disclosed herein, to a host cell disclosed in this document, or the composition disclosed in this document. The invention also includes a method for preventing a bleeding episode from occurring in a subject in need thereof, comprising administering an effective amount of a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding a chimeric molecule, a vector or set of vectors disclosed herein, to a host cell, disclosed in this document, or the composition disclosed in this document. In one embodiment of the invention, the bleeding episode is selected from a bleeding disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, skull injury (trauma capitis), gastrointestinal bleeding, intracranial hemorrhage , intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, pharyngeal hemorrhage, retroperitoneal hemorrhage, iliopsoas sheath hemorrhage, or any combination thereof. In another embodiment, a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding a chimeric molecule, a vector or set of vectors disclosed herein, a host cell disclosed herein, or a composition disclosed herein may be administered by a route selected from topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, oral administration, or any combination thereof.

The present disclosure also includes a method for producing a chimeric molecule, comprising transfecting one or more host cells with a polynucleotide disclosed herein or a vector disclosed herein, and expressing the chimeric molecule in the host cell. The method further includes isolating the chimeric molecule. In some embodiments, the FVIII activity of the chimeric molecule can be measured by activated partial thromboplastin time (APTT) analysis or by rotational thromboelastometry (ROTEM).

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Brief description of graphic materials

Fig. 1 illustrates an exemplary scheme of a chimeric molecule (FVIII-XTEN/remepoguMep VWF) containing two polypeptide chains - the first chain containing a VWF protein (e.g., the D' domain and the D3 domain of the VWF) linked to an Fc region via a thrombin-cleavable VWF linker, and a second chain containing the FVIII protein linked to the second Fc region via an FVIII linker. The FVIII protein contains one or more XTENs in different FVIII domains.

Fig. 2 illustrates different VWF constructs, with each construct containing a D' domain and a D3 domain linked to an Fc region via a thrombin-cleavable VWF linker, except for the control (ie, VWF-052). VWF-031 contains a 48 amino acid linker including the L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-034 contains a 288 amino acid XTEN sequence and a 35 amino acid linker including the L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-035 contains a 73 amino acid linker including the L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-036 contains a 98 amino acid linker including the L-V-P-R thrombin cleavage site (SEQ ID NO: 25). VWF-039 contains a 26 amino acid VWF linker comprising an L-V-P-R thrombin cleavage site (SEQ ID NO: 25) and a PAR1 interaction exosite motif. VWF-051 contains a 54 amino acid linker comprising the A-L-RP-R-V-V thrombin cleavage site (SEQ ID NO: 26). VWF-052 contains a 48 amino acid linker without any thrombin cleavage site (control). VWF-054 contains a 40 amino acid VWF linker including the a1 region of FVIII. VWF-055 contains a 34 amino acid VWF linker including the a2 region of FVIII. VWF-056 contains a 46 amino acid VWF linker including the a3 region of FVIII.

Fig. 3A illustrates the rate of thrombin-mediated cleavage in units of resonance position shift per second (RU/s) as a function of capture density in units of RU for VWF-Fc fusion constructs, ie. VWF-031, VWF-034, VWF-036, VWF-039, VWF-051 and VWF-052.

Fig. 3B illustrates the rate of thrombin-mediated cleavage in units of resonance position shift per second (RU/s) as a function of capture density in units of RU for VWF-Fc fusion constructs, ie. VWF-031, VWF-034, VWF-036, VWF-051 and VWF-052. In these experiments, each VWF-Fc fusion construct was captured at different densities and then exposed to a fixed concentration of human alpha thrombin. The slope of each curve in FIG. 3A and 3B directly depict the susceptibility of each construct to thrombin cleavage.

Fig. 4A illustrates the rate of thrombin-mediated cleavage in units of resonance position shift per second (RU/s) as a function of capture density in units of RU for VWF-Fc fusion constructs, ie. VWF-054, VWF-055 and VWF-056.

Fig. 4B illustrates the rate of thrombin-mediated cleavage in units of resonance position shift per second (RU/s) as a function of capture density in units of RU for VWF-Fc fusion constructs, ie. VWF-031, VWF-039, VWF-054, VWF-055 and VWF-056. In these experiments, each VWF-Fc fusion construct was captured at different densities and then exposed to a fixed concentration of human alpha thrombin. The slope of each curve in FIG. 4A and 4B directly depict the susceptibility of each construct to thrombin cleavage.

Fig. 5 illustrates the results of a linear regression analysis to determine the susceptibility of various VWF-Fc constructs - VWF-031, VWF-034, VWF-036, VWF-039, VWF-051, VWF052, VWF-054, VWF-055 and VWF-056 - to thrombin-mediated cleavage. Values are expressed in reciprocal seconds and represent the slopes of the curves illustrated in FIG. 3 and 4. The relative susceptibility of two different constructs is determined as the quotient of dividing the values of their respective slopes. The _slopevwF - ₀₃₉ / _slopevwF - ₀₃₁ ratio is 71, indicating that the VWF-Fc fusion construct VWF-039 is 71 times more sensitive to thrombin-mediated cleavage than VWF-031. The ratio vw _F - ₀ 55/slope vw _F - ₀₃₁ is 65, and the ratio vw _F - ₀ 5 ₁ /slope vw _F - ₀₃₁ is 1.8.

Fig. 6 illustrates the clotting time for various chimeric molecules in a HemA patient measured by whole blood assay by ROTEM. FVII155/VWF-031 contains two polypeptide chains - the first chain containing the BDD of FVIII connected to the Fc region, and the second chain containing the D' domain and the D3 domain of VWF connected to the Fc region through a minimal thrombin cleavage site (i.e. .L-V-P-R (SEQ ID NO: 25)). FVII155/VWF-039 contains two polypeptide chains - the first chain containing the BDD of FVIII connected to the Fc region, and the second chain containing the D' domain and the D3 domain of the VWF connected to the Fc region using a VWF linker containing L-V-P-R (SEQ ID NO: 25) and the PAR1 interaction exosite motif. FVII155/VWF-055 contains two polypeptide chains, the first chain containing the BDD of FVIII connected to the Fc region, and the second chain containing the D' domain and the D3 domain of the VWF connected to the Fc region using a VWF linker containing the a2 region from FVIII .

Fig. 7 illustrates a schematic of a typical FVIII-VWF heterodimer and constructs FVIII169, FVIII286, VWF057, VWF059, and VWF062. For example, the FVIII169 construct contains a FVIII protein with a B domain deletion with an R1648A substitution fused to an Fc region in which the XTEN sequence (e.g., AE288) is inserted at amino acid position 745, which corresponds to mature full-length FVIII

- 6 041588 (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-Fc). The FVIII286 construct contains a B domain deleted FVIII protein with an R1648 substitution fused to an Fc region in which the XTEN sequence (e.g., AE288) is inserted at amino acid position 745, corresponding to mature full-length FVIII, with an additional a2 region between FVIII and Fc (A1- a1-A2-a2-288XTEN-a3-A3-C1-C2-a2-Fc). VWF057 is a VWF-Fc fusion construct containing 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 that contains a thrombin site LVPR (LVPR) and GS linker (GS), with an XTEN sequence (ie 144XTEN) inserted between the D'D3 domain and the VWF linker (D'D3144XTEN-GS+LVPR-Fc). VWF059 is a VWF-Fc fusion construct containing 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 acid region region 2 (a2) as a VWF linker, with an XTEN sequence inserted between the D'D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct containing 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, with the XTEN sequence inserted between the domain D'D3 and Fc region (D'D3-144XTEN-Fc).

Fig. 8 illustrates the acute potency of FVIII-XTEN-Fc/D'D3-linker-Fc heterodimers (i.e., FVIII169/VWF034, FVIII169/VWF059, and FVIII169/VWF057) compared to B-domain-deleted FVIII (SQ BDD FVIII or BDD-rFVIII) or vehicle control in a clipped-tail HemA mouse model. BDD-rFVIII is indicated by circles and FVIII169/VWF034 is indicated by squares, FVIII169/VWF059 is indicated by triangles, FVIII169/VWF057 is indicated by open circles, and the carrier is indicated by inverted triangles. VWF034 is a VWF-Fc fusion construct that contains an Fc region connected to the D' domain and the D3 domain of the VWF via a VWF linker containing LVPR, with an XTEN sequence (i.e., 288XTEN) inserted between the D'D3 domain and VWF linker (D'D3-288XTEN-LVPR-Fc). Detailed descriptions of constructs FVIII169, VWF059 and VWF057 are provided elsewhere in this document. Median blood loss (µl) in mice after administration of a dose of 75 IU/kg of the construct in each experimental group is shown by horizontal lines.

Detailed description of the invention

The present invention relates to a chimeric molecule containing an XTEN sequence and a thrombin-cleavable linker that connects a VWF protein or FVIII protein to a heterologous moiety, such as a half-life prolonging moiety. The invention also provides a chimeric molecule containing two polypeptide chains - the first chain containing the VWF protein associated with a heterologous fragment, and the second chain containing the FVIII protein and the second heterologous fragment, and the chimeric molecule contains the XTEN sequence in the first or second polypeptide chains, and or the VWF protein or the FVIII protein (or both) is linked to the heterologous moiety via a VWF linker or an FVIII linker (or both). A thrombin-cleavable linker (VWF linker or FVIII linker) can be efficiently cleaved by thrombin at the site of injury where thrombin is readily available. Typical examples of chimeric molecules are illustrated in the present description and figures. In some embodiments, the invention relates to chimeric molecules having the structures shown, for example, in FIG. 1-7. In other embodiments, the invention provides a polynucleotide encoding the chimeric molecule constructs disclosed herein.

To ensure a clear understanding of the description and claims, the following definitions are provided.

I. Definitions.

It should be noted that objects described in the singular refer to one or more of the specified objects; for example, a nucleotide sequence is meant to refer to one or more nucleotide sequences. As such, the terms singular, one or more, and at least one may be used interchangeably herein. The term about is used herein with the meanings of approximately, roughly, about, or close to. When the term about is used in conjunction with a range of numerical values, it modifies that range by extending its boundaries to values above and below the specified numerical values. In general, the term about is used in this document to modify a numerical value by deviating by 10 percent from the specified value up or down (above or below).

The term polynucleotide or nucleotide is considered to encompass a single nucleic acid as well as a plurality of nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRPK) or plasmid DNA (pDNA). In certain embodiments of the invention, the polynucleotide contains a conventional phosphodiester bond or an unusual bond (eg, an amide bond, such as that present in peptide nucleic acids (PNA)). The term nucleic acid refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

- 7 041588

By isolated nucleic acid or polynucleotide is meant a nucleic acid, DNA or RNA molecule extracted from its natural environment. For example, a recombinant polynucleotide encoding a factor VIII polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partly or to a large extent) of other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. The isolated polynucleotides or nucleic acids in accordance with the present invention further include such molecules obtained by synthesis. In addition, the polynucleotide or nucleic acid may include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

As used herein, a coding region or coding sequence is a portion of a polynucleotide that consists of codons translated into amino acids. Although a stop codon (TAG, TGA, or TAA) is not typically translated into an amino acid, it may be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. areas. The boundaries of the coding region are typically defined by a start codon at the 5' end, encoding the amino end of the nascent polypeptide, and a translation termination codon at the 3' end, encoding the carboxyl end of the nascent polypeptide. Two or more coding regions of the present invention may be present in a single polynucleotide construct, eg in a single vector, or in separate polynucleotide constructs, eg in separate (different) vectors. It follows that one vector may contain only one coding region, or may contain two or more coding regions, for example, one vector may separately encode the first polypeptide chain and the second polypeptide chain of the chimeric molecule, as described below. In addition, the vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions fused or non-fused to the nucleic acid encoding the chimeric molecule of the invention. Heterologous coding regions include, without limitation, specialized elements or motifs such as a secretory signal peptide or a heterologous functional domain.

Certain proteins secreted by mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein upon initiation of export of the growing protein chain through the rough endoplasmic reticulum. Those of ordinary skill in the art are aware that signal peptides are typically attached to the N-terminus of a polypeptide and are cleaved from the full or full length polypeptide to form the secreted or mature form of the polypeptide. In certain embodiments, a native signal peptide is used, such as a FVIII signal peptide or a VWF signal peptide, or a functional derivative of such a sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, may be used. The term downstream of transcription refers to a nucleotide sequence that is located 3' to the end of a reference nucleotide sequence. In certain embodiments of the invention, downstream of the transcription of a nucleotide sequence refers to the sequence following the start of transcription. For example, the translation initiation codon of a gene is located downstream of the transcription start site.

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

As used herein, the term regulatory region refers to nucleotide sequences that are located upstream of transcription (5' non-coding sequences), within or downstream of transcription (3' non-coding sequences) of the coding region, and which have an effect on 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 the coding region is intended to be expressed in a eukaryotic cell, then the polyadenylation signal and transcription termination sequence will typically be located 3' to the coding sequence.

A polynucleotide that encodes for a gene product, such as a polypeptide, may include a promo

- 8 041588 torus and/or other transcription or translation control elements operably associated with one or more coding regions. In functional association, the coding region of a gene product, eg a polypeptide, is associated with one or more regulatory regions in such a manner as to bring expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are operably associated if induction of promoter function results in transcription of an mRNA encoding the gene product encoded by the coding region, and if the nature of the association between the promoter and the coding region does not interfere with the promoter's ability to direct expression of the gene product or interfere with transcriptional ability. DNA matrices. Transcriptional control elements other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may also be operably associated with the coding region to direct expression of the gene product.

Various transcriptional control regions are 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, cytomegalovirus promoter and enhancer segments (immediate early promoter, in combination with intron-A), simian virus 40 (early promoter), and retroviruses ( such as Rous sarcoma virus). Other transcriptional control regions include those isolated from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters induced by interferons or interleukins). Likewise, various translational control elements are known to those of ordinary skill in the art. These include, without limitation, ribosome binding sites, translation initiation and termination codons, and elements isolated from picornaviruses (specifically, the internal ribosome binding site, or IRES, also referred to as the CITE sequence).

The term expression as used herein refers to the process in which a polynucleotide produces a gene product, such as an RNA or a polypeptide. It includes, without limitation, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and translation of the mRNA into a polypeptide. Expression produces a gene product. As used herein, a gene product may either be a nucleic acid, such as messenger RNA, resulting from the transcription of a gene or a polypeptide that is translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

A vector refers to any vehicle for cloning and/or transferring a nucleic acid into a host cell. The vector may be a replicon to which another nucleic acid segment may be attached to effect replication of the attached segment. Replicon refers to any genetic element (eg, plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, ie. able to replicate under its own control. The term vector includes both viral and non-viral carriers for introducing a nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a vector of choice having complementary sticky ends.

Vectors can be genetically modified to encode selectable markers or reporters that allow selection or identification of cells with the included vector. Expression of selectable markers or reporters allows the identification and/or selection of host cells that include and express other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include: genes conferring resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, ie. anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), α-galactosidase (LacZ), α-glucuronidase (Gus), and the like. Selectable markers can also be considered reporters.

The term plasmid refers to an extrachromosomal element, often carrying a gene that is not

- 9 041588 being part of the central metabolism of the cell and usually in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences, genointegrating sequences, phage or nucleotide sequences, linear, circular or supercoiled, single or double stranded DNA or RNA, isolated from any source in which a number of nucleotide sequences have been linked or recombined into a unique construct capable of introducing the promoter fragment and the DNA sequence of the selected gene product, together with the corresponding 3' untranslated sequence, into the cell.

Eukaryotic viral vectors that can be used include, without limitation, adenovirus vectors, retroviral vectors, adeno-associated viral vectors, poxvirus, such as vaccinia vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

A cloning vector refers to a replicon, which is a single stretch of nucleic acid that replicates sequentially and which contains an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment can be attached to replicate the attached segment. Certain cloning vectors are capable of replication in one type of cell, such as bacteria, and expression in another, such as eukaryotic cells. Cloning vectors typically contain one or more sequences that can be used to select cells containing the vector and/or one or more multiple cloning sites to insert nucleic acid sequences of interest.

The term expression vector refers to a carrier designed to allow expression of the inserted nucleic acid sequence after introduction into a host cell. The inserted nucleic acid sequence is operably associated with regulatory regions as described above.

Vectors are introduced into host cells by methods well known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (fusion with lysosomes), gene gun, or vector - DNA carrier.

Culture, culture and cultivation, as used herein, means the incubation of cells under in vitro conditions that allow cells to grow or divide or maintain cells in a viable state. Cultured cells, as used herein, means cells that propagate in vitro.

As used herein, the term polypeptide is considered to encompass the singular form of a polypeptide as well as the plural form of a polypeptide, and refers to a molecule composed of monomers (amino acids) linked linearly 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 indicate a specific product length. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of a polypeptide, and the term polypeptide may be used instead of or interchangeably with any from these terms. The term polypeptide is also intended to refer to products of post-expression modification of a polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with unnatural amino acids. The polypeptide may be isolated from a natural biological source or obtained using recombinant technology, but not necessarily translated from the specified nucleic acid sequence. It can be obtained by any method, including chemical synthesis.

An isolated polypeptide or fragment, variant or derivative thereof refers to a polypeptide not found in its natural environment. No particular degree of purification is required. For example, an isolated polypeptide may simply be recovered from its native or natural environment. Recombinantly produced polypeptides and proteins 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 method.

The present invention also includes fragments or variants of polypeptides and any combination thereof. The terms fragment or variant with respect to the polypeptide binding domains or binding molecules of the present invention include any polypeptides that retain at least some properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulating activity at with respect to the FVIII variant, or FVIII binding activity with respect to the VWF protein) of the reference polypeptide. Frag

- 10 041588 polypeptides include proteolytic fragments, as well as fragments with deletions, in addition to specific antibody fragments described elsewhere in this document, but do not include natural full-length polypeptide (or mature polypeptide). Variants of the polypeptide-binding domains or binding molecules of the present invention include fragments as described above, as well as polypeptides with altered amino acid sequences as a result of amino acid substitutions, deletions or insertions. Variants may or may not occur naturally. Non-natural variants can be obtained using mutagenesis methods known in the art. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions or additions. The term VWF fragment or VWF fragments as used herein means any VWF fragments that interact with FVIII and retain at least one or more of the properties normally provided to FVIII by full-length VWF, such as preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes, which can lead to premature clearance, prevention of binding to FVIII clearance receptors that can bind naked FVIII but not VWF bound FVIII, and/or stabilization of FVIII heavy chain and light chain interactions. In a specific embodiment of the invention, the VWF fragment, as used herein, contains the D' domain and the D3 domain of the VWF protein, but does not include the A1 domain, A2 domain, A3 domain, D4 domain, B1 domain, B2 domain, B3 domain, domain C1, C2 domain, and CK domain of the VWF protein.

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

The term endogenous VWF, as used herein, refers to VWF molecules naturally present in plasma. The endogenous VWF molecule may be a multimer, but may be a monomer or a dimer. Endogenous VWF in plasma binds to FVIII and forms a non-covalent complex with FVIII. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is substituted for another amino acid from the same side chain family, that substitution is considered conservative. In another embodiment of the invention, an amino acid chain may be conservatively substituted for a structurally similar chain that differs in order and/or composition of the members of the side chain families.

As known in the art, sequence identity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with that of a second polypeptide. As discussed herein, whether a particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined with using methods and computer programs/applications known in the art, such as, without limitation, the BESTFIT program (Wisconsin Sequence Analysis Package (software package for sequence analysis, University of Wisconsin), version 8 for the Unix operating system, Genetics Computer Group , University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses Smith and Waterman's local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981)), to find the segment with the best homology between two sequences. When using BESTFIT or any other program to align sequences to determine if a certain sequence is, for example, 95% identical to the reference sequence according to the present invention, the parameters are set, of course, such that the percent identity is calculated from the full length of the reference sequence. polypeptide sequence, and that homology gaps of up to 5% of the total number of amino acids in the reference sequence are allowed.

As used herein, the amino acid corresponding to or equivalent to

- 11 041588 th amino acid in a VWF or FVIII protein sequence is determined by aligning to maximize identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify the equivalent amino acid in the second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

The fusion or chimeric molecule contains a first amino acid sequence linked to a second amino acid sequence to which it is not normally fused in nature. Amino acid sequences normally present in different proteins may be combined in a fusion polypeptide, or amino acid sequences normally present in the same protein may be placed in a different order in a fusion polypeptide, for example, by fusion of the factor VIII domain of the invention with domain of immunoglobulin Fc. The fusion protein is obtained, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired mutual arrangement. The chimeric protein may further comprise a second amino acid sequence associated with the first amino acid sequence via a covalent non-peptide bond or a non-covalent bond.

As used herein, the term half-life refers to the biological half-life of a particular polypeptide in vivo. The half-life can be represented as the time required to remove half of the amount administered to the subject from the circulation and/or other tissues of the animal. If a clearance curve for a given polypeptide is plotted as a function of time, then the curve is usually biphasic, with a fast a-phase and a longer P-phase. a-Phase typically corresponds to the balance of the administered polypeptide between the intra- and extravascular space and is determined in part by the size of the polypeptide. The β-phase typically corresponds to the catabolism of the polypeptide in the intravascular space. In some embodiments of the invention, the chimeric molecule of the invention is monophasic and thus has no alpha phase but only one beta phase. Thus, in certain embodiments of the invention, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

The term heterologous with respect to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is isolated from an entity other than the entity to which it is being compared. Thus, a heterologous polypeptide linked to a VWF protein means a polypeptide chain that is linked to a VWF protein and is not a naturally occurring part of the VWF protein. For example, a heterologous polynucleotide or antigen may be derived from different species, from different cell types of an individual, or from the same or different cell types from different individuals. The term bonded, fused, or joined as used herein refers to a first amino acid sequence or nucleotide sequence attached to a second amino acid sequence or nucleotide sequence (eg, via a peptide bond or a phosphodiester bond, respectively). The term covalently linked or covalent compound refers to a covalent bond, such as a disulfide bond, a peptide bond, or one or more amino acids, such as a linker, between two moieties linked together. The first amino acid or nucleotide sequence may be directly attached to the second amino acid or nucleotide sequence, or alternatively, an intermediate sequence may link the first sequence to the second sequence. The term linked, fused, or connected means not only the fusion of a first amino acid sequence with a second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or second amino acid sequence) between any two amino acids of the second amino acid sequence (or first amino acid sequences, respectively). In one embodiment of the invention, the first amino acid sequence can be attached to the second amino acid sequence using a peptide bond or a linker. The first nucleotide sequence may be attached to the second nucleotide sequence using a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for polypeptide chains) or a nucleotide or nucleotide chain (for nucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). A covalent bond is sometimes denoted as (-) or a dash.

As used herein, the term associated with refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment of the invention, the term associated with means a covalent non-peptide bond or a non-covalent bond. In some embodiments, such a relationship

- 12 041588 is denoted by a colon, i.e. (:). In another embodiment of the invention, it means a covalent bond, with the exception of the peptide bond. In other embodiments, the term covalently associated, as used herein, means an association between two moieties via a covalent bond, such as a disulfide bond, a peptide bond, or one or more amino acids (eg, a linker). For example, the amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with the thiol group of the second cysteine residue. In most natural IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 in the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, without limitation, peptide bond, metal bond, hydrogen bond, disulfide bond, sigma bond, pi bond, delta bond, glycosidic bond, agnostic bond, crooked bond, dipolar bond, pi-dative bond, double bond, triple bond, quadruple bond, five-fold bond, six-fold bond, conjugation, hyperconjugation, aromaticity, hapticity, or anti-bonding. Non-limiting examples of non-covalent bonds include ionic bond (e.g., cation-pi-bond or salt bond), metal bond, hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, low barrier hydrogen bond, or symmetrical hydrogen bond), van der Waals forces, London dispersion forces , mechanical bonding, halogen bonding, aurophilicity, intercalation, stacking, entropy forces, or chemical polarity.

As used herein, the term cleavage site or enzymatic cleavage site refers to a site recognized by an enzyme. In one embodiment, the polypeptide has an enzymatic cleavage site that is cleaved by an enzyme that is activated in the coagulation cascade, such that cleavage of such sites occurs at the site of clot formation. In another embodiment, the FVIII linker connecting the FVIII protein and the second heterologous fragment may contain a cleavage site. Typical examples of such sites include, for example, sites recognized by thrombin, factor XIa or factor Xa. Typical examples of FXIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO: 27) and SVSQTSKLTR (SEQ ID NO: 28). Typical examples of thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 29), TTKTKPR (SEQ ID NO: 30), LVPRG (SEQ ID NO: 31) and ALRPR (amino acids 1-5 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.

As used herein, the term processing site or intracellular processing site refers to a type of enzymatic cleavage site in a polypeptide that is targeted by enzymes that function after translation of the polypeptide. In one embodiment of the invention, such enzymes function during transport from the Golgi cavity to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before secreting the protein from the cell. Examples of such processing sites include, for example, sites targeted by the PACE/furin family of endopeptidases (where PACE is an acronym for Paired basic Amino Acid Cleaving Enzyme). These enzymes are localized in the Golgi membrane and cleave proteins from the carboxyl end of the Arg-[any residue]-(Lys or Arg)-Arg sequence motif. As used herein, the furin enzyme family includes, for example, PCSK1 (also known as PC1/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4) or PCSK7 (also known as PC7/LPC, PC8 or SPC7). Other processing sites are known in the art. The term processable linker as used herein means a linker containing an intracellular processing site.

The term furin refers to enzymes classified according to EC (Enzyme Classification) No. 3.4.21.75. Furin is a subtilisin-like proprotein convertase, also known as PACE (Paired basic Amino acid Cleaving Enzyme). Furin deletes regions of inactive precursor proteins, turning them into biologically active proteins. During intracellular transport, the propeptide is cleaved from the mature VWF molecule by the enzyme furin in the Golgi apparatus.

It should be understood that in constructs containing multiple processing or cleavage sites, such sites may be the same or different. Violation of hemostasis, 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 an impaired ability or inability to form a fibrin clot. Examples of such disorders include hemophilia. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or Christmas disease), and hemophilia C (factor XI deficiency, low bleeding tendency). Other disorders of hemostasis include, for example, von Willebrand disease, deficiency

- 13 041588 factor XI (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X or factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency of GPIb. GPIb, which is a VWF receptor, can be defective and lead to failure of primary clot formation (primary hemostasis) and an increased tendency to bleed) and Glanzmann-Negeli thrombasthenia (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), insufficient production of coagulating factors by the liver is observed; this may increase the risk of bleeding.

The chimeric molecules of the invention can be used prophylactically. As used herein, the term prophylactic treatment refers to administration of the molecule prior to a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is, or is to be, undergoing surgery. The chimeric protein of the invention may be administered before or after surgery as a prophylactic. The chimeric protein of the invention may be administered during or after surgery to arrest an acute bleeding episode. Surgery may include, without limitation, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

The chimeric molecule of the invention is also used for on-demand (also referred to as episodic) treatment. The term on-demand 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 provided to a subject at the onset of bleeding, such as after an injury, or when bleeding is expected, such as prior to surgery. In another aspect, on-demand treatment may be provided prior to activities that increase the risk of bleeding, such as contact sports.

As used herein, the term acute bleeding refers to an episode of bleeding, regardless of the underlying cause. For example, the subject may have an injury, uremia, an inherited bleeding disorder (eg, factor VII deficiency), a platelet disorder, or resistance due to the production of antibodies to blood clotting factors.

Treat, treatment, treating, as used herein, refer to, for example, reducing the severity of a disease or condition; reducing the duration of the course of the disease; alleviate one or more symptoms associated with the disease or condition; providing beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, or preventing one or more symptoms associated with the disease or condition. In one embodiment, the term "treat" or "treatment" refers to maintaining a subject at a minimum level of FVIII prior to administration of the next dose of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 IU/dl by administering a chimeric molecule of the invention. In another embodiment, treating or treating means maintaining a minimum level of FVIII prior to administration of the next dose, having a value between about 1 and about 20 IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL, about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9 and about 20 IU/dL dl or about 10 and about 20 IU/dl. Treatment or therapy for a disease or condition may also include maintaining a subject's FVIII activity 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% FVIII activity in a subject without hemophilia. The minimum pre-dose level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each subject.

II. chimeric molecules.

The chimeric molecules of the invention are designed to enhance the release of a VWF protein or FVIII protein from another moiety to which the VWF protein or FVIII protein is bound. The invention provides a thrombin-cleavable linker that can be rapidly and efficiently cleaved at the site of injury. In one aspect of the invention, the chimeric molecule may comprise a von Willebrand factor (VWF) protein, a heterologous fragment (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous fragment, the VWF linker comprising a polypeptide selected from: (i) the a2 region of factor VIII (FVIII); (ii) regions a1 of FVIII; (iii) a3 regions from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof, and the XTEN sequence is linked to a VWF protein, a heterologous fragment (H1), a VWF linker, or any combination thereof. In another aspect of the invention, the chimeric molecule may contain a first polypeptide chain that contains a VWF protein, a heterologous fragment (H1) and a VWF linker connecting the VWF protein and a heterologous fragment (H1), and a second polypeptide chain containing the FVIII protein and the XTEN sequence, and the linker The VWF in the first polypeptide chain contains: (i) the a2 region of FVIII; (ii) region a1 from FVIII; (iii) region a3 from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is

- 14 041588 aliphatic amino acid; or (v) any combination thereof, and the first polypeptide chain and the second polypeptide chain are associated with each other.

In other aspects of the invention, the chimeric molecule comprises a polypeptide chain containing a FVIII protein connected to a heterologous fragment via an FVIII linker, the FVIII linker comprising: (i) the a2 region of FVIII; (ii) region a1 from FVIII; (iii) region a3 from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination of them.

P.A. Chimeric molecules with VWF, XTEN, VWF linker.

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

In one embodiment, the chimeric molecule comprises a VWF protein, a heterologous fragment (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous fragment, wherein the XTEN sequence is located between the VWF protein and the VWF linker, and the VWF linker contains a polypeptide selected from: (i) a2 regions from factor VIII (FVIII); (ii) a1 regions from FVIII; (iii) a3 regions from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof. In another embodiment, the chimeric molecule contains a VWF protein, a heterologous fragment (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous fragment, wherein the XTEN sequence is located between the VWF linker and the heterologous fragment, and the VWF linker contains a polypeptide selected from: (i) a2 regions from FVIII; (ii) a1 regions from FVIII; (iii) a3 regions from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof. In other embodiments of the invention, the chimeric molecule further comprises a polypeptide chain containing the FVIII protein, wherein the first chain containing the VWF protein and the second chain containing the FVIII protein are associated with each other. In one example, the association may be a covalent association, such as a disulfide bond. In still other embodiments of the invention, the polypeptide chain containing the FVIII protein further comprises an additional XTEN sequence. An additional XTEN sequence may be linked to the N-terminus or C-terminus of the FVIII protein, or inserted between two adjacent FVIII amino acids. In other embodiments of the invention, the chain containing the FVIII protein additionally contains a second heterologous fragment (H2). In some embodiments of the invention, the FVIII protein is connected to the second heterologous fragment using a FVIII linker. In certain embodiments of the invention, the FVIII linker is identical to the VWF linker connecting the VWF protein and the heterologous fragment. In other embodiments, the FVIII linker is different from the VWF linker connecting the VWF protein and the heterologous fragment.

In certain embodiments of the invention, the chimeric molecule has the 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-X1L1-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:H1X1-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 linker; L2 is an optional FVIII linker; H1 is the first heterologous fragment; H2 denotes the second heterologous fragment; X1 denotes the sequence XTEN; X2 denotes an optional XTEN sequence; C is FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, with the XTEN sequence inserted between two adjacent FVIII amino acids; (-) denotes a peptide bond or one or more amino acids; and (:) denotes a covalent bond between H1 and H2.

In some embodiments, the FVIII protein in the chimeric molecule contains a third heterologous fragment (H3), which may be the XTEN sequence. In other embodiments, the FVIII protein of the chimeric molecule contains a fourth heterologous fragment (H4), which may be the XTEN sequence. In still other embodiments, the FVIII protein of the chimeric molecule contains a fifth heterologous fragment (H5), which may be an XTEN sequence. In other embodiments, the FVIII protein of the chimeric molecule contains a sixth heterologous fragment (H6), which may be the XTEN sequence. In certain embodiments of the invention, one or more of the third heterologous fragment (H3), the fourth heterologous fragment (H4), the fifth heterologous fragment (H5) and the sixth heterologous fragment (H6) are capable of increasing the half-life of the chimeric molecule. In some embodiments, a third heterologous fragment (H3), a fourth heterologous fragment (H4), a fifth heterologous fragment (H5), and a sixth heterologous fragment (H6) are attached to the C-terminus or N-terminus of FVIII, or inserted between two amino acids of the FVIII protein.

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II.B. Chimeric molecules with FVIII, XTEN, VWF protein, VWF linker.

The present invention also provides a chimeric molecule containing a first polypeptide chain that contains a VWF protein, a heterologous fragment (H1) and a VWF linker connecting the VWF protein and a heterologous fragment (H1), and a second polypeptide chain containing the FVIII protein and the XTEN sequence, and the linker The VWF in the first polypeptide chain contains: (i) the a2 region of FVIII; (ii) region a1 from FVIII; (iii) region a3 from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof, and the first polypeptide chain and the second polypeptide chain are associated with each other. In one embodiment, the XTEN sequence is fused to the N-terminus or C-terminus of the FVIII protein, or inserted between two adjacent FVIII amino acids. In another embodiment, the chimeric molecule further comprises an additional XTEN sequence that is linked to a VWF protein, a heterologous fragment, a VWF linker, or any combination thereof. In other embodiments of the invention, the chimeric molecule further comprises a second heterologous fragment (H2). In other embodiments of the invention, the second heterologous fragment of the chimeric molecule is connected to the FVIII protein, the XTEN sequence, or both. In other embodiments, the second heterologous fragment of the chimeric molecule is linked to the FVIII protein or XTEN sequence via an FVIII linker. In other embodiments, the FVIII linker is identical to the VWF linker. In some embodiments, the FVIII linker is different from the VWF linker. In certain embodiments of the invention, the chimeric molecule has the formula selected from: (i) V-L1-X1H1: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-L2C; (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) CX2-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:H1X1-L1-V; or (xii) C(X2)-L2-H2:H1-L1-X1-V; where V is a VWF protein; L1 is a VWF linker; L2 is an optional FVIII linker; H1 is the first heterologous fragment; H2 denotes the second heterologous fragment; X1 denotes an optional XTEN sequence; X2 denotes the sequence XTEN; C is FVIII protein; C(X2) is a FVIII protein fused to an XTEN sequence, with the XTEN sequence inserted between two adjacent FVIII amino acids; (-) denotes a peptide bond or one or more amino acids; and (:) denotes a covalent bond between H1 and H2. In one embodiment, the VWF linker and the FVIII linker may be the same. In another embodiment, the VWF linker and the FVIII linker are different. In certain embodiments, the FVIII protein of the chimeric molecule contains a third heterologous fragment (H3), which may be an XTEN sequence. In other embodiments, the FVIII protein of the chimeric molecule contains a fourth heterologous fragment (H4) which is the XTEN sequence. In still other embodiments, the FVIII protein of the chimeric molecule contains a fifth heterologous fragment (H5), which may be an XTEN sequence. In other embodiments, the FVIII protein contains a sixth heterologous fragment (H6), which may be an XTEN sequence. In certain embodiments of the invention, one or more of the third heterologous fragment (H3), the fourth heterologous fragment (H4), the fifth heterologous fragment (H5) and the sixth heterologous fragment (H6) are capable of increasing the half-life of the chimeric molecule. In some embodiments, a third heterologous fragment (H3), a fourth heterologous fragment (H4), a fifth heterologous fragment (H5), and/or a sixth heterologous fragment (H6) are attached to the C-terminus or N-terminus of FVIII or inserted between two amino acids of the protein FVIII.

P.S. Chimeric molecules with FVIII, XTEN and FVIII linker.

A chimeric molecule of the invention may contain a FVIII protein, an XTEN sequence, and a heterologous fragment attached via an FVIII linker that contains (i) an a2 region from FVIII; (ii) region a1 from FVIII; (iii) region a3 from FVIII; (iv) a thrombin cleavage site containing XV-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination of them. In certain embodiments of the invention, the chimeric molecule contains two polypeptide chains - the first chain containing the FVIII protein connected to the first Fc region using an FVIII linker, and the second chain containing the VWF protein (for example, the D' domain and the D3 domain of the VWF) connected with an Fc region, and the FVIII linker in the first polypeptide chain contains: (i) the a2 region of FVIII; (ii) region a1 from FVIII; (iii) region a3 from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof, and the first polypeptide chain and the second polypeptide chain are associated with each other, and the XTEN sequence is attached to the first polypeptide (for example, to the N-terminus or C-terminus of the FVIII protein, the linker or the first Fc region, or within FVIII protein), to a second polypeptide (eg, to the N-terminus or C-terminus of the VWF protein or Fc region, or within the FVIII protein), or both. In a particular embodiment, the linker in the first polypeptide chain contains the a2 region of FVIII.

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In certain embodiments of the invention, the chimeric molecule has the formula selected from: (i) V-L2-X2-H2: H1-L1-X1-C; (ii) V-X2-L2-H2: H1-L1-X1-C; (iii) V-L2-X2-H2: H1-X1-L1-C; (iv) V-X2L2-H2: H1-X1-L1-C; (v) V-L2-X2-H2: H1-L1-C(X1); (vi) V-X2-L2-H2: Hl-Ll-C(X1); (vii) C-X1-L1-H1: H2X2-L2-V; (viii) C-X1-L1-H1: H2-L2-X2-V; (ix) C-L1-X1-H1:H2-L2-X2-V; (x) C-L1-X1-H1:H2-L2-X2-V; (xi) C(X1)-L1-H1:H2-X2-L2-V; or (xii) C(X1)-L1-H1:H2-L2-X2-V, where V is a VWF protein; L1 is an FVIII linker; L2 denotes an optional VWF linker; H1 is the first heterologous fragment; H2 denotes the second heterologous fragment; X1 denotes an optional XTEN sequence; X2 denotes an optional XTEN sequence; C is FVIII protein; C(X1) is a FVIII protein fused to an XTEN sequence, with the XTEN sequence inserted between two adjacent FVIII amino acids; (-) denotes a peptide bond or one or more amino acids; and (:) denotes a covalent bond between H1 and H2, and wherein at least one XTEN sequence is present in the chimeric molecule. In one embodiment, the VWF linker and the FVIII linker are the same. In another embodiment, the VWF linker and the FVIII linker are different.

II.D. Components of chimeric molecules.

P.S.1. VWF linker or FVIII linker.

A VWF linker or FVIII linker suitable for a chimeric molecule of the invention is a thrombin-cleavable linker connecting a VWF protein to a heterologous moiety or a FVIII protein to a heterologous moiety. In one embodiment, the VWF linker or FVIII linker contains the a1 region of FVIII. In another embodiment, the VWF linker or FVIII linker contains the a2 region of FVIII. In other embodiments, the VWF linker or FVIII linker contains the a3 region of FVIII. In other embodiments, the VWF linker or FVIII linker comprises a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid.

In one embodiment, the VWF linker or FVIII linker comprises an al region that contains an amino acid sequence that is at least about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to a region from Met337 to Arg372, corresponding to a full-length mature FVIII, where the a1 region is capable of being cleaved by thrombin. In another embodiment, the VWF linker or FVIII linker comprises an al region that contains an amino acid sequence that is at least about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to amino acids 337-374, corresponding to full-length mature FVIII, where the a1 region is capable of being cleaved by thrombin. In other embodiments, the VWF linker or FVIII linker further comprises additional amino acids, such as one, two, three, four, five, ten or more. In a particular embodiment, the VWF linker or FVIII linker comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5). In some embodiments, the VWF linker or FVIII linker comprises an a2 region that contains an amino acid sequence that is at least about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to a region from Glu720 to Arg740 corresponding to a full-length mature FVIII, where the a2 region is capable of being cleaved by thrombin. In other embodiments, the VWF linker or FVIII linker comprises an a2 region that contains an amino acid sequence of at least about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99%, or 100% identical to amino acids 712-743 corresponding to full-length mature FVIII. In still other embodiments, the VWF linker or FVIII linker further comprises additional amino acids, such as one, two, three, four, five, ten or more. In a specific embodiment, the VWF linker contains ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4).

In certain embodiments, the VWF linker or FVIII linker comprises an a3 region that contains an amino acid sequence that is at least about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to a region from Glu1649 to Arg1689, corresponding to full-length mature FVIII, where the a3 region is capable of being cleaved by thrombin. In some embodiments, the VWF linker or FVIII linker comprises an a3 region that contains an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to amino acids 1649-1692 corresponding to full-length mature FVIII, where the a3 region is capable of being cleaved by thrombin. In other embodiments, the VWF linker or FVIII linker further comprises additional amino acids, such as one, two, three, four, five, ten or more. In a particular embodiment, the VWF linker or FVIII linker comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6).

In other embodiments, the VWF linker or FVIII linker comprises a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, wherein the PAR1 interaction exosite motif contains S-F-L-L-R-N (SEQ ID NO: 7). In some variants

The PAR1 interaction exosite motif further comprises an amino acid sequence selected from P, P-N, P-N-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 other embodiments, the aliphatic amino acid for the X-V-P-R thrombin cleavage site is selected from glycine, alanine, valine, leucine, or isoleucine. In a particular embodiment, the thrombin cleavage site contains L-V-P-R. In some embodiments, thrombin cleaves the VWF linker or FVIII linker faster than thrombin would cleave the thrombin cleavage site (e.g., L-V-P-R) if the thrombin cleavage site (L-V-P-R) replaced the VWF linker or FVIII linker, respectively (i.e., no motif exosite of PAR1 interaction). In some embodiments, thrombin cleaves the VWF linker or FVIII linker at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold. 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 (eg, L-V-P-R) if the thrombin cleavage site (eg, L-V-P-R) replaced the VWF linker or the FVIII linker.

In some embodiments, a VWF linker or FVIII linker comprising (i) an a1 region, (ii) an a2 region, (iii) an a3 region, or (iv) an XVPR thrombin cleavage site and a PAR1 interaction exosite motif further comprises one or more amino acids, having a length of at least about 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, 1000, 1200, 1400, 1600, 1800 or 2000 amino acids . In one embodiment, one or more amino acids comprise a gly peptide. In another embodiment of the invention, one or more amino acids contain GlyGly. In other embodiments of the invention, one or more amino acids contain IleSer. In still other embodiments, one or more amino acids comprise a gly/ser peptide. In other embodiments, one or more amino acids comprise a gly/ser peptide having the formula (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 some embodiments, one or more amino acids contain (Gly ₄ Ser) ₃ (SEQ ID NO: 89) or (Gly ₄ Ser) ₄ (SEQ ID NO: 90).

I.S.2. VWF protein.

VWF (also known as F8VWF) is a large multimeric glycoprotein present in plasma and produced constitutively in endothelium (Weibel-Palade bodies), megakaryocytes (platelet a-granules), and subendothelial connective tissue. The basic VWF monomer is a 2813 amino acid protein. 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 the platelet GPIb receptor, heparin, and/or possibly collagen), the A3 domain (which binds to collagen) , the C1 domain (in which the RGD domain binds to platelet integrin aIIbe3 when activated), and the cysteine knot domain at the C-terminus of the protein (which VWF shares with platelet growth factor (PDGF), transforming growth factor-β (TGFe), and β-chorionic human gonadotropin (eHCG)).

The term VWF protein as used herein includes, without limitation, full-length VWF protein or functional VWF fragments containing a D' domain and a D3 domain capable of inhibiting the binding of endogenous VWF to FVIII. In one embodiment, the 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, the VWF protein is not cleared by VWF clearance pathways. VWF proteins include derivatives, variants, mutants, or analogs that retain such VWF activities.

The 2813 amino acid monomeric sequence of human VWF has been deposited with Genbank under accession number _NP_000543.2__. The nucleotide sequence encoding the human VWF has been deposited with Genbank under accession number _NM_000552.3_. The nucleotide sequence of the human VWF is designated as SEQ ID NO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. All VWF domains are listed in Table. 1.

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Table 1. VWF sequences

VWF domains Amino acid sequence Signal peptide VWF (amino acids 1-22 SEQ GO NO: 2) 1 MIPARFAGVL LALALILPGT LC 22 VWF D1D2 region (amino acids 23-763 SEQ ID NO: 2) 23 AEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

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551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKR 763 Domain D'VWF 764 SLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDAT 866 D3 VWF domain ⁸⁶⁷ CST I_ GMAH YL T_F_D_G_ ДДХДДДДЕ CQ YyLyQDYCGS ⁹⁰¹ NPGT FRI LVG_ Д KGCJ HJ_S VK ДАKRVTILVE _GGEI ELFDGE VNVKRPMKDE 951 thfevvesgr_ ууудддбКАД ДДУУршуд ⁷ vvlkqt yqe KVCGLCGNFp 10 01 GIQNNDLT S S_ ДД QVEEDyДД A ^N _SWKVS SQ _CADT RKVP LD ЗЗРАТСНДД! 1051 MKQTMVDSS C_ ДДДT_S_DVF_Q_D _CNKLVD PE PY _LDVC IYDT CS CESIGDCACF 1101 GDTIAAYAHy_ CAQHGJVVTW ATATLCPQSC _EERNLRENGY ECEWRYNSCA 1151 PACQyTCQHP_ А ^Р ДА ^С АА ^С _УАУДНАНСР PG _KILDELLQTC ypPEDCPyCE 12 01 VAGRRFASGK_ КуТДДРЗДРД_HCQ АДСруу_NLTCEACQEP 1240 Al VWF domain 12 41 GGLWPPTDA 1251 PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV 1301 VDMMERLRIS QKWVRVAWE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA 1351 GSQVASTSEV LKYTLFQIFS KIDRPEASRI ALLLMASQEP QRMSRNFVRY 1401 VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1451 DEIVSYLCDL APEAPPPTLP PDMAQVTVG 1479 Nucleotide sequence (SEQ ID NO: 1) Full size VWF 1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTTGG CCCTCATTTTT 51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTTCTCGATTATTGGGGACTTCCA GAATGGCAAGAGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG

- 20 041588

451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTGACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGCACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCTCTCGAGACTG 1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAAGAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGGCTGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCGTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGAC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCRGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGAAGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGGAG TGCGGGACCCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC

- 21 041588

2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCAACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTCCATCAGGGCA 2501 AGGAGTATGCCCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGGGGAGTGCCAGTACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GCTTGGCCGG TACATCATTCTGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCTGCTTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGCATGGCAAGGT 3351 GGTGACTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGTGTGAGT GGCCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCCAGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG

- 22 041588

3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTCAACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG GGAGGCCTGG TGGTGCCTCCCACAGATGCC 3751 CCGGTGAGCC CCACCACTCT GTATGTGGAG GACATCTCGG AACCGCCGTT 3801 GCACGATTTC TACTGCAGCA GGCTACTGGA CCTGGTCTTC CTGCTGGATG 3851 GCTCCTCCAG GCTGTCCGAG GCTGAGTTTG AAGTGCTGAA GGCCTTTGTG 3901 GTGGACATGA TGGAGCGGCT GCGCATCTCC CAGAAGTGGG TCCGCGTGGC 3951 CGTGGTGGAG TACCACGACG GCTCCCACGC CTACATCGGG CTCAAGGACC 4001 GGAAGCGACC GTCAGAGCTG CGGCCGCATTG CCAGCCAGGT GAAGTATGCG 4051 GGCAGCCAGG TGGCCTCCAC CAGCGAGGTC TTGAAATACA CACTGTTCCA 4101 AATCTTCAGC AAGATCGACC GCCCTGAAGC CTCCCGCATC GCCCTGCTCC 4151 TGATGGCCAG CCAGGAGCCG CAACGGATGT CCCGGAACTT TGTCCGCTAC 4201 GTCCAGGGCC TGAAGAAGAA GAAGGTCATT GTGATCCCGG TGGGCATTGG 4251 GCCCCATGCC AACCTCAAGC AGATCCGCCT CATCGAGAAG CAGGCCCCTG 4301 AGAACAAGGC CTTCGTGCTG AGCAGTGTGG ATGAGCTGGA GCAGCAAAGG 4351 GACGAGATCG TTAGCTACCT CTGTGACCTT GCCCCTGAAG CCCCTCCTCC 4401 TACTCTGCCC CCCGACATGG CACAAGTCAC TGTGGGCCCG GGGCTCTTGG 4451 GGGTTTCGAC CCTGGGGCCC AAGAGGAACT CCATGGTTCT GGATGTGGCG 4501 TTCGTCCTGG AAGGATCGGA CAAAATTGGT GAAGCCGACT TCAACAGGAG 4551 CAAGGAGTTC ATGGAGGAGG TGATTCAGCG GATGGATGTG GGCCAGGACA 4601 GCATCCACGT CACGGTGCTG CAGTACTCCT ACATGGTGAC CGTGGAGTAC 4651 CCCTTCAGCG AGGCACAGTC CAAAGGGGAC ATCCTGCAGC GGGTGCGAGA 4701 GATCCGCTAC CAGGGCGGCA ACAGGACCAA CACTGGGCTG GCCCTGCGGT 4751 ACCTCTCTGA CCACAGCTTC TTGGTCAGCC AGGGTGACCG GGAGCAGGCG 4801 CCCAACCTGG TCTACATGGT CACCGGAAAT CCTGCCTCTGATGAGATCAA 4851 GAGGCCTGCCT GGAGACATCC AGGTGGTGCC CATTGGAGTG GGCCCTAATG 4901 CCAACGTGCA GGAGCTGGAG AGGATTGGCT GGCCCAATGCCCCTATCCTC 4951 ATCCAGGACT TTGAGACGCT CCCCCGAGAG GCTCCTGACC TGGTGCTGCA 5001 GAGGTGCTGC TCCGGAGAGG GGCTGCAGAT CCCCACCCTC TCCCCTGCAC 5051 CTGACTGCAG CCAGCCCCTG GACGTGATCC TTCTCCTGGA TGGCTCCTCC 5101 AGTTTCCCAG CTTCTTATTT TGATGAAATG AAGAGTTTCG CCAAGGCTTT 5151 CATTTCAAAAA GCCAATATAG GGCCTCGTCT CACTCAGGTG TCAGTGCTGC 5201 AGTATGGAAG CATCACCACC ATTGACGTGC CATGGAACGT GGTCCCGGAG

- 23 041588

5251 AAAGCCCATT TGCTGAGCCT TGTGGACGTC ATGCAGCGGG AGGGAGGCCC 5301 CAGCCAAATC GGGGATTGCCT TGGGCTTTGC TGTGCGATAC TTGACTTCAG 5351 AAATGCATGG TGCCAGGCCG GGAGCCTCAA AGGCGGTGGT CATCCTGGTC 5401 ACGGACGTCT CTGTGGATTC AGTGGATGCA GCAGCTGATG CCGCCAGGTC 5451 CAACAGAGTG ACAGTGTTCC CTATTGGAAT TGGAGATCGC TACGATGCAG 5501 CCCAGCTACG GATCTTGGCA GGCCCAGCAG GCGACTCCAA CGTGGTGAAG 5551 CTCCAGCGAA TCGAAGACCT CCCTACCATG GTCACCTTGGGCAATTCCTT 5601 CCTCCACAAA CTGTGCTCTG GATTTGTTAG GATTTGCATG GATGAGGATG 5651 GGAATGAGAA GAGGCCCGGG GACGTCTGGA CCTTGCCAGA CCAGTGCCAC 5701 ACCGTGACTT GCCAGCCAGA TGGCCAGACC TTGCTGAAGA GTCATCGGGT 5751 CAACTGTGAC CGGGGGCTGA GGCCTTCGTG CCCTAACAGC CAGTCCCCTG 5801 TTAAAGTGGAAGAGACTGT GGCTGCCGCT GGACCCTGCCC CTGYGTGTGC 5851 ACAGGCAGCT CCACTCGGCA CATCGTGACC TTTGATGGGC AGAATTTCAA 5901 GCTGACTGGC AGCTGTTTCTT ATGTCCTATTT TCAAAACAAGGAGCAGGACC 5951 TGGAGGTGAT TCTCCATAAT GGTGCCTGCA GCCCTGGAGC AAGGCAGGGC 6001 TGCATGAAAT CCATCGAGGT GAAGCACAGT GCCCTCTCCG TCGAGSTGCA 6051 CAGTGACATG GAGGTGACGG TGAATGGGAG ACTGGTCTCGTTCCTTTACG 6101 TGGGTGGGAA CATGGAAGTC AACGTTTATG GTGCCATCAT GCATGAGGTC 6151 AGATTCAATCACCTTGGTCACATCTTCACA TTCACTCCAC AAAACAATGA 6201 GTTCCAACTG CAGCTCAGCC CCAAGACTTT TGCTTCAAAG ACGTATGGTC 6251 TGTGTGGGAT CTGTGATGAG AACGGAGCCA ATGACTTCAT GCTGAGGGAT 6301 GGCACAGTCA CCACAGACTG GAAAACACTT GTTCAGGAAT GGACTGTGCA 6351 GCGGCCAGGG CAGACGTGCC AGCCCATCCT GGAGGAGCAG TGTCTTGTCC 6401 CCGACAGCTC CCACTGCCAG GTCCTCCTCT TACCACTGTT TGCTGAATGC 6451 CACAAGGTCC TGGCTCCAGC CACATTCTAT GCCATCTGCC AGCAGGACAG 6501 TTGCCACCAG GAGCAAGTGT GTGAGGTGAT CGCCTCTTAT GCCCACCTCT 6551 GTCGGACCAA CGGGGTCTGC GTTGACTGGA GGACACCTGA TTTCTGTGCT 6601 ATGTCATGCC CACCATCTCT GGTCTACAAC CACTGTGAGCATGGCTGTCC 6651 CCGGCACTGT GATGGCAACG TGAGCTCCTG TGGGGACCAT CCCTCCGAAG 6701 GCTGTTTCTG CCCTCCAGAT AAAGTCATGT TGGAAGGCAG CTGTGTCCCT 6751 GAAGAGGCCT GCACTCAGTG CATTGGTGAG GATGGAGTCC AGCACCAGTT 6801 CCTGGAAGCC TGGGTCCCGG ACCACCAGCC CTGTCAGATC TGCACATGCC

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6851 TCAGCGGGCG GAAGGTCAAC TGCACAACGC AGCCCTGCCC CACGGCCAAA 6901 GCTCCCCACGT GTGGCCTGTG TGAAGTAGCC CGCCTCCGCC AGAATGCAGA 6951 CCAGTGCTGC CCCGAGTATG AGTGTGTGTG TGACCCAGTG AGCTGTGACC 7001 TGCCCCCAGT GCCTCACTGT GAACGTGGCC TCCAGCCCAC ACTGACCACAC 7051 CCTGGCGAGT GCAGACCCAA CTTCACCTGC GCCTGCAGGA AGGAGGAGTG 7101 CAAAAGAGTG TSSSCCCCST CCTGCCCCCC GCACCGTTTG SSCASSSTTS 7151 GGAAGACCCA GTGCTGTGAT GAGTATGAGT GTGCCTGCAA CTGTGTCAAAC 7201 TCCACAGTGA GCTGTCCCCT TGGGTACTTG GCCTCAACCG CCACCAATGA 7251 CTGTGGCTGT ACCACAACCA CCTGCCTTCC CGACAAGGTG TGTGTCCACC 7301 GAAGCACCAT CTACCCTGTG GGCCAGTTCT GGGAGGAGGG CTGCGATGTG 7351 TGCACCTGCA CCGACATGGA GGATGCCGTG ATGGGCCTCC GCGTGGCCCA 7401 GTGCTCCCAG AAGCCCTGTG AGGACAGCTG TCGGTCGGGC TTCACTTACG 7451 TTTCTGCATGA AGGCGAGTGC TGTGGAAGGT GCCTGCCATC TGCCTGTGAG 7501 GTGGTGACTG GCTCACCGCG GGGGGACTCC CAGTCTTTCCT GGAAGAGTGT 7551 CGGCTCCCAG TGGGCCTCCC CGGAGAACCC CTGCCTCATC AATGAGTGTG 7601 TCCGAGTGAA GGAGGAGGTC TTTATASAAS AAAGGAACGT CTCCTGCCCC 7651 CAGCTGGAGG TCCCTGTCTG CCCCTCGGGC TTTCAGCTGA GCTGTAAGAC 7701 CTCAGCGTGC TGCCCAAGCT GTCGCTGTGA GCGCATGGAG GCCTGCATGC 7751 TCAATGGCAC TGTCATTGGG CCCGGGAAGA CTGTGATGAT CGATGTGTGC 7801 ACGACCTGCC GCTGCATGGT GCAGGTGGGG GTCATCTCTG GATTCAAGCT 7851 GGAGTGCAGG AAGACCACCT GCAACCCCTG CCCCCTGGGT TACAAGGAAG 7901 AAAATAACAC AGGTGAATGT TGTGGGAGAT GTTTGCCTAC GGCTTGCACC 7951 ATTCAGCTAA GAGGAGGACA GATCATGACA CTGAAGCGTG ATGAGACGCT 8001 CCAGGATGGC TGTGATACTC ACTTCTGCAA GGTCAATGAG AGAGGAGAGT 8051 ACTTCTGGGA GAAGAGGGTC ACAGGCTGCC CACCCTTTGA TGAACACAAG 8101 TGTCTTGCTG AGGGAGGTAA AATTATGAAA ATTCCAGGCA CCTGCTGTGA 8151 CACATGTGAG GAGCCTGAGT GCAACGACAT CACTGCCAGG CTGCAGTATG 8201 TCAAGGTGGG AAGCTGTAAG TCTGAAGTAG AGGTGGATAT CCACTACTGC 8251 CAGGGCAAAT GTGCCAGCAA AGCCATGTAC TCCATTGACA TCAACGATGT 8301 GCAGGACCAG TGCTCCTGCT GCTCTCCGAC ACGGACGGAG CCCATGCAGG 8351 TGGCCCTGCA CTGCACCAAT GGCTCTGTTG TGTACCATGA GGTTCTCAAT 8401 GCCATGGAGT GCAAATGCTC CCCCAGGAAG TGCAGCAAGT GA

A VWF protein, as used herein, may comprise a D' domain and a VWF D3 domain, wherein the VWF protein binds to FVIII and inhibits binding of endogenous VWF (full length VWF) to FVIII. The VWF protein comprising a D' domain and a D3 domain may further comprise 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, CK domain, one or more fragments thereof, or any combination thereof. In one embodiment, the VWF protein comprises, consists essentially of, or consists of: (1) VWF D' and D3 domains or fragments thereof; (2) VWF D1, D' and D3 domains or fragments thereof; (3) VWF D2, D' and D3 domains or fragments thereof; (4) VWF D1, D2, D' and D3 domains or fragments thereof; or (5) VWF D1, D2, D', D3 or A1 domains or fragments thereof. The VWF protein described herein does not contain a VWF clearance receptor binding site. The VWF protein of the present invention may contain any other sequence associated with or fused to the VWF protein. For example, the VWF protein described herein may further comprise a signal peptide.

In one embodiment, the VWF protein binds to or is associated with the FVIII protein. Through binding to or association with the FVIII protein, the VWF protein of the invention can protect FVIII from protease cleavage and FVIII activation, stabilize the heavy chain and light chain.

- 25 041588 FVIII chain and prevents clearance of FVIII by phagocytic receptors. In another embodiment, the VWF protein binds to or associates with the FVIII protein and blocks or prevents the binding of the FVIII protein to the phospholipid and activated protein C. By preventing or inhibiting the binding of the FVIII protein to endogenous full-length VWF, the VWF protein of the invention reduces the clearance of FVIII by clearance receptors. endogenous VWF and thus increases the half-life of the FVIII protein. The increase in the half-life of the FVIII protein is thus caused by the association of the FVIII protein with the VWF protein with the missing VWF clearance receptor binding site and thereby shielding and/or protecting the FVIII protein from endogenous VWF which contains the VWF clearance receptor binding site. The FVIII protein associated with or protected by the VWF protein may also allow the FVIII protein to be recycled. By eliminating the receptor binding sites of the VWF clearance pathway in the full-length VWF molecule, the FVIII/VWF heterodimers of the invention are protected from the VWF clearance pathway, further increasing the half-life of FVIII.

In one embodiment, the VWF protein of the present invention comprises a D' domain and a VWF D3 domain, wherein the D' domain is at least about 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100 % identical to amino acids 764-866 of SEQ ID NO: 2, and the VWF protein prevents endogenous VWF from binding to FVIII. In another embodiment, the VWF protein comprises a D' domain and a VWF D3 domain, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867- 1240 SEQ ID NO: 2, and the VWF protein prevents endogenous VWF from binding to FVIII. In some embodiments, a VWF protein described herein comprises, consists essentially of, or consists of a D' domain and a VWF D3 domain that are at least 60, 70, 80, 85, 90, 95, 96 , 97, 98, 99, or 100% identical to amino acids 764-1240 of SEQ ID NO: 2, and the VWF protein prevents endogenous VWF from binding to FVIII. In other embodiments, the VWF protein contains, consists essentially of, or consists of at least 60, 70, 80, 85, 90, 95, 96, 97, 98 D1, D2, D', and D3 domains. , 99 or 100% identical to amino acids 23-1240 of SEQ ID NO: 2, and the VWF protein prevents endogenous VWF from binding to FVIII. In still other embodiments, the VWF protein further comprises an operably linked signal peptide. In some embodiments, a VWF protein of the invention consists essentially of or consists of (1) a D'D3 domain, a D1D'D3 domain, a D2D'D3 domain, or a D1D2D'D3 domain, and (2) an additional VWF sequence comprising up to about 10 amino acids (for example, any sequence from amino acid 764-1240 of SEQ ID NO: 2 to amino acid 764-1250 of SEQ ID NO: 2), up to about 15 amino acids (for example, any sequence from amino acid 764-1240 of SEQ ID NO: 2 up to amino acid 764-1255 of SEQ ID NO: 2), up to about 20 amino acids (for example, any sequence from amino acid 764-1240 of SEQ ID NO: 2 to amino acid 764-1260 of SEQ ID NO: 2), up to about 25 amino acids (for example, any sequence from amino acid 764-1240 of SEQ ID NO: 2 to amino acid 764-1265 of SEQ ID NO: 2), or up to about 30 amino acids (e.g., any sequence from amino acid 764-1240 of SEQ ID NO: 2 to amino acid 764-1260 of SEQ ID NO: 2). In a particular embodiment, a VWF protein containing or consisting essentially of a D' domain and a D3 domain is not amino acids 764-1274 of SEQ ID NO: 2 or a full-length mature VWF. In some embodiments, the D1D2 domain is expressed in a trans configuration with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in a cis configuration with the D'D3 domain.

In other embodiments, the VWF protein containing D'D3 domains associated with D1D2 domains further comprises an intracellular processing site, e.g. expression. Non-limiting examples of intracellular processing sites are disclosed elsewhere in this document.

In other embodiments, the VWF protein contains a D' domain and a D3 domain, but does not contain an amino acid sequence selected from (1) amino acids 1241-2813 of SEQ ID NO: 2, (2) amino acids 1270 to amino acids 2813 of SEQ ID NO: 2 , (3) from amino acid 1271 to amino acid 2813 of SEQ ID NO: 2, (4) from amino acid 1272 to amino acid 2813 of SEQ ID NO: 2, (5) from amino acid 1273 to amino acid 2813 of SEQ ID NO: 2, (6) from amino acids 1274 to amino acids 2813 of SEQ ID NO: 2, or any combination thereof.

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

In some embodiments, the VWF protein of the invention contains a D' domain and a D3 domain, but does not contain at least one VWF domain selected from (1) an A1 domain, (2) an A2 domain, (3) an A3 domain, (4) D4 domain, (5) B1 domain, (6) B2 domain, (7) B3 domain, (8) C1 domain, (9) C2 domain, (10) CK domain, (11) CK domain and C2 domain, (12 ) CK domain, C2 domain and C1 domain, (13) to

- 26 041588 CK domain, C2 domain, C1 domain, B3 domain, (14) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, (15) CK domain, C2 domain, C1 domain, B3 domain, B2 domain and B1 domain, (16) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain and D4 domain, (17) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, domain D4 and A3 domain, (18) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain and A2 domain, (19) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain, A2 domain and A1 domain, or (20) any combination thereof.

In other embodiments, the VWF protein contains D'D3 domains and one or more domains or modules. Examples of such domains or modules include, without limitation, the domains and modules disclosed in Zhour et al., Blood, April 6, 2012 Web Publication: DOI 10.1182/blood-2012-01405134. For example, a VWF protein may comprise a D'D3 domain and one or more domains or modules selected from A1 domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL4 module, C1 module, C2 module, C3 module, C4 module, C5 module, C5 module, C6 module, or any combination thereof.

In certain embodiments, the VWF protein of the invention forms a multimer, such as a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or higher order multimers. In other embodiments, the VWF protein is a monomer containing 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 unable to form a disulfide bond or form a dimer or multimer. In another embodiment, the amino acid substitution is within the D' domain and the D3 domain. In a specific embodiment, the VWF protein of the invention contains at least one amino acid substitution at residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. The at least one amino acid substitution can be any amino acids that do not occur naturally in wild-type VWFs. For example, the amino acid substitution may be any amino acid other than cysteine, such as isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine or histidine. In another example, the amino acid substitution contains one or more amino acids that prevent or inhibit the formation of multimers by VWF proteins.

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

In certain embodiments of the invention, the VWF protein suitable for use herein can be further modified to improve its interaction with FVIII, for example, to improve the binding affinity for FVIII. As a non-limiting example, the VWF protein contains a serine residue at residue position corresponding to amino acid 764 of SEQ ID NO: 2 and a lysine residue at residue position corresponding to amino acid 773 of SEQ ID NO: 2. Residues 764 and/or 773 may contribute to affinity binding of VWF proteins to FVIII. In other embodiments of the invention, the VWF protein suitable for use according to the invention may have other modifications, for example, the protein may be pegylated, glycosylated, gekylated, or polysialylated.

P.S.3. heterologous fragment.

The heterologous fragment that can be linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker can be a heterologous polypeptide or a heterologous non-polypeptide fragment. In certain embodiments of the invention, the heterologous fragment is a molecule that increases the half-life, which is known in the art and contains a polypeptide, a non-polypeptide fragment, or a combination of both. A heterologous polypeptide fragment may contain a FVIII protein, an immunoglobulin constant region or a portion thereof, albumin or a fragment thereof, an albumin-binding fragment, transferrin or a fragment thereof, a PAS sequence, a HAP sequence, a derivative or variant thereof, a C-terminal peptide (CTP) of the β-subunit of the chorionic human gonadotropin, or any combination thereof. In some embodiments, the non-polypeptide binding moiety comprises polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combination thereof. In certain embodiments of the invention, one, two, three, or more heterologous moieties may be present, which may all be the same or different molecules.

P.S.3.a Immunoglobulin constant region or part of it.

The constant region of an immunoglobulin consists of domains designated as CH domains (Constant Heavy) (CH1, CH2, etc.). Depending on the isotype, (ie, IgG, IgM, IgA, IgD, or IgE), the constant region may consist of three or four CH domains. The constant regions of some isotypes (eg, IgG) also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

The immunoglobulin constant region or portion thereof for producing the chimeric protein of the present invention can be obtained from a number of different sources. In some embodiments, the immunoglobulin constant region or portion thereof is isolated from human immunoglobulin. It should be understood, however, that an immunoglobulin constant region or portion thereof may be isolated from an immunoglobulin of another mammalian species, including, for example, a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate species (e.g., chimpanzee, macaque). ). In addition, the immunoglobulin constant region, or a portion thereof, can be isolated from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment of the invention, the human IgG1 isotype is used. Various immunoglobulin constant region gene sequences (eg, human constant region gene sequences) are available in the form of publicly available depositories. Constant region domain sequences can be selected to have a particular effector function (or lack a particular effector function), or with a particular modification to reduce immunogenicity. A large number of antibody sequences and antibody-coding genes have been published, and useful Ig constant region sequences (eg, hinge, CH2 and/or CH3 sequences, or portions thereof) can be isolated from such sequences using methods generally recognized in the art. Genetic material obtained using any of the methods described above can then be modified or synthesized to obtain the polypeptides of the present invention. Additionally, it should be understood that the scope of this invention covers alleles, variants and mutations of constant region DNA sequences. Immunoglobulin constant region sequences or portions thereof can be cloned, for example, using polymerase chain reaction (PCR) and primers selected to amplify the domain of interest. To clone an immunoglobulin constant region sequence or portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymphatic cells, reverse transcribed into DNA, and antibody genes can be amplified by CR. PCR amplification methods are described in detail in US Pat. Nos. 4,683,195; 4683202; 4800159; 4965188; and, for example, in PCR Protocols: A Guide to Methods and Applications Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989 Gene 77:51; Horton et al. 1993 Methods Enzymol. 217:270). An immunoglobulin constant region as used herein may include all of the domains and the hinge region, or portions thereof. In one embodiment, the immunoglobulin constant region, or portion thereof, comprises a CH2 domain, a CH3 domain, and a hinge region, i.e. Fc region or FcRn binding partner. As used herein, the term Fc region is defined as the portion of a polypeptide corresponding to the Fc region of native immunoglobulin, i. resulting from the dimeric association of the corresponding Fc domains of its two heavy chains. The native Fc region forms a homodimer with another Fc region. In one embodiment, the Fc region refers to the portion of a single immunoglobulin heavy chain starting at the hinge region just downstream of the upstream papain cleavage site (i.e., residue 216 in IgG, counting 114 as the first residue of the heavy chain constant region) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain contains at least a hinge domain, a CH2 domain, and a CH3 domain. The Fc region of an immunoglobulin constant region, depending on the immunoglobulin isotype, may include CH2, CH3, and CH4 domains, as well as a hinge region. Chimeric proteins containing the immunoglobulin Fc region have several desirable properties for chimeric proteins, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors, such as neonatal Fc receptor (FcRn) (US Pat. Nos. 6,086,875; 6,485,726; The immunoglobulin constant region, or a portion thereof, may be a FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the intestinal cavity, lung airways, nasal surfaces, vaginal surfaces, colonic and rectal surfaces (US Pat. No. 6,485,726). The FcRn binding partner is the part of the immunoglobulin that binds to the FcRn. 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 pH, actively transports IgG transcellularly from the cavity to serum, and then releases IgG at the relatively higher pH found in interstitial fluids . It is expressed in adult epithelial tissue (US Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1), including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), epithelium proximal renal tubules (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358), as well as nasal epithelium, vaginal surfaces, and bile duct surfaces.

FcRn binding partners suitable for use in the present invention encompass molecules that can specifically bind to the FcRn receptor, including whole IgG, IgG Fc fragment, and other fragments that include the entire binding region of the FcRn receptor. The IgG Fc region that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The main site of contact between Fc and FcRn is located near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are on the same Ig heavy chain. FcRn binding partners include whole IgG, an 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. All references to the amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are based on the system of Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

Fc regions or FcRn binding partners associated with FcRn can be efficiently moved across epithelial barriers by FcRn, thereby providing a non-invasive means for systemically administering the desired therapeutic molecule. Additionally, fusion proteins containing an Fc region or an FcRn binding partner are endocytosed by FcRn expressing cells. But instead of serving as markers for degradation, such fusion proteins are released back into circulation, thereby increasing the in vivo half-life of such proteins. In certain embodiments, parts of the immunoglobulin constant region are an Fc region or FcRn binding partner that typically associate, via disulfide bonds and other non-specific interactions, with another Fc region or other FcRn binding partner to form higher order dimers and multimers.

An FcRn binding partner region is a molecule, or portion thereof, that can be specifically bound by the FcRn receptor, followed by active transport by the FcRn Fco region. Specifically bound refers to two molecules forming a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, in contrast to non-specific binding, which generally has low affinity with moderate to high capacity. Typically, binding is considered specific if the affinity constant KA is greater than 106 M ^-1 , or greater than 108 M ^-1 . If necessary, non-specific binding can be reduced without significantly affecting specific binding by changing the binding conditions. Appropriate binding conditions such as molecular concentration, solution ionic strength, temperature, binding time, blocking agent concentration (eg, serum albumin, milk casein), etc., can be optimized by the skilled artisan using conventional techniques.

A variety of 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 by reference in in full.

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

In certain embodiments, the heterologous fragment linked to the VWF protein via a VWF linker or linked to the FVIII protein via an FVIII linker is albumin or a functional fragment thereof. In some embodiments, the albumin fused to the VWF protein is covalently associated with the albumin fused to the FVIII protein. Human serum albumin (HSA or HA), a 609 amino acid protein in its full-length form, is responsible for a significant portion of serum osmotic pressure and also acts as a carrier for endogenous and exogenous ligands. The term albumin, as used herein, includes full length albumin, or a functional fragment, variant, derivative, or analog thereof. Examples of albumin or fragments or variants thereof are disclosed in U.S. Patent Publication No. 2008/0194481 A1, 2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1 or 2008/0153751 A1 or PCT Application Publication No. 2008/033413 A2, 20092 A1 or 2007/021494 A2, which are incorporated herein by reference in their entirety.

P.S.3.s. Albumin binding fragment.

In certain embodiments, the heterologous fragment linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is an albumin-binding fragment that contains an albumin-binding peptide, a bacterial albumin-binding domain, an albumin-binding antibody fragment, or any combination thereof. For example, the albumin-binding protein can be a bacterial albumin-binding protein, an antibody, or an antibody fragment including an antibody domain (see US Pat. No. 6,696,245). The albumin-binding protein, for example, may be a bacterial albumin-binding domain, such as that of streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73-29 041588

83). Other examples of albumin-binding peptides that can be used as a conjugation partner are, for example, peptides having the consensus sequence CysXaa1-Xaa2-Xaa ₃ -Xaa ₄ -Cys, where Xaa1 is Asp, Asn, Ser, Thr or Trp; Xaa2 is Asn, Gln, His, Ile, Leu or Lys; Xaa3 is Ala, Asp, Phe, Trp or Tyr; and Xaa4 is Asp, Gly, Leu, Phe, Ser, or Thr as described in US 2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

II.C.3.d. PAS sequence.

In other embodiments, the heterologous fragment linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is a PAS sequence. In one embodiment, the chimeric molecule comprises the VWF protein described herein fused to a PAS sequence via a VWF linker. In another embodiment, the chimeric molecule of the invention comprises a first strand containing a VWF protein fused to a PAS sequence via a VWF linker and a second strand containing a FVIII protein and an additional optional PAS sequence, wherein the PAS sequence shields or constricts the VWF binding site of the FVIII protein. , thereby inhibiting or preventing the interaction of the FVIII protein with endogenous VWF. The two PAS sequences can be covalently associated with each other.

The PAS sequence, as used herein, means an amino acid sequence containing predominantly alanine and serine residues, or containing predominantly alanine, serine and proline residues, and the amino acid sequence has a random coil conformation under physiological conditions. Accordingly, a PAS sequence is a functional block, amino acid polymer, or cassette of sequences containing, consisting essentially of, or consisting of alanine, serine and proline, which can be used as part of a heterologous fragment in a chimeric protein. That said, the skilled artisan will appreciate that the amino acid polymer can also assume a random coil conformation if residues other than alanine, serine, and proline are added to the PAS sequence as a minor component. The term minor component, as used herein, means that amino acids other than alanine, serine and proline can be added to the PAS sequence up to a certain extent, for example up to about 12%, i. about 12 of the 100 amino acids of the PAS sequence, up to about 10%, i. e. about 10 of the 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 out of 100 amino acids, about 6%, i.e. about 6 out of 100 amino acids, about 5%, i.e. about 5 out of 100 amino acids, about 4%, i.e. about 4 out of 100 amino acids, about 3%, i.e. about 3 out of 100 amino acids, about 2%, i.e. about 2 out of 100 amino acids, about 1%, i.e. about 1 in 100 amino acids. The amino acids other than alanine, serine and proline may be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val.

Under physiological conditions, a stretch of the PAS sequence forms a random coil conformation and thus may mediate increased in vivo and/or in vitro stability of the VWF factor or coagulation activity protein. Since the random coil domain itself does not form a stable structure or function, the biological activity mediated by the VWF protein or the FVIII protein to which it is associated is largely preserved. In other embodiments, the PAS sequences that form the random coil domain are biologically inert, especially with respect to plasma proteolysis, immunogenicity, isoelectric point/electrostatic properties, cell surface receptor binding, or internalization of the invention, but remain biodegradable, allowing clear advantages over synthetic polymers such as PEG.

Non-limiting examples of random coil conformation PAS sequences include amino acid sequences selected from the group consisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 32), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 33), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 34), APSSPSPSAPSSPSPASPS (SEQ ID NO: 35), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 36), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 37), and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 38), or any combination thereof. Additional examples of PAS sequences are known, for example, from US Patent Publication No. 2010/0292130 A1 and PCT Application Publication No. WO 2008/155134 A1.

P.S.3.e. NAR sequence.

In certain embodiments, the heterologous moiety linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is a glycine-rich homoamino acid polymer (HAP). The HAP sequence may comprise a repeating glycine sequence having a length of 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. In one embodiment of the invention, the HAP sequence is capable of increasing

- 30 041588 to calculate the half-life of the fragment fused to or associated with the HAP sequence. Non-limiting examples of a HAP sequence include, without limitation, (Gly)n, (Gly ₄ Ser) _n , or S(Gly ₄ Ser)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, 11, 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 its fragment.

In certain embodiments, the heterologous moiety linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is transferrin or a fragment thereof. Any transferrin may be used to prepare the chimeric molecules of the invention. As an example, wild-type human Tf (Tf) is a 679 amino acid, about 75 kDa (excluding glycosylation) protein with two major domains, N (about 330 amino acids) and C (about 340 amino acids), which apparently formed as a result of gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847, and S95936 (www.ncbi.nlm.nih.gov/), which are all incorporated herein by reference in their entirety. Transferrin contains two domains, the N-domain and the C-domain. The N domain contains two subdomains, the N1 domain and the N2 domain, and the C domain contains two subdomains, the C1 domain and the C2 domain.

In one embodiment, the transferrin moiety of the chimeric molecule includes a spliced transferrin variant. In one example, the spliced transferrin variant may be a human transferrin splicing variant, such as Genbank accession number AAA61140. In another embodiment, the transferrin portion of the chimeric molecule comprises one or more transferrin sequence domains, such as an N domain, a C domain, an N1 domain, an N2 domain, a C1 domain, a C2 domain, or any combination thereof.

II.C.3.g. A polymer such as polyethylene glycol (PEG).

In other embodiments, the heterologous moiety linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is a soluble polymer known in the art, including, but not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran or polyvinyl alcohol. A heterologous moiety, such as a soluble polymer, may be attached to any position in the chimeric molecule.

In certain embodiments, the chimeric molecule comprises a VWF protein linked to a heterologous moiety (eg, Fc region) via a VWF linker, the VWF protein being further linked to a PEG. In another embodiment of the invention, the chimeric molecule contains a VWF protein connected to the Fc region using a VWF linker and a FVIII protein that are associated with each other, and the FVIII protein is connected to PEG.

The invention also provides chemically modified derivatives of the chimeric molecule of the invention, which may provide additional benefits such as increased solubility, stability and circulation time of the polypeptide, or reduced immunogenicity (see US Pat. No. 4,179,337). The chemical moieties for modification may be selected from water-soluble polymers, including, without limitation, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, or polyvinyl alcohol. The 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 chemical moieties attached.

The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycol, in one embodiment of the invention, 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., duration of desired sustained release, effect, if any, on biological activity, ease of handling, degree of antigenicity or lack thereof, and other known effects of polyethylene glycol exposure to the protein or analogue). For example, polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 90000, 10000, 10000. 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20,000, 25000, 30000, 35000, 40000, 45000, 45000, 45000, 45000 , 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000 or 100000 kDa.

In some embodiments of the invention, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in US patent No. 5643575; Morpurgo et al, Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al, Bioconjug. Chem. 10:638-646 (1999), which are all incorporated herein by reference in their entirety.

The number of polyethylene glycol fragments attached to each chimeric molecule (i.e. the degree

- 31 041588 substitutions) may also vary. For example, the pegylated proteins of the invention can be associated with an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. Likewise, the average degree of substitution (may matter) in a range such as 1-3, 2-4, 3-5, 4-6, 57, 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 fragments per protein molecule. Methods for determining the degree of substitution are described, for example, in Delgado et al, Crit. Rev. Them. Drug Carrier Sys. 9:249-304 (1992).

In other embodiments, the FVIII protein used in the invention is conjugated to one or more polymers. The polymer may be water soluble and covalently or non-covalently attached to Factor VIII or other Factor VIII conjugated moieties. Non-limiting examples of the polymer may be polyalkylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyoxazoline, or polyacryloylmorpholine. Additional types of polymer-conjugated FVIII are disclosed in US Pat. No. 7,199,223.

P.S.3.K Hydroxyethyl starch (HES).

In certain embodiments, the heterologous moiety linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is a polymer, such as hydroxyethyl starch (HES) or a derivative thereof.

Hydroxyethyl starch (HES) is a derivative of natural amylopectin and is degraded in the body by alpha-amylase. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95% by weight. HES exhibits beneficial biological properties and is used as a bulk blood substitute and in clinical hemodilution therapy (Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987); and Weidler et al., Arzneim.-Forschung/Drug Res ., 41, 494-498 (1991)).

Amylopectin contains glucose fragments, with alpha-1,4 glycosidic bonds present in the main chain, and alpha-1,6 glycosidic bonds at the branch nodes. The physicochemical properties of this molecule are predominantly determined by the type of glycosidic bonds. Due to single-strand breaks of alpha-1,4-glycosidic bonds, helical structures are formed with about six glucose monomers per turn. Physico-chemical as well as biochemical properties of the polymer can be modified by substitution. The introduction of the hydroxyethyl group can be carried out by alkaline hydroxyethylation. By varying the reaction conditions, one can exploit the different reactivity of the corresponding hydroxyl group in the unsubstituted glucose monomer towards hydroxyethylation. Due to this, a qualified specialist is able, to a limited extent, to influence the nature of the substitution.

HES is predominantly characterized by molecular weight distribution and degree of substitution. The degree of substitution, referred to as DS, refers to molar substitution as known to those skilled in the art. See Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), cited above, especially p. 273. In one embodiment, the hydroxyethyl starch has an average molecular weight (weight average) of 1 to 300 kDa, 2 to 200 kDa, 3 to 100 kDa, or 4 to 70 kDa. The hydroxyethyl starch may further have a molar degree of substitution from 0.1 to 3, preferably from 0.1 to 2, more preferably from 0.1 to 0.9, preferably from 0.1 to 0.8, and a ratio between C2:C6 substitution in the range of values from 2 to 20 with respect to hydroxyethyl groups. A non-limiting example of HES having an average molecular weight of about 130 kDa is HES with a degree of substitution from 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, the HES with an average molecular weight of about 130 kDa is a VOLUVEN® product from Fresenius. VOLUVEN® is an artificial colloid used, for example, for volumetric replacement, used for therapeutic indications in the treatment and prevention of hypovolemia. VOLUVEN has an average molecular weight of 130,000+/-20,000 Da, a molar substitution of 0.4, and a C2:C6 ratio of about 9:1. In other embodiments, the hydroxyethyl starch average molecular weight ranges are, for example, 4 to 70 kDa, or 10 to 70 kDa, or 12 to 70 kDa, or 18 to 70 kDa, or 50 to 70 kDa, or 4 to 50 kDa, or 10 to 50 kDa, or 12 to 50 kDa, or 18 to 50 kDa, or 4 to 18 kDa, or 10 to 18 kDa, or 12 to 18 kDa, or 4 to 12 kDa, or 10 to 12 kDa, or 4 to 10 kDa. In yet other embodiments, the average molecular weight of the hydroxyethyl starch used is in the range of greater than 4 kDa to less than 70 kDa, such as about 10 kDa, or in the range of 9 to 10 kDa, or 10 to 11 kDa, or 9 to 11 kDa or about 12 kDa or in the range of 11 to 12 kDa) or 12 to 13 kDa or 11 to 13 kDa or about 18 kDa or in the range of 17 to 18 kDa or 18 to 19 kDa, or 17 to 19 kDa, or about 30 kDa, or in the range of 29 to 30 or 30 to 31 kDa, or about 50 kDa, or in the range of 49 to 50 kDa, or 50 to 51 kDa, or from 49 to 51 kDa. In certain embodiments, the heterologous fragment may be mixtures of hydroxyethyl starches having different average molecular weights and/or different degrees of substitution and/or different C2:C6 substitution ratios. Thus, it can be used

- 32 041588 c hydroxyethyl starches having different average molecular weights and different degrees of substitution and different ratios of C2:C6 substitution, or having different average molecular weights and different degrees of substitution and the same or approximately the same ratios of C2:C6 substitution, or having different average molecular weights weight and the same or approximately the same degree of substitution and different ratios of C2:C6 substitution, or having the same or approximately the same average molecular weight and different degrees of substitution and different ratios of C2:C6 substitution, or having different average molecular weights and the same or approximately the same degrees of substitution and the same or about the same C2:C6 substitution ratios, or having the same or about the same average molecular weights and different degrees of substitution, and the same or about the same C2:C6 substitution ratios, or having the same or about the same average molecular weight and the same or about one equal degrees of substitution and different ratios of C2:C6 substitution, or having approximately the same average molecular weight and approximately the same degree of substitution and approximately the same ratio of C2:C6 substitution.

ILC3.i. Polysialic acids (PSA).

In certain embodiments, the non-polypeptide heterologous fragment linked to the VWF protein via a VWF linker or to the FVIII protein via an FVIII linker is a polymer, such as polysialic acids (PSAs) or a derivative thereof. Polysialic acids (PSAs) are natural, straight-chain polymers of sialic acid produced by certain bacterial strains and in mammals in certain cells. Roth J., 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 obtained in various degrees of polymerization from n=about 80 or more sialic acid residues down to n=2, by limited acid hydrolysis, or by enzymatic degradation with neuraminidase, or by fractionation of natural bacterially produced forms of the polymer. The composition of different polysialic acids also varies in such a way that there are homopolymeric forms, i.e. alpha-2,8 linked polysialic acid containing capsular polysaccharide of E. coli strain K1 and group B meningococci, which is also present in the embryonic form of the neural cell adhesion molecule (N-CAM). There are also heteropolymeric forms such as the alternating alpha-2,8 alpha-2,9 polysialic acid of E. coli strain K92 and the group C polysaccharides of N. meningitidis. Sialic acid may also be present in alternating copolymers with monomers other than sialic acid, such as the W135 group or the Y group of N. meningitidis. Polysialic acids have important biological functions including avoiding immune and complement system attack by pathogenic bacteria and 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) 11427-11431 , although polysialic acid receptors are not known in mammals. Alpha-2,8-linked polysialic acid of E. coli strain K1 is also known as colominic acid and is used (in various lengths) to illustrate the present invention. Various methods for attaching or conjugating polysialic acids to a polypeptide have been described (for example, see US Pat.

I.S.4. XTEN sequence.

As used herein, the XTEN sequence refers to extended length polypeptides with non-natural, substantially non-repetitive sequences that consist predominantly of small hydrophilic amino acids, with a sequence that has little or no secondary or tertiary structure under physiological conditions. As a partner of the chimeric protein, XTEN can serve as a carrier providing certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when coupled to the VWF protein or FVIII protein of the invention to produce the chimeric protein. Such desirable properties include, without limitation, improved pharmacokinetic parameters and solubility characteristics. As used herein, XTEN specifically excludes antibodies or antibody fragments such as single chain antibodies or light chain or heavy chain Fc fragments.

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

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The XTEN sequence of the invention may comprise one or more sequence motifs 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% sequence identity to the motif, wherein the motif contains, consists essentially of, or consists of 4-6 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.

In some embodiments, the XTEN contains non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90, or about 91, or about 92, or about 93, or about 94, or about 95, or about 96, or about 97, or about 98, or about 99, or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from the same family of motifs selected from Table. 2A to form a family sequence. As used herein, family means that an XTEN has motifs selected from only one of the motif categories in Table 1. 2A; those. XTEN AD, AE, AF, AG, AM, AQ, BC, or BD, and that any other amino acids in XTEN that do not belong to family motifs are chosen to achieve the desired property, such as allowing restriction site incorporation by coding nucleotides, incorporation cleavable sequence, or achieving a better association with FVIII or VWF. In some embodiments of the XTEN families, the XTEN sequence contains a plurality of units of non-overlapping motif sequences of the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD, and the resulting XTEN shows the range of homology values described above. In other embodiments of the invention, XTEN contains a plurality of motif sequence units from two or more motif families from Table. 2A. Such sequences can be selected to achieve the desired physical/chemical characteristics, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repeatability, which are provided by the amino acid composition of the motifs described in more detail below. In the embodiments of the invention described above in this paragraph, the motifs included in the XTEN can be selected and assembled using the methods described herein to obtain an XTEN consisting of from about 36 to about 3000 amino acid residues.

Table 2A. Sequence motifs

XTEN, consisting of 12 amino acids, and a family of motifs

Motif family* Motif Sequence AD GESPGGSSGSES (SEQ W NO: 49) AD GSEGSSGPGESS (SEQ W NO: 50) AD GSSESGSSEGGP (SEQ W NO: 51) AD GSGGEPSESGSS (SEQ W NO: 52) AE, AM GSPAGSPTSTEE (SEQ W NO: 53) AE, AM, AQ GSEPATSGSETP (SEQ W NO: 54) AE, AM, AQ GTSESATPESGP (SEQ W NO: 55) AE, AM, AQ GTSTEPSEGSAP (SEQ W NO: 56) AF, AM GSTSESPSGTAP (SEQ ID NO: 57) AF, AM GTSTPESGSASP (SEQ GO NO: 58) AF, AM GTSPSGESTAP (SEQ GO NO: 59) AF, AM GSTSSTAESPGP (SEQ GO NO: 60) AG, AM GTPGSGTASSSP (SEQ GO NO: 61) AG, AM GSSTPSGATGSP (SEQ GD NO: 62) AG, AM GSSPSASTGTGP (SEQ GO NO: 63) AG, AM GASPGTSSTGSP (SEQ GO NO: 64) AQ GEPAGSPTSTSE (SEQ GO NO: 65) AQ GTGEPSSTPASE (SEQ ID NO: 66) AQ GSGPSTESAPTE (SEQ GO NO: 67) AQ GSETPSGPSETA (SEQ GO NO: 68) AQ GPSETSTSEPGA (SEQ ID NO: 69) AQ GSPSEPTEGTSA (SEQ ID NO: 70) sun GSGASEPTSTEP (SEQ ID NO: 71) sun GSEPATSGTEPS (SEQ GD NO: 72) sun GTSEPSTSEPGA (SEQ ID NO: 73) sun GTSTEPSEPGSA (SEQ ID NO: 74) BD GSTAGSETSTEA (SEQ GO NO: 75) BD GSETATSGSETA (SEQ GO NO: 76) BD GTSESATSESGA (SEQ GO NO: 77) BD GTSTEASEGSAS (SEQ GO NO: 78)

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Denotes individual motif sequences which, when used together with various permutations, result in XTEN family sequences may also be of various lengths for insertion into or association with 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 property or function to be achieved in the fusion protein. Depending on the intended property or function, XTEN may be a short or intermediate length sequence, or a longer sequence that can serve as carriers. In certain embodiments, XTEN includes short segments of about 6 to about 99 amino acid residues, with intermediate lengths of about 100 to about 399 amino acid residues, and even longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. . Thus, an XTEN inserted into or associated with a FVIII or VWF may be about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500 or up to about 3000 amino acid residues. In other embodiments, the XTEN sequences are about 6 to about 50, about 50 to 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 up to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues. The exact length of the XTEN inserted into or associated with the FVIII or VWF may vary without undesirably affecting the activity of the FVIII or VWF. In one embodiment, one or more of the XTENs used herein are 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 sequences in the family XTEN; those. AD, AE, AF, AG, AM, AQ, BC or BD.

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% sequence identical, selected from the group consisting of AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288,

AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504,

AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684,

AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875,

АЕ912, АМ923, АМ1318, ВС864, BD864, АЕ948, АЕ1044, АЕ1140, АЕ1236, АЕ1332, АЕ1428, АЕ1524, АЕ1620, АЕ1716, АЕ1812, АЕ1908, АЕ2004А, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908 and AG2004. See US 2010-0239554 A1. 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 combinations thereof. In a particular embodiment, the XTEN sequence is AE288. Amino acid sequences for certain XTEN sequences according to the invention are given in table. 2B.

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Table 2B. XTEN sequences

XTEN Amino acid sequence AE42 SEQ ID NO: 39 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS AE72 SEQ ID NO: 40 GAP TSESATPESG PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG TSTEPSEGSA PGASS AE144 SEQ ID NO: 41 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESA PESGPGSEPATSGSETPGTSTEPSEGSAP AG144 SEQ ID NO: 42 GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA STGTGPGTPGSGTASSSPGSSTPSGATGSP AE288 SEQ ID NO: 43 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AG288 SEQ ID NO: 44 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS AE576 SEQ ID NO: 45 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG576 SEQ ID NO: 46 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSG ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AE864 SEQ ID NO: 47 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AG864 SEQ ID NO: 48 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTP SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

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In those embodiments of the invention where less than 100% of the amino acids in the XTEN component used consist of 4, 5 or 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E ) and proline (P), or less than 100% of sequences consisting of sequences of motifs from the table. 2A or sequences XTEN table. 2B, and the other amino acid residues of XTEN are selected from any of the other 14 natural L-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 about 99% hydrophilic amino acids. XTEN amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) are either scattered throughout the XTEN sequence, or located within or between sequence motifs, or concentrated in one or more short stretches of the XTEN sequence, for example, to create a linker between XTEN and other components; for example, the VWF protein. Where the XTEN component contains amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), preferably less than about 2% or less about 1% of the amino acids are hydrophobic residues such that the resulting sequences generally have no secondary structure, eg, no more than 2% alpha helices or 2% beta sheets, as determined by the methods disclosed herein. Hydrophobic residues that are less favorable for XTEN construction include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, it is possible to design XTEN sequences that contain less than 5, or less than 4, or less than 3, or less than 2, or less than 1%, or none of the following amino acids: cysteine (to avoid disulfide bonding and oxidation), methionine (in to avoid oxidation), asparagine and glutamine (to avoid deamidation). Thus, in some embodiments, an XTEN containing amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) has a sequence with less than 5 % of residues involved in the formation of alpha helices and beta sheets, when measured by the Chow-Fasman algorithm, and at least 90%, or at least about 95% or more form a random coil, when measured by the GOR algorithm.

In additional embodiments of the invention, the XTEN sequence used in the invention affects the physical or chemical property, for example pharmacokinetic, of the chimeric protein of the present invention. The XTEN sequence used in the present invention may exhibit one or more of the following preferred properties: conformational flexibility, increased water solubility, a high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii. In a particular embodiment, the XTEN sequence associated with the FVIII protein of this invention improves pharmacokinetic properties, such as longer terminal phase half-life or increased area under the curve (AUC), so that the chimeric protein described herein remains in vivo. over a longer period of time compared to wild-type FVIII. In additional embodiments, the XTEN sequence used in this invention improves pharmacokinetic properties, such as a longer terminal phase half-life or increased area under the curve (AUC), so that the FVIII protein remains in vivo for a longer period of time compared to with wild-type FVIII.

Various methods and methods of analysis can be used to determine the physical/chemical properties of proteins containing the XTEN sequence. Such methods include, without limitation, analytical centrifugation, EPR, HPLC ion exchange, HPLC size exclusion chromatography, HPLC reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC ion exchange, HPLC size exclusion chromatography. , IR, NMR, Raman spectroscopy, Refractometry and UV/Visible spectroscopy. Additional methods are disclosed in Amau et al, Prot Expr and Purif 48, 1-13 (2006).

Additional examples of XTEN sequences that can be used in accordance with the present invention are disclosed in US Patent Publication Nos. , or international patent publications No. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, WO 2011028344 A2 or WO2013123457 A1, or international applications No. PCT / US24913 / US24913.

I.S.5. FVIII protein.

FVIII protein, as used herein, means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise indicated. The term FVIII protein includes a functional fragment, variant, analog, or derivative thereof that retains the function of the full-length wild-type factor VIII in the coagulation pathway. The FVIII protein is used interchangeably with a FVIII or FVIII polypeptide (or protein). Examples of FVIII functions include, without limitation, the ability to activate coagulation, the ability to act as a cofactor for factor IX, or the ability to form a tenase complex with factor IX in the presence of Ca ²⁺ and phospholipids, which then converts factor X to the activated form Xa. . The FVIII protein can be human, porcine, canine, rat or mouse FVIII protein. In addition, comparisons between FVIII derived from humans and from other species have identified conserved residues that are likely required for its function (Cameron et al., Thromb. Haemost. 79:317-22 (1998);

US 6251632).

A number of tests are available to evaluate the function of the coagulation system: activated partial thromboplastin time (APTT) test, chromogenic test, rotational thromboelastometry (ROTEM) test, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often Claus method), platelet count, platelet function testing (often PFA-100), TCT, bleeding time, mixing test (is the abnormality corrected if the patient's plasma is mixed with normal plasma), coagulation factor tests, antiphospholipid antibodies, D- dimer, genetic tests (eg, factor V Leiden, prothrombin G20210A mutation), dilute Russell's viper venom time (dRWT), various platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, such as ROTEM®), or euglobulin lysis time (ELT).

The APTT test is a performance indicator that measures the effectiveness of both the intrinsic (also called contact activation pathway) and general coagulation pathways. This test is commonly used to measure the clotting activity of commercially available recombinant blood clotting factors such as FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

The ROTEM analysis provides information on the kinetics of hemostasis in general: clotting time, clot formation, clot stability and lysis. Various parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or a large number of factors that influence these interactions. This analysis can provide a holistic picture of secondary hemostasis. The FVIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified variants. Examples of human FVIII sequences (full length) are shown as subsequences in SEQ ID NO: 16 or 18.

Table 3 Full-length FVIII (FVIII signal peptide underlined;

the heavy chain of FVIII is underlined with a double line; The B-domain is shown in italics; and FVIII light chain are shown in normal font) Signal peptide: (SEQ ID NO: 15) MQIELSTCFFLCLLRFCFS

Mature factor VIII (SEQ ID NO: 16)*

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSWYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQ AEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDDOTSOREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCL TYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTOTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKM HTWGYWRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHROASLEISPITFLTAOTLLMDLGQFLL FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDWRFDDDNSPSFIQIRSVAKKHPKTWVHY IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLL IIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM ERDLASGLIGPLLICYKESVDORGNOIMSDKRNVILFSVFDENRSWYLTENIORFLPNPAGVOLEDPEFQASNIMHS

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INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCH NSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQTVSRHPSTRQKQFNATTIPENDIEKTD PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDM VFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGK KSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTN KTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQ QKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKWVGKG EFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENWLPQIHTVTGTKNFMKNLFLLST RQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQ RSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRS PLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLG TSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANR PGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACE SNHAIAAINEGQ WRETEVWAA0GRFERI,CS0WPVI,ARH0REITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNI MVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVH SGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYI MDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLI GEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPM IIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHY SIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPWNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLY

Table 4. Nucleotide sequence encoding full-length FVIII (SEQ ID NO: 17)*

661 ATG CAAATAGAGC TCTCCACCTG

721 CTTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC

781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG

841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC

901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT

961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA

1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC

1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT

1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC

1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA

1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC

1321 ACAGACCTTG CACAAATTTA TACTACTTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA

1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC

1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATTGCCA

1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT

1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC

1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT

1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG

1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA

1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT

1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA

1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG

1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT

2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT

2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC

2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG

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2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT 2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG 2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA 2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA 2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT 2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC 2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA 2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC 3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC 3061 T GACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA 3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT 3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC 3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT 3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA 3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG 3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC 3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG 3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901 AATGCTT ATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC 3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC 4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT 4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA 4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT 4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT 4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA 4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA 4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA 4741 AAGGATAATT GT GGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC 4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA 5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC 5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC 5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT 5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA 5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA 5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581 AATAGCAGCA ATAAATGA GG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA 5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC 5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG 5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA 5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG 6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA

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6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT

6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC

6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA

6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC

6301 AGGCCTGATT GGACCCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG

6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG

6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA

6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT

6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG

6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA

6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT

6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG

6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC

6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA

6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC

6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC

7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG

7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA

7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT

7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG

7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA

7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG

7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG

7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA

7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA

7561 TCAGTGGACT CTCTTTTTTC AGAAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC

7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA

7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA

7741 GGACCTCTAC * Underlined nucleic acids encode a signal peptide.

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

The human FVIII gene has been 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 312:330337 (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. 4757006). The amino acid sequence of FVIII was determined from cDNA as shown in US Pat. human factor V as described in US Pat.

The porcine FVIII sequence is published in Toole, J. J., et al, Proc. Natl. Acad. sci. USA 83:5939-5942 (1986). Additionally, the complete sequence of porcine cDNA resulting from PCR amplification of the FVIII sequence from a porcine spleen cDNA library has been described in Healey, J. F., et al, Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII having substitutions in all domains, all subunits, and specific amino acid sequences have been disclosed in US Pat. No. 5,364,771 to Lollar and Runge and in WO 93/20093. Recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII and chimeric FVIII with porcine A1 and/or A2 domains replacing the corresponding human domains have been described in WO 94/11503. US Pat. No. 5,859,204 to Lollar, J. S. also discloses porcine cDNA and deduced amino acid sequences. US Pat. No. 6,458,563 discloses a porcine FVIII with a B domain deletion.

US Pat. No. 5,859,204 to Lollar, J. S. describes functional FVIII mutants having reduced antigenicity and reduced immunoreactivity. US Pat. No. 6,376,463 to Lollar, J. S. also describes FVIII mutants having reduced immunoreactivity. US Publication No. 2005/0100990, to Saenko et al., describes functional mutations in the A2 domain of FVIII. In one embodiment, the FVIII protein (or FVIII-related portion of a chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence represented by amino acids 1-1438 of SEQ ID NO: 18 or amino acids 1-2332 of SEQ ID NO: 16 (no signal sequence), wherein FVIII has clotting activity, e.g., activates factor IX as a cofactor to convert factor X to activated factor X. FVIII (or the FVIII portion of the chimeric protein) may be identical to the FVIII amino acid sequence represented by amino acids 1-1438 of SEQ ID NO: 18 or amino acids 1-2332 of SEQ ID NO: 16 (no signal sequence). The FVIII protein may further comprise a signal sequence.

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The B domain of FVIII, as used herein, is the same as the B domain known in the art, defined by the identity of internal amino acid sequences and proteolytic cleavage sites, eg Ser741-Arg1648 residues of full-length human FVIII. Other domains of human FVIII are defined by the following amino acid residues: A1 - Ala1-Arg372 residues; A2 - Ser373-Arg740 residues; A3 residues Ser1690Asn2019; C1 - Lys2020-Asn2172 residues; C2 - Ser2173-Tyr2332 residues. The A3-C1-C2 sequence includes Ser1690-Tyr2332 residues. The remainder of the sequence, residues Glu1649-Arg1689, is commonly referred to as the a3 acid region. The boundary positions of all domains, including B domains, for porcine, murine and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted (factor VIII with deletion of the B domain or BDD of FVIII). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII) having the same sequence as the factor VIII portion of the sequence in Table 1. 5. (BDD FVIII heavy chain underlined with a double line; B-domain in italics; and BDD FVIII light chain in regular type).

Table 5. BDD FVIII (SEQ ID NO: 18)

ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSWYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQ AEVYDTWITL·KNMΆSHPVSL·HAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVL·KENGPMASDPL·CL· TYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTOTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKM HTVNGYVNRLHjPGLIGCHRKYjyYW^ FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDWRFDDDNSPSFIQIRSVAKKHPKTWVHY lAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLL IIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM ERDLASGLIGPLLICYKESVDORGNOIMSDKRNVILFSVFDENRSWYLTENIORFLPNPAGVOLEDPEFQASNIMHS INGYVFDSLOLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCH NaDFRNRGMITU^LKySSCDKNTGDYYEDimLD^^ DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKWFQEFTDG SFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWK VQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENM ERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVF FGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATW SPSKARLHLQGRSNAWRPQWNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV KVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

Table 6. Nucleotide sequence encoding BDD FVIII (SEQ ID NO: 19)*

661 A TGCAAATAGA GCTCTCCACC TGCTTCTTTC

721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC

781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC

841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAAG ACTCTGTTTG

901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC

961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG

1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG

1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG

1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC

1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC

1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT

1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA

- 42 041588

1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC 1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG 1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG 1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221 C AAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061 ATACCAT ATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901 GCATGGGCAG CA ATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG 4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA 4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741 CAAAAGAGTG GCTGCAAG TG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA 4921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041 GCGAGGCACA GGACCTCTAC * подчеркнутые нуклеиновые кислоты кодируют сигнальный пептид.

FVIII с делецией В-домена может иметь полные или частичные делеции, раскрытые в патентах США № 6316226, 6346513, 7041635, 5789203, 6060447, 5595886, 6228620, 5972885, 6048720, 5543502, 5610278, 5171844, 5112950, 4868112 и 6458563. В некоторых embodiments, the FVIII sequence with the B domain deletion of the present invention contains any of the deletions disclosed from column 4, line 4 to column 5, line 28, and in examples 1-5 of US patent No. 6316226 (also in US 6346513). In another embodiment, Factor VIII with a B domain deletion is Factor VIII with a B domain deletion of S743/Q1638 (SQ BDD FVIII) (e.g., Factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g. amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 16, i.e. SEQ ID NO: 18). In some embodiments, the B domain deletion FVIII of the present invention has the deletion disclosed in column 2, lines 26-51, and examples 5-8 of US Pat. In some embodiments, factor VIII with a B domain deletion has the deletion described from column 1, line 25 to column 2, line 40 of US Pat. No. 5,972,885; in column 6, lines 1-22, and example 1 of US patent No. 6048720; in column 2, lines 17-46,

- 43 041588 US patent No. 5543502; from column 4, line 22, to column 5, line 36, US patent No. 5171844; in column 2, lines 55-68, figure 2 and example 1 of US patent No. 5112950; from column 2, line 2, to column 19, line 21, and in table 2 of US patent No. 4868112; from column 2, line 1, to column 3, line 19, from column 3, line 40, to column 4, line 67, from column 7, line 43, to column 8, line 26, and from column 11, line 5, to column 13, line 39, US Pat. No. 7,041,635; or in column 4, lines 25-53, US Pat. No. 6,458,563.

In some embodiments, FVIII with a B domain deletion has a deletion of most of the B domain, but contains amino-terminal B domain sequences that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains, as disclosed in WO 91/ 09122. In some embodiments, the FVIII with the B domain deletion is constructed with a deletion of amino acids 747-1638, i. e. essentially, with a complete deletion of the B-domain. Hoeben R.C., et al. J Biol. Chem. 265 (13): 7318-7323 (1990). Factor VIII with a B domain deletion may also contain a deletion of FVIII amino acids 771-1666 or amino acids 868-1562. Meulien P., et al. ProteinEng. 2(4): 301-6 (1988). Additional B domain deletions that are part of the invention include: deletion of amino acids 982-1562 or 760-1639 (Toole et al, Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797-1562 ( Eaton, et al Biochemistry (1986) 25:8343-8347)), 741-1646 (Kaufman (PCT Publication No. WO 87/04187)), 747-1560 (Sarver, et al, DNA (1987) 6:553 -564)), 741-1648 (Pasek (PCT Application No. 88/00831)), or 816-1598 or 741-1648 (Lagner (Behring Inst. Mitt. (1988) No 82:1625, EP 295597)). In other embodiments, the FVIII BDD comprises a FVIII polypeptide comprising B-domain fragments retaining one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or optionally 943, which correspond to the amino acid sequence of the full-length sequence FVIII. Examples of B domain fragments include 226 amino acids or 163 amino acids of the B domain as disclosed in Miao, H.Z., et al, Blood 103(a): 3412-3419 (2004), Kasuda, A, et al, J. Thromb. haemost. 6: 1352-1359 (2008) and Pipe, S.W., et al., J. Thromb. haemost. 9: 2235-2242 (2011) (i.e., the first 226 amino acids or 163 amino acids of the B domain are retained). In some embodiments, the partial B domain FVIII is FVIII198. FVIII198 is a single chain FVIIIFc-226N6 molecule containing a partial B domain. 226 denotes the N-terminal amino acid 226 of the B domain of FVIII, and N6 denotes the six N-glycosylation sites in the B domain. In still other embodiments, the FVIII BDD further comprises a point mutation at residue position 309 (Phe to Ser) to improve expression of the FVIII BDD protein. See Miao, H.Z., et al., Blood 103(a): 3412-3419 (2004). In still other embodiments, the FVIII BDD comprises a FVIII polypeptide containing a portion of the B domain but lacking one or more furin cleavage sites (eg, Arg1313 and Arg 1648). See Pipe, S.W., et al., J. Thromb. haemost. 9:2235-2242 (2011). Each of the above deletions can be made in any sequence of FVIII.

A FVIII protein suitable for use in the present invention may include FVIII having one or more additional heterologous sequences or chemical or physical modifications therein that do not affect the coagulative activity of FVIII. 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 pairs of amino acid residues in the FVIII protein. Such inserts in the FVIII protein do not affect the clotting activity of FVIII or the function of FVIII. In one embodiment, the insert improves the pharmacokinetic properties of the FVIII protein (eg, half-life). In another embodiment, insertions may be made at more than two, three, four, five, or six sites.

In one embodiment, FVIII is cleaved immediately after arginine at amino acid position 1648 (in full-length factor VIII or SEQ ID NO: 16), amino acid 754 (in factor VIII with a B domain deletion of S743/Q1638 or SEQ ID NO: 16), or the corresponding arginine residue (in other embodiments), resulting in a heavy chain and a light chain. In another embodiment, the FVIII comprises a heavy chain and a light chain that are linked or associated via a metal ion mediated non-covalent bond. In other embodiments, FVIII is a single chain FVIII that has not been cleaved immediately after arginine at amino acid 1648 (in full length FVIII or SEQ ID NO: 16), amino acid 754 (in FVIII with the S743/Q1638 B domain deletion or SEQ ID NO: 18), or the corresponding arginine residue (in other embodiments). Single chain FVIII may contain one or more amino acid substitutions. In one embodiment, the amino acid substitution is at residue 1648, residue 1645, or both, of the full-length mature Factor VIII polypeptide (SEQ ID NO: 16) or residue 754, residue 751, or both, of Factor VIII SQ BDD (SEQ ID NO: 18). The amino acid substitution may be any amino acid other than arginine, such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine or taurine. FVIII can additionally be cleaved by thrombin and then activated as FVIIIa, serving

- 44 041588 cofactor for activated factor IX (FIXa). Activated FIX together with activated FVIII forms the Xase complex and converts factor X to activated factor X (FXa). For activation, FVIII is cleaved by thrombin after three arginine residues, at amino acid positions 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the FVIII sequence with a B domain deletion), resulting in cleavage of FVIIIa, which has 50 kDa A1, 43 kDa A2 chains. and 73 kDa A3-C1C2. In one embodiment, the FVIII protein suitable for use in the present invention is an inactive FVIII. In another embodiment, the FVIII protein is an activated FVIII. A protein having a FVIII polypeptide associated with or associated with a VWF protein may contain a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16 or 18, wherein the sequence has FVIII clotting activity, eg, activates factor IX as a cofactor to convert factor X to activated factor X (FXa).

In some embodiments, the FVIII protein further comprises one or more heterologous fragments fused to the C-terminus or N-terminus of the FVIII protein or inserted between two adjacent amino acids in the FVIII protein. In other embodiments, the heterologous fragments 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 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 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 increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4 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 times, at least about 11 times, or at least about 12 times, compared to wild-type FVIII.

Other exemplary FVIII variants are also disclosed in US Publication No. US2013/0017997 published January 17, 2013, International Publication No. WO 2013/122617 published August 22, 2013, or International Publication No. WO 2014/011819 published January 16, 2014 or International Publication No. WO2013123457 A1, or International Application No. PCT/US2013/049989.

III. Polynucleotides, vectors and methods of preparation.

The invention also provides a polynucleotide encoding the chimeric molecule described herein. Where the VWF protein is connected to the heterologous fragment via a VWF linker and to the FVIII protein and the XTEN sequence in the chimeric protein as a single polypeptide chain, the invention relates to a single polynucleotide encoding a single polypeptide chain. In cases where the chimeric protein contains first and second polypeptide chains, the first polypeptide chain containing the VWF protein, the XTEN sequence and the first heterologous fragment (for example, the first Fc region) attached using the VWF linker, and the second polypeptide chain containing the protein FVIII and a second heterologous fragment (eg, a second Fc region), the polynucleotide may comprise a first nucleotide region and a second nucleotide region. In one embodiment of the invention, the first nucleotide region and the second nucleotide region are in the same polynucleotide. In another embodiment of the invention, the first nucleotide region and the second nucleotide region are in two different polynucleotides (eg, different vectors). In certain embodiments, the invention relates to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, wherein the first nucleotide strand encodes a VWF protein, an XTEN sequence, a VWF linker, and a heterologous fragment of a chimeric protein, and the second nucleotide strand encodes a FVIII protein and a second heterologous fragment. . In some embodiments, the present invention relates to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, wherein the first nucleotide strand encodes a VWF protein and a heterologous fragment of a chimeric protein, and the second nucleotide strand encodes an FVIII protein connected to the second heterologous fragment using an FVIII linker. , wherein at least one XTEN sequence is fused to the chimeric protein. In other embodiments, the invention relates to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, wherein the first nucleotide strand encodes a VWF protein, a VWF linker, and a heterologous fragment of a chimeric protein, and the second nucleotide strand encodes a FVIII protein, a FVIII linker, and a second heterologous fragment. , wherein at least one XTEN sequence is fused to the chimeric protein.

- 45 041588

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

In certain embodiments of the invention, the invention includes a set of polynucleotides comprising a first nucleotide sequence encoding a VWF protein containing a D' domain and a VWF D3 domain fused to a first heterologous fragment using a VWF linker, a second nucleotide sequence encoding an FVIII protein, and a second heterologous fragment, and a third nucleotide sequence encoding a D1 domain and a D2 domain of a VWF, wherein the XTEN sequence is present in the first strand or in the second strand. In this embodiment, the D1 domain and the D2 domain are expressed separately (not associated with the D'D3 domain of the VWF protein) to ensure proper disulfide bond formation and folding of the D'D3 domains. Expression of the D1D2 domain can occur in the cis or trans configuration.

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

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

In one embodiment of the invention, the coding sequence of 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 in such a manner as to allow each constituent nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a manner as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5' sequence of gene expression results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not introduce a frameshift mutation, (2) does not affects the ability of the promoter region to direct transcription of the coding sequence, or (3) does not affect the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence will be operably linked to a nucleic acid coding sequence if the gene expression sequence is capable of causing transcription of that nucleic acid coding sequence such that the resulting transcript is translated into the desired protein or polypeptide.

A gene expression control sequence, as used herein, is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, that promotes efficient transcription and translation of the coding nucleic acid to which it is operably linked. The gene expression control sequence may, for example, be a mammalian promoter or a viral promoter such as a constitutive or inducible promoter. Mammalian constitutive promoters include, without limitation, the promoters of the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Typical examples of viral promoters that function constitutively in eukaryotic cells include, for example, promoters of cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, long terminal repeats (LTR) of the Moloney leukemia virus and other retroviruses, and the herpes simplex virus thymidine kinase promoter. Other constitutive promoters are known to those of ordinary skill in the art. Promoters suitable for use as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

In general, a gene expression control sequence will include, as appropriate, 5'-non-transcribed and 5'-non-translated sequences involved in the initiation of transcription and translation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, and the like. . In particular, such 5' non-transcribed sequences will include a promoter region that includes a promoter sequence to control transcription of the operably linked coding nucleic acid. Gene expression sequences optionally include enhancer sequences or, if necessary, upstream activator sequences.

Viral vectors include, without limitation, the nucleic sequences of the following viruses: a retrovirus such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor factor, and Rous sarcoma virus; adenovirus, adeno-associated virus; viruses of the SV40 type; polyomaviruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; poliomyelitis virus; and an RNA virus such as a retrovirus. Other vectors well known in the art can easily be used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses whose life cycle involves reverse transcription of viral genomic RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are replication-deficient retroviruses (ie, capable of directing the synthesis of desired proteins but unable to produce infectious particles). Such genetically modified retroviral expression vectors are widely used in highly efficient in vivo gene transduction. Standard protocols for the production of replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with a plasmid, producing recombinant retroviruses with a packaging cell line, harvesting viral particles from tissue culture media, and infecting target cells with viral particles) are given in Kriegler, M ., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment of the invention, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated virus can be genetically engineered to be replication-deficient and capable of infecting a wide range of cell types and species. Additionally, it has the advantages of heat resistance and lipid solvent resistance; high frequency of transduction in cells of different genealogy, including hematopoietic cells; and no inhibition of superinfection, thereby allowing for multiple series of transduction. As reported, adeno-associated virus can integrate site-specifically into human cellular DNA, thereby minimizing the possibility of insertional mutagenesis and variability in expression patterns of inserted retroviral infection genes. In addition, wild-type adeno-associated virus infections have been traced in tissue cultures for more than 100 passages in the absence of selection pressure, suggesting that adeno-associated virus genomic integration is a relatively stable phenomenon. The adeno-associated virus can also function extrachromosomally.

Other vectors include plasmid vectors. Plasmid vectors are described in detail in the art 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 the past few years, plasmid vectors have been found to be particularly preferred for delivering genes to cells in vivo due to their inability to replicate within the host and integrate into its genome. Such plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded in the plasmid. Some commonly used plasmids available from commercial vendors include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, part number V87020; pcDNA4/myc-His, part number V86320; and pBudCE4.1, part number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and/or add specific DNA fragments.

In one insect-based expression system that can be used to produce proteins of the invention, Autographa californica polyhidrosis virus (AcNPV) is used as a vector for expression of foreign genes. The virus is grown in Spodoptera frugiperda cells.

- 47 041588

The coding sequence can be cloned into non-essential regions (eg polyhedron gene) of the virus and placed under the control of the ACNPV promoter (eg polyhedron promoter). Successful insertion of a coding sequence will result in the inactivation of the polyhedron gene and the production of a non-occluded recombinant virus (ie, a virus lacking the protein coat encoded by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells that express the inserted gene. (See, for example, Smith et al. (1983) J Virol 46:584; US Pat. No. 4,215,051). Additional examples of this expression system can be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publishing. Assoc. & Wiley Interscience.

Another system that can be used to express the proteins of the invention is the glutamine synthetase gene expression system, also referred to as the GS expression system (Lonza Biologies PLC, Berkshire UK). This expression system is described in detail in US Pat. No. 5,981,216.

A number of virus-based expression systems can be used in mammalian host cells. Where an adenovirus is used as the expression vector, the coding sequence may be ligated to an adenovirus transcription/translation control complex, such as a late promoter and a tri-leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, the 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.

To improve production efficiency, polynucleotides can be designed to encode multiple protein units of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (eg, by treatment with a suitable enzyme) to isolate the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in suitable viral expression systems, translational control of each mRNA-encoded polypeptide occurs within the transcript; for example, an internal ribosome binding site, IRES. Thus, the polycistronic construct directs the transcription of a single large polycistronic mRNA, which in turn directs the translation of multiple individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and can significantly increase the yield of polypeptides driven by a single promoter.

Vectors used for transformation will typically contain a selectable marker used to identify transformants. In bacterial systems, they may include a gene for resistance to an antibiotic such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include genes conferring drug resistance such as neomycin, hygromycin and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. A review of selectable markers is by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass, and selection of selectable markers is readily available to one of ordinary skill in the art.

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. When using the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, in which case a dicistronic mRNA is produced. Constructs of this type are known in the art (eg US Pat. No. 4,713,339).

Expression vectors can encode labels that allow easy purification of the recombinantly produced protein. Examples include, without limitation, the pUR278 vector (Ruther et al. (1983) EMBO J 2:1791) in which the coding sequences for the protein to be expressed can be ligated into a vector in frame with the lac z coding region such that a labeled fusion protein; pGEX vectors can be used to express the proteins of the invention tagged with glutathione S transferase (GST). Such proteins are usually soluble and can be easily purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (by thrombin or factor Xa protease or PRESCISSION PRONEASE™ (Pharmacia, Peapack, N. J.)) for ease of label removal after purification.

The expression vector or vectors are then transfected or co-transfected into a suitable target cell that expresses the polypeptides. Transfection methods known in the art

- 48 041588 techniques include, without limitation, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841) and the use of liposome-based reagents. Various host-expression vector systems can be used to express the proteins described herein, including both prokaryotic and eukaryotic cells. These include, without limitation, microorganisms such as bacteria (eg, E. coli) transformed with recombinant bacteriophage or plasmid DNA expression vectors containing a suitable coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungal expression vectors containing a suitable coding sequence; insect cell systems infected with recombinant virus (eg, baculovirus) expression vectors containing a suitable coding sequence; plant cell systems infected with recombinant virus expression vectors (eg cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (eg Ti plasmids) containing a suitable coding sequence; or animal cell systems, including mammalian cells (eg, HEK 293, CHO, Cos, HeLa, HKB11 and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell having a defined nucleus. Animal eukaryotic cells include cells from vertebrates, such as mammals, and cells from invertebrates, such as insects. Eukaryotic plant cells may include, in particular, without limitation, yeast cells. A eukaryotic cell is different from a prokaryotic cell such as bacteria. In certain embodiments of the invention, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, without limitation, 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, cat no. 11631-017, or cages 293-F, cat. no. 11625-019, from Invitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cell is a PER.C6 cell, which is a human cell line isolated 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) (eg, CHO-Ki; 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.) (eg 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). In one embodiment, a plasmid encoding a VWF protein, VWF linker, heterologous fragment, or chimeric protein of the invention further includes a selectable marker, such as zeocin resistance, and is transfected into HEK 293 cells to produce the chimeric protein. In still other embodiments of the invention, the transfected cells are stably transfected. Such cells can be selected and maintained as a stable cell line using conventional techniques known to those skilled in the art. Host cells containing the DNA constructs of the protein are grown in a suitable nutrient medium. As used herein, the term suitable growth medium means a medium containing nutrients required for cell growth. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the media may contain one or more selection factors. Optionally, the media may contain calf serum or fetal bovine serum (FCS). In one embodiment, the media are substantially free of IgG. The culture medium is typically selected for cells containing the DNA construct, for example by drug or essential nutrient deficiency supplemented with a selectable marker on the DNA construct, or co-transfected with the DNA construct. Cultured mammalian cells are usually grown in commercially available serum-containing or serum-free media (eg, 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 choice of medium suitable for the particular cell line used is readily available to those of ordinary skill in the art.

- 49 041588

To co-express the two polypeptide chains of a chimeric molecule as described herein, host cells are cultured under conditions that allow both chains to be expressed. As used herein, cultivation refers to maintaining the viability of cells in vitro for at least a certain amount of time. Maintenance may optionally include an increase in the population of living cells. For example, cells maintained in culture may be population static, yet viable and capable of producing the desired product, such as a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include proper selection of culture media, media additives, temperature, pH, oxygen saturation, and the like. For commercial purposes, cultivation may involve the use of any of various types of scaling systems, including shake flasks, roller bottles, hollow fiber bioreactors, stirred tank bioreactors, airlift bioreactors, wave bioreactors, and others.

Cell culture conditions are also chosen to allow association of the first strand and second strand of the chimeric molecule. Conditions to allow expression of the chimeric molecule may include the presence of a vitamin K source. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 medium (Invitrogen, Carlsbad, CA) or OptiCHO medium (Invitrogen, Carlsbad, CA) supplemented with 4 mM glutamine. In one aspect, the present invention relates to a method for expressing, obtaining or producing a chimeric protein, comprising: a) transfecting a host cell with a polynucleotide encoding a chimeric molecule, and b) culturing the host cell in a culture medium under conditions suitable for expressing the chimeric molecule, in which the chimeric molecule is expressed.

In additional embodiments of the invention, the protein product containing the chimeric molecule is secreted into the medium. The medium is separated from the cells, concentrated, filtered and then passed through two or three affinity chromatography columns, such as a protein A column and one or two anion exchange columns.

In certain aspects, the present invention relates to a chimeric polypeptide obtained by the methods described in this document.

In vitro production allows scaling up to produce large amounts of the desired modified polypeptides of the invention. Techniques for cultivating mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift or continuously stirred reactor, or immobilized or retained cell culture, e.g., in hollow fibers, microcapsules, on agarose microbeads. or ceramic cartridges. If necessary and/or desired, polypeptide solutions can be purified by conventional chromatographic techniques, for example gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC), DEAE-cellulose chromatography, or affinity chromatography.

The invention also includes a method for improving the FVIII activity of a chimeric FVIII protein containing a VWF protein fused to a first heterologous fragment and an XTEN sequence and an FVIII protein fused to a second heterologous fragment, comprising inserting a VWF linker between the VWF protein and the first heterologous fragment, wherein the linker The VWF contains a polypeptide selected from: (i) the a2 region of factor VIII (FVIII); (ii) regions a1 of FVIII; (iii) a3 regions from FVIII; (iv) a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 3) and a PAR1 interaction exosite motif, where X is an aliphatic amino acid; or (v) any combination thereof. In some embodiments, FVIII activity is measured by activated partial thromboplastin time (APTT) analysis or by rotational thromboclastometry (ROTEM).

IV. Pharmaceutical composition.

Compositions containing a chimeric molecule of the present invention may contain a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and/or excipients to facilitate the processing of the active compounds into preparations intended for delivery to the site of action.

The pharmaceutical composition may be formulated for parenteral administration (ie intravenous, subcutaneous, or intramuscular) by bolus injection. Compositions for injection may be presented in unit dosage forms, for example in ampoules or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain adjuvants such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in the form of a powder for reconstitution with a suitable vehicle, such as pyrogen-free water.

Suitable compositions for parenteral administration also include aqueous solutions of the active compounds in water-soluble form, for example water-soluble salts. In addition, suspensions of the active compounds may be administered as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil,

- 50 041588 or esters of synthetic fatty acids, such as ethyl oleate or triglycerides. Aqueous suspensions for injection 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 molecules of the invention for delivery to cells or the interstitial space. Typical examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and retarding agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments of the invention, the composition contains isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments of the invention, the compositions contain pharmaceutically acceptable substances, such as wetting agents or small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the active ingredients.

Compositions of the invention may be in various forms, including, for example, liquids (eg solutions for injection and infusion), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the route of administration and therapeutic application.

The composition may be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structures suitable for providing high drug concentrations. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in a suitable diluent with one or a combination of the ingredients listed above, as appropriate, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization to obtain a powder of the active ingredient plus any additional desired ingredient from a pre-sterile filtered solution. Proper fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying agent, such as monostearate salts and gelatin, in the compositions.

The composition of the active ingredient may be formulated using a formulation or sustained release device. Examples of such compositions and devices include implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, for example ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparing such compositions 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.

Depot formulations for injection can be made by forming microencapsulated drug matrices in biodegradable polymers such as polylactide polyglycolide. Depending on the ratio of drug to polymer, and the nature of the polymer used, the rate of drug release can be controlled. Other typical examples of biodegradable polymers are polyorthoesters and polyanhydrides. Depot formulations for injection can also be prepared by entrapping the drug in liposomes or microemulsions.

Additional active compounds may be included in the compositions. In one embodiment, a chimeric molecule of the invention is formulated with another coagulation factor, or variant, fragment, analog, or derivative thereof. For example, clotting factor includes, without limitation, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF), or activated forms of any of the above materials. The hemostatic clotting factor may also include antifibrinolytic drugs, eg epsilon aminocaproic acid, tranexamic acid.

Dosing regimens can be adjusted to provide optimal desired responses. For example, a single bolus may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased depending on the requirements of the therapeutic situation. Compositions for parenteral administration are preferably formulated as unit dosage forms for ease of administration and dosage uniformity. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

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

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, glycerin, propylene glycol, water, ethanol, and the like. The composition may also contain pH buffering agents and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition may be in the form of tablets or capsules prepared by conventional methods. The composition may also be prepared as a liquid, such as a syrup or suspension. The liquid may include suspending agents (eg, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (eg, lecithin or gum arabic), non-aqueous vehicles (eg, almond oil, butyric esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (eg. , methyl or propyl p-hydroxybenzoates or sorbic acid). Formulations may also include flavoring agents, coloring agents and sweeteners.

Alternatively, the composition may be a dry product for reconstitution with water or other suitable vehicle.

For buccal administration, the composition may be in the form of tablets or lozenges according to conventional protocols.

For administration by inhalation, compounds for use in accordance with the present invention are typically delivered as a nebulized aerosol with or without excipients, or as a nebulized aerosol from a pressurized pack or nebulizer, optionally with a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a metering device can be obtained by providing a valve to deliver a metered amount. Capsule and cartridge formulations, for example made from 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. The pharmaceutical composition may 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.

The chimeric molecule of the invention can be produced in vivo in a mammal, such as a patient, (for whom) the use of a gene therapy approach to treat a bleeding disease or disorder selected from a bleeding disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage , muscle hemorrhage, oral hemorrhage, trauma, skull injury, gastrointestinal hemorrhage, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, pharyngeal hemorrhage, retroperitoneal hemorrhage, or hemorrhage into the sheath of the iliopsoas muscle will be therapeutically useful. In one embodiment, the bleeding-related disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This provides for the introduction of a suitable chimeric molecule encoding a nucleic acid operably linked to suitable expression control sequences. In certain embodiments of the invention, such sequences are included in a viral vector. Suitable viral vectors for such gene therapy include adenovirus vectors, lentiviral vectors, baculovirus vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia vectors, herpes simplex virus vectors, and adeno-associated viral (AAV) vectors. The viral vector may be a replication defective viral vector. In other embodiments, the adenoviral vector has a deletion in its E1 gene or E3 gene. Where an adenoviral vector is used, the mammal may not be exposed to the nucleic acid encoding the selectable marker gene. In other embodiments, the sequences are included in a non-viral vector known to those skilled in the art.

VI. Methods of using the chimeric protein.

The present invention further provides a method for reducing the frequency or severity of a bleeding episode in a subject in need thereof using a chimeric molecule of the invention. A typical method includes administering to a subject in need thereof a therapeutically effective amount of a chimeric molecule of the invention. In other aspects, the invention includes

- 52 041588 a method for preventing the occurrence of a bleeding episode in a subject in need thereof, using a chimeric molecule according to the invention. In other aspects, a composition containing DNA encoding a recombinant protein of the invention may be administered to a subject in need thereof. In certain aspects of the invention, a cell expressing a chimeric molecule of the invention may be administered to a subject in need thereof. 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 containing a nucleic acid encoding a chimeric molecule, (iv) a cell containing an isolated nucleic acid encoding a chimeric molecule and/or a vector containing a nucleic acid encoding a chimeric molecule, or (v) a combination thereof, and the pharmaceutical compositions additionally contain an acceptable excipient or carrier. A bleeding episode may be caused by or result from a clotting disorder. A blood clotting disorder can also be called a coagulopathy. In one example, a blood clotting disorder that can be treated with a pharmaceutical composition as described herein is hemophilia or von Willebrand's disease (vWD). In another example, a blood coagulation disorder that can be treated with a pharmaceutical composition according to the present invention is hemophilia A. In some embodiments, the type of bleeding associated with the bleeding condition is selected from hemarthrosis, muscle bleeding, oral bleeding, hemorrhage , hemorrhages in the muscles, hemorrhages in the oral cavity, trauma, damage to the skull, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, bleeding in the central nervous system, bleeding in the pharynx, bleeding in the retroperitoneal space, hemorrhage in sheath of the iliopsoas muscle, or any combination thereof. In other embodiments of the invention, a subject suffering from a bleeding condition is in need of surgical treatment, including, for example, surgical prophylaxis or perioperative medical care. In one example, surgery is selected from minor surgery and major surgery. Typical examples of surgical procedures include tooth extraction, tonsillectomy, inguinal hernia repair, synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, joint replacement surgery (e.g. total knee replacement, hip replacement, etc.). ), cardiac surgery and caesarean section. In another example, the subject is receiving concomitant treatment with factor IX. Because the compounds of the invention are capable of activating FIXa, they can be used to pre-activate a FIXa polypeptide prior to administering FIXa to a subject. The methods of the invention may be practiced on a subject in need of prophylactic or on-demand treatment. Pharmaceutical compositions containing a chimeric molecule of the invention may be formulated for any suitable route of administration, including, for example, topical (eg, transdermal or ocular), oral, buccal, nasal, vaginal, rectal, or parenteral administration. The term parenteral, as used herein, includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial, and intraperitoneal injection, as well as any similar injection or infusion technique. 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 conventional binders and carriers such as triglycerides. After a detailed description of the present invention, it can be better understood with reference to the following examples, given in this document by way of illustration only and not limiting the invention. All patents and publications cited in this document are expressly incorporated herein by reference.

Examples

The following materials and methods were used in the examples, unless otherwise indicated. Materials and methods.

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

Example 1 Evaluation of Thrombin-Mediated D'D3 Release by Various VWF Constructs.

This example evaluates the kinetics of thrombin-mediated D'D3 release at 37°C with various VWF constructs illustrated in FIG. 2. Biocore experiments were carried out with con

- 53 041588 VWF-Fc structures containing different thrombin-cleavable linkers between the D'D3 domain of VWF and Fc. The ultimate goal is to use the information obtained from thrombin hydrolysis of VWF-Fc in relation to the FVIII-VWF heterodimers described herein. All VWF-D'D3 constructs were passed over the chip to achieve protein binding densities in the range of 100-700 RU. After binding the VWF construct to the chip, 5 units/ml of thrombin are injected into the volume on the surface for 5 minutes. The Fc remains bound to the chip while the D'D3 in the cleavable constructs is released. The rate (RU/c) versus capture density (RU) was plotted as illustrated in FIG. 3 and 4. The rate of cleavage is proportional to the initial capture density, and the slope of the curve is a measure of the susceptibility of each construct to cleavage by thrombin.

Fig. 3 illustrates that VWF-052 (having no thrombin cleavage site in the linker region) is not cleaved by thrombin as expected. The rate for VWF-039 (LVPR with PAR1 site) is comparable to the rate of FVIII cleavage (data not shown). Thus, VWF-039 serves as a reference point for the complete release of D'D3 from Fc. Slope ratios for the various VWF-Fc constructs versus VWF-039 were used to determine the efficiency of thrombin digestion. VWF-039 (LVPR with PAR1 site) cleaves thrombin about 70-80 times faster than VWF-031 (LVPR). VWF-51 (ALRPRVV) breaks down 1.8 times faster than VWF-031 (LVPR). VWF-034 containing 288 XTEN near (along) the LVPR site showed slower cleavage compared to VWF-031. VWF-Fc constructs were also generated by introducing different acidic regions (a1, a2 and a3) of the FVIII protein into the linker region. VWF-055, containing an a2 region between D'D3 and the Fc region, exhibits thrombin cleavage similar to construct VWF-039. As illustrated in FIG. 4, VWF-054 (a1 region) and VWF-056 (a3 region) showed about 5 times slower thrombin cleavage.

Fig. 5 illustrates the slopes of the thrombin cleavage curves for different VWF constructs. From these results, the acidic region 2(a2) of FVIII is a highly efficient thrombin cleavage site and was incorporated into FVIII-VWF heterodimers as described herein.

Example 2 Evaluation of the haemostatic capacity of FVIII/VWFD'D3 heterodimers by ROTEM analysis of whole blood from a patient with hemophilia A (HemA).

FVIII/VWFD'D3 heterodimers containing various thrombin-cleavable linkers were evaluated by ROTEM (rotational thromboelastometry) analysis of whole blood from a hemophilia A (HemA) donor for their ability to influence hemostasis. A whole blood sample was taken from a donor with severe bleeding hemophilia A, using sodium citrate as an anticoagulant. 40 min after blood sampling, FVIII/VWFD'D3 heterodimer variants containing different thrombin-cleavable linkers - FVIII155/VWF031 (48 amino acids, LVPR site), FVIII155/VWF039 (26 amino acids, LVPR+PAR1 site), FVIII155/VWF055 ( 34 amino acids, a2 from FVIII) were diluted in a whole blood sample to a final concentration of 100%, 30%, 10% and 3% of normal as measured by FVIII chromogenic assay. Immediately after the addition of the FVIII/VWFD'D3 heterodimers, the ROTEM reaction was started by adding CaCl ₂ . Clotting time (time to reach 2 mm amplitude from the start of the test) was recorded with the instrument and plotted against FVIII concentration in the samples (FIG. 6). It has been hypothesized that the more potent FVIII/VWFD'D3 heterodimer will induce a faster clotting process, thus resulting in shorter clotting times compared to the less potent FVIII/VWFD'D3 heterodimer. As shown in FIG. 6, samples supplemented with the FVIII/VWF039 heterodimer had the shortest clotting time at all concentrations tested, and samples supplemented with the FVIII/VWF031 heterodimer had the longest clotting time at all concentrations. The clotting time of samples with the addition of the FVIII155/VWF055 heterodimer has intermediate values. Thus, hemostatic capacity changes in the order FVIII155/VWF039>FVIII155/VWF055>FVIII155/VWF031. Since the only difference between these three molecules is the thrombin-cleavable linkers between the VWF protein and the Fc region, the results indicate that the linker containing the LVPR site and the PAR1 interaction exosite motif and FVIII a2 region functions better than the LVPR site alone.

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

FVIII-XTEN/VWF heterodimeric constructs were transfected into HEK293F cells using three plasmids: the first expressed FVIII-XTEN-Fc, the second expressed VWF-XTEN-Fc, and the third expressed PACE. For transfection, a standard polyethyleneimine (PEI) protocol was used, and after 5 days of transfection, tissue culture medium was harvested. Various combinations of FVIII-VWF heterodimers were isolated from the media. Purified protein activity was tested using both chromogenic (two-step) and APTT (one-step) coagulation assays using standard protocols. Introduction of acidic region 2(a2) of FVIII either between FVIII and Fc or between D'D3 and Fc (as shown in Table 7A and Figure 7) improves the APTT activity of the FVIII-VWF heterodimer as shown in Table 7A. 7C. For example, the FVIII169/VWF059 heterodimer has an a2 thrombin cleavage site in the D'D3-Fc linker region and has better APTT activity than FVIII169/VWF057,

- 54 041588 which contains the thrombin site LVPR in the linker D'D3Fc, as shown in table. 7C. Similarly, inclusion of the a2 region between FVIII and Fc increases the one-step clotting activity of the heterodimer as seen in the improved ratio of chromogenic and APTT assay results for

FVIII286 / VWF059 and FVIII286 / VWF062, as shown in table. 7B.

Table 7A

Sequence No. construct Linker length between FVIII and Fc (amino acids) Thrombin site in the linker 1 FVIII169 - absent 2 FVIII286 32 FVin-a2 Sequence No. construct Linker length between D'D3 and Fc (amino acids) Thrombin site in the linker 1 VWF057 144AE XTEN+ 35+ LVPR LVPR 2 VWF059 144AE XTEN+ 32 FVin-a2 3 VWF062 144AE XTEN absent

Table 7B

Constructs Chromogenic/APTT ratio FVIII169/VWF057 2.51 FVIII169/VWF059 1.67 FVIII169/VWF062 2.7 FVIII286/VWF059 0.69 FVIII286/VWF062 0.83

Table 7C

Constructs Chromo-specific activity (IU/pmol) APTT-specific activity (IU/pmol) FVIII169/VWF057 1.60 0.65 FVIII169/VWF059 1.60 0.90 FVIII169/VWF062 0.87 0.32 FVIII286/VWF059 1.35 1.96 FVIII286/VWF062 1.08 1.33

Example 4 Acute Efficacy of FVIII-XTEN-Fc/D'D3-XTEN-Fc Heterodimers in a HemA Mice Bleeding Model with Tail Clipping.

The acute efficacy of heterodimers containing different thrombin-cleavable linkers was assessed using a tail-tipped HemA mouse bleeding model.

Male HemA mice aged 8-12 weeks were randomized into 5 experimental groups and received a single intravenous injection of SQ BDD-FVIII, rFVIII169/VWF034, rFVIII169/VWF057, rFVIII169/VWF059, or vehicle solution, respectively. To mimic episodic treatment with FVIII (to restore to 50-100% normal plasma FVIII levels), the selected treatment dose of FVIII was 75 IU/kg as measured by FVIII APTT activity. At this dosage level, all FVIII variants tested will restore approx. 70% of normal plasma FVIII activity in mice 5 minutes post-dose.

The procedure for trimming the tip of the tail was carried out as follows. Briefly, mice were anesthetized with a 50 mg/kg ketamine/0.5 mg/kg dexmedetomidine cocktail prior to tail injury and placed on a heating pad at 37° C. to maintain body temperature. The tails of mice were then immersed in saline heated to 37° C. for 10 minutes to expand the lateral vein. After vein dilatation, FVIII variants or vehicle solution were injected through the tail vein and the tip of the tail was then cut 5 mm from the tip using a No. 11 straight blade scalpel 5 min post dose. The outflowing blood was collected in 13 ml of saline at 37°C for 30 minutes, and the volume of blood loss was determined by the change in the weight of the collected blood tube: volume of blood lost = (tube weight at the end of the determination - initial weight + 0.10) ml. Statistical analysis is carried out using the t-test (Kolmogorov-Smirnov test) and one-way analysis of variance (Kruskal-Wallis test, post hoc analysis: Dunn's test with multiple comparisons). The blood loss volume for each individual animal in the studies was plotted as shown in FIG. 8. Significant reduction in blood loss volume was observed for all FVIII experimental groups compared to vehicle treated animals (p<0.05, Table 8). A similar reduction in blood loss was observed for all experimental heterodimer groups compared with BDD-FVIII administration (p > 0.5, Table 8), suggesting that heterodimer molecules could potentially be as effective as SQ BDD-FVIII in treatment as needed.

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Table 8. β-value for the Kolmogorov-Smirnov test

FVIII169/VWF034 FVIII169/VWF057 FVIII169/VWF059 BDD-FVIII 0.7591 0.9883 0.5176 Carrier 0.0006 0.0006 0.0266

Nucleotide sequence of pSYN VWF057 (D'D3-Fc VWF with LVPR thrombin site in linker) (SEQ ID NO: 79)

1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTTT 51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTACTCTC CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GOTGTATSTA GAAACTGAGG CTGGGTACTA CAAGCGTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTTCCT CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCGC CAGTACCCTGC TGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT C CAT GT CAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGAC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAG CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG

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1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGAG TGCGGGACC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGGCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAAA ACTGTCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CAT GT GT GT G ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GCTTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACTGG AGGACGGCCA CATTGTGCCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG ATATCGGGCG CGCCAACATC AGAGAGCGCC 3751 ACCCCTGAAA GTGGTCCCGG GAGCGAGCCA GCCACATCTG GGTCGGAAAC 3801 GCCAGGCACA AGTGAGTCTG CAACTCCCGA GTCCGGACCT GGCTCCGAGC 3851 CTGCCACTAG CGGCTCCGAG ACTCCGGGAA CTTCCGAGAG CGCTACACCA 3901 GAAAGCGGAC CCGGAACCAG TACCGAACCT AGCGAGGGCT CTGCTCCGGG 3951 CAGCCCAGCC GGCTCTCCCTA CATCCACGGA GGAGGGCACT TCCGAATCCG 4001 CCACCCCGGA GTCAGGGCCA GGATCTGAAC CCGCTACCTC AGGCAGTGAG 4051 ACGCCAGGAA CGAGCGAGTC CGCTACACCG GAGAGTGGGC CAGGGAGCCC 4101 TGCTGGATCT CCTACGTCCA CTGAGGAAGG GTCACCAGCG GGCTCGCCCA 4151 CCAGCACTGA AGAAGGTGCC TCGAGCGGCG GTGGAGGATC CGGTGGCGGG 4201 GGATCCGGTG GCGGGGGATC CGGTGGCGGG GGATCCGGTG GCGGGGGATC 4251 CGGTGGCGGG GGATCCCTGG TCCCCCGGGG CAGCGGAGGC GACAAAACTC 4301 ACACATGCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG ACCGTCAGTC 4351 TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 4401 TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA 4451 AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG 4501 CCGCGGGAGG AGCAGTACAAA CAGCACGTAC CGTGTGGTCA GCGTCCTAC 4551 CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT 4601 CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA 4651 GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 4701 GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAA GGCTTTCTATC 4751 CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAC 4801 TACAAGACCA CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA 4851 CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT 4901 4951 CATGCTCCGT CTCTCCCTGT GATGCATGAG CTCCGGGTAA GCTCTGCACA AT GA ACCACTACAC GCAGAAGAGC

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Protein sequence of pSYN VWF057 (D'D3-Fc VWF with LVPR thrombin site in linker): the underlined region shows the linker region containing thrombin-cleaved LVPR (SEP ID NO: 80)

1 51 MIPARFAGVL YSFAGYCSYL LALALILPGT LAGGCQKRSF LCAEGTRGRS SIIGDFQNGK STARCSLFGS RVSLSVYLGE DFVNTFDGSM FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFGE VNVKRPMKDE 951 THFEWESGR YIILLLGKAL SWWDRHLSI SWLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKWTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP ISGAPTSESA 1251 TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE TPGTSESATP 1301 ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP GSEPATSGSE 1351 TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGA SSGGGGSGGG 1401 GSGGGGSGGG GSGGGGSGGG GSGGGGSLVVP RGSGGDKTHT CPPCPAPELL 1451 GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH 1501 NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 1551 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG 1601 QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 1651 YTQKSLSLSP GK*

Nucleotide sequence of pSYN VWF059 (D'D3-Fc VWF with acid region 2 (a2) thrombin site in linker) (SEQ ID NO: 81)

ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGTCTGG CCCTCATTTT

GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC

101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG

151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA

201 ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC

251 TCTCCGTGTA TCTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT

301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG

351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCGTGTCC GGTGAGGCCT

401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG

451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT

501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC

551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT

601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT

651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT

701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTTGT GGCCCTGTGT

751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC

801 CCTCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG

851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG

901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT

951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG

1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCCTGC

1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG

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1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AG CCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCA AGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG ATATCGGGCG CGCCAACATC AGAGAGCGCC 3751 ACCCCTGAAA GTGGTCCCGG GAGCGAGCCA GCCACATCTG GGTCGGAAAC 3801 GCCAGGCACA AGTGAGTCTG CAACTCCCGA GTCCGGACCT GGCTCCGAGC 3851 CTGCCACTAG CGGCTCCGAG ACTCCGGGAA CTTCCGAGAG CGCTACACCA 3901 GAAAGCGGAC CCGGAACCAG TACCGAACCT AGCGAGGGCT CTGCTCCGGG 3951 CAGCCCAGCC GGCTCTCCTA CATCCACGGA GGAGGGCACT TCCGAATCCG 4001 CCACCCCGGA GTCAGGGCCA GGATCTGAAC CCGCTACCTC AGGCAGTGAG 4051 ACGCCAGGAA CGAGCGAGTC CGCTACACCG GAGAGTGGGC CAGGGAGCCC 4101 TGCTGGATCT CCTACGTCCA CTGAGGAAGG GTCACCAGCG GGCTCGCCCA 4151 CCAGCACTGA AGAAGGTGCC TCGATATCTG ACAAGAACAC TGGTGATTAT 4201 TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA GTAAAAACAA 4251 TGCCATTGAA CCAAGAAGCT TCTCTGACAA AACTCACACA TGCCCACCGT

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4301 GCCCAGCTCC AGAACTCCTG GGCGGACCGT CAGTCTTCCT CTTCCCCCCA

4351 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT

4401 GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG

4451 TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG

4501 TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA

4551 CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC

4601 CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA

4651 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA

4701 GGTCAGCCTG ACTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG

4751 TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT

4801 CCCGTGTTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT

4851 GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC

4901 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG

4951 ggtaaatga

Protein sequence pSYN VWF059 (D'D3-Fc VWF with LVPR thrombin site in linker) - the underlined region shows the a2 region (SEQ ID NO: 82)

1 51 MIPARFAGVL YSFAGYCSYL LALALILPGT LAGGCQKRSF LCAEGTRGRS SIIGDFQNGK STARCSLFGS RVSLSVYLGE DFVNTFDGSM FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFGE VNVKRPMKDE 951 THFEWESGR YIILLLGKAL SWWDRHLSI SWLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKWTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP ISGAPTSESA 1251 TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE TPGTSESATP 1301 ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP GSEPATSGSE 1351 TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGA SISDKNTGDY 1401 YEDSYEDISA YLLSKNNAIE PRSFSDKTHT CPPCPAPELL GGPSVFLFPP 1451 KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 1501 YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE 1551 PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 1601 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP 1651 GK*

Nucleotide sequence of pSYN VWF062 (D'D3-Fc VWF without thrombin site in linker) (SEQ ID NO: 83)

ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGTCTGG CCCTCATTTT

GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC

101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG

151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA

201 ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC

251 TCTCCGTGTA TCTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT

301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG

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351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCGTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTTCCT CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCC TTGCGCCAGG ACTGTCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCGC CAGTACCCTGC TGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT CCATT GT CAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGAC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGAGTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGAG TGCGGGACC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGGCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAAA ACTGTCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CAT GT GT GT G ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GCTTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACTGG AGGACGGCCA CATTGTGCCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG

- 61 041588

3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG ATATCGGGCG CGCCAACATC AGAGAGCGCC 3751 ACCCCTGAAA GTGGTCCCGG GAGCGAGCCA GCCACATCTG GGTCGGAAAC 3801 GCCAGGCACA AGTGAGTCTG CAACTCCCGA GTCCGGACCT GGCTCCGAGC 3851 CTGCCACTAG CGGCTCCGAG ACTCCGGGAA CTTCCGAGAG CGCTACACCA 3901 GAAAGCGGAC CCGGAACCAG TACCGAACCT AGCGAGGGCT CTGCTCCGGG 3951 CAGCCCAGCC GGCTCTCCCTA CATCCACGGA GGAGGGCACT TCCGAATCCG 4001 CCACCCCGGA GTCAGGGCCA GGATCTGAAC CCGCTACCTC AGGCAGTGAG 4051 ACGCCAGGAA CGAGCGAGTC CGCTACACCG GAGAGTGGGC CAGGGAGCCC 4101 TGCTGGATCT CCTACGTCCA CTGAGGAAGG GTCACCAGCG GGCTCGCCCA 4151 CCAGCACTGA AGAAGGTGCC TCGAGCGACA AAACTCACAC ATGCCCACCG 4201 TGCCCAGCTC CAGAACTCCT GGGCGGACCG TCAGTCTTCC TCTTCCCCCC 4251 AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG 4301 TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC 4351 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA 4401 GTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG 4451 ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAAA CAAAGCCCTC 4501 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA 4551 ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC 4601 AGGTCAGCCCT GACCTGCCTG GTCAAAAGGCT TCTATCCCAG CGACATCGCC 4651 GTGGAGTGGG AGAGCAATGG GCAGCGGAG AACAACTACA AGACCACGCC 4701 TCCCGTGTTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 4751 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 4801 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 4851 GGGTAAATGA

Protein sequence pSYN VWF062 (D'D3-Fc VWF without thrombin site in linker) (SEQ ID NO: 84)

1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFGE VNVKRPMKDE 951 THFEWESGR YIILLLGKAL SWWDRHLSI SWLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKWTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP ISGAPTSESA 1251 TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE TPGTSESATP 1301 ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP GSEPATSGSE 1351 TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGA SSDKTHTCPP 1401 CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY 1451 VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL 1501 PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA 1551 VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM

- 62 041588

1601 HEALHNHYTQ KSLSLSPGK*

Nucleotide sequence of pSYN FVIII 286 (FVIII-Fc with additional a2 region between FVIII and Fc) (SEQ ID NO: 85)

ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG

CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG

101 ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT

151 CCTAGAGTGC CAAAATCTTT TCCATTCAAC ACTCAGTCG TGTACAAAAA

201 GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC GCTAAGCCAA

251 GGCCACCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301 GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT

351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG

401 ATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT

451 GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC

501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG

551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601 GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT

651 TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT

701 CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG

751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG

801 CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG

851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

901 CGCCAGGCTA GCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC

951 ACTCTTGATG GACCTTTGGAC AGTTTCCTACT GTTTTGTCAT ATCTCTTCCC

1001 ACCAACATGA TGGCATGGAA GCTTATTGTCA AAGTAGACAG CTGTCCAGAG

1051 GAACCCCAAC TACGATGAA ΑΑΑΎΑΆΎΰΑΆ GAAGCGGAAG ACTATGATGA

1101 TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT GATGACAACT

1151 CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT

1201 TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT

1251 AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG

1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAAA AAGTCCGATT TATGGCATAC

1351 ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT

1401 CTTGGGACCCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT

1451 TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT

1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT

1551 GAAGGATTTT CCAATTCTGC CAGGAGAAAT ATTCAAATAT AATGGACAG

1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC

1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT

1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC

1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG

1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC

1851 AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC

1901 ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG

1951 CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT

2001 CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG

2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG

2101 ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG

2151 GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA

2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG

2251 AGTAAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACGGCGCGCC

2301 AGGTACCTCA GAGTCTGCTA CCCCCGAGTC AGGGCCAGGA TCAGAGCCAG

2351 CCACCTCCGG GTCTGAGACA CCCGGGACTT CCGAGAGTGC CACCCCTGAG

2401 TCCGGACCCG GGTCCGAGCC CGCCACTTCC GGCTCCGAAA CTCCCGGCAC

2451 AAGCGAGAGC GCTACCCCAG AGTCAGGACC AGGAACATCT ACAGAGCCCT

2501 CTGAAGGCTC CGCTCCAGGG TCCCCAGCCG GCAGTCCCAC TAGCACCGAG

2551 GAGGGAACCT CTGAAAGCGC CACACCCGAA TCAGGGCCAG GGTCTGAGCC

2601 TGCTACCAGC GGCAGCGAGA CACCAGGCAC CTCTGAGTCC GCCACACCAG

2651 AGTCCGGACC CGGATCTCCC GCTGGGAGCC CCACCTCCAC TGAGGAGGGA

2701 TCTCCTGCTG GCTCTCCAAC ATCTACTGAG GAAGGTACCT CAACCGAGCC

2751 ATCCGAGGGA TCAGCTCCCG GCACCTCAGA GTCGGCAACC CCGGAGTCTG

2801 GACCCGGAAC TTCCGAAAGT GCCACACCAG AGTCCGGTCC CGGGACTTCA

2851 GAATCAGCAA CACCCGAGTC CGGCCCTGGG TCTGAACCCG CCACAAGTGG

- 63 041588

2901 TAGTGAGACA CCAGGATCAG AACCTGCTAC CTCAGGGTCA GAGACACCCG 2951 GATCTCCGGC AGGCTCACCA ACCTCCACTG AGGAGGGCAC CAGCACAGAA 3001 CCAAGCGAGG GCTCCGCACC CGGAACAAGC ACTGAACCCA GTGAGGGTTC 3051 AGCACCCGGC TCTGAGCCGG CCACAAGTGG CAGTGAGACA CCCGGCACTT 3101 CAGAGAGTGC CACCCCCGAG AGTGGCCCAG GCACTAGTAC CGAGCCCTCT 3151 GAAGGCAGTG CGCCAGCCTC GAGCCCACCA GTCTTGAAAC GCCATCAAGC 3201 TGAAATAACT CGTACTACTC TTCAGTCAGA TCAAGAGGAA ATCGATTATG 3251 ATGATACCAT ATCAGTTGAA ATGAAGAAGG AAGATTTTGA CATTTATGAT 3301 GAGGATGAAA ATCAGAGCCC CCGCAGCTTT CAAAAGAAAA CACGACACTA 3351 TTTTATTGCT GCAGTGGAGA GGCTCTGGGA TTATGGGATG AGTAGCTCCC 3401 CACATGTTCT AAGAAACAGG GCTCAGAGTG GCAGTGTCCC TCAGTTCAAG 3451 AAAGTTGTTT TCCAGGAATT TACTGATGGC TCCTTTACTC AGCCCTTATA 3501 CCGTGGAGAA CTAAATGAAC ATTTGGGACT CCTGGGGCCA TATATAAGAG 3551 CAGAAGTTGA AGATAATATC ATGGTAACTT TCAGAAATCA GGCCTCTCGT 3601 CCCTATTCCT TCTATTCTAG CCTTATTTCT TATGAGGAAG ATCAGAGGCA 3651 AGGAGCAGAA CCTAGAAAAA ACTTTGTCAA GCCTAATGAA ACCAAAACTT 3701 ACTTTTGGAA AGTGCAACAT CATATGGCAC CC ACTAAAGA TGAGTTTGAC 3751 TGCAAAGCCT GGGCTTATTT CTCTGATGTT GACCTGGAAA AAGATGTGCA 3801 CTCAGGCCTG ATTGGACCCC TTCTGGTCTG CCACACTAAC ACACTGAACC 3851 CTGCTCATGG GAGACAAGTG ACAGTACAGG AATTTGCTCT GTTTTTCACC 3901 ATCTTTGATG AGACCAAAAG CTGGTACTTC ACTGAAAATA TGGAAAGAAA 3951 CTGCAGGGCT CCCTGCAATA TCCAGATGGA AGATCCCACT TTTAAAGAGA 4001 ATTATCGCTT CCATGCAATC AATGGCTACA TAATGGATAC ACTACCTGGC 4051 TTAGTAATGG CTCAGGATCA AAGGATTCGA TGGTATCTGC TCAGCATGGG 4101 CAGCAATGAA AACATCCATT CTATTCATTT CAGTGGACAT GTGTTCACTG 4151 TACGAAAAAA AGAGGAGTAT AAAATGGCAC TGTACAATCT CTATCCAGGT 4201 GTTTTTGAGA CAGTGGAAAT GTTACCATCC AAAGCTGGAA TTTGGCGGGT 4251 GGAATGCCTT ATTGGCGAGC ATCTACATGC TGGGATGAGC ACACTTTTTC 4301 TGGTGTACAG CAATAAGTGT CAGACTCCCC TGGGAATGGC TTCTGGACAC 4351 ATTAGAGATT TTCAGATTAC AGCTTCAGGA CAATATGGAC AGTGGGCCCC 4401 AAAGCTGGCC AGACTTCATT ATTCCGGATC AATCAATGCC TGGAGCACCA 4451 AGGAGCCCTT TTCTTGGATC AAGGTGGATC TGTTGGCACC AATGATTATT 4501 CACGGCATCA AGACCCAGGG TGCCCGTCAG AAGTTCTCCA GCCTCTACAT 4551 CTCTCAGTTT ATCA TCATGT ATAGTCTTGA TGGGAAGAAG TGGCAGACTT 4601 ATCGAGGAAA TTCCACTGGA ACCTTAATGG TCTTCTTTGG CAATGTGGAT 4651 TCATCTGGGA TAAAACACAA TATTTTTAAC CCTCCAATTA TTGCTCGATA 4701 CATCCGTTTG CACCCAACTC ATTATAGCAT TCGCAGCACT CTTCGCATGG 4751 AGTTGATGGG CTGTGATTTA AATAGTTGCA GCATGCCATT GGGAATGGAG 4801 AGTAAAGCAA TATCAGATGC ACAGATTACT GCTTCATCCT ACTTTACCAA 4851 TATGTTTGCC ACCTGGTCTC CTTCAAAAGC TCGACTTCAC CTCCAAGGGA 4901 GGAGTAATGC CTGGAGACCT CAGGTGAATA ATCCAAAAGA GTGGCTGCAA 4951 GTGGACTTCC AGAAGACAAT GAAAGTCACA GGAGTAACTA CTCAGGGAGT 5001 AAAATCTCTG CTTACCAGCA TGTATGTGAA GGAGTTCCTC ATCTCCAGCA 5051 GTCAAGATGG CCATCAGTGG ACTCTCTTTT TTCAGAATGG CAAAGTAAAG 5101 GTTTTTCAGG GAAATCAAGA CTCCTTCACA CCTGTGGTGA ACTCTCTAGA 5151 CCCACCGTTA CTGACTCGCT ACCTTCGAAT TCACCCCCAG AGTTGGGTGC 5201 ACCAGATTGC CCTGAGGATG GAGGTTCTGG GCTGCGAGGC ACAGGACCTC 5251 TACGACAAGA ACACTGGTGA TTATTACGAG GACAGTTATG AAGATATTTC 5301 AGCATACTTG CTGAGTAAAA ACAATGCCAT TGAACCAAGA AGCTTCTCTG 5351 ACAAAACTCA CACATGCCCA CCGTGCCCAG CTCCAGAACT CCTGGGCGGA5401 CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC 5451 CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC 5501 CTGAGGTCAA GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC 5551 AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG 5601 CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT 5651 GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC 5701 AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC 5751 CCGGGATGAG CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG 5801 GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG 5851 GAGAACAACT ACAAGACCAC GCCTCCCGTG TTGGACTCCG ACGGCTCCTT 5901 CTTCCTCTAC AGCAAGCTCA CCGTGGAGAA GAGCAGGTGG CAGCAGGGGA 5951 ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG 6001 CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGA

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Protein sequence pSYN FVIII 286 (FVIII-Fc with additional a2 region between FVIII and Fc; in bold and underline) (SEQ ID NO: 86)

ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSWYKKTL

FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTWITLKN MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA

201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL

351 TDSEMDWRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG

451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD

501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP

551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG

601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS

651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR

701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGAPGT

751 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE

801 SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT

851 SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE

901 GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE

951 TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP

1001 GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP PVLKRHQAEI

1051 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI

1101 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKWFQEFTD GSFTQPLYRG

1151 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA

1201 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG

1251 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR

1301 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN

1351 ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC

1401 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL

1451 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ

1501 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR

1551 LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF

1601 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS

1651 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPWNSLDPP

1701 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKNTGDYY EDSYEDISAY

1751 LLSKNNAIEP RSFSDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT

01 PEVTCVWDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRWSVL

1851 TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD

1901 ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

1951 YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K*

Nucleotide sequence of FVIII 169 (SEQ ID NO: 87)

ATGCA AATAG AGCTC TCCAC CTGCT TCTTT CTGTG CCTTT TGCGA TTCTG

CTTTA GTGCC ACCAG AAGAT ACTAC CTGGG TGCAG TGGAA CTGTG ATGGG

101 ASTAT ATGCA AAGTG ATCTC GGTGA GCTGC CTGTG GACGC AAGAT TTCCT

151 CCTAG AGTGC CAAAA TCTTT TCCAT TCAAC ACCTC AGTCG TGTAC AAAAA

201 GACTC TGTTT GTAGA ATTCA CGGAT CACCT TTTCA ACATC GCTAA GCCAA

251 GGCCA CCCTG GATGG GTCTG CTAGG TCCTA CCATC CAGGC TGAGG TTTAT

301 GATAC AGTGG TCATT ACACT TAAGA ACATG GCTTC CCATC CTGTC AGTCT

351 TCATG CTGTT GGTGT ATCCT ACTGG AAAGC TTCTG AGGGA GCTGA ATATG

401 ATGAT CAGAC CAGTC AAAGG GAGAA AGAAG ATGAT AAAGT CTTCC CTGGT

451 GGAAG CCATA CATAT GTCTG GCAGG TCCTG AAAGA GAATG GTCCA ATGGC

501 CTCTG ACCCA CTGTG CCTTA CCTAC TCATA TCTTT CTCAT GTGGA CCTGG

551 TAAAA GACTT GAATT CAGGC CTCAT TGGAG CCCTA CTAGT ATGTA GAGAA

601 GGGAG TCTGG CCAAG GAAAA GACAC AGACC TTGCA CAAAT TTATA CTACT

651 TTTTG CTGTA TTTGA TGAAG GGAAA AGTTG GCACT CAGAA ACAAA GAACT

701 CCTTG ATGCA GGATA GGGAT GCTGC ATCTG CTCGG GCCTG GCCTA AAATG

751 CACAC AGTCA ATGGT TATGT AAACA GGTCT CTGCC AGGTC TGATT GGATG

801 CCACA GGAAA TCAGT CTATT GGCAT GTGAT TGGAA TGGGC ACCAC TCCTG

851 AAGTG CACTC AATAT TCCTC GAAGG TCACA CATTT CTTGT GAGGA ASSAT

- 65 041588

901 CGCCA GGCTA GCTTG GAAAT CTCGC CAATA ACTTT CCTTA CTGCT CAAAC

951 ACTCT TGATG GACCT TGGAC AGTTT CTACT GTTTT GTCAT ATCTC TTCCC

1001 ACCAA CATGA TGGCA TGGAA GCTTA TGTCA AAGTA GACAG CTGTC CAGAG

1051 GAACC CCAAC TACGA ATGAA AAATA ATGAA GAAGC GGAAG ASTAT GATGA

1101 TGATC TTACT GATTC TGAAA TGGAT GTGGT CAGGT TTGAT GATGA CAACT

1151 CTCCT TCCTT TATCC AAATT CGCTC AGTTG CCAAG AAGCA TCCTA AAACT

1201 TGGGT ACATT ACATT GCTGC TGAAG AGGAG GACTG GGACT ATGCT CCCTT

1251 AGTCC TCGCC CCCGA TGACA GAAGT TATAA AAGTC AATAT TTGAA CAATG

1301 GCCCT CAGCG GATTG GTAGG AAGTA CAAAA AAGTC CGATT TATGG CATAC

1351 ACAGA TGAAA CCTTT AAGAC TCGTG AAGCT ATTCA GCATG AATCA GGAAT

1401 CTTGG GACCT TTACT TTATG GGGAA GTTGG AGACA CACTG TTGAT TATAT

1451 TTAAG AATCA AGCAA GCAGA CCATA TAACA TCTAC CCTCA CGGAA TCACT

1501 GATGT CCGTC CTTTG TATTC AAGGA GATTA CCAAA AGGTG TAAAA CATTT

1551 GAAGG ATTTT CCAAT TCTGC CAGGA GAAAT ATTCA AATAT AAATG GACAG

1601 TGACT GTAGA AGATG GGCCA ACTAA ATCAG ATCCT CGGTG CCTGA CCCGC

1651 TATTA CTCTA GTTTC GTTAA TATGG AGAGA GATCT AGCTT CAGGA CTCAT

1701 TGGCC CTCTC CTCAT CTGCT ACAAA GAATC TGTAG ATCAA AGAGG AAACC

1751 AGATA ATGTC AGACA AGAGG AATGT CATCC TGTTT TCTGT ATTTG ATGAG

1801 AACCG AAGCT GGTAC CTCAC AGAGA ATATA CAACG CTTTC TCCCC AATCC

1851 AGCTG GAGTG CAGCT TGAGG ATCCA GAGTT CCAAG CCTCC AACAT CATGC

1901 ACAGC ATCAA TGGCT ATGTT TTTGA TAGTT TGCAG TTGTC AGTTT GTTTG

1951 CATGA GGTGG CATAC TGGTA CATTC TAAGC ATTGG AGCAC AGACT GACTT

2001 CCTTT CTGTC TTCTT CTCTG GATAT ACCTT CAAAC ACAAA ATGGT CTATG

2051 AAGAC ACACT CACCC TATTC CCATT CTCAG GAGAA ACTGT CTTCA TGTCG

2101 ATGGA AAACC CAGGT CTATG GATTC TGGGG TGCCA CAACT CAGAC TTTCG

2151 GAACA GAGGC ATGAC CGCCT TACTG AAGGT TTCTA GTTGT GACAA GAACA

2201 CTGGT GATTA TTACG AGGAC AGTTA TGAAG ATATT TCAGC ATACT TGCTG

2251 AGTAA AAACA ATGCC ATTGA ACCAA GAAGC TTCTC TCAAA ACGGC GCGCC

2301 AGGTA CCTCA GAGTC TGCTA CCCCC GAGTC AGGGC CAGGA TCAGA GCCAG

2351 CCACC TCCGG GCTTG AGACA CCCGG GACTT CCGAG AGTGC CACCC CTGAG

2401 TCCGG ACCCG GGTCC GAGCC CGCCA CTTCC GGCTC CGAAA CTCCC GGCAC

2451 AAGCG AGAGC GCTAC CCCAG AGTCA GGACC AGGAA CATCT ACAGA GCCCT

2501 CTGAA GGCTC CGCTC CAGGG TCCCC AGCCG GCAGT CCCAC TAGCA CCGAG

2551 GAGGG AACCT CTGAA AGCGC CACAC CCGAA TCAGG GCCAG GGTCT GAGCC

2601 TGCTA CCAGC GGCAG CGAGA CACCA GGCAC CTCTG AGTCC GCCAC ACCAG

2651 AGTCC GGACC CGGAT CTCCC GCTGG GAGCC CCACC TCCAC TGAGG AGGGA

2701 TCTCC TGCTG GCTCT CCAAC ATCTA CTGAG GAAGG TACCT CAACC GAGCC

2751 ATCCG AGGGA TCAGC TCCCG GCACC TCAGA GTCGG CAACC CCGGA GTCTG

2801 GACCC GGAAC TTCCG AAAGT GCCAC ACCAG AGTCC GGTCC CGGGA CTTCA

2851 GAATC AGCAA CACCC GAGTC CGGCC CTGGG TCTGA ACCCG CCACA AGTGG

2901 TAGTG AGACA CCAGG ATCAG AACCT GCTAC CTCAG GGTCA GAGAC ACCCG

2951 GATCT CCGGC AGGCT CACCA ACCTC CACTG AGGAG GGCAC CAGCA CAGAA

3001 CCAAG CGAGG GCTCC GCACC CGGAA CAAGC ACTGA ACCCA GTGAG GGTTC

3051 AGCAC CCGGC TCTGA GCCGG CCACA AGTGG CAGTG AGACA CCCGG CACTT

3101 CAGAG AGTGC CACCC CCGAG AGTGG CCCAG GCACT AGTAC CGAGC CCTCT

3151 GAAGG CAGTG CGCCA GCCTC GAGCC CACCA GTCTT GAAAC GCCAT CAAGC

3201 TGAAA TAACT CGTAC TACTC TTCAG TCAGA TCAAG AGGAA ATCGA TTATG

3251 ATGAT ASSAT ATCAG TTGAA ATGAA GAAGG AAGAT TTTGA CATTT ATGAT

3301 GAGGA TGAAA ATCAG AGCCC CCGCA GCTTT CAAAA GAAAA CACGA CACTA

3351 TTTTA TTGCT GCAGT GGAGA GGCTC TGGGA TTATG GGATG AGTAG CTCCC

3401 CACAT GTTCT AAGAA ACAGG GCTCA GAGTG GCAGT GTCCC TCAGT TCAAG

3451 AAAGT TGTTT TCCAG GAATT TACTG ATGGC TCCTT TACTC AGCCC TTATA

3501 CCGTG GAGAA CTAAA TGAAC ATTTG GGACT CCTGG GGCCA TATAT AAGAG

3551 CAGAA GTTGA AGATA ATATC ATGGT AACTT TCAGA AATCA GGCCT CTCGT

3601 CCCTA TTCCT TCTAT TCTAG CCTTA TTTCT TATGA GGAAG ATCAG AGGCA

3651 AGGAG CAGAA CCTAG AAAAA ACTTT GTCAA GCCTA ATGAA ACCAA AACTT

3701 ACTTT TGGAA AGTGC AACAT CATAT GGCAC CCACT AAAGA TGAGT TTGAC

3751 TGCAA AGCCT GGGCT TATTT CTCTG ATGTT GACCT GGAAA AAGAT GTGCA

3801 CTCAG GCCTG ATTGG ACCCC TTCTG GCTTG CCACA CTAAC ACACT GAACC

3851 CTGCT CATGG GAGAC AAGTG ACAGT ACAGG AATTT GCTCT GTTTT TCACC

3901 ATCTT TGATG AGACC AAAAG CTGGT ACTTC ACTGA AAATA TGGAA AGAAA

3951 CTGCA GGGCT CCCTG CAATA TCCAG ATGGA AGATC CCACT TTTAA AGAGA

4001 ATTAT CGCTT CCATG CAATC AATGG STAS TAATG GATAC ACTAC CTGGC

4051 TTAGT AATGG CTCAG GATCA AAGGA TTCGA TGGTA TCTGC TCAGC ATGGG

- 66 041588

4101 CAGCA ATGAA AACAT CCATT CTATT CATTT CAGTG GACAT GTGTT CACTG 4151 TAG GA AAAAA AGAGG AGTAT AAAAT GGCAC TGTAC AATCT CTATC CAGGT 4201 GTTT TGAGA CAGTG GAAAT GT-TAG CATCC AAAGC TGGAA TTTGG CGGGT 4251 GGAAT GCCTT ATTGG CGAGC AT STA CATGC TGGGA TGAGC ACACT TTTTC 4301 TGGTG TACAG CAATA AGTGT CAGAC TCCCC TGGGA ATGGC TTCTG GACAC 4351 AT TAG AGATT TTCAG AT TAG AGCTT CAGGA CAATA TGGAC AGTGG GCCCC 4401 AAAGC TGGCC AGACT TCATT ATTCC GGATC AATCA ATGCC TGGAG SASS 4451 AGGAG CCCTT TTCTT GGATC AAGGT GGATC TGTTG GCACC AATGA TTATT 4501 CACGG CATC A AGACC CAGGG TGCCC GTCAG AAGTT CTCCA GCCTC TACAT 4551 CTCTC AGTTT AT CAT CAT GT ATAGT CTTGA TGGGA AGAAG TGGCA GACTT 4601 PBX GA GGAAA TTCCA CTGGA ACCTT AATGG TCTTC TTTGG CAATG TGGAT 4651 TCATC TGGGA TAAAA CACAA TATTT TTAAC CCTCC AATTA TTGCT CGATA 4701 CATCC GTTTG CACCC AACTC AT TAT AGCAT TCGCA GCACT CTTCG CATGG 4751 AGTTG ATGGG CTGTG ATTTA AATAG TTGCA GCATG CCATT GGGAA TGGAG 4801 AGTAA AGCAA TATCA GATGC ACAGA TTACT GCTTC ATCCT ACTTT ACCAA 4851 TAT GT TTGCC ACCTG GTCTC CTTCA AAAGC TCGAC TTCAC CTCCA AGGGA 4901 GGAGT AATGC CTGGA GACCT CAGGT GAATA AT CCA AAAGA GTGGC TGCAA 4951 GTGGA CTTCC AGAAG ACAAT GAAAG TCACA GGAGT AACTA CTCAG GGAGT 5001 AAAAT CTCTG CTTAC CAGCA TGTAT GTGAA GGAGT TCCTC ATCTC CAGCA 5051 GTCA GATGG CCATC AGTGG ACTCT CTTTT TTCAG AATGG CAAAG TAAAG 5101 GTTT TCAGG GAAAT CAAGA CTCCT TCACA CCTGT GGTGA ACTCT CTAGA 5151 CCCAC CGTTA CTGAC TCGCT ACCTT CGAAT TCACC CCCAG AGTTG GGTGC 5201 ACC AG ATTGC CCTGA GGATG GAGGT TCTGG GCTGC GAGGC ACAGG ACCTC 5251 TAG GA CAAAA CTCAC ACATG CCCAC CGTGC CCAGC TCCAG AACTC CTGGG 5301 CGGAC CGTCA GTCTT CCTCT TCCCC CCAAA AC CCA AGGAC ACCCT CAT GA 5351 TCTCC CGGAC CCCTG AGGTC ACATG CGTGG TGGTG GACGT GAGCC ACGAA 5401 GACCC TGAGG TCAAG TTCAA CTGGT ACGTG GACGG CGTGG AGGTG CATAA 5451 TGCCA AGACA AAGCC GCGGG AGGAG CAGTA CAACA GCACG TACCG TGTGG 5501 TCAGC GTCCT CACCG TCCTG SASS GGACT GGCTG AATGG CAAGG AGTAC 5551 AAGTG CAAGG TCTCC AACAA AGCCC TCCCA GCCCC CATCG AGAAA ASCAT 5601 CTCCA AAGCC AAAGG GCAGC CCCGA GAACC ACAGG TGTAC ACCCT GCCCC 5651 CATCC CGGGA TGAGC TGACC AAGAA CCAGG TCAGC CTGAC CTGCC TGGTC 5701 AAAGG CTTCT ATCCC AGCGA CATCG CCGTG GAGTG GGAGA GCAAT GGGCA 5751 GCCGG AGAAC AACTA CAAGA CCACG CCTCC CGTGT TGGAC TCCGA CGGCT 5801 CCTTC TTCCT STAS GCAAG CTCAC CGTGG ACAAG AGCAG GTGGC AGCAG 5851 5901 GGGAA CACGC CGTCT AGAAG TCTCA AGCCT TGCTC CTCCC CGTGA TGTCT TGCAT CCGGG GAGGC TAAAT TCTGC GA ACAAC CACTA

Protein sequence of FVIII 169 (SEQ ID NO: 88)

101

151

201

251

301

351

401

451

501

551

601

651

701

751

801

851

901

951

1001

1051

MQIELSTCFF PRVPKSFPFN DTWITLKNM GSHTYVWQVL GSLAKEKTQT HTVNGYVNRS RQASLEISPI EPQLRMKNNE WVHYIAAEEE TDETFKTREA DVRPLYSRRL YYSSFVNMER NRSWYLTENI HEVAYWYILS MENPGLWILG SKNNAIEPRS SGPGSEPATS EGTSESATPE SPAGSPGTSPGSA

LCLLRFCFSA TSWYKKTLF ASHPVSLHAV KENGPMASDP LHKFILLFAV LPGLIGCHRK TFLTAQTLLM EAEDYDDDLT DWDYAPLVLA IQHESGILGP PKGVKHLKDF DLASGLIGPL QRFLPNPAGV IGAQTDFLSV CHNSDFRNRG FSQNGAPGTS GSETPGTSES SGPGSEPATS EGTSTEPSEG SEPATSGSET VAEDLIT

TRRYYLGAVE VEFTDHLFNI GVSYWKASEG LCLTYSYLSH FDEGKSWHSE SVYWHVIGMG DLGQFLLFCH DSEMDWRFD PDDRSYKSQY LLYGEVGDTL PILPGEIFKY LICYKESVDQ QLEDPEFQAS FFSGYTFKHK MTALLKVSSC ESATPESGPG ATPESGPGEE SETPGTSSDPA SAPGTSESAT PGSEPATS

LSWDYMQSDL AKPRPPWMGL AEYDDQTSQR VDLVKDLNSG TKNSLMQDRD TTPEVHSIFL ISSHQHDGME DDNSPSFIQI LNNGPQRIGR LIIFKNQASR KWTVTVEDGP RGNQIMSDKR NIMHSINGYV MVYEDTLTLF DKNTGDYYYED SEPATSGDYYED SEPATSGDYYED TEPSEGSAPG ATPESGPGSPDTSP PETSPGPGTSPE ID

GELPVDARFP LGPTIQAEVY EKEDDKVFPG LIGALLVCRE AASARAWPKM EGHTFLVRNH AYVKVDSCPE RSVAKKHPKT KYKKVRFMAY PYNIYPHGIT TKSDPRCLTR NVILFSVFDE FDSLQLSVCL PFSGETVFMS SYEDISAYLL PGTSESATPE SPAGSPTSTE AGDISPTSTEEG ATPESGTSPGTS SGTEMKTEDFTSKE

- 67 041588

1101 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1151 KWFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1201 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1251 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1301 IFDEKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1351 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1401 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1451 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1501 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1551 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME

1601 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1651 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK

1701 VFQGNQDSFT PWNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1751 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE

1801 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY

1851 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

1901 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

1951 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

Nucleotide sequence of VWF034 (SEQ ID NO: 91)

ATGAT TCCTG CCAGA TTTGC CGGGG TGCTG CTTGC TCTGG CCCTC ATTTT

GCCAG GGACC CTTTG TGCAG AAGGA ACTCG CGGCA GGTCA TCCAC GGCCC

101 GATGC AGCCT TTTCG GAAGT GACTT CGTCA ACACC TTTGA TGGGA GCATG

151 TACAG CTTTG CGGGA TACTG CAGTT ACCTC CTGGC AGGGG GCTGC CAGAA

201 ACGCT CCTTC TCGAT TATTG GGGAC TTCCA GAATG GCAAG AGAGT GAGCC

251 TCTCC GTGTA TCTTG GGGAA TTTTT TGACA TCGAT TTGTT TGTCA ATGGT

301 ACCGT GACAC AGGGG GACCA AAGAG TCTCC ATGCC CTATG CCTCC AAAGG

351 GCTGT ATCTA GAAAC TGAGG CTGGG TACTA CAAGC TGTCC GGTGA GGCCT

401 ATGGC TTTGT GGCCA GGATC GATGG CAGCG GCAAC TTTCA AGTCC TGCTG

451 TCAGA CAGAT ACTTC AACAA GACCT GCGGG CTGTG TGGCA ACTTT AACAT

501 CTTTG CTGAA GATGA CTTTA TGACC CAAGA AGGGA CCTTG ACCTC GGACC

551 CTTAT GACTT TGCCA ACTCA TGGGC TCTGA GCAGT GGAGA ACAGT GGTGT

601 GAACG GGCAT CTCCT CCCAG CAGCT CATGC AACAT CTCCT CTGGG GAAAT

651 GCAGA AGGGC CTGTG GGAGC AGTGC CAGCT TCTGA AGAGC ACCTC GGTGT

701 TTGCC CGCTG CCACC CTCTG GTGGA CCCCG AGCCT TTTGT GGCCC TGTGT

751 GAGAA GACTT TGTGT GAGTG TGCTG GGGGG CTGGA GTGCG CCTGC CCTGC

801 CCTCC TGGAG TACGC CCGGA CCTGT GCCCA GGAGG GAATG GTGCT GTACG

851 GCTGG ACCGA CCACA GCGCG TGCAG CCCAG TGTGC CCTGC TGGTA TGGAG

901 TATAG GCAGT GTGTG TCCCC TTGCG CCAGG ACCTG CCAGA GCCTG CACAT

951 CAATG AAATG TGTCA GGAGC GATGC GTGGA TGGCT GCAGC TGCCC TGAGG

1001 GACAG CTCCT GGATG AAGGC CTCTG CGTGG AGAGC ACCGA GTGTC CCTGC

1051 GTGCA TTCCG GAAAG CGCTA CCCTC CCGGC ACCTC CCTCT CTCGA GACTG

1101 CAACA CCTGC ATTTG CCGAA ACAGC CAGTG GATCT GCAGC AATGA AGAAT

1151 GTCCA GGGGA GTGCC TTGTC ACTGG TCAAT CCCAC TTCAA GAGCT TTGAC

1201 AACAG ATACT TCACC TTCAG TGGGA TCTGC CAGTA CCTGC TGGCC CGGGA

1251 TTGCC AGGAC CACTC CTTCT CCATT GTCAT TGAGA CTGTC CAGTG TGCTG

1301 ATGAC CGCGA CGCTG TGTGC ACCCG CTCCG TCACC GTCCG GCTGC CTGGC

1351 CTGCA CAACA GCCTT GTGAA ACTGA AGCAT GGGGC AGGAG TTGCC ATGGA

1401 TGGCC AGGAC ATCCA GCTCC CCCTC CTGAA AGGTG ACCTC CGCAT CCAGC

1451 ATASA GTGAC GGCCT CCGTG CGCCT CAGCT ACGGG GAGGA CCTGC AGATG

1501 GACTG GGATG GCCGC GGGAG GCTGC TGGTG AAGCT GTCCC CCGTC TATGC

1551 CGGGA AGACC TGCGG CCTGT GTGGG AATTA CAATG GCAAC CAGGG CGACG

1601 ACTTC CTTAC CCCCT CTGGG CTGGC GGAGC CCCGG GTGGA GGACT TCGGG

1651 AACGC CTGGA AGCTG CACGG GGACT GCCAG GACCT GCAGA AGCAG CACAG

1701 CGATC CCTGC GCCCT CAACC CGCGC ATGAC CAGGT TCTCC GAGGA GGCGT

1751 GCGCG GTCCT GACGT CCCCC ACATT CGAGG CCTGC CATCG TGCCG TCAGC

1801 CCGCT GCCCT ACCTG CGGAA CTGCC GCTAC GACGT GTGCT CCTGC TCGGA

1851 CGGCC GCGAG TGCCT GTGCG GCGCC CTGGC CAGCT ATGCC GCGGC CTGCG

1901 CGGGG AGAGG CGTGC GCGTC GCGTG GCGCG AGCCA GGCCG CTGTG AGCTG

1951 AACTG CCCGA AAGGC CAGGT GTACC TGCAG TGCGG GACCC CCTGC AACCT

2001 GACCT GCCGC TCTCT CTCTT ACCCG GATGA GGAAT GCAAT GAGGC CTGCC

2051 TGGAG GGCTG CTTCT GCCCC CCAGG GCTCT ACATG GATGA GAGGG GGGAC

2101 TGCGT GCCCA AGGCC CAGTG CCCCT GTTAC TATGA CGGTG AGATC TTCCA

- 68 041588

2151 GCCAG AAGAC ATCTT CTCAG АССАТ CACAC CATGT GCTAC TGTGA GGATG 2201 GCTTC ATGCA CTGTA GGATG AGTGG AGTCC CCGGA AGCTT GCTGC CTGAC 2251 GCTGT CCTCA GCAGT CCCCT GTCTC ATCGC AGCAA AAGGA GCCTA TCCTG 2301 TCGGC CCCCC ATGGT CAAGC TGGTG TGTCC CGCTG ACAAC CTGCG GGCTG 2351 AAGGG CTCGA GTGTA CCAAA ACGTG CCAGA АСТАТ GACCT GGAGT GGATG 2401 AGCAT GGGCT GTGTC TCTGG CTGCG TCTGC CCCCC GGGCA TGGTG CGGCA 2451 TGAGA ACAGA TGTGT GGCCC TGGAA AGGTG TCCCT GCTTC CATCA GGGCA 2501 AGGAG TATGC CGCTG GAGAA ACAGT GAAGA TTGGC TGGAA CACTT GTGTC 2551 TGTCG GGACC GGAAG TGGAA CTGCA CAGAC CATGT GTGTG ATGCC ACGTG 2601 CTCGA GGATG GGCAT GGCCC ACTAC CTGAC CTTCG ACGGG CTCAA ATACC 2651 TGTTC CCCGG GGAGT GCCAG TACGT TCTGG TGCAG GATTA CTGCG GCAGT 2701 AACCC TGGGA CCTTT CGGAT CCTAG TGGGG AATAA GGGAT GCAGC CACCC 2751 CTCAG TGAAA TGCAA GAAAC GGGTC АССАТ CCTGG TGGAG GGAGG AGAGA 2801 TTGAG CTGTT TGACG GGGAG GTGAA TGTGA AGAGG CCCAT GAAGG ATGAG 2851 ACTCA CTTTG AGGTG GTGGA GTCTG GCCGG TACAT CATTC TGCTG CTGGG 2901 CAAAG CCCTC TCCGT GG TCT GGGAC CGCCA CCTGA GCATC TCCGT GGTCC 2951 TGAAG CAGAC ATACC AGGAG AAAGT GTGTG GCCTG TGTGG GAATT TTGAT 3001 GGCAT CCAGA ACAAT GACCT САССА GCAGC AACCT CCAAG TGGAG GAAGA 3051 CGCTG TGGAG TTTGG GAACT CCTGG AAAGT GAGCT CGCAG TGTGG TGAGA 3101 CCAGA AAAGT GCCTC TGGAC TCATC CCCTG CCACC TGCCA TAACA ACATC 3151 ATGAA GCAGA CGATG GTGGA TTCCT CCTGT AGAAT CCTTA CCAGT GACGT 3201 CTTCC AGGAC TGCAA CAAGC TGGTG GACCC CGAGC CATAT CTGGA TGTCT 3251 GCATT TACGA CACCT GCTCC TGTGA GTCCA TTGGG GACTG CGCCG CATTC 3301 TGCGA САССА TTGCT GCCTA TGCCC ACGTG TGTGC CCAGC ATGGC AAGGT 3351 GGTGA CCTGG AGGAC GGCCA CATTG TGCCC CCAGA GCTGC GAGGA GAGGA 3401 ATCTC CGGGA GAACG GGTAT GAGGC TGAGT GGCGC TATAA CAGCT GTGCA 3451 CCTGC CTGTC AAGTC ACGTG TCAGC ACCCT GAGCC ACTGG CCTGC CCTGT 3501 GCAGT GTGTG GAGGG CTGCC ATGCC CACTG CCCTC CAGGG AAAAT CCTGG 3551 ATGAG CTTTT GCAGA CCTGC GTTGA CCCTG AAGAC TGTCC AGTGT GTGAG 3601 GTGGC TGGCC GGCGT TTTGC CTCAG GAAAG AAAGT CACCT TGAAT CCCAG 3651 TGACC CTGAG CACTG CCAGA TTTGC CACTG TGATG TTG TC AACCT CACCT 3701 GTGAA GCCTG CCAGG AGCCG ATATC GGGTA CCTCA GAGTC TGCTA CCCCC 3751 GAGTC AGGGC CAGGA TCAGA GCCAG CCACC TCCGG GTCTG AGACA CCCGG 3801 GACTT CCGAG AGTGC CACCC CTGAG TCCGG ACCCG GGTCC GAGCC CGCCA 3851 CTTCC GGCTC CGAAA CTCCC GGCAC AAGCG AGAGC GCTAC CCCAG AGTCA 3901 GGACC AGGAA CATCT ACAGA GCCCT CTGAA GGCTC CGCTC CAGGG TCCCC 3951 AGCCG GCAGT CCCAC TAGCA CCGAG GAGGG AACCT CTGAA AGCGC CACAC 4001 CCGAA TCAGG GCCAG GGTCT GAGCC TGCTA CCAGC GGCAG CGAGA САССА 4051 GGCAC CTCTG AGTCC GCCAC ACCAG AGTCC GGACC CGGAT CTCCC GCTGG 4101 GAGCC CCACC TCCAC TGAGG AGGGA TCTCC TGCTG GCTCT CCAAC ATCTA 4151 CTGAG GAAGG TACCT CAACC GAGCC ATCCG AGGGA TCAGC TCCCG GCACC 4201 TCAGA GTCGG CAACC CCGGA GTCTG GACCC GGAAC TTCCG AAAGT GCCAC 4251 ACCAG AGTCC GGTCC CGGGA CTTCA GAATC AGCAA CACCC GAGTC CGGCC 4301 CTGGG TCTGA ACCCG CCACA AGTGG TAGTG AGACA CCAGG ATCAG AACCT 4351 GCTAC CTCAG GGTCA GAGAC ACCCG GATCT CCGGC AGGCT CACCA ACCTC 4401 CACTG AGGAG GGCAC GAGGA GAGAA CCAAG GGAGG GCTCC GCACC CGGAA 4451 CAAGC ACTGA ACCCA GTGAG GGTTC AGCAC CCGGC TCTGA GCCGG CCACA 4501 AGTGG CAGTG AGACA CCCGG CACTT CAGAG AGTGC CACCC CCGAG AGTGG 4551 CCCAG GCACT AGTAC CGAGC CCTCT GAAGG CAGTG CGCCA GATTC TGGCG 4601 GTGGA GGTTC CGGTG GCGGG GGATC CGGTG GCGGG GGATC CGGTG GCGGG 4651 GGATC CGGTG GCGGG GGATC CCTGG TCCCC CGGGG CAGCG GAGGC GACAA 4701 AACTC ACACA TGCCC ACCGT GCCCA GCTCC AGAAC TCCTG GGCGG ACCGT 4751 CAGTC TTCCT CTTCC CCCCA AAACC CAAGG ACACC CTCAT GATCT CCCGG 4801 ACCCC TGAGG TCACA TGCGT GGTGG TGGAC GTGAG CCACG AAGAC CCTGA 4851 GGTCA AGTTC AACTG GTACG TGGAC GGCGT GGAGG TGCAT AATGC CAAGA 4901 CAAAG CCGCG GGAGG AGCAG TACAA CAGCA CGTAC CGTGT GGTCA GCGTC 4951 CTCAC CGTCC TGCAC CAGGA CTGGC TGAAT GGCAA GGAGT ACAAG TGCAA 5001 GGTCT CCAAC AAAGC CCTCC CAGCC CCCAT CGAGA AAACC ATCTC CAAAG 5051 CCAAA GGGCA GCCCC GAGAA CCACA GGTGT ACACC CTGCC CCCAT CCCGG 5101 GATGA GCTGA CCAAG AACCA GGTCA GCCTG ACCTG CCTGG TCAAA GGCTT 5151 CTATC CCAGC GACAT CGCCG TGGAG TGGGA GAGCA ATGGG CAGCC GGAGA 5201 ACAAC TACAA GACCA CGCCT CCCGT GTTGG ACTCC GACGG CTCCT TCTTC 5251 CTCTA CAGCA AGCTC ACCGT GGACA AGAGC AGGTG GCAGC AGGGG AACGT 5301 CTTCT CATGC TCCGT GATGC ATGAG GCTCT GCACA ACCAC TACAC GCAGA

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Claims (20)

5351 AGAGC CTCTC CCTGT CTCCG GGTAA ATGA Protein sequence VWF034 (SEQ ID NO: 92) 1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFGE VNVKRPMKDE 951 THFEWESGR YIILLLGKAL SWWDRHLSI SWLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKWTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP ISGTSESATP 1251 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES 1301 GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP 1351 GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT 1401 SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP 1451 ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTSTEPS GSAPGSEPAT 1501 SGSETPGTSE SATPESGPGT STEPSEGSAP DIGGGGGSGG GGSLVPRGSG 1551 GDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE 1601 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY 1651 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 1701 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 1751 GNVFSCSVMH EALHNHYTQK SLSLSPGK* The foregoing description of the specific embodiments discloses the general meaning of the invention so fully that outsiders will be able, using the knowledge available to those skilled in the art, to easily modify and/or adapt for various applications such specific embodiments of the invention without undue experimentation, without deviating from the general ideas of the present invention. Thus, such adaptations and modifications are considered to be within the meaning and scope of the equivalents of the disclosed embodiments based on the description and guidance provided herein. It should be understood that the phraseology or terminology in this document is used for purposes of description and not limitation, so that the terminology or phraseology of this application specification should be considered by the skilled artisan in light of the description and guidance. Other embodiments of the invention will be apparent to those skilled in the art from the description of the application and the practice of the invention disclosed herein. It should be understood that the description of the application and the examples are to be considered as illustrative only, and the actual scope and essence of the invention is defined by the following claims. All patents and publications cited in this document are incorporated herein by reference in their entirety. This application claims priority over U.S. Provisional Application No. 61/840872, filed June 28, 2013. The contents of the above application are incorporated herein by reference in their entirety. CLAIM 1. A chimeric molecule capable of binding to the FVIII protein and containing a von Willebrand factor (VWF) protein, a heterologous fragment (H1), an extended recombinant polypeptide sequence (XTEN) and a VWF linker connecting the VWF protein to the heterologous fragment, the VWF protein containing the D domain ' and D3 VWF domain; wherein the VWF linker contains the a2 region of factor VIII (FVIII); wherein the XTEN sequence is linked to a VWF protein, a heterologous fragment (H1), a VWF linker, or any combination thereof. 2. A chimeric molecule according to claim 1, characterized in that the XTEN sequence connects the VWF protein to a VWF linker or the VWF linker to a heterologous fragment. 3. A chimeric molecule according to claim 1 or 2, further comprising a second polypeptide chain that contains the FVIII protein, wherein the first polypeptide chain is a chimeric molecule consisting of a VWF protein, a heterologous fragment (H1), and both chains are associated with each other. 4. Chimeric molecule according to claim 3, characterized in that the FVIII protein additionally contains an additional XTEN sequence. - 70 041588 5. Chimeric molecule according to claim 3 or 4, characterized in that the second polypeptide chain additionally contains a second heterologous fragment (H2). 6. A chimeric molecule capable of binding to the FVIII protein and containing a first polypeptide chain that contains the VWF protein, a heterologous fragment (H1) and a VWF linker connecting the VWF protein and a heterologous fragment (H1), and a second polypeptide chain containing the FVIII protein and the sequence XTEN, wherein the VWF protein contains a D' domain and a VWF D3 domain; moreover, the VWF linker in the first polypeptide chain contains the a2 region of factor VIII (FVIII); wherein the first polypeptide chain and the second polypeptide chain are associated with each other. 7. A chimeric molecule according to claim 6 containing an additional XTEN sequence that is fused to a VWF protein, a heterologous fragment, a VWF linker, or any combination thereof. 8. A chimeric molecule according to claim 6 or 7, containing a second heterologous fragment (H2) linked to the FVIII protein, the XTEN sequence, or both. 9. A chimeric molecule according to any one of claims 1 to 8, characterized in that at least one of the XTEN sequences contains 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, approx. about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. 10. Chimeric molecule according to any one of claims 1 to 9, characterized in that at least one XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144, 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 and SEQ ID NO: 42. 11. Chimeric molecule according to any one of claims 1 to 10, characterized in that the VWF linker contains the specified a2 region, which contains an amino acid sequence of at least about 90, about 95 or 100% identical to the section from Glu720 to Arg740 corresponding to full-length FVIII, and the a2 region is able to be cleaved by thrombin. 12. A chimeric molecule according to claim 11, characterized in that the VWF linker contains an a2 region containing SEQ ID NO: 4. 13. A chimeric molecule according to any one of claims 1 to 12, characterized in that (a) the D' domain of the VWF protein contains an amino acid sequence of at least about 90, about 95, about 96, about 97, about 98, about 99 or 100% identical to amino acids 764-866 of the sequence of SEQ ID NO: 2; (b) the D3 domain of the VWF protein contains an amino acid sequence that is at least about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to amino acids 8671240 of SEQ ID NO: 2; or both (a) and (b). 14. A chimeric molecule according to any one of claims 1-13, characterized in that the VWF protein sequence contains amino acids 764-1240 of SEQ ID NO: 2. 15. Chimeric molecule according to claim 14, characterized in that the VWF protein further comprises a VWF domain selected from the group consisting of a D1 domain, a D2 domain, D1 and D2 domains, an A1 domain, an A2 domain, an A3 domain, a D4 domain, a domain B1, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, and any combination thereof. 16. A chimeric molecule according to any one of claims 1 to 15, characterized in that the VWF protein consists of: (1) VWF D' and D3 domains or fragments thereof; (2) VWF D1, D' and D3 domains or fragments thereof; (3) VWF D2, D' and D3 domains or fragments thereof; (4) VWF D1, D2, D' and D3 domains or fragments thereof; or (5) VWF D1, D2, D', D3 and A1 domains or fragments thereof. 17. A chimeric molecule according to any one of claims 1 to 16, characterized in that the heterologous fragment (H1) contains an immunoglobulin constant region or a portion thereof; the fact that the second heterologous fragment (H2) contains the constant region of the immunoglobulin or its plot; or that H1 and H2 both contain an immunoglobulin constant region. 18. A chimeric molecule according to claim 16, characterized in that the first heterologous fragment (H1) contains an Fc region; in that the second heterologous fragment (H2) contains an Fc region; or that H1 and H2 both contain an Fc region. 19. A chimeric molecule according to any one of claims 5 and 8-18, characterized in that the first heterologous fragment (H1) is an Fc region and the second heterologous fragment (H2) is an Fc region, wherein H1 and H2 are associated with each other. another through a covalent bond. 20. A polynucleotide encoding a chimeric molecule according to any one of claims 1 to 19 or its complementary sequence. -