Thrombin cleavable linker with xten and its uses thereof

Abstract

To provide a chimeric molecule comprising a VWF protein fused to a heterologous moiety via a VWF linker.SOLUTION: The present invention provides an efficient VWF linker that can be cleaved in the presence of thrombin. The chimeric molecule can further comprise a polypeptide chain comprising a FVIII protein and a second heterologous moiety, and a chain comprising a VWF protein and a chain comprising a FVIII protein are associated with each other. The method also comprises a method for using a nucleotide, a vector, a host cell, and a chimeric protein.SELECTED DRAWING: None

Description

[Background technology]

[0001] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding coagulation factor VIII (FVIII) and affects 1-2 of every 10,000 live male births. Graw et al., Nat. Rev. Genet. 6(6):488-501 (2005). Patients with hemophilia A can be treated with infusions of purified or recombinantly produced FVIII. However, all commercially available FVIII products are known to have a half-life of approximately 8-12 hours, necessitating frequent intravenous administration to patients. See Weiner MA and Cairo, MS, Pediatric Hematology Secrets, Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried to extend FVIII half-life. For example, approaches in development to extend the half-life of coagulation factors include PEGylation, glycopegylation, and conjugation to albumin. See Dumont et al., Blood. 119(13):3024-3030 (published online January 13, 2012). However, regardless of the protein engineering used, long-acting FVIII products currently in development have improved half-lives, but half-life has been reported to be limited to only about a 1.5- to 2-fold improvement in preclinical animal models. (Id.) Consistent results have been demonstrated in humans, for example, rFVIIIFc has been reported to improve half-life by up to about 1.7-fold compared to ADVATE® in patients with hemophilia A. (Id.) Thus, the half-life increase, despite being a small improvement, is comparable to other therapies. 1 / 2 This may indicate the presence of a limiting factor. Due to the inconvenience caused by frequent administration and the dosing regimen, there remains a need to develop FVIII products that require less frequent administration, i.e., FVIII products that have a half-life longer than the 1.5-2 half-life limit. [Prior art documents] [Non-patent literature]

[0002] [Non-Patent Document 1] Graw et al., Nat. Rev. Genet. 6(6):488-501(2005) [Non-Patent Document 2] Weiner MAand Cairo, MS, Pediatric Hematology Secrets, Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001 [Non-Patent Document 3] Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8(2008) [Non-Patent Document 4] Dumont et al.,Blood.119(13):3024-3030(January 13, 2012) Summary of the Invention [Means for solving the problem]

[0003] The present invention is directed to a chimeric molecule comprising a von Willebrand factor (VWF) protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein with the heterologous moiety, the VWF linker comprising (i) the a2 region from factor VIII (FVIII), (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof. The chimeric molecule comprises a selected polypeptide, and the XTEN sequence is connected to a VWF protein, a heterologous moiety (H1), a VWF linker, or any combination thereof. In one embodiment, the XTEN sequence connects the VWF protein to the VWF linker, or the VWF linker to the heterologous moiety. In another embodiment, the chimeric molecule further comprises a second polypeptide chain comprising a FVIII protein, and the first and second polypeptide chains associate with each other. In other embodiments, the FVIII protein in the chimeric molecule further comprises an additional XTEN sequence. The additional XTEN sequence can be attached to the N-terminus or C-terminus of the FVIII protein, or can be inserted between two adjacent FVIII amino acids. In still other embodiments, the second polypeptide chain further comprises a second heterologous moiety (H2).

[0004] The present disclosure also includes a chimeric molecule comprising a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting the VWF protein and the heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein the VWF linker in the first polypeptide chain comprises (i) an a2 region from FVIII, (ii) an a1 region from FVIII, (iii) an a3 region from FVIII, (iv) an XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, where X is an aliphatic amino acid, a thrombin cleavage site, or (v) any combination thereof, and the first polypeptide chain and the second polypeptide chain associate with each other. In one embodiment, the XTEN sequence is connected 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 attached to the VWF protein, the heterologous moiety, the VWF linker, or any combination thereof. In another embodiment, the chimeric molecule further comprises a second heterologous moiety (H2). In yet another embodiment, the second heterologous moiety is attached to the FVIII protein, the XTEN sequence, or both.

[0005] In the chimeric molecules of the present disclosure, the XTEN sequence attached to either the VWF protein, the VWF linker, the FVIII protein, or any other component within the chimeric molecule can be 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 650 amino acids, about 660 amino acids, about 670 amino acids, about 680 amino acids, about 690 amino acids, about 700 amino acids, about 710 amino acids, about 720 amino acids, about 730 amino acids, about 740 amino acids, about 750 amino acids, about 760 amino acids, about 770 amino acids, about 780 amino acids, about 790 amino acids, about 800 amino acids, about 810 amino acids, about 820 amino acids, about 830 amino acids, about 840 amino acids, about 850 amino acids, about 860 amino acids, about 870 amino acids, about 880 amino acids, about 890 amino acids, about 900 amino acids, about 910 amino acids, about 920 amino acids, about 930 amino acids, about 940 amino acids, about 950 amino acids, about 960 amino acids, about 970 amino acids, about 980 amino acids, about 990 amino acids, about 1000 amino acids, about 1010 amino acids, about 1020 amino acids, about 1030 In some embodiments, the XTEN polypeptide comprises about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. In some embodiments, the XTEN polypeptide is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. In other embodiments, the XTEN polypeptide is selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:45, SEQ ID NO:44, SEQ ID NO:41, SEQ ID NO:48, SEQ ID NO:46, SEQ ID NO:44, or SEQ ID NO:42.

[0006] In other embodiments, the additional XTEN sequence in the chimeric molecule 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 380 amino acids, about 390 amino acids, about 400 amino acids, about 410 amino acids, about 420 amino acids, about 430 amino acids, about 440 amino acids, about 450 amino acids, about 460 amino acids, about 470 amino acids, about 480 amino acids, about 490 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, about 600 amino acids, about 610 amino acids, about 620 amino acids, about 630 amino acids, about 640 amino acids, about 650 amino acids, about 660 amino acids, about 670 amino acids, about 680 amino acids, about 690 amino acids, about 700 amino acids, about 710 amino acids, about 720 amino acids, about 750 amino acids, about 760 amino acids, about 770 amino acids, about 780 amino acids, about 790 amino acids, about 800 amino acids, about 810 amino acids, about 820 amino acids, about 830 amino acids, about 840 amino acids, about 850 amino acids, about 860 amino acids, about 870 amino acids, about 880 amino acids, about 890 amino acids, about 900 amino acids, about 910 amino acids, about 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 The XTEN polypeptide comprises 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 some 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.

[0007] In one embodiment, a VWF linker useful for connecting a VWF protein and a heterologous moiety in a chimeric molecule comprises an a2 region that comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu720 to Arg740 corresponding to full-length FVIII, the a2 region being capable of being cleaved by thrombin. In a particular embodiment, the a2 region comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4). In another embodiment, a VWF linker useful for connecting a VWF protein and a heterologous moiety comprises an a1 region that comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Met337 to Arg372 corresponding to full-length FVIII, the a1 region being capable of being cleaved by thrombin. In some embodiments, the a1 region comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5).

[0008] In other embodiments, a VWF linker useful for connecting a VWF protein and a heterologous moiety comprises an a3 region that comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu1649 to Arg1689 corresponding to full-length FVIII, the a3 region being capable of being cleaved by thrombin. In certain embodiments, the a3 region comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6).

[0009] In yet another embodiment, a VWF linker useful for connecting a VWF protein and a heterologous moiety comprises a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, the PAR1 non-active site interaction motif comprising SFLLRN (SEQ ID NO: 7). In one embodiment, the PAR1 non-active site interaction motif further comprises a sequence selected from P, PN, PND, PNDK (SEQ ID NO: 8), PNDKY (SEQ ID NO: 9), PNDKYE (SEQ ID NO: 10), PNDKYEP (SEQ ID NO: 11), PNDKYEPF (SEQ ID NO: 12), PNDKYEPFW (SEQ ID NO: 13), PNDKYEPFWE (SEQ ID NO: 14), PNDKYEPFWED (SEQ ID NO: 20), PNDKYEPFWEDE (SEQ ID NO: 21), PNDKYEPFWEDEE (SEQ ID NO: 22), PNDKYEPFWEDEES (SEQ ID NO: 23), or any combination thereof. In another embodiment, the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine. In certain embodiments In this case, the VWF linker comprises GGLVPRSFLLRNPNDKYEPFWEDEES (SEQ ID NO: 24).

[0010] In certain embodiments, thrombin cleaves the VWF linker faster than thrombin cleaves the thrombin cleavage site when it is replaced with the VWF linker in the chimeric molecule, in other embodiments, thrombin cleaves the VWF linker at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times faster than thrombin cleaves the thrombin cleavage site when it is replaced with the VWF linker in the chimeric molecule.

[0011] In some embodiments, the VWF linker further comprises one or more amino acids having a length of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. In one example, the one or more amino acids comprise a gly peptide. In another example, the one or more amino acids comprise a GlyGly. In other examples, the one or more amino acids comprise a gly / ser peptide. In some examples, the gly / ser peptide has 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 certain examples, the (Gly4Ser)n linker is (Gly4Ser)3 (SEQ ID NO:89) or (Gly4Ser)4 (SEQ ID NO:90).

[0012] A VWF protein useful in the chimeric molecule of the invention can comprise a D' domain and a D3 domain of VWF, the D' domain and the D3 domain being capable of binding to a FVIII protein. In one embodiment, the D' domain of the VWF protein comprises 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 D3 domain of the VWF protein comprises an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO:2. In another embodiment, the VWF protein contains at least one amino acid substitution at residues corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO:2. In still other embodiments, an amino acid other than cysteine ​​is substituted within the sequence of the VWF protein for residues corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO:2. In yet other embodiments, the sequence of the VWF protein comprises amino acids 764-1240 of SEQ ID NO:2. In certain embodiments, the VWF protein further comprises the D1 domain, the D2 domain, or the D1 and D2 domains of VWF. In some embodiments, the VWF protein further comprises a VWF domain selected from the A1 domain, the A2 domain, the A3 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, or any combination thereof. In other embodiments, the VWF protein is selected from the group consisting of (1) the D' and D3 domains of VWF, or fragments thereof, (2) the D1, D', and D3 domains of VWF, or fragments thereof, (3) the D2, D', and D3 domains of VWF, or fragments thereof, (4) the D1, D2, D', and D3 domains of VWF, or fragments thereof, or (5) the D1, D2, D', D3, and A1 domains of VWF, or fragments thereof. In still other embodiments, the VWF protein further comprises a signal peptide of VWF. In yet other embodiments, the VWF protein is PEGylated, glycosylated, HESylated, or polysialylated. The term "PEGylated" refers to having polyethylene glycol (PEG) on the protein, the term "glycosylated" refers to having glycosylation on the protein, the term "HESylated" refers to having hydroxyethyl starch (HES) on the protein, and the term "polysialylated" refers to having polysialic acid (PSA) on the protein. Examples of PEG, HES, and PSA are provided elsewhere herein.

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

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

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

[0016] In certain embodiments, the FVIII protein is linked to the second heterologous moiety by a FVIII linker. In one embodiment, the second linker is a cleavable linker. In another embodiment, the FVIII linker is the same as the VWF linker. In another embodiment, the FVIII linker is different from the VWF linker.

[0017] In some embodiments, the chimeric molecule of the invention comprises: (a) V-L1-X1-H1:H2 -L2-X2-C, (b) V-X1-L1-H1:H2-L2-X2-C, (c) V-L1-X1-H1:H2-X2-L2-C, (d) V-X1- L1-H1:H2-X2-L2-C, (e)V-L1-X1-H1:H2-L2-C(X2), (f)V-X1-L1-H1:H2-L2-C(X2 ), (g)C-X2-L2-H2:H1-X1-L1-V, (h)C-X2-L2-H2:H1-L1-X1-V, (i)C-L2-X2-H2:H 1-L1-X1-V, (j)C-L2-X2-H2:H1-L1-X1-V, (k)C(X2)-L2-H2:H1-X1-L1-V, or (l)C (X2)-L2-H2: includes a formula selected from H1-L1-X1-V, where V is a VWF protein, L1 is a VWF linker, L2 is an optional FVIII linker, H1 is a first heterologous moiety, H2 is a second heterologous moiety, X1 is an XTEN sequence, X2 is an optional XTEN sequence, C is a FVIII protein, C(X2) is a FVIII protein fused to an XTEN sequence, the XTEN sequence being inserted between two adjacent FVIII amino acids, (-) is a peptide bond or one or more amino acids, and (:) is a covalent bond between H1 and H2.

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

[0019] In the chimeric molecule of the present invention, the VWF protein is capable of inhibiting or preventing the binding of endogenous VWF to the FVIII protein.

[0020] In certain embodiments, the FVIII protein in the chimeric molecule can include a third heterologous moiety (H3). The third heterologous moiety (H3) can be an XTEN sequence. In other embodiments, the FVIII protein includes a fourth heterologous moiety (H4). The fourth heterologous moiety (H4) can be an XTEN sequence. In some embodiments, the FVIII protein includes a fifth heterologous moiety (H5). The fifth heterologous moiety can be an XTEN sequence. In other embodiments, the FVIII protein includes a sixth heterologous moiety (H6). The sixth heterologous moiety can be an XTEN sequence. In certain embodiments, 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) can extend the half-life of the chimeric molecule. In other embodiments, the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are attached to the C-terminus or N-terminus of FVIII or are inserted between two amino acids of the FVIII protein. In still other embodiments, one or more of the third heterologous moiety, the fourth heterologous moiety, the fifth heterologous moiety, and the sixth heterologous moiety are 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 512 amino acids, about 520 amino acids, about 532 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, about 600 amino acids, about 610 amino acids, about 620 amino acids, about 630 amino acids, about 640 amino acids, about 650 amino acids, about 660 amino acids, about 670 amino acids, about 680 amino acids, about 700 amino acids, about 720 amino acids, about 760 amino acids, about 780 amino acids, about 790 amino acids, about 800 amino acids, about 810 amino acids, about 820 amino acids, about 830 amino acids, about 840 amino acids, about 850 amino acids, about 860 amino acids, about 870 amino acids, about 880 amino acids, about 900 amino acids, about 920 In some embodiments, the length may be selected from one or more of the following: about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. For example, the XTEN sequence of the third heterologous portion, the fourth heterologous portion, the fifth heterologous portion, or the sixth heterologous portion can be selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. More specifically, the XTEN sequence 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.

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

[0022] The present disclosure also provides a polynucleotide or set of polynucleotides that encodes the chimeric molecule or its complementary sequence. The polynucleotide or set of polynucleotides can further comprise a polynucleotide chain that encodes PC5 or PC7.

[0023] Further included are vectors or sets of vectors comprising the polynucleotide or set of polynucleotides, and one or more promoters operably linked to the polynucleotide or set of polynucleotides. In some embodiments, the vector or set of vectors can further comprise an additional polynucleotide strand encoding PC5 or PC7.

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

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

[0026] Further included is a method for reducing the frequency or severity of bleeding episodes in a subject in need thereof, comprising administering to a subject an effective amount of a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding the chimeric molecule, a vector or set of vectors disclosed herein, a host cell disclosed herein, or a composition disclosed herein. The present invention also includes a method for preventing the occurrence 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 the chimeric molecule, a vector or set of vectors disclosed herein, a host cell disclosed herein, or a composition disclosed herein. In one embodiment, the bleeding episode is from a bleeding coagulopathy, hemarthrosis, muscle bleeding, oral bleeding, major bleeding, major bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, or any combination thereof. In another embodiment, the chimeric molecule disclosed herein, the polynucleotide or set of polynucleotides encoding the chimeric molecule, the vector or set of vectors disclosed herein, or the host cell disclosed herein, or the composition disclosed herein may be administered by a route selected from topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, oral administration, and any combination thereof.

[0027] The present disclosure also includes a method for making 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 cells. The method further comprises isolating the chimeric molecule. In some embodiments, the FVIII activity of the chimeric molecule can be measured by aPTT assay or ROTEM assay. In certain embodiments, for example, the following are provided: (Item 1) A chimeric molecule comprising a von Willebrand factor (VWF) protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting said VWF protein to said heterologous moiety, said VWF linker comprising: i. the a2 region from factor VIII (FVIII); ii. the a1 region from FVIII; iii. the a3 region from FVIII; iv. a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid; or v. any combination thereof; The chimeric molecule, wherein the XTEN sequence is attached to the VWF protein, the heterologous moiety (H1), the VWF linker, or any combination thereof. (Item 2) 2. The chimeric molecule of item 1, wherein the XTEN sequence connects the VWF protein to the VWF linker or the VWF linker to the heterologous moiety. (Item 3) 3. The chimeric molecule according to item 1 or 2, further comprising a second polypeptide chain comprising a FVIII protein, wherein the first polypeptide chain and the second polypeptide chain are associated with each other. (Item 4) 4. The chimeric molecule of item 3, wherein the FVIII protein further comprises an additional XTEN sequence. (Item 5) 5. The chimeric molecule of item 4, wherein the additional XTEN sequence is attached to the N-terminus or C-terminus of the FVIII protein or inserted between two adjacent FVIII amino acids. (Item 6) 6. The chimeric molecule according to any one of items 3 to 5, wherein the second polypeptide chain further comprises a second heterologous moiety (H2). (Item 7) A chimeric molecule comprising a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting said VWF protein and said heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein said VWF linker in said first polypeptide chain is i. the a2 region from FVIII, ii. the a1 region from FVIII; iii. the a3 region from FVIII; iv. a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid; or v. any combination thereof; The chimeric molecule, wherein the first polypeptide chain and the second polypeptide chain are associated with each other. (Item 8) 8. The chimeric molecule of item 7, wherein the XTEN sequence is attached to the N-terminus or C-terminus of the FVIII protein or inserted between two adjacent FVIII amino acids. (Item 9) 9. The chimeric molecule of item 7 or 8, further comprising an additional XTEN sequence connected to the VWF protein, the heterologous moiety, the VWF linker, or any combination thereof. (Item 10) 10. The chimeric molecule according to any one of items 7 to 9, further comprising a second heterologous moiety (H2). (Item 11) 11. The chimeric molecule of item 10, wherein the second heterologous moiety is attached to the FVIII protein, the XTEN sequence, or both. (Item 12) The XTEN sequence is selected from the group consisting 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 800 amino acids, about 810 amino acids, about 820 amino acids, about 830 amino acids, about 840 amino acids, about 850 amino acids, about 860 amino acids, about 870 amino acids, about 880 amino acids, about 890 amino acids, about 900 amino acids, about 910 amino acids, about 920 amino acids, about 930 amino acids, about 940 amino acids, about 950 amino acids, about 960 amino acids, about 970 amino acids, about 980 amino acids, about 990 amino acids, about 1000 amino acids, about 1010 amino acids, about 1020 amino acids, about 1030 amino acids, about 1040 amino acids, about 1050 amino acids, about 1060 amino acids, about 1070 amino acids, about 1080 amino acids, about 1090 amino acids, about 1100 amino acids, about 1120 amino acids, about 1130 amino acids, about 1140 amino acids, about 1150 amino acids, about 1160 amino acids, about 1170 amino acids, about 11 12. The chimeric molecule according to any one of items 1 to 11, comprising 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. (Item 13) 13. The chimeric molecule of any one of items 1 to 12, wherein the XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. (Item 14) 14. The chimeric molecule according to any one of items 1 to 13, wherein the XTEN sequence 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. (Item 15) The additional XTEN sequence is selected from the group consisting 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 800 amino acids, about 810 amino acids, about 820 amino acids, about 830 amino acids, about 840 amino acids, about 850 amino acids, about 860 amino acids, about 870 amino acids, about 880 amino acids, about 890 amino acids, about 900 amino acids, about 910 amino acids, about 920 amino acids, about 930 amino acids, about 940 amino acids, about 950 amino acids, about 960 amino acids, about 970 amino acids, about 980 amino acids, about 990 amino acids, about 1000 amino acids, about 1010 amino acids, about 1020 amino acids, about 1030 amino acids, about 1040 amino acids, about 1050 amino acids, about 1060 amino acids, about 1070 amino acids, about 1080 amino acids, about 1090 amino acids, about 1100 amino acids, about 1120 amino acids, about 1130 amino acids, about 1140 amino acids, about 1150 amino acids, about 1160 amino acids, about 1170 amino acids, about 1 The chimeric molecule according to any one of Items 4 to 6 and 9 to 11, comprising 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. (Item 16) 16. The chimeric molecule of any one of items 4 to 6, 9 to 11, and 15, wherein the additional XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. (Item 17) 17. The chimeric molecule according to any one of items 4 to 6, 9 to 11, and 15 to 16, wherein the additional XTEN sequence 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. (Item 18) 18. The chimeric molecule according to any one of items 1 to 17, wherein the VWF linker comprises the a2 region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu720 to Arg740 corresponding to full-length FVIII, and the a2 region is capable of being cleaved by thrombin. (Item 19) 19. The chimeric molecule of item 18, wherein the a2 region comprises SEQ ID NO:4. (Item 20) The chimeric molecule according to any one of Items 1 to 17, wherein the VWF linker comprises the a1 region that contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% identical to Met337 to Arg372 corresponding to full-length FVIII, and the a1 region is capable of being cleaved by thrombin. (Item 21) 21. The chimeric molecule of item 20, wherein the a1 region comprises SEQ ID NO:5. (Item 22) 18. The chimeric molecule according to any one of items 1 to 17, wherein the VWF linker comprises 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 Glu1649 to Arg1689 corresponding to full-length FVIII, and the a3 region is capable of being cleaved by thrombin. (Item 23) 23. The chimeric molecule of item 22, wherein the a3 region comprises SEQ ID NO:6. (Item 24) 18. The chimeric molecule according to any one of items 1 to 17, wherein the VWF linker comprises the thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and the PAR1 non-active site interacting motif, and the PAR1 non-active site interacting motif comprises SFLLRN (SEQ ID NO: 7). (Item 25) The PAR1 non-active site interaction motif is P, PN, PND, PND- 25. The chimeric molecule according to any one of items 1 to 17, further comprising a sequence selected from the group consisting of K (SEQ ID NO: 8), PNDKY (SEQ ID NO: 9), PNDKYE (SEQ ID NO: 10), PNDKYEP (SEQ ID NO: 11), PNDKYEPF (SEQ ID NO: 12), PNDKYEPFW (SEQ ID NO: 13), PNDKYEPFWE (SEQ ID NO: 14), PNDKYEPFWED (SEQ ID NO: 20), PNDKYEPFWEDE (SEQ ID NO: 21), PNDKYEPFWEDEE (SEQ ID NO: 22), PNDKYEPFWEDEES (SEQ ID NO: 23), or any combination thereof. (Item 26) 26. The chimeric molecule according to any one of items 1 to 17, 24 and 25, wherein the aliphatic amino acids are selected from glycine, alanine, valine, leucine, or isoleucine. (Item 27) 27. The chimeric molecule according to any one of items 1 to 26, wherein the VWF linker is cleaved by thrombin faster than the thrombin cleavage site consisting of LVPR (SEQ ID NO: 25) is cleaved by thrombin. (Item 28) 28. The chimeric molecule according to any one of items 1 to 27, wherein the VWF linker comprises SEQ ID NO: 24. (Item 29) 28. The chimeric molecule of item 27, wherein the VWF linker is cleaved by thrombin at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times faster than the thrombin cleavage site consisting of LVPR (SEQ ID NO: 25) is cleaved by thrombin. (Item 30) 30. The chimeric molecule of any one of items 1 to 29, wherein the VWF linker further comprises one or more amino acids, and wherein the VWF linker has a length of at least about 1, 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. (Item 31) 31. The chimeric molecule of item 30, wherein the one or more amino acids comprise a gly peptide. (Item 32) 32. The chimeric molecule according to item 30 or 31, wherein the one or more amino acids comprises GlyGly. (Item 33) 31. The chimeric molecule of item 30, wherein the one or more amino acids comprise a gly / ser peptide. (Item 34) 34. The chimeric molecule of item 33, wherein the gly / ser peptide has 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. (Item 35) 35. The chimeric molecule of item 34, wherein the (Gly4Ser)n linker is (Gly4Ser)3 (SEQ ID NO: 89) or (Gly4Ser)4 (SEQ ID NO: 90). (Item 36) 36. The chimeric molecule according to any one of items 1 to 35, wherein the VWF protein comprises the D' and D3 domains of VWF, and the D' and D3 domains are capable of binding to a FVIII protein. (Item 37) 37. The chimeric molecule of item 36, wherein the D' domain of the VWF protein comprises an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO:2. (Item 38) 38. The chimeric molecule of item 36 or 37, wherein the D3 domain of the VWF protein comprises an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO:2. (Item 39) 39. The chimeric molecule according to any one of items 36 to 38, wherein the VWF protein contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO:2. (Item 40) 40. The chimeric molecule according to any one of items 1 to 39, wherein within the sequence of the VWF protein an amino acid other than cysteine ​​is substituted with a residue corresponding to residue 1099, residue 1142 or both residues 1099 and 1142 of SEQ ID NO: 2. (Item 41) 40. The chimeric molecule according to any one of items 1 to 39, wherein the sequence of the VWF protein comprises amino acids 764 to 1240 of SEQ ID NO: 2. (Item 42) 42. The chimeric molecule according to any one of items 36 to 41, wherein the VWF protein further comprises the D1 domain, the D2 domain, or the D1 and D2 domains of VWF. (Item 43) 43. The chimeric molecule according to any one of items 36 to 42, wherein the VWF protein further comprises a VWF domain selected from the group consisting of A1 domain, A2 domain, A3 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, or any combination thereof. (Item 44) 44. The chimeric molecule according to any one of items 36 to 43, wherein the VWF protein essentially consists of or consists of (1) the D' and D3 domains of VWF, or fragments thereof, (2) the D1, D', and D3 domains of VWF, or fragments thereof, (3) the D2, D', and D3 domains of VWF, or fragments thereof, (4) the D1, D2, D', and D3 domains of VWF, or fragments thereof, or (5) the D1, D2, D', D3, and A1 domains of VWF, or fragments thereof. (Item 45) 45. The chimeric molecule according to any one of items 1 to 44, further comprising a signal peptide of VWF. (Item 46) 46. ​​The chimeric molecule according to any one of items 1 to 45, wherein the VWF protein is PEGylated, glycosylated, HESylated, or polysialylated. (Item 47) 47. The chimeric molecule according to any one of items 1 to 46, wherein the heterologous moiety (H1) is capable of extending the half-life of the chimeric molecule. (Item 48) The heterologous moiety (H1) is selected from the group consisting of an immunoglobulin constant region or a part thereof, albumin, albumin-binding polypeptide, PAS, and the C-terminus of the human chorionic gonadotropin β subunit. 48. The chimeric molecule of item 47, comprising a peptide (CTP), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin-binding small molecule, or any combination thereof. (Item 49) 49. The chimeric molecule of item 48, wherein the immunoglobulin constant region or portion thereof comprises an FcRn binding partner. (Item 50) 49. The chimeric molecule of item 48, wherein the immunoglobulin constant region or portion thereof comprises an Fc region. (Item 51) 48. The chimeric molecule according to any one of items 1 to 47, wherein the heterologous moiety (H1) comprises a clearance receptor or a fragment thereof, and the clearance receptor blocks binding of FVIII protein to a FVIII clearance receptor. (Item 52) 52. The chimeric molecule of item 51, wherein the clearance receptor is low density lipoprotein receptor-related protein 1 (LRP1), or a FVIII-binding fragment thereof. (Item 53) 53. The chimeric molecule according to any one of items 6 and 10 to 52, wherein the second heterologous moiety comprises an immunoglobulin constant region or a portion thereof, albumin, an albumin binding polypeptide, PAS, the C-terminal peptide of human chorionic gonadotropin β subunit (CTP), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding small molecule, or any combination thereof. (Item 54) 54. The chimeric molecule according to any one of items 6 and 10 to 53, wherein the second heterologous moiety (H2) is capable of extending the half-life of the FVIII protein. (Item 55) 55. The chimeric molecule of item 54, wherein the second heterologous moiety (H2) comprises a polypeptide, a non-polypeptide moiety, or both. (Item 56) 56. The chimeric molecule of item 54 or 55, wherein the second heterologous moiety (H2) comprises an immunoglobulin constant region or a portion thereof. (Item 57) 57. The chimeric molecule of paragraph 56, wherein the second heterologous moiety comprises an FcRn binding partner. (Item 58) 57. The chimeric molecule of claim 56, wherein the second heterologous moiety comprises a second Fc region. (Item 59) 59. The chimeric molecule according to any one of items 6 and 10 to 58, wherein the first heterologous moiety and the second heterologous moiety are associated with each other. (Item 60) 60. The chimeric molecule of item 59, wherein the association between the first polypeptide chain and the second polypeptide is a covalent bond. (Item 61) 60. The chimeric molecule of item 59, wherein the association between the first heterologous moiety and the second heterologous moiety is a disulfide bond. (Item 62) 59. The chimeric molecule according to any one of items 6 and 10 to 58, wherein the first heterologous moiety is an FcRn binding partner and the second heterologous moiety is an FcRn binding partner. (Item 63) Item 6. The first heterologous moiety is an Fc region and the second heterologous moiety is an Fc region. 59. The chimeric molecule according to any one of claims 10 to 58. (Item 64) 59. The chimeric molecule according to any one of items 6 and 10 to 58, wherein the FVIII protein is linked to the second heterologous moiety by a FVIII linker. (Item 65) 65. The chimeric molecule of item 64, wherein the FVIII linker is a cleavable linker. (Item 66) 66. The chimeric molecule according to item 64 or 65, wherein the FVIII linker is identical to the VWF linker. (Item 67) 66. The chimeric molecule according to item 64 or 65, wherein the FVIII linker is different from the VWF linker. (Item 68) (a) V-L1-X1-H1:H2-L2-X2-C; (b) V-X1-L1-H1:H2-L2-X2-C; (c) V-L1-X1-H1:H2-X2-L2-C; (d) V-X1-L1-H1:H2-X2-L2-C; (e) V-L1-X1-H1:H2-L2-C(X2); (f) V-X1-L1-H1:H2-L2-C(X2); (g) C-X2-L2-H2:H1-X1-L1-V, (h) C-X2-L2-H2:H1-L1-X1-V, (i) C-L2-X2-H2:H1-L1-X1-V; (j) C-L2-X2-H2:H1-L1-X1-V, (k) C(X2)-L2-H2:H1-X1-L1-V, or (l) C(X2)-L2-H2:H1-L1-X1-V; wherein V is the VWF protein; L1 is the VWF linker, L2 is any FVIII linker; H1 is the first heterologous moiety; H2 is the second heterologous moiety; C is the FVIII protein, C(X2) is the FVIII protein fused to the XTEN sequence, and the XTEN sequence is inserted between two adjacent FVIII amino acids; (-) is a peptide bond or one or more amino acids, and 68. The chimeric molecule according to any one of items 6 and 10 to 67, wherein (:) is a covalent bond between the H1 and the H2. (Item 69) 69. The chimeric molecule according to any one of items 6 and 10 to 68, wherein the VWF protein inhibits or prevents binding of endogenous VWF to the FVIII protein. (Item 70) 70. The chimeric molecule according to any one of items 6 and 10 to 69, wherein the FVIII protein comprises a third heterologous moiety (H3). (Item 71) 71. The chimeric molecule of item 70, wherein the third heterologous portion (H3) is an XTEN sequence. (Item 72) 72. The chimeric molecule according to item 70 or 71, wherein the FVIII protein comprises a fourth heterologous moiety (H4). (Item 73) 73. The chimeric molecule of item 72, wherein the fourth heterologous moiety (H4) is an XTEN sequence. (Item 74) 74. The chimeric molecule of item 72 or 73, wherein the FVIII protein comprises a fifth heterologous moiety (H5). (Item 75) 75. The chimeric molecule of item 74, wherein the fifth heterologous portion is an XTEN sequence. (Item 76) 76. The chimeric molecule of item 74 or 75, wherein the FVIII protein comprises the sixth heterologous moiety (H6). (Item 77) 77. The chimeric molecule of item 76, wherein the sixth heterologous portion is an XTEN sequence. (Item 78) 78. The chimeric molecule according to item 76 or 77, wherein one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are capable of extending the half-life of the chimeric molecule. (Item 79) 78. The chimeric molecule according to item 76 or 77, wherein the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are attached to the C-terminus or N-terminus of FVIII or are inserted between two amino acids of the FVIII protein. (Item 80) wherein one or more of the XTEN sequences 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 amino acids, about 828 amino acids, 78. The chimeric molecule of any one of paragraphs 71, 73, 75, and 77, comprising a length selected from one or more of 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. (Item 81) 81. The chimeric molecule of any one of paragraphs 71, 73, 75, 77, and 80, wherein the XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144. (Item 82) 82. The chimeric molecule of item 81, wherein the XTEN sequence 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. (Item 83) 83. The chimeric molecule of any one of paragraphs 1 to 82, wherein the half-life of the chimeric molecule is extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild-type FVIII. (Item 84) 84. A polynucleotide or a set of polynucleotides encoding the chimeric molecule according to any one of items 1 to 83 or its complementary sequence. (Item 85) 85. The method according to item 84, further comprising a polynucleotide sequence encoding PC5 or PC7. A polynucleotide or a set of polynucleotides. (Item 86) 86. A vector or a set of vectors comprising a polynucleotide or a set of polynucleotides according to item 84 or 85 and one or more promoters operably linked to said polynucleotide or said set of polynucleotides. (Item 87) 87. The vector or set of vectors according to item 86, further comprising an additional polynucleotide strand encoding PC5 or PC7. (Item 88) A host cell comprising a polynucleotide or set of polynucleotides according to item 86 or 87, or a vector or set of vectors according to item 86 or 87. (Item 89) 89. The host cell according to item 88, which is a mammalian cell. (Item 90) 90. The host cell of item 89, wherein the mammalian cell is selected from a HEK293 cell, a CHO cell, or a BHK cell. (Item 91) A pharmaceutical composition comprising the chimeric molecule according to any one of items 1 to 83, the polynucleotide or polynucleotide according to item 84 or 85, the vector or set of vectors according to item 86 or 87, or the host cell according to any one of items 88 to 90, and a pharma- ceutically acceptable carrier. (Item 92) 92. The composition of claim 91, wherein the chimeric molecule has an extended half-life compared to a wild-type FVIII protein. (Item 93) 93. The composition of item 91 or 92, wherein the half-life of the chimeric molecule is extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild-type FVIII. (Item 94) A method for reducing the frequency or severity of bleeding episodes in a subject in need thereof, comprising administering an effective amount of the chimeric molecule according to any one of items 1 to 83, the polynucleotide according to item 84 or 85, the vector according to item 86 or 87, the host cell according to any one of items 88 to 90, or the composition according to any one of items 91 to 93. (Item 95) A method for preventing the occurrence of a bleeding episode in a subject in need thereof, comprising administering an effective amount of the chimeric molecule according to any one of items 1 to 83, the polynucleotide according to item 84 or 85, the vector according to item 86 or 87, the host cell according to any one of items 88 to 90, or the composition according to any one of items 91 to 93. (Item 96) 96. The method of item 94 or 95, wherein said bleeding episode is from a bleeding coagulopathy, hemarthrosis, muscle bleeding, oral bleeding, major bleeding, major bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, or any combination thereof. (Item 97) The method according to any one of items 95 to 96, wherein the chimeric molecule according to any one of items 1 to 83, the polynucleotide according to item 84 or 85, the vector according to item 86 or 87, the host cell according to item 88 to 90, or the composition according to any one of items 91 to 93 is administered by a route selected from topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, oral administration, and any combination thereof. (Item 98) A method for producing a chimeric molecule, the method comprising transfecting one or more host cells with a polynucleotide according to item 84 or 85, or a vector according to item 86 or 87, and expressing the chimeric molecule in the host cells. (Item 99) 99. The method of claim 98, further comprising isolating the chimeric molecule. (Item 100) A method for improving FVIII activity of a chimeric FVIII protein comprising a VWF protein, a heterologous portion (H1), and a VWF linker connecting said VWF protein and said heterologous portion (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein said VWF linker in said first polypeptide chain is i. the a2 region from FVIII, ii. the a1 region from FVIII; iii. the a3 region from FVIII; iv. a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid; or v. any combination thereof; (Item 101) 101. The method of claim 100, wherein the FVIII activity is measured by aPTT assay or ROTEM assay. (Item 102) A chimeric molecule comprising two polypeptide sequences, wherein a first polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to FVIII169 (SEQ ID NO: 88), and a second polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to VWF059 (SEQ ID NO: 82). (Item 103) A chimeric molecule comprising two polypeptide sequences, wherein a first polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to FVIII286 (SEQ ID NO: 86), and a second polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to VWF059 (SEQ ID NO: 82). (Item 104) A chimeric molecule comprising two polypeptide sequences, wherein a first polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to FVIII286 (SEQ ID NO: 86), and a second polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to VWF062 (SEQ ID NO: 84). (Item 105) A chimeric molecule comprising two polypeptide sequences, wherein a first polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to FVIII286 (SEQ ID NO: 86), and a second polypeptide sequence comprises an amino acid sequence at least about 80%, about 90%, about 95%, or 100% identical to VWF057 (SEQ ID NO: 80). (Item 106) A polynucleotide or polynucleotides encoding the chimeric molecule according to any one of items 102 to 105. (Item 107) A polynucleotide encoding a nucleotide sequence that is at least about 80%, about 90%, about 95%, or 100% identical to VWF057 (SEQ ID NO:79). (Item 108) 108. The polynucleotide of Item 107, further comprising a second polynucleotide comprising a second nucleotide sequence at least about 80%, about 90%, about 95%, or 100% identical to FVIII169 (SEQ ID NO: 87). (Item 109) 108. The polynucleotide of item 107, further comprising a second polynucleotide comprising a second nucleotide sequence at least about 80%, about 90%, about 95%, or 100% identical to FVIII286 (SEQ ID NO: 85). (Item 110) 110. The polynucleotide according to item 108 or 109, wherein the first polynucleotide and the second polynucleotide are on the same vector or on two different vectors. (Item 111) 102. A method for reducing the frequency or severity of bleeding episodes in a subject in need thereof, comprising administering an effective amount of the chimeric molecule according to any one of items 102 to 105, or the polynucleotide(s) according to any one of items 106 to 110. [Brief description of the drawings]

[0028] [Figure 1] 1 shows an exemplary diagram of a chimeric molecule (FVIII-XTEN / VWF heterodimer) comprising two polypeptide chains, where the first chain comprises a VWF protein (e.g., the D' and D3 domains of VWF) fused to an Fc region via a thrombin-cleavable VWF linker, and the second chain comprises a FVIII protein fused to a second Fc region via a FVIII linker. The FVIII protein comprises one or more XTENs within various domains of FVIII. [Diagram 2]Various VWF constructs are shown, each of which, except for the control (i.e., VWF-052), contains a D' domain and a D3 domain fused to the Fc region via a thrombin-cleavable VWF linker. VWF-031 contains a 48 amino acid linker containing the thrombin cleavage site of LVPR (SEQ ID NO: 25). VWF-034 contains an XTEN sequence with 288 amino acids and a 35 amino acid linker containing the thrombin cleavage site of LVPR (SEQ ID NO: 25). VWF-035 contains a 73 amino acid linker containing the thrombin cleavage site of LVPR (SEQ ID NO: 25). VWF-036 contains a 98 amino acid linker containing the thrombin cleavage site of LVPR (SEQ ID NO: 25). VWF-039 contains a 26 amino acid VWF linker containing the thrombin cleavage site of LVPR (SEQ ID NO: 25) and a PAR1 non-active site interaction motif. VWF-051 contains a 54 amino acid linker that includes the thrombin cleavage site of ALRPRVV (SEQ ID NO: 26). VWF-052 contains a 48 amino acid linker that does not have any thrombin cleavage site (control). VWF-054 contains a 40 amino acid VWF linker that includes the a1 region from FVIII. VWF-055 contains a 34 amino acid VWF linker that includes the a2 region from FVIII. VWF-056 contains a 46 amino acid VWF linker that includes the a3 region from FVIII. [Diagram 3] Figure 3A shows the thrombin-mediated cleavage rate of VWF-Fc fusion constructs, namely VWF-031, VWF-034, VWF-036, VWF-039, VWF-051 and VWF-052, in units of resonance units per second (RU / s), as a function of capture density in units of RU. Figure 3B shows the thrombin-mediated cleavage rate of VWF-Fc fusion constructs, namely VWF-031, VWF-034, VWF-036, VWF-051 and VWF-052, in units of resonance units per second (RU / s), as a function of capture density in units of RU. In these experiments, each VWF-Fc fusion construct was captured at various densities and subsequently exposed to a constant concentration of human α-thrombin. The slope of each curve in Figures 3A and 3B directly reflects the susceptibility of each construct to thrombin cleavage. [Figure 4] Figure 4A shows the thrombin-mediated cleavage rate of VWF-Fc fusion constructs, namely VWF-054, VWF-055, and VWF-056, in units of resonance units per second (RU / s), as a function of capture density in units of RU. Figure 4B shows the thrombin-mediated cleavage rate of VWF-Fc fusion constructs, namely VWF-031, VWF-039, VWF-054, VWF-055, and VWF-056, in units of resonance units per second (RU / s), as a function of capture density in units of RU. In these experiments, each VWF-Fc fusion construct was captured at various densities and subsequently exposed to a constant concentration of human α-thrombin. The slope of each curve in Figures 4A and 4B directly reflects the susceptibility of each construct to thrombin cleavage. [Diagram 5] Figure 4 shows 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, VWF-052, VWF-054, VWF-055, and VWF-056, to thrombin-mediated cleavage. Values ​​are expressed in units of inverse seconds and reflect the slope of the curves shown in Figures 3 and 4. The relative susceptibility of two different constructs is obtained from the quotient of their respective slopes. Slope VWF-039 / slope VWF-031 is 71, indicating that the VWF-Fc fusion construct VWF-039 is 71 times more susceptible to thrombin-mediated cleavage than VWF-031. The slope VWF-055 / slope VWF-031 is 65, and the slope VWF-051 / slope VWF-031 is 1.8. [Figure 6]1 shows the clotting time of various chimeric molecules in HemA patients measured by whole blood ROTEM assay. FVII155 / VWF-031 comprises two polypeptide chains, a first chain comprising BDD FVIII fused to an Fc region and a second chain comprising the D' and D3 domains of VWF fused to an Fc region via a minimal thrombin cleavage site (i.e., LVPR (SEQ ID NO: 25)). FVII155 / VWF-039 comprises two polypeptide chains, a first chain comprising BDD FVIII fused to an Fc region and a second chain comprising the D' and D3 domains of VWF fused to an Fc region via LVPR (SEQ ID NO: 25) and a VWF linker comprising a PAR1 non-active site interaction motif. FVII155 / VWF-055 comprises two polypeptide chains, a first chain comprising the BDD FVIII fused to an Fc region and a second chain comprising the D' and D3 domains of VWF fused to the Fc region via a VWF linker comprising the a2 region from FVIII. [Figure 7]Illustrated are diagrams of representative FVIII-VWF heterodimers, as well as FVIII169, FVIII286, VWF057, VWF059, and VWF062 constructs. For example, the FVIII169 construct comprises a B domain deleted FVIII protein with a R1648A substitution fused to the Fc region, with an XTEN sequence (e.g., AE288) inserted at amino acid 745, corresponding to mature full-length FVIII (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-Fc). The FVIII286 construct comprises a B domain deleted FVIII protein with an R1648 substitution fused to an Fc region with an XTEN sequence (e.g., AE288) inserted at amino acid 745 corresponding to mature full length FVIII, with an additional a2 region between FVIII and Fc (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-a2-Fc). VWF057 is a VWF-Fc fusion construct comprising a D'D3 domain of a VWF protein (having two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A) ​​linked to an Fc region via a VWF linker comprising an LVPR thrombin site ("LVPR") and a GS linker ("GS"), and an XTEN sequence (i.e., 144XTEN) is inserted between the D'D3 domain and the VWF linker (D'D3-144XTEN-GS+LVPR-Fc). VWF059 is a VWF-Fc fusion construct comprising a D'D3 domain of a VWF protein (having two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A) ​​linked to an Fc region via an acidic region 2 (a2) region as a VWF linker, and an XTEN sequence is inserted between the D'D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct that contains 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, and an XTEN sequence is inserted between the D'D3 domain and the Fc region (D'D3-144XTEN-Fc). [Figure 8]1 shows the rapid efficacy 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 the HemA mouse tail transection model. BDD-rFVIII is shown as a circle, while FVIII169 / VWF034 is shown as a square, FVIII169 / VWF059 is shown as a triangle, FVIII169 / VWF057 is shown as an open circle, and vehicle is shown as an inverted triangle. VWF034 is a VWF-Fc fusion construct that contains the D' and D3 domains of VWF fused to the Fc region via a VWF linker containing LVPR, and an XTEN sequence (i.e., 288XTEN) is inserted between the D'D3 domain and the VWF linker (D'D3-288XTEN-LVPR-Fc). Details of the constructs of FVIII169, VWF059, and VWF057 are provided elsewhere herein. The median blood loss (uL) of mice following administration of 75 IU / kg of construct in each treatment group is indicated by a horizontal line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention is directed to chimeric molecules comprising an XTEN sequence and a thrombin cleavable linker connecting a VWF protein or a FVIII protein with a heterologous moiety, e.g., a half-life extending moiety. The present invention also provides chimeric molecules comprising two polypeptide chains, a first chain comprising a VWF protein fused to a heterologous moiety, and a second chain comprising a FVIII protein and a second heterologous moiety, the chimeric molecules comprising an XTEN sequence in the first or second polypeptide chain, and either the VWF protein or the FVIII protein (or both) is fused to the heterologous moiety via a VWF linker or a FVIII linker (or both). The thrombin cleavable linker (VWF linker or FVIII linker) can be efficiently cleaved by thrombin at the injury site where thrombin is readily available. Exemplary chimeric molecules are illustrated in the present description and figures. In some embodiments, the present invention relates to chimeric molecules having structures as shown, for example, in Figures 1-7. In other embodiments, the present invention relates to polynucleotides encoding the chimeric molecule constructs disclosed herein.

[0030] In order to provide a clear understanding of the specification and claims, the following definitions are provided below.

[0031] I. Definition It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "a nucleotide sequence" refers to one or more of that entity. A "nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0032] The term "about" is used herein to mean approximately, roughly, in the region, and within that region. When the term "about" is used in conjunction with a numerical range, it also means The range is modified by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent upward or downward (higher or lower).

[0033] The term "polynucleotide" or "nucleotide" is intended to encompass single nucleic acids as well as multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, polynucleotides contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present within a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained within a vector is considered isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides that are maintained in a heterologous host cell or that are purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules that are produced synthetically. In addition, a polynucleotide or nucleic acid can contain regulatory elements, such as a promoter, enhancer, ribosomal binding site, or transcription termination signal.

[0034] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide consisting of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is typically not translated into amino acids but may be considered to be part of the coding region, however, any flanking sequences, e.g., promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are typically determined by a start codon at the 5' end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention are present within a single polynucleotide construct, e.g., on a single vector, or within separate polynucleotide constructs, e.g., on separate (different) vectors. Thus, a single vector can contain only a single coding region or can include two or more coding regions, e.g., a single vector can separately encode a first polypeptide chain and a second polypeptide chain of a chimeric molecule as described below. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to the nucleic acid encoding the chimeric molecule of the invention. Heterologous coding regions include, without limitation, specialized elements or motifs such as secretory signal peptides or heterologous functional domains.

[0035] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein upon initiation of transport of the growing protein chain across the rough endoplasmic reticulum. Those skilled in the art will recognize that signal peptides are generally fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, such as the FVIII signal peptide or the VWF signal peptide, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide, such as the human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, may be used.

[0036] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to a sequence following the start of translation. For example, the translation start codon of a gene is located downstream of the start site of translation.

[0037] The term "upstream" refers to a nucleotide sequence located 5' relative to a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence refers to a coding region or a sequence located 5' from the start of translation. For example, most promoters are located upstream of the translation start site.

[0038] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that influence the transcription, RNA processing, stability, and translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences will usually be located 3' to the coding sequence.

[0039] A polynucleotide encoding a gene product, e.g., a polypeptide, can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. In operably associated, a coding region for a gene product, e.g., a polypeptide, is operably associated with one or more regulatory regions in such a manner that expression of the gene product is under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if the introduction of promoter function results in transcription of an mRNA that encodes the gene product encoded by the coding region, and if the nature of the binding between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the gene product or the ability of the DNA template to be transcribed. Other transcription control elements other than a promoter, e.g., enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0040] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as promoter and enhancer segments from cytomegalovirus (immediate early promoter combined with intron-A), Simian Virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences that are capable of controlling gene expression in eukaryotic cells. Further suitable transcription control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0041] Similarly, various translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

[0042] The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product, e.g., an RNA or a polypeptide. It can be expressed as messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (SRNA), or a polypeptide. Expression includes, without limitation, transcription into small interfering RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids that have undergone post-transcriptional modification, e.g., polyadenylation or splicing, or polypeptides that have undergone post-translational modification, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0043] "Vector" refers to any vehicle for cloning and / or transporting a nucleic acid into a host cell. A 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 (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of in vivo replication, i.e., capable of replication under its own control. The term "vector" includes both viral and non-viral vehicles for introducing nucleic acid into cells in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector may be accomplished by ligation of an appropriate polynucleotide fragment into a selected vector having complementary cohesive termini.

[0044] Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), etc. Selectable markers can also be considered as reporters.

[0045] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genome-integrating sequences, phages or nucleotide sequences of single- or double-stranded linear, circular or supercoiled DNA or RNA from any source, many of which are combined or engineered into a unique structure that allows the introduction of a promoter fragment and a DNA sequence for a selected gene product, along with appropriate 3' untranslated sequences, into a cell.

[0046] Eukaryotic viral vectors that may be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxviruses, such as vaccinia virus vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0047] A "cloning vector" is a unit length of nucleic acid that is replicated sequentially, such as a plasmid, phage, or other nucleic acid segment to which another nucleic acid segment can be attached so as to bring about replication of the attached segment. A cloning vector refers to a "replicon" that contains an origin of replication, such as a plasmid, a plasmid p53, or a cosmid. A particular cloning vector is capable of replication in one cell type, e.g., bacteria, and expression in another cell type, e.g., a eukaryotic cell. Cloning vectors typically contain one or more sequences that can be used for selection of cells that contain one or more multiple cloning sites for insertion of the vector and / or nucleic acid sequence of interest.

[0048] The term "expression vector" refers to a vehicle designed to allow for the expression of an inserted nucleic acid sequence after insertion into a host cell, the inserted nucleic acid sequence being placed in operative association with a regulatory region as described above.

[0049] Vectors are introduced into host cells by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or a DNA vector transporter.

[0050] "Culture", "culturing" and "culturing" as used herein refer to incubating cells under in vitro conditions that permit cell growth or division or maintaining cells alive. "Cultured cells" as used herein refer to cells that have been propagated in vitro.

[0051] As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain(s) of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain(s) of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced recombinantly, but is not necessarily translated from a specified nucleic acid sequence. It may be purified in any manner, including chemical synthesis.

[0052] An "isolated" polypeptide, or a fragment, variant, or derivative thereof, refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by suitable techniques.

[0053] Further included in the present invention are fragments or variants of the polypeptides, and any combination thereof. The term "fragment" or "variant", when referring to a polypeptide binding domain or a binding molecule of the present invention, includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding domain, or FcRn binding affinity for an Fc variant, coagulant activity for a FVIII variant, or FVIII binding activity for a VWF protein). Polypeptide fragments include proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments described elsewhere herein, but are not limited to naturally occurring fragments. The present invention does not include full-length polypeptides (or mature polypeptides) of the above-mentioned form. Variants of the polypeptide binding domain or binding molecule of the present invention include fragments as described above, and also polypeptides having modified amino acid sequences by amino acid substitutions, deletions, insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0054] The term "VWF fragment" or "s" as used herein refers to any VWF fragment that interacts with FVIII and retains at least one or more properties normally provided to FVIII by full-length VWF, such as preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that may lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and / or stabilizing FVIII heavy and light chain interactions. In a particular embodiment, "VWF fragment" as used herein includes the D' and D3 domains of the VWF protein, but does not include the A1, A2, A3, D4, B1, B2, B3, C1, C2, and CK domains of the VWF protein.

[0055] The term "half-life limiting factor" or "FVIII half-life limiting factor" as used herein refers to a factor that prevents the half-life of a FVIII protein from being longer than 1.5-fold or 2-fold compared to wild-type FVIII (e.g., ADVATE® or REFACTO®). For example, full-length or mature VWF can act as a FVIII half-life limiting factor by triggering FVIII and VWF complexes to be cleared from the system by 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 bound to a FVIII protein is a FVIII half-life limiting factor.

[0056] The term "endogenous VWF" as used herein refers to a VWF molecule that is naturally present in plasma. Endogenous VWF molecules may be multimeric, but not monomeric or dimeric. Endogenous VWF in plasma binds to FVIII and forms a non-covalent complex with FVIII.

[0057] "Conservative amino acid substitution" refers to an amino acid residue that is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0058] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. As discussed herein, it is understood that any particular polypeptide has a similar sequence to another polypeptide. Whether a sequence is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a polypeptide of the present invention can be determined by using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence in accordance with the present invention, the parameters are set, of course, so that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and so that differences in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0059] As used herein, an "amino acid corresponding to" or "equivalent amino acid" in a VWF or FVIII protein sequence is identified by alignment to maximize identity or similarity between a first and a second VWF or FVIII sequence. The number used to recognize 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.

[0060] A "fusion" or "chimeric" molecule comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. Amino acid sequences that are normally present in separate proteins can be linked in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new arrangement in a fusion polypeptide, e.g., a fusion of a Factor VIII domain of the present invention with an immunoglobulin Fc domain. Fusion proteins are created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0061] As used herein, the term "half-life" refers to a particular polypeptide biological half-life in vivo. Half-life can be represented by the time required for half of the amount administered to a subject to be cleared from the circulation and / or other tissues in the animal. When the clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid alpha phase and a longer beta phase. The alpha phase typically represents the equilibrium state of the administered polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The beta phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, the chimeric molecules of the present invention are monophasic, and thus have no alpha phase, but only a single beta phase. Thus, in certain embodiments, as used herein, the term half-life refers to the half-life of a polypeptide in the beta phase. The typical beta phase half-life of a human antibody in humans is 21 days.

[0062] The term "heterologous" when applied to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is derived from a different entity than the entity to which it is being compared. Thus, a heterologous polypeptide bound to a VWF protein refers to a polypeptide chain that is bound to a VWF protein and that is not a naturally occurring part of the VWF protein. For example, a heterologous polynucleotide or antigen may be derived from a different species. , from different cell types in one individual, or from the same or different types of cells in another individual.

[0063] The term "linked", "fused" or "connected" as used herein refers to a first amino acid sequence or nucleotide sequence joined to a second amino acid sequence or nucleotide sequence (e.g., via a peptide bond or phosphodiester bond, respectively). The term "covalently linked" or "covalent linkage" refers to a covalent bond, e.g., a disulfide bond, a peptide bond, or one or more amino acids between two moieties that are joined together, e.g., a linker. The first amino acid or nucleotide sequence can be directly connected to the second amino acid or nucleotide sequence, or alternatively, an intervening sequence can connect the first sequence to the second sequence. The term "linked", "fused" or "connected" refers not only to the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or second amino acid sequence) within any two amino acids of the second amino acid sequence (or first amino acid sequence). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (in a polypeptide chain), or a nucleotide or nucleotide chain (in a nucleotide chain), or any chemical moiety (in both polypeptide and polynucleotide chains). Covalent bonds are sometimes indicated as (-) or hyphens.

[0064] As used herein, the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" refers to a covalent bond, a non-peptide bond, or a non-covalent bond. In some embodiments, the association is indicated by a colon, i.e., (:). In another embodiment, it refers to a covalent bond other than a peptide bond. In other embodiments, the term "covalently associated with" as used herein refers to an association between two moieties by a covalent bond, e.g., a disulfide bond, a peptide bond, one or more amino acids (e.g., a linker). For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma (σ) bonds, pi (π) bonds, delta (δ) bonds, glycosidic bonds, agnostic bonds, bent bonds, dipolar bonds, pi-back bonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, hapticity, or antibonds. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilic, intercalation, stacking, entropic forces, or chemical polarity.

[0065] As used herein, "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. In one embodiment, a polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during the coagulation cascade, such that cleavage of such a site occurs at the site of thrombus formation. In another embodiment, the FVIII protein and the second The FVIII linker connecting the heterologous moieties can include a cleavage site. Exemplary such sites include, for example, those recognized by thrombin, factor XIa, or factor Xa. Exemplary FXIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO: 27) and SVSQTSKLTR (SEQ ID NO: 28). Exemplary thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 29), TTKIKPR (SEQ ID NO: 30), LVPRG (SEQ ID NO: 31), and ALRPR (amino acids 1-5 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."

[0066] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide that is the target of an enzyme that functions after translation of the polypeptide. In one embodiment, such an enzyme functions during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave the polypeptide prior to secretion of the protein from the cell. Examples of such processing sites include those targeted by, for example, PACE / furin (PACE is an acronym for Paired basic Amino acid Cleaving Enzyme) members of the endopeptidases. These enzymes localize to the Golgi membrane and cleave proteins on the carboxy-terminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein, the "furin" family of enzymes includes, for example, PCSK1 (also known as PC1 / Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known as PC7 / LPC, PC8, or SPC7). Other processing sites are known in the art. The term "processable linker" as used herein refers to a linker that includes an intracellular processing site.

[0067] The term "furin" refers to the enzyme corresponding to the EC number 3.4.21.75. Furin is a subtilisin-like proprotein convertase, also known as PACE (Paired basic amino acid cleaving enzyme). Furin removes segments of inactive precursor proteins and converts them into biologically active proteins. During its intracellular transport, the propeptide is cleaved from the mature VWF molecule by the furin enzyme in the Golgi.

[0068] It will be understood that in constructs that include more than one processing or cleavage site, such sites may be the same or different.

[0069] Hemostatic disorder, as used herein, refers to an inherited or acquired condition characterized by a tendency to bleed, either spontaneously or as a result of trauma, due to an impaired ability or inability to form fibrin clots. Examples of such disorders include hemophilia, the three major forms of which are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency, or "Christmas disease"), and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, and Bernard-Soulier syndrome, which is a defect or deficiency of GPIb. The receptor for VWF, GPIb, may be defective, resulting in defects in primary clot formation (primary hemostasis) and increased bleeding tendency, as well as the platelet aplasia of Glanzman and Naegeli. In liver failure (acute and chronic), there is insufficient production of clotting factors by the liver, which may increase the risk of bleeding.

[0070] The chimeric molecule of the present invention can be used prophylactically. As used herein, "prophylactic treatment" refers to administration of the molecule before a bleeding episode. In one embodiment, the subject in need of general hemostatic medicine is undergoing or about to undergo surgery. The chimeric protein of the present invention can be administered as a prophylactic drug before or after surgery. The chimeric protein of the present invention can be administered during or after surgery to control acute bleeding episodes. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0071] The chimeric molecules of the present invention are also used for on-demand (also referred to as "episodic") treatment. The term "on-demand treatment" or "episodic treatment" refers to the administration of chimeric molecules in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one embodiment, on-demand (episodic) treatment can be administered to a subject when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another embodiment, on-demand treatment can be administered before an activity that increases the risk of bleeding, such as contact sports.

[0072] As used herein, the term "acute bleeding" refers to bleeding episodes regardless of underlying cause.For example, subject may have trauma, uremia, genetic bleeding disease (e.g., factor VII deficiency), platelet disorder, tolerance due to the development of antibodies against coagulation factors.

[0073] Treat, treatment, treating, as used herein, refers to, for example, reducing the severity of a disease or condition, reducing the duration of a disease process, alleviating one or more symptoms associated with a disease or condition, providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition, or preventing one or more symptoms associated with a disease or condition. In one embodiment, "treating" or "treatment" means maintaining a FVIII trough level in a subject at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL by administering a chimeric molecule of the invention. In another embodiment, treating or treatment means maintaining FVIII trough levels at about 1 to about 20 IU / dL, about 2 to about 20 IU / dL, about 3 to about 20 IU / dL, about 4 to about 20 IU / dL, about 5 to about 20 IU / dL, about 6 to about 20 IU / dL, about 7 to about 20 IU / dL, about 8 to about 20 IU / dL, about 9 to about 20 IU / dL, or about 10 to about 20 IU / dL. Treating or treating a disease or condition also includes maintaining FVIII activity in a subject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophilic subject. The minimum trough level required for treatment can be determined by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0074] II. Chimeric molecules The chimeric molecules of the present invention are designed to improve the release of VWF or FVIII proteins from another moiety to which they are fused. The present invention provides a thrombin cleavable linker that can be rapidly and efficiently cleaved at the site of injury. In one embodiment of the present invention, the chimeric molecule is a von Willebrand The polypeptide can comprise a factor VIII (FVIII) protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous moiety, the VWF linker comprising a polypeptide selected from (i) the a2 region from factor VIII (FVIII), (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof, and the XTEN sequence is connected to the VWF protein, the heterologous moiety (H1), the VWF linker, or any combination thereof. In another embodiment of the invention, the chimeric molecule can comprise a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting the VWF protein and the heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein the VWF linker in the first polypeptide chain comprises (i) the a2 region from FVIII, (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof, and wherein the first polypeptide chain and the second polypeptide chain associate with each other.

[0075] In other embodiments of the invention, the chimeric molecule comprises a polypeptide chain comprising a FVIII protein fused to a heterologous moiety via a FVIII linker, the FVIII linker comprising (i) the a2 region from FVIII, (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof.

[0076] II.A. Chimeric molecules with VWF, XTEN and VWF linker The present invention provides chimeric molecules 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 from FVIII, (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof.

[0077] In one embodiment, the chimeric molecule comprises a VWF protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous moiety, wherein the XTEN sequence is located between the VWF protein and the VWF linker, and the VWF linker comprises a polypeptide selected from (i) the a2 region from factor VIII (FVIII), (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof. In another embodiment, the chimeric molecule comprises a VWF protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein to the heterologous moiety, wherein the XTEN sequence is located between the VWF linker and the heterologous moiety, and the VWF linker comprises a polypeptide selected from (i) the a2 region from FVIII, (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, wherein X is an aliphatic amino acid, or (v) any combination thereof.

[0078] In another embodiment, the chimeric molecule further comprises a polypeptide chain comprising a FVIII protein, wherein the first chain comprising the VWF protein and the second chain comprising the FVIII protein are associated with each other. In one example, the association is by a covalent bond, e.g., a disulfide bond, association. In still other embodiments, the polypeptide chain comprising the FVIII protein further comprises an additional XTEN sequence. The additional XTEN sequence can be attached to the N-terminus or C-terminus of the FVIII protein, or can be inserted between two adjacent FVIII amino acids. In still other embodiments, the chain comprising the FVIII protein further comprises a second heterologous moiety (H2). In some embodiments, the FVIII protein is fused to the second heterologous moiety via a FVIII linker. In certain embodiments, the FVIII linker is the same as the VWF linker that connects the VWF protein and the heterologous moiety. In other embodiments, the FVIII linker is different from the VWF linker that connects the VWF protein and the heterologous moiety.

[0079] In certain embodiments, the chimeric molecule comprises: (i) V-L1-X1-H1:H2-L2-X2-C, (ii) V-X1-L1-H1:H2-L2-X2-C, (iii) V-L1-X1-H1:H2-X2-L2-C, (iv) V-X1-L1-H1:H2-X2-L2-C, (v) V-L1-X1-H1:H2-L2-C ( X2), (vi)V-X1-L1-H1:H2-L2-C(X2), (vii)C-X2-L2-H2:H1-X1-L1-V, (viii)C-X2-L2-H2 :H1-L1-X1-V, (ix)C-L2-X2-H2:H1-L1-X1-V, (x)C-L2-X2-H2:H1-L1-X1-V, (xi)C(X2)-L wherein V is a VWF protein, L1 is a VWF linker, L2 is an optional FVIII linker, H1 is a first heterologous moiety, H2 is a second heterologous moiety, X1 is an XTEN sequence, X2 is an optional XTEN sequence, C is a FVIII protein, C(X2) is a FVIII protein fused to an XTEN sequence, the XTEN sequence is inserted between two adjacent FVIII amino acids, (-) is a peptide bond or one or more amino acids, and (:) is a covalent bond between H1 and H2.

[0080] In some embodiments, the FVIII protein in the chimeric molecule comprises a third heterologous moiety (H3), which may be an XTEN sequence. In other embodiments, the FVIII protein in the chimeric molecule comprises a fourth heterologous moiety (H4), which may be an XTEN sequence. In still other embodiments, the FVIII protein in the chimeric molecule comprises a fifth heterologous moiety (H5), which may be an XTEN sequence. In yet other embodiments, the FVIII protein in the chimeric molecule comprises a sixth heterologous moiety (H6), which may be an XTEN sequence. In certain embodiments, one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are capable of extending the half-life of the chimeric molecule. In some embodiments, the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are attached to the C-terminus or N-terminus of FVIII or are inserted between two amino acids of the FVIII protein.

[0081] II.B. Chimeric molecule having FVIII, XTEN, VWF protein and VWF linker The present invention also provides a chimeric molecule comprising a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting the VWF protein and the heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein the VWF linker in the first polypeptide chain comprises (i) an a2 region from FVIII, (ii) an a1 region from FVIII, (iii) an a3 region from FVIII, (iv) an XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, where X is an aliphatic amino acid, a thrombin cleavage site, or (v) any combination thereof, and wherein the first and second polypeptide chains associate with each other. In one embodiment, the XTEN sequence is connected to the N-terminus or C-terminus of the FVIII protein. In another embodiment, the chimeric molecule further comprises an additional XTEN sequence connected to the VWF protein, the heterologous moiety, the VWF linker, or any combination thereof. In another embodiment, the chimeric molecule further comprises a second heterologous moiety (H2). In another embodiment, the second heterologous moiety of the chimeric molecule is connected to the FVIII protein, the XTEN sequence, or both. In still other embodiments, the second heterologous moiety is connected to the FVIII protein or the XTEN sequence via a FVIII linker. In yet other embodiments, the FVIII linker is the same as the VWF linker. In some embodiments, the FVIII linker is different from the VWF linker.

[0082] In certain embodiments, the chimeric molecule comprises: (i) V-L1-X1-H1:H2-L2-X2-C, (ii) V-X1-L1-H1:H2-L2-X2-C, (iii) V-L1-X1-H1:H2-X2-L2-C, (iv) V-X1-L1-H1:H2-X2-L2-C, (v) V-L1-X1-H1:H2-L2-C ( X2), (vi)V-X1-L1-H1:H2-L2-C(X2), (vii)C-X2-L2-H2:H1-X1-L1-V, (viii)C-X2-L2-H2 :H1-L1-X1-V, (ix)C-L2-X2-H2:H1-L1-X1-V, (x)C-L2-X2-H2:H1-L1-X1-V, (xi)C(X2)-L 2-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 a first heterologous moiety, H2 is a second heterologous moiety, X1 is an optional XTEN sequence, X2 is an XTEN sequence, C is a FVIII protein, C(X2) is a FVIII protein fused to an XTEN sequence, the XTEN sequence is inserted between two adjacent FVIII amino acids, (-) is a peptide bond or one or more amino acids, and (:) is a covalent bond between H1 and H2. In one embodiment, the VWF linker and the FVIII linker can be the same. In another embodiment, the VWF linker and the FVIII linker are different.

[0083] In certain embodiments, the FVIII protein of the chimeric molecule comprises a third heterologous portion (H3) that can be an XTEN sequence. In other embodiments, the FVIII protein of the chimeric molecule comprises a fourth heterologous portion (H4) that is an XTEN sequence. In still other embodiments, the FVIII protein of the chimeric molecule comprises a fifth heterologous portion (H5) that can be an XTEN sequence. In yet other embodiments, the FVIII protein in the chimeric molecule comprises a sixth heterologous portion (H6) that can be an XTEN sequence. In certain embodiments, one or more of the third heterologous portion (H3), the fourth heterologous portion (H4), the fifth heterologous portion (H5), and the sixth heterologous portion (H6) can extend the half-life of the chimeric molecule. In some embodiments, the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and / or the sixth heterologous moiety (H6) are attached to the C-terminus or N-terminus of FVIII or are inserted between two amino acids of the FVIII protein.

[0084] II.C. Chimeric molecules with FVIII, XTEN, and a FVIII linker The chimeric molecule of the invention can comprise a FVIII protein, an XTEN sequence, and a heterologous moiety fused by a FVIII linker that includes (i) the a2 region from FVIII, (ii) the a1 region from FVIII, (iii) the a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interacting motif, where X is an aliphatic amino acid, or (v) any combination thereof. In certain embodiments, the chimeric molecule can comprise two polypeptide chains, a first chain comprising a FVIII protein fused to a first Fc region via a FVIII linker, and a VWF protein (e.g., the D' and D3 domains of VWF) fused to the Fc region. and a second chain comprising a FVIII linker comprising: (i) an a2 region from FVIII, (ii) an a1 region from FVIII, (iii) an a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, where X is an aliphatic amino acid, or (v) any combination thereof, and the first and second polypeptide chains are associated with each other, and the XTEN sequence is attached to the first polypeptide (e.g., the N-terminus or C-terminus of the FVIII protein, the linker, or the first Fc region, or within the FVIII protein), the second polypeptide (e.g., the N-terminus or C-terminus of the VWF protein or the Fc region, or within the FVIII protein), or both. In certain embodiments, the linker in the first polypeptide chain comprises the a2 region from FVIII.

[0085] In certain embodiments, the chimeric molecule comprises: (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-X2-L2-H2:H1-X1-L1-C, (v) V-L2-X2-H2:H1-L1-C(X1), (vi) V-X2-L2-H2:H1-L1-C(X1), (vii)C-X1-L1-H1:H2-X2-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) a formula selected from C(X1)-L1-H1:H2-L2-X2-V, where V is a VWF protein, L1 is a FVIII linker, L2 is an optional VWF linker, H1 is a first heterologous moiety, H2 is a second heterologous moiety, X1 is an optional XTEN sequence, X2 is an optional XTEN sequence, C is a FVIII protein, C(X1) is a FVIII protein fused to an XTEN sequence, the XTEN sequence is inserted between two adjacent FVIII amino acids, (-) is a peptide bond or one or more amino acids, (:) is a covalent bond between H1 and H2, and 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.

[0086] II.D. Components of Chimeric Molecules II.C.1. VWF linker or FVIII linker The VWF or FVIII linker useful in the chimeric molecule of the present invention is a thrombin cleavable linker that fuses the VWF protein to a heterologous moiety or the FVIII protein to a heterologous moiety. In one embodiment, the VWF or FVIII linker comprises the a1 region of FVIII. In another embodiment, the VWF or FVIII linker comprises the a2 region of FVIII. In another embodiment, the VWF or FVIII linker comprises the a3 region of FVIII. In yet another embodiment, the VWF or FVIII linker comprises a thrombin cleavage site that comprises XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, where X is an aliphatic amino acid.

[0087] In one embodiment, the VWF or FVIII linker comprises an a1 region that comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to Met337-Arg372 corresponding to full-length mature FVIII, and the a1 region is capable of being cleaved by thrombin. In another embodiment, the VWF or FVIII linker comprises an a1 region that comprises an amino acid sequence 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, and the a1 region is capable of being cleaved by thrombin. In other embodiments, the VWF or FVIII linker further comprises additional amino acids, for example, 1, 2, 3, 4, 5, 10 or more. In certain embodiments, the VWF or FVIII linker comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5).

[0088] In some embodiments, the VWF or FVIII linker comprises an a2 region that comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to Glu720 to Arg740 corresponding to full-length mature FVIII, and the a2 region is capable of being cleaved by thrombin. In other embodiments, the VWF or FVIII linker comprises an a2 region that comprises an amino acid sequence 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 to 743 corresponding to full-length mature FVIII. In still other embodiments, the VWF or FVIII linker further comprises additional amino acids, e.g., 1, 2, 3, 4, 5, 10 or more. In a particular embodiment, the VWF linker comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4).

[0089] In certain embodiments, the VWF linker or FVIII linker comprises an a3 region that comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to Glu1649-Arg1689 corresponding to full-length mature FVIII, and the a3 region is capable of being cleaved by thrombin. In some embodiments, the VWF linker or FVIII linker comprises an a3 region that comprises an amino acid sequence 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, and the a3 region is capable of being cleaved by thrombin. In other embodiments, the VWF linker or FVIII linker further comprises additional amino acids, for example, 1, 2, 3, 4, 5, 10 or more. In certain embodiments, the VWF linker or FVIII linker comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6).

[0090] In other embodiments, the VWF linker or FVIII linker comprises a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, and the PAR1 non-active site interaction motif comprises SFLLRN (SEQ ID NO: 7). In some embodiments, the PAR1 non-active site interaction motif further comprises an amino acid sequence selected from P, PN, PND, PNDK (SEQ ID NO: 8), PNDKY (SEQ ID NO: 9), PNDKYE (SEQ ID NO: 10), PNDKYEP (SEQ ID NO: 11), PNDKYEPF (SEQ ID NO: 12), PNDKYEPFW (SEQ ID NO: 13), PNDKYEPFWE (SEQ ID NO: 14), PNDKYEPFWED (SEQ ID NO: 20), PNDKYEPFWEDE (SEQ ID NO: 21), PNDKYEPFWEDEE (SEQ ID NO: 22), PNDKYEPFWEDEES (SEQ ID NO: 23), or any combination thereof. In other embodiments, the aliphatic amino acid of the thrombin cleavage site comprising XVPR is selected from glycine, alanine, valine, leucine, or isoleucine. In certain embodiments, the thrombin cleavage site comprises the LVPR. In some embodiments, thrombin cleaves the VWF linker or FVIII linker faster than thrombin cleaves the thrombin cleavage site (e.g., LVPR) when the thrombin cleavage site (LVPR) is replaced with a VWF linker or FVIII linker, respectively (i.e., without the PAR1 non-active site interaction motif). In some embodiments, thrombin cleaves the VWF linker or FVIII linker at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times faster than thrombin cleaves the thrombin cleavage site (e.g., LVPR) when the thrombin cleavage site (e.g., LVPR) is replaced with a VWF linker or FVIII linker.

[0091] In some embodiments, the VWF linker or FVIII linker comprising (i) the a1 region, (ii) the a2 region, (iii) the a3 region, or (iv) the thrombin cleavage site XVPR and PAR1 non-active site interacting motif is at least about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 310, 320, 330, 340, 350, 360, 370, 380, 390, , 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 length. In one embodiment, the one or more amino acids comprise a gly peptide. In another embodiment, the one or more amino acids comprise a GlyGly. In another embodiment, the one or more amino acids comprise an IleSer. In yet another embodiment, the one or more amino acids comprise a gly / ser peptide. In still other embodiments, the one or more amino acids comprise a gly / ser peptide having the formula of (Gly4Ser)n or S(Gly4Ser)n, where n is a positive integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the one or more amino acids comprise (Gly4Ser)3 (SEQ ID NO:89) or (Gly4Ser)4 (SEQ ID NO:90).

[0092] II.C.2. VWF protein VWF (also known as F8VWF) is a large multimeric glycoprotein present in plasma and constitutively produced in endothelial cells (within Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2813 amino acid protein. All monomers contain a number of specific domains with specific functions: the D' / D3 domain (binds factor VIII), the A1 domain (binds platelet GPIb receptors, heparin, and / or possibly collagen), the A3 domain (binds collagen), the C1 domain (RGD domain binds platelet integrin αIIbβ3 when it is activated), and a "cysteine ​​knot" domain at the C-terminal end of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ), and β-human chorionic gonadotropin (βHCG)).

[0093] The term "VWF protein" as used herein includes, but is not limited to, a full-length VWF protein or a functional VWF fragment comprising the D' and D3 domains that is 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 the VWF clearance pathway. The VWF protein includes derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0094] The 2813 monomer amino acid sequence for human VWF is reported in Genbank under the accession number _NP_000543.2__. Nucleotide sequence encoding human VWF is reported in Genbank under the accession number __NM_000552.3_. The nucleotide sequence of human VWF is designated as SEQ ID NO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. The domains of VWF are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0095] As used herein, the VWF protein can include the D' and D3 domains of VWF, and the VWF protein binds to FVIII and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF protein including the D' and D3 domains can include the A1 domain, A2 domain, A3 domain, D1 domain, D2 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, or any combination thereof. In one embodiment, the VWF protein comprises, consists essentially of, or consists of: (1) the D' and D3 domains of VWF, or a fragment thereof; (2) the D1, D', and D3 domains of VWF, or a fragment thereof; (3) the D2, D', and D3 domains of VWF, or a fragment thereof; (4) the D1, D2, D', and D3 domains of VWF, or a fragment thereof; or (5) the D1, D2, D', D3, or A1 domains of VWF, or a fragment thereof. The VWF proteins described herein do not contain a VWF clearance receptor binding site. The VWF proteins of the present invention can include any other sequence that is linked or fused to the VWF protein. For example, the VWF proteins described herein can further include a signal peptide.

[0096] In one embodiment, the VWF protein binds to or associates with the FVIII protein. By binding to or associating with the FVIII protein, the VWF protein of the present invention can protect FVIII from protease cleavage and FVIII activation, stabilize the heavy and light chains of FVIII, and prevent the clearance of FVIII by scavenger receptors. In another embodiment, the VWF protein binds to or associates with the FVIII protein, blocking or preventing the binding of FVIII protein to phospholipids and activated protein C. By preventing or inhibiting the binding of FVIII protein to endogenous full-length VWF, the VWF protein of the present invention reduces the clearance of FVIII by endogenous VWF clearance receptors, thus extending the half-life of the FVIII protein. Thus, the extended half-life of the FVIII protein is due to the association of the FVIII protein with the VWF protein, which lacks a VWF clearance receptor binding site, thereby shielding and / or protecting the FVIII protein from endogenous VWF, which contains a VWF clearance receptor binding site. The FVIII protein bound to or protected by the VWF protein also allows the FVIII protein to be recycled. By eliminating the VWF clearance pathway receptor binding site in the full-length VWF molecule, the FVIII / VWF heterodimer of the present invention is shielded from the VWF clearance pathway, further extending the FVIII half-life.

[0097] In one embodiment, the VWF protein of the invention comprises a D' domain and a D3 domain of VWF, the D' domain being 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 D3 domain of VWF, the D3 domain being at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO: 2, and the VWF protein prevents endogenous VWF from binding to FVIII. In some embodiments, the VWF proteins described herein comprise, consist essentially of, or consist of the D' and D3 domains of VWF that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 764-1240 of SEQ ID NO: 2, and the VWF proteins prevent endogenous VWF from binding to FVIII. In other embodiments, the VWF proteins comprise, consist essentially of, or consist of the D1, D2, D' and D3 domains that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 23-1240 of SEQ ID NO: 2, and the VWF proteins prevent endogenous VWF from binding to FVIII. In still other embodiments, the VWF proteins further comprise a signal peptide operably linked thereto.

[0098] In some embodiments, the VWF protein of the present invention comprises: (1) a D'D3 domain; 2), up to about 15 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:2 to amino acids 764-1255 of SEQ ID NO:2), up to about 20 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:2 to amino acids 764-1260 of SEQ ID NO:2), up to about 25 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:2 to amino acids 764-1265 of SEQ ID NO:2), or up to about 30 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:2 to amino acids 764-1260 of SEQ ID NO:2). In certain embodiments, the VWF protein comprising or consisting essentially of the D' and D3 domains is not amino acids 764-1274 of SEQ ID NO:2 or is full-length mature VWF. In some embodiments, the D1D2 domains are expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domains are expressed in cis with the D'D3 domain.

[0099] In other embodiments, the VWF protein comprising a D'D3 domain linked to a D1D2 domain further comprises an intracellular processing site that allows cleavage of the D1D2 domain from the D'D3 domain upon expression, e.g., a PACE (furin) or PC5 processing site. Non-limiting examples of intracellular processing sites are described elsewhere herein.

[0100] In yet another embodiment, the VWF protein comprises the D' domain and the D3 domain but does not comprise an amino acid sequence selected from (1) amino acids 1241 to 2813 of SEQ ID NO: 2, (2) amino acids 1270 to 2813 of SEQ ID NO: 2, (3) amino acids 1271 to 2813 of SEQ ID NO: 2, (4) amino acids 1272 to 2813 of SEQ ID NO: 2, (5) amino acids 1273 to 2813 of SEQ ID NO: 2, (6) amino acids 1274 to 2813 of SEQ ID NO: 2, or any combination thereof.

[0101] In still other embodiments, the VWF protein of the invention comprises, consists essentially of, or consists of an amino acid sequence corresponding to the D', D3, and A1 domains, which 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 binding of endogenous VWF to FVIII. In certain embodiments, the VWF protein is not amino acids 764-1274 of SEQ ID NO: 2.

[0102] In some embodiments, the VWF protein of the present invention comprises a D' domain and a D3 domain, but is not limited to: (1) an A1 domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2 domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CK domain, (11) a CK domain and a C2 domain, (12) a CK domain, a C2 domain, and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, and a B3 domain, (14) a CK domain, a C2 domain, a C1 domain, a B3 domain, and a B domain. 2 domain, (15) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, and B1 domain, (16) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, and D4 domain, (17) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, and A3 domain, (18) CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain, and A2 domain, (19) CK domain, C2 domain, C1 (20) does not contain at least one VWF domain selected from the group consisting of the B3 domain, the B2 domain, the B1 domain, the D4 domain, the A3 domain, the A2 domain, and the A1 domain, or any combination thereof.

[0103] In yet other embodiments, the VWF protein comprises a D'D3 domain and one or more domains or modules. Examples of such domains or modules include, but are not limited to, those disclosed in Zhour et al., Blood, published online April 6, 2012, DOI 10.1182 / blood-2012-01-405134. For example, the VWF protein can comprise a D'D3 domain and one or more domains or modules selected from an A1 domain, an A2 domain, an A3 domain, a D4N module, a VWD4 module, a C8-4 module, a TIL-4 module, a C1 module, a C2 module, a C3 module, a C4 module, a C5 module, a C5 module, a C6 module, or any combination thereof.

[0104] In certain embodiments, the VWF protein of the present invention forms multimers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, or higher multimers. In other embodiments, the VWF protein is a monomer with only one VWF protein. In some embodiments, the VWF protein of the present invention can have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF protein can contain amino acid substitutions, deletions, additions, or modifications such that the VWF protein cannot form disulfide bonds or form dimers or multimers. In another embodiment, the amino acid substitutions are in the D' and D3 domains. In certain embodiments, the VWF protein of the present invention contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. The at least one amino acid substitution can be any amino acid that does not naturally occur in wild-type VWF. For example, the amino acid substitution can be any amino acid other than cysteine, such as isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, or histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the VWF protein from forming multimers.

[0105] In some embodiments, the VWF protein includes an amino acid substitution from cysteine ​​to alanine at residue 336 (residue 1099 of SEQ ID NO:2), which corresponds to the D'D3 domain of VWF, an amino acid substitution from cysteine ​​to alanine at residue 379 (residue 1142 of SEQ ID NO:2), which corresponds to the D'D3 domain of VWF, or both.

[0106] In certain embodiments, the VWF protein useful herein may be further modified to improve its interaction with FVIII, e.g., to improve binding affinity to FVIII. As a non-limiting example, the VWF protein includes a serine residue at a residue corresponding to amino acid 764 of SEQ ID NO:2 and a lysine residue at a residue corresponding to amino acid 773 of SEQ ID NO:2. Residues 764 and / or 773 may contribute to the binding affinity of the VWF protein to FVIII. In other embodiments, the VWF protein useful in the present invention may have other modifications, e.g., the protein may be PEGylated, glycosylated, HESylated, or polysialylated.

[0107] II.C.3. Dissimilar parts The heterologous moiety, which may be fused to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker, may be a heterologous polypeptide or a heterologous non-polypeptide. In certain embodiments, the heterologous moiety is a half-life extending molecule known in the art, and includes a polypeptide, a non-polypeptide moiety, or a combination of both. The heterologous polypeptide moiety can include a FVIII protein, an immunoglobulin constant region or a portion thereof, albumin or a fragment thereof, an albumin binding moiety, a transferrin or a fragment thereof, a PAS sequence, an HAP sequence, a derivative or a variant thereof, a C-terminal peptide (CTP) of the human chorionic gonadotropin β subunit, or any combination thereof. In some embodiments, the non-polypeptide binding moiety includes polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combination thereof. In certain embodiments, there can be one, two, three or more heterologous moieties, each of which can be the same or different molecules.

[0108] II.C.3.a Immunoglobulin constant regions or portions thereof Immunoglobulin constant regions are composed of domains that exhibit CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA IgD, or IgE), the constant region may be composed of three or four CH domains. Some isotype (e.g., IgG) constant regions also contain a hinge region. Janeway See et al. 2001, Immunobiology, Garland Publishing, NY, NY.

[0109] The immunoglobulin constant region or a 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 a portion thereof is derived from a human immunoglobulin. However, it is understood that the immunoglobulin constant region or a portion thereof can be derived from the immunoglobulin of another mammalian species, including rodents (e.g., mouse, rat, rabbit, guinea pig), or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, the immunoglobulin constant region or a portion thereof can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgG1 is used.

[0110] A variety of immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domain sequences can be selected with (or lacking) specific effector functions or with specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be derived from these sequences using art-recognized techniques. The genetic material obtained using any of the foregoing methods can then be modified or synthesized to obtain the polypeptides of the invention. It will be further understood that the scope of the invention encompasses alleles, variants, and mutants of the constant region DNA sequences.

[0111] Immunoglobulin constant region or partial sequences can be cloned, for example, using polymerase chain reaction and primers selected to amplify the domain of interest. To clone immunoglobulin constant region or partial sequences from an antibody, mRNA can be isolated from hybridoma, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody genes amplified by PCR. PCR amplification methods are described in U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, and in, for example, "PCR Protocols: A Guide to Methods and Applications" Innis et al. ds., Academic Press, San Diego, CA (1990); et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270).

[0112] As used herein, an immunoglobulin constant region can include all domains and a hinge region, or a portion thereof. In one embodiment, an immunoglobulin constant region or a portion thereof includes a CH2 domain, a CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[0113] As used herein, the term "Fc region" is defined as that portion of a polypeptide that corresponds to the Fc region of a naturally occurring immunoglobulin, i.e., formed by association of the Fc domains of each of the two heavy chains of a dimer. A naturally occurring Fc region forms a homodimer with another Fc region.

[0114] In one embodiment, "Fc region" refers to that portion of a single immunoglobulin heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, considering the first residue of the heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2, and CH3 domains.

[0115] The Fc region of an immunoglobulin constant region can include CH2, CH3, and CH4 domains, as well as a hinge region, depending on the immunoglobulin isotype. Chimeric proteins that include an immunoglobulin Fc region confer several desirable properties to the chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613, WO03 / 077834, US2003-0235536A1), which are incorporated herein by reference in their entirety.

[0116] Immunoglobulin constant regions or portions thereof can be FcRn binding partners. FcRn is active in adult epithelial tissues and is expressed in the lumen of the intestine, the lung airways, the nasal surface, the vaginal surface, the colon, and the rectum (U.S. Patent No. 6,485,726). An FcRn binding partner is a portion of an immunoglobulin that binds to FcRn.

[0117] FcRn receptors have been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG at a relatively low pH (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE), actively transports IgG transcellularly from the lumen toward the serosal membrane, and then releases IgG at the relatively high pH found in interstitial fluid. It is expressed in adult epithelial tissues, including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358), as well as nasal epithelium, the vaginal surface, and the bile duct surface (U.S. Pat. Nos. 6,485,726, 6,030,613, 6,086,875, WO 03 / 077834, US 2003-0235536 A1).

[0118] FcRn binding partners useful in the present invention include molecules that can be specifically bound by the FcRn receptor, including whole IgG, Fc fragments of IgG, and other fragments that contain the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor is It has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major interface of Fc with FcRn is near the junction of the CH2 and CH3 domains. The Fc-FcRn contacts are all within a single Ig heavy chain. FcRn binding partners include whole IgG, Fc fragments of IgG, and other fragments of IgG that contain the complete binding region of FcRn. The major interfaces include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain, and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All references to amino acid numbering of immunoglobulins, or immunoglobulin fragments or regions, are based on Kabat et al. 1991, Sequences Based on the Proteins of Immunological Interest, USDapartment of Public Health, Bethesda, Md.

[0119] Fc regions or FcRn binding partners that are bound to FcRn can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means for systemically administering desired therapeutic molecules. Furthermore, fusion proteins that contain Fc regions or FcRn binding partners are endocytosed by cells that express FcRn. However, instead of being marked for degradation, these fusion proteins are recycled back into the circulation, thus increasing the in vivo half-life of these proteins. In certain embodiments, a portion of an immunoglobulin constant region is an Fc region or FcRn binding partner that typically associates with another Fc region or another FcRn binding partner through disulfide bonds and other non-specific interactions to form dimers and higher order multimers.

[0120] An FcRn binding partner region is a molecule or portion thereof that is specifically bound by the FcRn receptor, resulting in active transport of the Fc region by the FcRn receptor. Specifically bound refers to the formation of a relatively stable complex between two molecules under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, as distinguished from nonspecific binding, which usually has low affinity with low to moderate capacity. Typically, binding occurs with an affinity constant KA of 10 or greater. 6 M -1 When it is higher, or 10 8 M -1 When it is higher, it is considered specific. If necessary, non-specific binding can be reduced by varying the binding conditions without substantially affecting the specific binding. Suitable binding conditions, such as the concentration of the molecules, the ionic strength of the solution, the temperature, the time allowed for binding, the concentration of blocking agents (e.g., serum albumin, milk casein), and the like, can be optimized by one of skill in the art using routine techniques.

[0121] Numerous mutants, fragments, variants, and derivatives are described, for example, in PCT Publication Nos. WO2011 / 069164 A2, WO2012 / 006623 A2, WO2012 / 006635 A2, or WO2012 / 006633 A2, all of which are incorporated by reference in their entirety.

[0122] II.C.3.b. Albumin, or a fragment or variant thereof In certain embodiments, the heterologous moiety that is linked to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is albumin or a functional fragment thereof. In some embodiments, the albumin that is fused to the VWF protein is covalently associated with the albumin that is fused to the FVIII protein.

[0123] Human serum albumin (HSA, or HA), a 609 amino acid protein in its full-length form, is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. The term "albumin" as used herein , full length albumin, or a functional fragment, variant, derivative, or analogue thereof. Examples of albumin, or a fragment or variant thereof, are disclosed in U.S. Patent Application Publication Nos. 2008 / 0194481 A1, 2008 / 0004206 A1, 2008 / 0161243 A1, 2008 / 0261877 A1, or 2008 / 0153751 A1, or PCT International Application Publication Nos. 2008 / 033413 A2, 2009 / 058322 A1, or 2007 / 021494 A2, which are incorporated herein by reference in their entireties.

[0124] II.C.3.c. Albumin-binding moiety In certain embodiments, the heterologous moiety that is linked to VWF protein via VWF linker or to FVIII protein via FVIII linker is an albumin binding moiety, and includes 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 that includes a domain antibody (see U.S. Patent No. 6,696,245).The albumin binding protein can be, for example, a bacterial albumin binding domain, such as one of the streptococcal protein G (Konig, T. and Skerra, A. (1998) J.Immunol.Methods 218,73-83). Other examples of albumin binding peptides that can be used as conjugation partners are those having the consensus sequence Cys-Xaa1-Xaa2-Xaa3-Xaa4-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, for example, in U.S. Patent Application No. 2003 / 0069395, or by Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

[0125] II.C.3.d. PAS sequence In another embodiment, the heterologous moiety that is linked to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a PAS sequence. In one embodiment, the chimeric molecule comprises a VWF protein as described herein fused to a PAS sequence via a VWF linker. In another embodiment, the chimeric molecule of the present invention comprises a first chain comprising a VWF protein fused to a PAS sequence via a VWF linker and a second chain comprising a FVIII protein and an additional optional PAS sequence, which guards or protects the VWF binding site on the FVIII protein, thereby inhibiting or preventing the interaction of the FVIII protein with endogenous VWF. The two PAS sequences may be covalently associated with each other.

[0126] A PAS sequence, as used herein, refers to an amino acid sequence that contains primarily alanine and serine residues or primarily alanine, serine, and proline residues, which forms a random coil structure under physiological conditions. Thus, a PAS sequence is a component, amino acid polymer, or sequence cassette that contains, consists essentially of, or consists of alanine, serine, and proline that can be used as part of a heterologous portion in a chimeric protein. However, those skilled in the art will recognize that an amino acid polymer can also form a random coil structure when residues other than alanine, serine, and proline are added as minor components in a PAS sequence. The term "minor components" as used herein refers to amino acids other than alanine, serine, and proline that are present to a certain extent, for example, up to about 12%, i.e., 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 of the 100 amino acids, up to about 8%, i.e., about 10 of the 100 amino acids of the PAS sequence, up to about 10 ... That is, about 8 of 100 amino acids, about 6%, i.e., about 6 of 100 amino acids, about 5%, i.e., about 5 of 100 amino acids, about 4%, i.e., about 4 of 100 amino acids, about 3%, i.e., about 3 of 100 amino acids, about 2%, i.e., about 2 of 100 amino acids, about 1%, i.e., about 1 of 100 amino acids may be added to the PAS sequence. Amino acids different from 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.

[0127] Under physiological conditions, the PAS sequence stretches form random coil structures, which can mediate increased in vivo and / or in vitro stability for VWF factor or coagulation activity proteins. Since the random coil domain has no stable structure or function of its own, the biological activity mediated by the VWF or FVIII protein to which it is fused is essentially preserved. In another embodiment, the PAS sequences forming the random coil domains are biologically inert, particularly with respect to proteolysis in plasma, immunogenicity, isoelectric point / electrostatic effects, binding to cell surface receptors, or internalization, yet are biodegradable, offering distinct advantages over synthetic polymers such as PEG.

[0128] Non-limiting examples of PAS sequences that form random coil structures include amino acid sequences selected from the group consisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 32), AAPASPAPAAPSAPAAPS (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 AAAAPAAASAAASAPSAAA (SEQ ID NO: 38), or any combination thereof. Additional examples of PAS sequences are known, for example, from U.S. Patent Application Publication No. 2010 / 0292130 A1 and PCT Application Publication No. WO2008 / 155134 A1.

[0129] II.C.3.e. HAP sequence In certain embodiments, the heterologous moiety that is linked to the VWF protein via the VWF linker or to the FVIII protein via the FVIII linker is a homoamino acid polymer rich in glycine (HAP). The HAP sequence can comprise a repeat sequence of glycine with 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, the HAP sequence can extend the half-life of the moiety that is fused or linked to the HAP sequence. Non-limiting examples of HAP sequences include (Gly) n , (Gly4Ser) n , or S(Gly4Ser) nExamples of suitable nucleotide sequences include, but are not limited to, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See, e.g., Schlapschy M et al., Protein Eng. Design Selection, 20:273-284 (2007).

[0130] II.C.3.f. Transferrin or fragments thereof In certain embodiments, the VWF protein is linked via a VWF linker or via F The heterologous moiety that is linked to the FVIII protein via the FVIII linker is transferrin or a fragment thereof. Any transferrin can be used to make the chimeric molecule of the present invention. As an example, wild-type human Tf (Tf) is a 679 amino acid protein of approximately 75 KDa (not including glycosylation) with two major domains, N (about 330 amino acids) and C (about 340 amino acids), which are believed to be derived from gene duplication. See GenBank Accession Nos. NM001063, XM002793, M12530, XM039845, XM039847, and S95936 (www.ncbi.nlm.nih.gov / ), all of which are incorporated herein by reference in their entirety. Transferrin comprises two domains, N domain and C domain. The N domain comprises two subdomains, N1 domain and N2 domain, and the C domain comprises two subdomains, C1 domain and C2 domain.

[0131] In one embodiment, the transferrin portion of the chimeric molecule comprises a transferrin splice variant. In one example, the transferrin splice variant can be a splice variant of human transferrin, e.g., Genbank accession AAA61140. In another embodiment, the transferrin portion of the chimeric molecule comprises one or more domains of the transferrin sequence, e.g., the N domain, the C domain, the N1 domain, the N2 domain, the C1 domain, the C2 domain, or any combination thereof.

[0132] II.C.3.g. Polymers, e.g., polyethylene glycol (PEG) In other embodiments, the heterologous moiety attached to the VWF protein via the VWF linker or to the FVIII protein via the FVIII linker is a soluble polymer known in the art, including but not limited to polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, or polyvinyl alcohol. Heterologous moieties, such as soluble polymers, can be attached at any position within the chimeric molecule.

[0133] In certain embodiments, the chimeric molecule comprises a VWF protein fused to a heterologous moiety (e.g., an Fc region) via a VWF linker, which is further linked to PEG. In another embodiment, the chimeric molecule comprises a VWF protein fused to an Fc region via a VWF linker and a FVIII protein that are associated with each other, which is linked to PEG.

[0134] Further provided by the present invention are chemically modified derivatives of the chimeric molecules of the present invention that may provide additional advantages such as increased solubility, stability, and circulation time of the polypeptide, or reduced immunogenicity (see U.S. Pat. No. 4,179,337). Chemical moieties for modification may be selected from water-soluble polymers, including, but not limited to, polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol. Chimeric molecules may be modified at random positions within the molecule, or at the N- or C-terminus, or at defined positions within the molecule, and may contain one, two, three or more attached chemical moieties.

[0135] The polymers can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, in one embodiment, the molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing. Other sizes can be used depending on the desired profile (e.g., the desired duration of sustained release, efficacy, biological activity, if any, ease of handling, degree or lack of antigenicity, and other known effects of polyethylene glycol on proteins or analogs). For example, polyethylene glycol can be about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 2300, 2400, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 21000, 22000, 23000, 00, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000 , 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0136] In some embodiments, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575, Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996), Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999), and Caliceti et al. al., Bioconjug. Chem. 10:638-646 (1999), each of which is incorporated herein by reference in its entirety.

[0137] The number of polyethylene glycol moieties attached to each chimeric molecule (i.e., the degree of substitution) can also vary. For example, the PEGylated proteins of the present invention can be attached to an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. Similarly, the average degree of substitution can be in the range of 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule, etc. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

[0138] In other embodiments, the FVIII protein used herein is conjugated with one or more polymers. The polymers may be water-soluble and may be covalently or non-covalently attached to factor VIII or other moieties conjugated to factor VIII. Non-limiting examples of polymers may be poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine). Additional types of polymer-conjugated FVIII are disclosed in U.S. Patent No. 7,199,223.

[0139] II.C.3.h. Hydroxyethyl starch (HES) In certain embodiments, the heterologous moiety that is attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a polymer, such as hydroxyethyl starch (HES) or a derivative thereof.

[0140] Hydroxyethyl starch (HES) is a derivative of naturally occurring amylopectin and is degraded by α-amylase in the body. HES is a substituted derivative of the carbohydrate polymer amylopectin and is present in corn starch at concentrations of up to 95% by weight. HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinic (Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), and Weidler et al., Arzneim.-Forschung / Drug Res., 41, 494-498 (1991)).

[0141] Amylopectin contains glucose moieties with α-1,4-glycosidic bonds in the main chain and α-1,6-glycosidic bonds at the branching sites. The physicochemical properties of the molecule are mainly determined by the type of glycosidic bond. Due to the discontinuous α-1,4-glycosidic bonds, a helical structure with about six glucose monomers per turn is produced. The physicochemical as well as biochemical properties of the polymer can be modified by substitution. The introduction of hydroxyethyl groups can be achieved by alkaline hydroxyethylation. By adapting the reaction conditions, it is possible to take advantage of the different reactivity of each hydroxyl group in the unsubstituted glucose monomer with respect to hydroxyethylation. This fact allows the skilled person to influence the substitution pattern to a limited extent.

[0142] HES is mainly characterized by its molecular weight distribution and degree of substitution. The degree of substitution, indicated as DS, refers to molar substitution and is known to those skilled in the art. See Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), as cited above, especially p. 273.

[0143] In one embodiment, the hydroxyethyl starch has an average molecular weight (weight average) of 1-300 kD, 2-200 kD, 3-100 kD, or 4-70 kD. The hydroxyethyl starch may further exhibit a molar degree of substitution of 0.1-3, preferably 0.1-2, more preferably 0.1-0.9, preferably 0.1-0.8, and a ratio between C2:C6 substitutions in the range of 2-20, with respect to the hydroxyethyl groups. Non-limiting examples of HES with an average molecular weight of about 130 kD are HES with a degree of substitution of 0.2-0.8, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferably 0.4-0.7, such as 0.4, 0.5, 0.6, or 0.7. In a particular embodiment, the HES with an average molecular weight of about 130 kD is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid used in blood volume replacement therapy, for example, with therapeutic indications for the treatment of hypovolemia, and therapeutic applications in the treatment and prevention of hypovolemia. VOLUVEN® is characterized by an average molecular weight of 130,000+ / -20,000 D, a molar substitution of 0.4, and a C2:C6 ratio of about 9:1. In other embodiments, the average molecular weight range of the hydroxyethyl starch is, for example, 4-70 kD, or 10-70 kD, or 12-70 kD, or 18-70 kD, or 50-70 kD, or 4-50 kD, or 10-50 kD, or 12-50 kD, or 18-50 kD, or 4-18 kD, or 10-18 kD, or 12-18 kD, or 4-12 kD, or 10-12 kD, or 4-10 kD. In still other embodiments, the average molecular weight of the hydroxyethyl starch used is in the range of more than 4 kD and less than 70 kD, such as about 10 kD, or in the range of 9-10 kD or 10-11 kD or 9-11 kD, or about 12 kD, or in the range of 11-12 kD) or 12-13 kD or 11-13 kD, or about 18 kD, or in the range of 17-18 kD or 18-19 kD or 17-19 kD, or about 30 kD, or in the range of 29-30, or in the range of 30-31 kD, or about 50 kD, or in the range of 49-50 kD or 50-51 kD or 49-51 kD.

[0144] In certain embodiments, the heterogeneous portion may be a mixture of hydroxyethyl starches having different average molecular weights and / or different degrees of substitution and / or different C2:C6 substitution ratios. Thus, the mixture of hydroxyethyl starches may have different average molecular weights, different degrees of substitution and different C2:C6 substitution ratios, or different average molecular weights, different degrees of substitution and the same or approximately the same C2:C6 substitution ratios, or different average molecular weights, the same or approximately the same degree of substitution and different C2:C6 substitution ratios, or different average molecular weights, the same or approximately the same degree of substitution and the same or approximately the same C2:C6 substitution ratios, or different average molecular weights, the same or approximately the same degree of substitution and the same or approximately the same C2:C6 substitution ratios, or the same or approximately the same average molecular weights and different degrees of substitution and different C2:C6 substitution ratios. Polyolefins having the same or approximately the same degree of substitution and the same or approximately the same C2:C6 substitution ratio, or having the same or approximately the same average molecular weight, the same or approximately the same degree of substitution and different C2:C6 substitution ratios, or approximately the same average molecular weight, approximately the same degree of substitution and approximately the same C2:C6 substitution ratios may be used.

[0145] II.C.3.i. Polysialic acid (PSA) In certain embodiments, the non-polypeptide heterologous moiety attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a polymer, such as polysialic acid (PSA) or a derivative thereof. Polysialic acid (PSA) is a naturally occurring unbranched polymer of sialic acid produced by certain bacterial strains and in certain mammalian cells. Roth J., et al. (1993) in Polysialic Acid: From Microbes to Man, eds. Roth J., Rutishauser U., Troy FA (Birkhauser Verlag, Basel, Switzerland), p p 335-348. They can be produced with various degrees of polymerization, ranging from n=about 80 or more sialic acid residues to n=2, by limited acid hydrolysis, or by digestion with neuraminidase, or by fractionation of the natural bacterial form of the polymer. The composition of the different polysialic acids is also diverse, so that homopolymeric forms, i.e., α-2,8-linked polysialic acids, exist, including the capsular polysaccharides of E. coli K1 and group B Neisseria meningitidis, which are also found in the embryonic neuronal cell adhesion molecule (N-CAM). Heteropolymeric forms also exist, such as the alternating α-2,8 α-2,9 polysialic acid of E. coli K92, and the group C polysaccharides of Neisseria meningitidis. Sialic acids can also be found in alternating copolymers with monomers other than sialic acid, such as groups W135 or Y of Neisseria meningitidis. Polysialic acid has important biological functions, including evasion of immune and complement systems by pathogenic bacteria, and regulation of glial cell adhesion of immature neurons during fetal development (the polymer has an anti-adhesive function) Cho and Troy, PNAS, USA, 91 (1994) 11427-11431, but there are no known receptors for polysialic acid in mammals. The α-2,8-linked polysialic acid of E. coli K1 strain is also known as "colominic acid" and is used (in various lengths) to illustrate the present invention. Various methods of binding or conjugating polysialic acid to polypeptides have been described (see, for example, U.S. Pat. No. 5,846,951, WO-A-0187922, and US2007 / 0191597 A1, which are incorporated herein by reference in their entireties).

[0146] II.C.4. XTEN sequences. As used herein, "XTEN sequence" refers to a length-extended polypeptide having a non-naturally occurring, substantially non-repetitive sequence composed primarily of small hydrophilic amino acids, with a sequence that has low order structure or no secondary or tertiary structure under physiological conditions. As a chimeric protein partner, XTEN can impart certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties and function as a carrier when combined with the VWF or FVIII protein of the present invention to create a chimeric protein. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility properties. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as single chain antibodies or Fc fragments of the light or heavy chains.

[0147] In some embodiments, the XTEN sequences of the invention are peptides or polypeptides having more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. Thus, XTEN is a peptide or polypeptide having more than about 20 to about 3000 amino acid residues, more than 30 to about 2500 residues, more than 40 to about 2000 residues, more than 50 to about 1500 residues, more than 60 to about 1000 residues, more than 70 to about 900 residues, more than 80 to about 800 residues, more than 90 to about 700 residues, more than 100 to about 600 residues, more than 110 to about 500 residues, or more than 120 to about 400 residues.

[0148] The XTEN sequences of the invention can include one or more sequence motifs of 9-14 amino acid residues, or an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, which motif comprises, consists essentially of, or consists of 4-6 amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). See US2010-0239554 A1.

[0149] In some embodiments, the XTEN comprises non-overlapping sequence motifs, where 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 sequences are composed of multiple units of non-overlapping sequences selected from a single motif family selected from Table 2A, resulting in a family sequence. As used herein, "family" means that the XTEN has motifs selected only from a single motif category from Table 2A, i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids within the XTEN that are not from a family motif are selected to allow for the incorporation of a restriction enzyme site with the coding nucleotides, the incorporation of a cleavage sequence, or to achieve better binding to FVIII or VWF. In some embodiments of the XTEN family, the XTEN sequence comprises multiple units of non-overlapping sequence motifs from the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, and the resulting XTENs exhibit the ranges of homology described above. In other embodiments, the XTENs comprise multiple units of motif sequences from two or more of the motif families of Table 2A. These sequences can be selected to achieve desired physical / chemical properties conferred by the amino acid composition of the motif, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repetition, as described in more detail below. In the embodiments described above in this paragraph, the motifs incorporated into the XTENs can be selected and constructed using the methods described herein to achieve XTENs of about 36 to about 3000 amino acid residues. [Table 2A]

[0150] XTEN can have lengths that vary depending on the insertion or attachment to FVIII or VWF or any other component of the chimeric molecule. In one embodiment, the length of the XTEN sequence(s) is selected based on the property or function to be achieved within the fusion protein. Depending on the intended property or function, XTEN can be short or medium length sequences, or longer sequences that can function as carriers. In certain embodiments, XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN inserted or attached to FVIII or VWF can have a length of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues in length. The XTEN may be 0 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The exact length of the XTEN inserted or attached to FVIII or VWF may vary without adversely affecting the activity of FVIII or VWF. In one embodiment, one or more of the XTENs used herein have a length of 36 amino acids, 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids and may be selected from one or more of the XTEN family sequences, i.e., AD, AE, AF, AG, AM, AQ, BC, or BD.

[0151] In some embodiments, the XTEN sequences used in the present invention are AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE 396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, A F576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1 044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US2010-0239554 A1.

[0152] In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of AE42, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144, and any combination thereof. In another embodiment, the XTEN sequence is selected from the group consisting of AE42, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144, and any combination thereof. In a particular embodiment, the XTEN sequence is AE288. The amino acid sequences of certain XTEN sequences of the invention are shown in Table 2B. [Table 2B-1] [Table 2B-2] [Table 2B-3]

[0153] In those embodiments in which less than 100% of the amino acids of the XTEN component used consist of 4, 5, or 6 amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), or less than 100% of the sequence consists of a sequence motif of Table 2A or an XTEN sequence of Table 2B, the other amino acid residues of the XTEN are selected from any of the other 14 naturally occurring L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. XTEN amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) are either interspersed throughout the XTEN sequence, or are located within or between sequence motifs, or are concentrated within one or more short stretches of the XTEN sequence, e.g., to create linkers between the XTEN and other components, e.g., VWF protein. When an XTEN component includes amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids are hydrophobic residues, such that the resulting sequence generally lacks secondary structure, e.g., does not have more than 2% alpha helices or more than 2% beta sheets, as determined by the methods disclosed herein. Hydrophobic residues that are less favored in the structure of XTENs include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, XTEN sequences can be designed to contain less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or none of the following amino acids: cysteine ​​(to avoid disulfide formation and oxidation), methionine, cyclohexyl 1 ... Thionine (to avoid oxidation), Asparagine, and Glutamine (to avoid deamidation). Thus, in some embodiments, XTENs containing other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) have sequences with less than 5% of residues contributing to alpha helices and beta sheets as measured by the Chou-Fasman algorithm, and at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.

[0154] In further embodiments, the XTEN sequence used in the present invention affects the physical or chemical properties, such as pharmacokinetic properties, of the chimeric protein of the present invention.The XTEN sequence used in the present invention can exhibit one or more of the following advantageous properties: structural flexibility, enhanced water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or improved hydrodynamic (or Stokes) radius.In certain embodiments, the XTEN sequence attached to the FVIII protein in the present invention increases pharmacokinetic properties, such as longer terminal half-life or increased area under the curve (AUC), thereby allowing the chimeric protein described herein to remain in vivo for a longer period of time compared to wild-type FVIII.In further embodiments, the XTEN sequence used in the present invention increases pharmacokinetics, such as longer terminal half-life or increased area under the curve (AUC), thereby allowing the FVIII protein to remain in vivo for a longer period of time compared to wild-type FVIII.

[0155] A variety of methods and assays can be used to determine the physical / chemical properties of proteins containing XTEN sequences. Such methods include, but are not limited to, analytical centrifugation, EPR, ion-exchange HPLC, size-exclusion HPLC, reversed-phase HPLC, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, ion-exchange HPLC, size-exclusion HPLC, 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).

[0156] Additional examples of XTEN sequences that can be used in accordance with the present invention are described in U.S. Patent Publication Nos. 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1, or 2011 / 0172146 A1, or International Patent Publication Nos. WO2010091122 A1, WO2010144502 A2, WO2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2, or WO2013123457 A1, or as disclosed in International Application No. PCT / US2013 / 049989.

[0157] II.C.5. FVIII protein As used herein, "FVIII protein" means a FVIII polypeptide functional in its normal role in coagulation, unless otherwise specified. The term FVIII protein includes functional fragments, variants, analogs, and derivatives thereof that retain the function of full-length wild-type factor VIII in the coagulation pathway. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include the ability to activate coagulation, function as a cofactor for factor IX, and inhibit Ca. 2+These include, but are not limited to, the ability to form a tenase complex with factor IX in the presence of phospholipids and then convert factor X to the activated form Xa. The FVIII protein can be a human, porcine, canine, rat, or mouse FVIII protein. In addition, comparisons between FVIII from humans and other species have identified conserved residues that may be required for function (Cameron et al., Thro mb.Haemost.79:317-22(1998);US6,251,632).

[0158] Many tests are available to analyze the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM assay, prothrombin time (PT) test (also used to determine the INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether the abnormality is corrected when the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g. factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), various platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g. ROTEM®), or euglobulin lysis time (ELT).

[0159] The aPTT test is a performance indicator that measures the effectiveness of both the "intrinsic" coagulation pathway (also called the contact activation pathway) and the common coagulation pathway. This test is widely used to measure the coagulation activity of commercially available recombinant coagulation factors, such as FVIII or FIX. It is used in combination with the prothrombin time (PT), which measures the extrinsic pathway.

[0160] ROTEM analysis provides information on the entire dynamics of hemostasis: clotting time, clot formation, clot stability, and lysis. The different parameters within thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many factors that affect the interaction of these. This assay can provide a complete picture of secondary hemostasis.

[0161] FVIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants, and modified versions. An example of a human FVIII sequence (full length) is shown as a subsequence within SEQ ID NO: 16 or 18.

[0162] [Table 3-1] [Table 3-2] Table 4. Nucleotide sequence encoding full length FVIII (SEQ ID NO: 17)* [Table 4-1] [Table 4-2] [Table 4-3] *The underlined nucleic acid encodes the signal peptide.

[0163] FVIII polypeptides include full-length FVIII, full-length FVIII without a Met at the N-terminus, mature FVIII (without a 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, the FVIII variant comprises a B domain deletion, whether a partial or complete deletion.

[0164] The human FVIII gene was isolated and expressed in mammalian cells (Toole, JJ, et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, WI, et al., Nature 312:330-337 (1984); Vehar, GA, et al., Nature 312:337-342 (1984)). 84), WO87 / 04187, WO88 / 08035, WO88 / 03558, and U.S. Pat. No. 4,757,006). The FVIII amino acid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199. In addition, partial or complete B-domain deleted FVIII are shown in U.S. Pat. Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B-domain is replaced with the human factor V B-domain as shown in U.S. Pat. No. 5,004,803. The cDNA sequence encoding human factor VIII and the amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively, of U.S. Pat. No. 7,211,559.

[0165] The porcine FVIII sequence is published in Toole, JJ, et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). In addition, the complete porcine cDNA sequence obtained from PCR amplification of FVIII sequence from a pig spleen cDNA library is reported in Healey, JF, et al., Blood 88:4209-4214 (1996). Human / porcine hybrid FVIII with substitutions of all domains, all subunits, and specific amino acid sequences was disclosed in U.S. Patent No. 5,364,771 by Lollar and Runge, and in WO93 / 20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII, as well as chimeric FVIII with porcine A1 and / or A2 domains replaced with the corresponding human domains, were reported in WO94 / 11503. US Patent No. 5,859,204, Lollar, JS also discloses a porcine cDNA and deduced amino acid sequence. US Patent No. 6,458,563 discloses a B-domain deleted porcine FVIII.

[0166] U.S. Patent No. 5,859,204 to Lollar, JS reports functional mutants of FVIII with reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463 to Lollar, JS also reports mutants of FVIII with reduced immunoreactivity. U.S. Application Publication No. 2005 / 0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

[0167] In one embodiment, the FVIII protein (or the FVIII portion of the chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 18, or amino acids 1 to 2332 of SEQ ID NO: 16 (without a signal sequence), and the FVIII has a coagulation activity, e.g., activates factor IX as a cofactor and converts factor X to activated factor X. The FVIII (or the FVIII portion of the chimeric protein) may be identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 18, or amino acids 1 to 2332 of SEQ ID NO: 16 (without a signal sequence). The FVIII protein may further comprise a signal sequence.

[0168] The "B-domain" of FVIII, as used herein, is the same as the B-domain known in the art, defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741 to Arg1648 of full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1 to Arg372; A2, residues Ser373 to Arg740; A3, residues Ser1690 to Asn2019; C1, residues Lys2020 to Asn2172; C2, residues Ser2173 to Tyr2332. The A3-C1-C2 sequence includes residues Ser1690 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is commonly referred to as the a3 acidic region. The locations of all the boundaries of the domains, including the B-domain, for porcine, mouse, and canine FVIII are also known in the art. In this embodiment, the B domain of FVIII is deleted ("B-domain deleted factor VIII" or "BDD FVIII"). One example of a BDD FVIII is REFACTO® (recombinant BDD FVIII), which has a sequence identical to the factor VIII portion of the sequence in Table 5. (BDD FVIII heavy chain is double underlined, the B domain is italicized, and the BDD FVIII light chain is in plain text). Table 5. BDD FVIII (SEQ ID NO: 18) [Table 5-1] [Table 5-3] Table 6. Nucleotide sequence encoding BDD FVIII (SEQ ID NO: 19)* [Table 6-1] [Table 6-2] *The underlined nucleic acid encodes the signal peptide.

[0169] A "B-domain deleted FVIII" may have a complete or partial deletion as disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In some embodiments, a B-domain deleted FVIII sequence of the invention comprises any one of the deletions disclosed in column 4, line 4, column 5, line 28, and Examples 1-5 of US Pat. No. 6,316,226 (also in US Pat. No. 6,346,513). In another embodiment, the B-domain deleted factor VIII is S743 / Q1638 B-domain deleted factor VIII (SQ BDD FVIII) (e.g., a factor VIII having a deletion of amino acids 744 to 1637, e.g., a factor VIII having amino acids 1 to 743 and amino acids 1638 to 2332 of SEQ ID NO: 16, i.e., SEQ ID NO: 18). In some embodiments, the B-domain deleted FVIII of the invention has a deletion as disclosed in column 2, lines 26-51 and Examples 5-8 of U.S. Patent No. 5,789,203 (also U.S. Patent No. 6,060,447, U.S. Patent No. 5,595,886, and U.S. Patent No. 6,228,620). In some embodiments, the B-domain deleted FVIII has a deletion as disclosed in column 1, line 25 to column 2, line 40 of U.S. Patent No. 5,972,885, column 6, lines 1-22 and Example 1 of U.S. Patent No. 6,048,720, column 2, lines 17-46 of U.S. Patent No. 5,543,502, column 4, lines 22 to column 5, line 36 of U.S. Patent No. 5,112,950, column 2, lines 5 No. 4,868,112; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of U.S. Pat. No. 7,041,635; or column 4, lines 25 to 53 of U.S. Pat. No. 6,458,563.

[0170] In some embodiments, the B-domain deleted FVIII has most of the B domain deleted, but contains two polyclonal antibodies of the primary translation product, as disclosed in WO91 / 09122. It still contains the amino-terminal sequence of the B domain, which is essential for in vivo proteolytic processing into peptide chains. In some embodiments, the B-domain deleted FVIII is constructed by deleting amino acids 747-1638, i.e., virtually the complete deletion of the B domain. Hoeben RC, et al. J. Biol. Chem. 265(13):7318-7323(1990). The B-domain deleted factor VIII may also contain deletions of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4):301-6(1988). Additional B domain deletions which are part of the present invention include deletions of amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5939-5942), 797-1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347), 741-1646 (Kaufman (PCT Published Application 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. 82:16-25, EP 295597). In other embodiments, the BDD FVIII comprises a FVIII polypeptide comprising a fragment of the B-domain that retains one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or, optionally, 943, corresponding to the amino acid sequence of the full-length FVIII sequence.Examples of B-domain fragments include 226 or 163 amino acids of the B-domain as disclosed in Miao, HZ, et al., Blood 103(a):3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6:1352-1359 (2008), and Pipe, SW, et al., J. Thromb. Haemost. 9:2235-2242 (2011) (i.e., the first 226 or 163 amino acids of the B-domain are retained). In some embodiments, the FVIII with a partial B-domain is FVIII198. FVIII198 is a partial B-domain that includes a single chain FVIIIFc molecule-226N6. 226 represents the N-terminal 226 amino acids of the FVIII B-domain, and N6 represents the six N-glycosylation sites within the B-domain. In still other embodiments, the BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve expression of the BDD FVIII protein. See Miao, HZ, et al., Blood 103(a):3412-3419 (2004). In still other embodiments, the BDD FVIII comprises a FVIII polypeptide that contains a portion of the B-domain but does not contain one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, SW, et al., J. Thromb. Haemost. 9:2235-2242 (2011). Each of the foregoing deletions can be made within any FVIII sequence.

[0171] The FVIII protein useful in the present invention can comprise FVIII having one or more additional heterologous sequences or chemical or physical modifications therein, which do not affect FVIII coagulation activity.Such heterologous sequences or chemical or physical modifications can be fused to the C-terminus or N-terminus of FVIII protein, or can be inserted between one or more of two amino acid residues in FVIII protein.Such insertions in FVIII protein do not affect FVIII coagulation activity or FVIII function.In one embodiment, the insertions improve the pharmacokinetic properties (e.g., half-life) of FVIII protein.In another embodiment, the insertions can be 2, 3, 4, 5, or more than 6 sites.

[0172] In one embodiment, FVIII is amplified by amino acid 1648 (full length factor VIII or In another embodiment, the FVIII comprises a heavy chain and a light chain that are linked or associated by a metal ion-mediated non-covalent bond.

[0173] In other embodiments, the FVIII is a single-chain FVIII that is not truncated immediately after the arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 16), amino acid 754 (in S743 / Q1638 B-domain deleted FVIII or SEQ ID NO: 18), or the corresponding arginine residue (in other variants). The single-chain FVIII may contain one or more amino acid substitutions. In one embodiment, the amino acid substitutions are at residue 1648, residue 1645, or both of the full-length mature factor VIII polypeptide (SEQ ID NO: 16), or at residues corresponding to residue 754, residue 751, or both of the SQ BDD factor VIII (SEQ ID NO: 18). The amino acid substitutions can be any amino acid other than arginine, for example, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine.

[0174] FVIII can be further cleaved by thrombin and then activated as FVIIIa, which functions as a cofactor for activated factor IX (FIXa). Activated FIX then forms an Xase complex with activated FVIII, converting factor X to activated factor X (FXa). For activation, FVIII is cleaved by thrombin after three arginine residues at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deleted FVIII sequence), which cleavage produces FVIIIa with 50 kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chains. In one embodiment, the FVIII protein useful in the present invention is inactive FVIII. In another embodiment, the FVIII protein is activated FVIII.

[0175] A protein having a FVIII polypeptide bound to or associated with a VWF protein can comprise a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16 or 18, which sequence has FVIII coagulation activity, e.g., activating factor IX as a cofactor to convert factor X to activated factor X (FXa).

[0176] In some embodiments, the FVIII protein further comprises one or more heterologous moieties 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 moiety comprises an amino acid sequence of at least about 50 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, at least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 550 amino acids, at least about 600 amino acids, at least about 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 at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, less than wild-type FVIII. In some embodiments, the length of the sequence is at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold longer.

[0177] Other exemplary FVIII variants are also disclosed in U.S. Publication No. US2013 / 0017997, published January 17, 2013, International Publication No. WO2013 / 122617, published August 22, 2013, or International Publication No. WO2014 / 011819, published January 16, 2014, or International Publication No. WO2013123457 A1, or International Application No. PCT / US2013 / 049989.

[0178] III. Polynucleotides, Vectors, Host Cells, and Methods of Production Further provided herein is a polynucleotide encoding the chimeric molecule described herein. When the VWF protein is linked to the heterologous moiety via a VWF linker and to the FVIII protein and XTEN sequence within the chimeric protein as a single polypeptide chain, the present invention focuses on a single polynucleotide encoding a single polypeptide chain. When the chimeric protein comprises a first and a second polypeptide chain, the first polypeptide chain comprises a VWF protein, an XTEN sequence, and a first heterologous moiety (e.g., a first Fc region) via a VWF linker, and the second polypeptide chain comprises a FVIII protein and a second heterologous moiety (e.g., a second Fc region), the polynucleotide can comprise a first nucleotide region and a second nucleotide region. In one embodiment, the first nucleotide region and the second nucleotide region are on the same polynucleotide. In another embodiment, the first nucleotide region and the second nucleotide region are on two different polynucleotides (e.g., different vectors). In certain embodiments, the present invention is directed to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, the first nucleotide strand encoding a VWF protein, an XTEN sequence, a VWF linker, and a heterologous portion of a chimeric protein, and the second nucleotide strand encoding a FVIII protein and a second heterologous portion. In some embodiments, the present invention is directed to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, the first nucleotide strand encoding a VWF protein and a heterologous portion of a chimeric protein, and the second nucleotide strand encoding a FVIII protein fused to a second heterologous portion via a FVIII linker, and at least one XTEN sequence fused to the chimeric protein.In another embodiment, the present invention is directed 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 portion of a chimeric protein, and the second nucleotide strand encodes a FVIII protein, a FVIII linker, and a second heterologous portion, wherein at least one XTEN sequence is fused to the chimeric protein.

[0179] 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) encoding a protein convertase. The protein convertase may be selected from proprotein convertase subtilisin / kexin type 5 (PCSK5 or PC5), proprotein convertase subtilisin / kexin type 7 (PCSK7 or PC5), yeast Kex 2, proprotein convertase subtilisin / kexin type 3 (PACE or PCSK3), or a combination of two or more thereof. In some embodiments, the protein convertase is PACE, PC5, or PC7. In certain embodiments, the protein convertase is PC5 or PC7. See International Application No. PCT / US2011 / 043568, which is incorporated herein by reference. In another embodiment, the protein convertase is PACE / furin.

[0180] In a particular embodiment, the present invention comprises a set of polynucleotides comprising a first nucleotide sequence encoding a VWF protein comprising the D' and D3 domains of VWF fused to a first heterologous moiety via a VWF linker, a second nucleotide sequence encoding a FVIII protein and a second heterologous moiety, and a third nucleotide sequence encoding the D1 and D2 domains of VWF, wherein the XTEN sequence is present in either the first or second chain. In this embodiment, the D1 and D2 domains are expressed separately (not linked to the D' and D3 domains of VWF protein) for proper disulfide bond formation and folding of the D' and D3 domains. The D1 and D2 domains expression can be either cis or trans.

[0181] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, for replication and translation when introduced into a suitable host cell. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0182] The expression vector of the present invention will contain a polynucleotide that encodes the chimeric molecule.

[0183] In one embodiment, the coding sequence for the chimeric molecule is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in a manner that places the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if the introduction of a promoter in the 5' gene expression sequence results in transcription of the coding sequence, and if the nature of the link between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence is operably linked to a coding nucleic acid sequence when the gene expression sequence is capable of effecting the transcription of that nucleic acid sequence such that the resulting transcript is translated into a desired protein or polypeptide.

[0184] As used herein, a gene expression control sequence is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination, that promotes efficient transcription and translation of an operably linked coding nucleic acid. A gene expression control sequence can be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters from cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus, and other retroviruses long terminal repeats (LTRs), as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the present 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 skilled in the art.

[0185] In general, gene expression control sequences will include, as appropriate, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as the TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed sequences will include a promoter region comprising a promoter sequence for transcriptional control of an operably linked coding nucleic acid. Gene expression sequences will optionally include enhancer sequences or upstream activator sequences, if desired.

[0186] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; RNA viruses such as adenovirus, adeno-associated virus, SV40-type viruses, polyoma virus, Epstein-Barr virus, papilloma virus, herpes virus, vaccinia virus, polio virus, and retroviruses. Other vectors known in the art can be easily 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 genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are retroviruses that are replication-deficient (i.e., capable of directing the synthesis of desired proteins but unable to manufacture infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses, including the steps of incorporating the exogenous genetic material into a plasmid, transfecting the plasmid into a packaging cell line, producing recombinant retrovirus by the packaging cell line, harvesting the viral particles from tissue culture medium, and infecting target cells with the viral particles, are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990), and Murry, E.J., Methods in Molecular Biology, Vol. 7, Humanaa Press, Inc., Cliffton, N.J. (1991).

[0187] In one embodiment, the virus is adeno-associated virus, a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication-deficient and can infect a wide range of cell types and species. It has further advantages such as heat and aliphatic solvent stability, high transduction frequency in cells of various lineages, including hematopoietic cells, and no superinfection inhibition, thus allowing multiple transduction. Reportedly, adeno-associated virus can integrate into human cell DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and the variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infection has been performed in tissue culture for more than 100 passages in the absence of selection pressure, meaning that adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also function in an extrachromosomal manner.

[0188] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those of skill 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, it has been discovered that plasmid vectors are particularly advantageous for delivering genes to cells in vivo because of their inability to replicate and integrate within the host genome. However, these plasmids with promoters compatible with the host cell are capable of expressing peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020, pcDNA3.1 / hygro, catalog number V87020, pcDNA4 / myc-His, catalog number V86320, and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids are well known to those of skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0189] In one insect expression system that can be used to produce the proteins of the invention, the AcNPV nuclear polyhidrosis virus is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence is cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an ACNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the coding sequence will result in the inactivation of the polyhedrin gene and the production of non-occluded recombinant viruses (i.e., viruses lacking the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted genes are expressed. (See, e.g., Smith et al. (1983) J Virol 46:584; U.S. Pat. No. 4,215,051.) Further examples of this expression system are found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

[0190] 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 Biologics PLC, Berkshire UK). This expression system is described in detail in U.S. Patent No. 5,981,216.

[0191] In mammalian host cells, a number of viral-based expression systems can be utilized. In the case where adenovirus is used as an expression vector, the coding sequence can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the peptide in an infected host. See, e.g., Logan & Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5K promoter can be used. 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 See Sci USA 79:4927.

[0192] To increase the efficiency of production, a polynucleotide can be designed to encode multiple units of a protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with an appropriate enzyme) to recover the polypeptide units. This allows for the production of multiple polypeptides driven by a single promoter. When used in a suitable viral expression system, the translation of each polypeptide encoded by the mRNA is directed internally within the transcript, for example by an internal ribosome entry site, IRES. Thus, polycistronic constructs direct 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.

[0193] The vectors used for transformation will usually contain a selectable marker that is used to identify transformants. In bacterial systems, this can include antibiotic resistance genes such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker can be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen CC et al. (1983) Proc Natl Acad Sci USA 80:2495-9. Selectable markers are reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable marker is well within the level of ordinary skill in the art.

[0194] The selectable marker can be introduced into the cell at the same time as the gene of interest on a separate plasmid, or they can be inserted on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest can be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. This type of construct is known in the art (e.g., U.S. Pat. No. 4,713,339).

[0195] The expression vector can encode a tag that allows easy purification of recombinantly produced proteins. Examples include, but are not limited to, the vector pUR278 (Ruther et al. (1983) EMBO J 2:1791), in which the coding sequence for the expressed protein can be ligated into the vector in frame with the lacZ coding region so that a tagged fusion protein is produced; pGEX vectors can be used to express the proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are 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 vector contains a cleavage site (thrombin, or factor Xa protease, or PRESCISSION PROTEASE™ (Pharmacia, Peapack, NJ)) for easy removal of the tag after purification.

[0196] The expression vector or vectors are then transfected or co-transfected into appropriate target cells which express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host-expression vector systems can be utilized to express the proteins described herein, including both prokaryotic and eukaryotic cells. These include microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing the appropriate coding sequences, recombinant endonucleases containing the appropriate coding sequences, and recombinant endonucleases containing the appropriate coding sequences. Examples of suitable coding sequences include, but are not limited to, yeast or filamentous fungi transformed with yeast or fungal expression vectors, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the appropriate coding sequences, plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the appropriate coding sequences, and animal cell systems including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

[0197] In one embodiment, the host cell is a eukaryotic cell. As used herein, eukaryotic cell refers to any animal or plant cell that has a definitive nucleus. Animal eukaryotic cells include vertebrate, e.g., mammalian, and invertebrate, e.g., insect, cells. Plant eukaryotic cells can specifically include, but are not limited to, yeast cells. Eukaryotic cells are distinct from prokaryotic cells, e.g., bacteria.

[0198] In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, a human embryonic kidney cell line. HEK 293 cells are available from the American Type Culture Collection, Manassas, VA as CRL-1533, and from Invitrogen, Carlsbad, Calif. as 293-F cells, catalog number 11631-017, or 293-F cells, catalog number 11625-019. In some embodiments, the mammalian cell is a PER.C6® cell, a human cell derived from the retina. PER.C6® cells are available from Crucell, Leiden, The Netherlands. In other embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. CHO cells are available from the American Type Culture Collection, Manassas, VA. (e.g., CHO-K1, CCL-61). In still other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from the American Type Culture Collection, Manassas, Va. (e.g., CRL-1632). In some embodiments, the mammalian cell is an HKB11 cell, a hybrid cell line of HEK293 cells and a human B cell line. Mei et al., Mol. Biotechnol. 34(2):165-78 (2006).

[0199] In one embodiment, a plasmid encoding the VWF protein, VWF linker, heterologous moiety, or chimeric protein of the present invention further comprises a selectable marker, e.g., zeocin resistance, and is transfected into HEK 293 cells for production of the chimeric protein.

[0200] In yet other embodiments, the transfected cells are stably transfected. These cells may be selected and maintained as stable cell lines using conventional techniques known to those of skill in the art.

[0201] The host cells containing the DNA construct of the protein are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" refers to a medium containing nutrients required for cell growth. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the medium may contain bovine calf serum or fetal calf serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium generally selects for cells containing the DNA construct, for example, by drug selection or deprivation of an essential nutrient that is supplemented by a selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbad, Calif.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.). Selection of the appropriate medium for the particular cell line used is within the level of ordinary skill in the art.

[0202] To co-express the two polypeptide chains (echains) of a chimeric molecule as described herein, the host cells are cultured under conditions that allow for the expression of both chains. As used herein, culturing refers to maintaining living cells in vitro for at least a period of time. Maintaining can include, but does not have to include, an increase in the number of living cells. For example, cells maintained in culture can be static in number, but still viable and capable of producing a desired product, e.g., a recombinant protein or a recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include the appropriate selection of media, media supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing can include the use of any of a variety of types of scale-up systems, including shake flasks, roller bottles, hollow fiber bioreactors, stirred tank bioreactors, airlift bioreactors, Wave bioreactors, and the like.

[0203] Cell culture conditions are also selected to allow the association of the first and second chains in the chimeric molecule. Conditions allowing 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.

[0204] In one aspect, the invention is directed to a method for expressing, making, or producing a chimeric protein comprising a) transfecting a host cell with a polynucleotide encoding the chimeric molecule, and b) culturing the host cell under conditions suitable for expressing the chimeric molecule and in a culture medium in which the chimeric molecule is expressed.

[0205] In a further embodiment, the protein product containing the chimeric molecule is secreted into the medium, which is separated from the cells, concentrated, filtered, and then passed over two or three affinity columns, e.g., a Protein A column and one or two anion exchange columns.

[0206] In certain aspects, the present invention relates to chimeric polypeptides produced by the methods described herein.

[0207] In vitro production allows for scale-up to obtain large quantities of the desired modified polypeptide of the invention. Techniques for the cultivation of mammalian cells under tissue culture conditions are known in the art and include homogenous suspension culture, e.g., in airlift reactors or continuous stirred reactors, or immobilized or encapsulated cell culture, e.g., in hollow fibers, in microcapsules, on agarose microbeads or porcelain cartridges. If necessary and / or desired, solutions of the polypeptide may be purified by conventional chromatographic methods, e.g., gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, or the like. The product can be purified by affinity chromatography (HIC, chromatography on DEAE-cellulose, or affinity chromatography.

[0208] The present invention also includes a method for improving FVIII activity of a chimeric FVIII protein comprising a VWF protein fused to a first heterologous moiety and an XTEN sequence and a FVIII protein fused to a second heterologous moiety, the method comprising inserting a VWF linker between the VWF protein and the first heterologous moiety, the VWF linker comprising a polypeptide selected from (i) an a2 region from factor VIII (FVIII), (ii) an a1 region from FVIII, (iii) an a3 region from FVIII, (iv) a thrombin cleavage site comprising XVPR (SEQ ID NO: 3) and a PAR1 non-active site interaction motif, where X is an aliphatic amino acid, (v) any combination thereof. In some embodiments, the FVIII activity is measured by aPTT assay or ROTEM assay.

[0209] IV. Pharmaceutical Compositions Compositions containing the chimeric molecules of the present invention may contain suitable pharma- ceutically acceptable carriers, e.g., they may contain excipients and / or auxiliary agents that facilitate processing of the active compounds into preparations designed for delivery to the site of action.

[0210] The pharmaceutical composition can be formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular) by bolus injection.The preparation for injection can be in unit dosage form, for example, in ampoules, or in multi-dose containers with added preservatives.The composition can take such form as suspension, solution, or emulsion in oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.Alternatively, the active ingredient can be in powder form for constitution with suitable vehicle, for example, pyrogen-free water.

[0211] Suitable formulations for parenteral administration also include aqueous solutions of the active compound in water-soluble form, e.g., water-soluble salts. In addition, suspensions of the active compound as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, e.g., sodium carboxymethylcellulose, sorbitol, and dextran. Optionally, the suspension may also contain stabilizers. Liposomes may also be used to encapsulate the molecules of the invention for delivery to cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments, the composition includes an isotonic agent, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions contain pharma- ceutically acceptable substances such as wetting agents, which enhance the shelf life or effectiveness of the active ingredient, or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers.

[0212] The compositions of the present invention may be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the mode of administration and therapeutic application.

[0213] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of ingredients listed above, if required, followed by filtered sterilization. Generally, dispersions contain the active ingredient in the form of a salt. It is prepared by incorporating it into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, for example, monostearate salts and gelatin, in the composition.

[0214] The active ingredient can be formulated with sustained release preparations or devices.Examples of such preparations or devices include implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.The method for preparing such preparations and devices is known in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0215] Injectable depot formulations can be made by forming microencapsulated matrices of the drug 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 exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions.

[0216] Supplementary active compounds can be incorporated into the composition. In one embodiment, the chimeric molecule of the present invention is formulated with another coagulation factor, or its variant, fragment, analog, or derivative. For example, the coagulation factor includes, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF), or any of the above-mentioned activated forms. The hemostatic coagulation factor can also include antifibrinolytic agents, such as eprosin-amino-caproic acid, tranexamic acid.

[0217] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single pill may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0218] In addition to the active compound, liquid dosage forms may contain inactive ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.

[0219] Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by E. W. Martin. Excipients Some examples include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc. The composition can also contain pH buffering agents, and wetting or emulsifying agents.

[0220] For oral administration, the pharmaceutical composition can be in the form of tablets or capsules prepared by conventional methods.The composition can also be prepared as liquid, for example, syrup or suspension.The liquid can contain suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid).The preparation can also contain flavorings, colorings, and sweetening agents.Alternatively, the composition can be present as a dry product for constitution with water or another suitable vehicle.

[0221] For buccal administration, the composition may take the form of a tablet or lozenge following conventional protocols.

[0222] For administration by inhalation, the compound for use according to the present invention is conveniently delivered in the form of aerosol that is nebulized with or without excipients, or in the form of aerosol spray from a pressurized pack or nebulizer, optionally with a propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas.In the case of pressurized aerosol, dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin, for use in inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base, for example, lactose or starch.

[0223] The pharmaceutical compositions may also be formulated for rectal administration as suppositories or retention enemas, e.g., containing cocoa butter or other conventional suppository bases, such as glycerides.

[0224] V. Gene Therapy The chimeric molecule of the present invention can be produced in vivo in a mammal, for example, a human patient, and is beneficial to treat using a gene therapy approach to the treatment of bleeding diseases or disorders selected from bleeding clotting disorders, hemarthrosis, muscle bleeding, oral bleeding, major bleeding, major bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the iliopsoas sheath. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This involves the administration of a suitable chimeric molecule-encoding nucleic acid operably linked to a suitable expression control sequence. In certain embodiments, these sequences are incorporated into a viral vector. Suitable viral vectors for such gene therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, and adeno-associated virus (AAV) vectors. The viral vector can be a replication-defective viral vector. In other embodiments, the adenoviral vector has a deletion in its E1 or E3 gene. When an adenoviral vector is used, the mammal may not be exposed to a nucleic acid encoding a selectable marker gene. In other embodiments, the sequence is incorporated into a non-viral vector known to those skilled in the art. do.

[0225] VI. Methods of Use of Chimeric Proteins The present invention further provides a method for reducing the frequency or severity of bleeding episodes in a subject in need thereof using the chimeric molecule of the present invention. An exemplary method comprises administering a therapeutically effective amount of the chimeric molecule of the present invention to a subject in need thereof. In another embodiment, the present invention comprises a method for preventing the occurrence of bleeding episodes in a subject in need thereof using the chimeric molecule of the present invention. In another embodiment, a composition comprising DNA encoding a recombinant protein of the present invention can be administered to a subject in need thereof. In certain embodiments of the present invention, a cell expressing the chimeric molecule of the present invention can be administered to a subject in need thereof. In certain embodiments of the present invention, a pharmaceutical composition comprises (i) a chimeric molecule, (ii) an isolated nucleic acid encoding the chimeric molecule, (iii) a vector comprising a nucleic acid encoding the chimeric molecule, (iv) a cell comprising an isolated nucleic acid encoding the chimeric molecule, and / or a vector comprising a nucleic acid encoding the chimeric molecule, or (v) a combination thereof, the pharmaceutical composition further comprising an acceptable excipient or carrier.

[0226] Bleeding episodes can be caused or derived from blood clotting disorders. Blood clotting disorders can also be referred to as coagulopathy. In one example, the blood clotting disorder that can be treated with the pharmaceutical composition of the present disclosure is hemophilia or von Willebrand's disease (vWD). In another example, the blood clotting disorder that can be treated with the pharmaceutical composition of the present disclosure is hemophilia A.

[0227] In some embodiments, the type of bleeding associated with the bleeding condition is selected from hemarthrosis, muscle bleeding, oral bleeding, major bleeding, major bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal space bleeding, retroperitoneal space bleeding, iliopsoas sheath bleeding, or any combination thereof.

[0228] In other embodiments, the patient suffering from the bleeding condition is in need of treatment for surgery, including, for example, surgical prophylaxis or perioperative management. In one example, the surgery is selected from minor or major surgery. Exemplary surgical procedures include tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, joint replacement surgery (e.g., total knee replacement, hip replacement, etc.), cardiac surgery, and cesarean section.

[0229] In another example, the subject is co-treated with factor IX. The compounds of the present invention are capable of activating FIXa and can therefore be used to pre-activate FIXa polypeptides before administering FIXa to the subject.

[0230] The methods of the invention can be practiced on subjects in need of preventative or on-demand treatment.

[0231] Pharmaceutical compositions containing the chimeric molecules of the present invention can be formulated for any suitable mode of administration, including, for example, topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal, or parenteral administration.

[0232] 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. For example, it can be a suspension, emulsion, sustained release formulation, cream, gel, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0233] Having now described the invention in detail, the same will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention. All patents and publications mentioned within this specification are expressly incorporated by reference. EXAMPLES

[0234] Throughout the examples, the following materials and methods were used unless otherwise stated.

[0235] Materials and Methods In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (e.g., antibody technology), and standard techniques in electrophoresis. See, e.g., 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 See Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996), Antibodies: A Laboratory Manual, Harlow et al., CS. HLPress, Pub. (1999), and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

[0236] Example 1. Evaluation of thrombin-mediated D'D3 release of various VWF constructs This example evaluates the kinetics of thrombin-mediated D'D3 release at 37°C of various VWF constructs described in Figure 2. Biocore experiments were performed with VWF-Fc constructs containing different thrombin-cleavable linkers between the D'D3 domain of VWF and the Fc. The ultimate goal is to apply the information gleaned from VWF-Fc thrombin digestion to the FVIII-VWF heterodimer as described herein. All VWF-D'D3 constructs were run on the chip, achieving capture densities of protein ranging from 100 to 700 RU. After the VWF constructs were captured on the chip, 5 U / ml of thrombin was injected across the surface for 5 min. The D'D3 in the cleavable constructs is released while the Fc remains bound to the chip. The velocity (RU / sec) versus capture density (RU) was plotted as shown in Figures 3 and 4. The cleavage rate was proportional to the starting capture density, while the slope provided a measure of the susceptibility of each construct to thrombin cleavage.

[0237] Figure 3 shows that VWF-052 (which does not have a thrombin cleavage site within the linker region) is not cleaved by thrombin, as predicted. The rate of VWF-039 (LVPR with PAR1 site) is comparable to the FVIII cleavage rate (data not shown). For this reason, VWF-039 served as a benchmark for complete D'D3 release from Fc. Ratios of slopes of various VWF-Fc constructs with respect to VWF-039 were used to determine the efficiency of thrombin cleavage. VWF-039 (LVPR with PAR1 site) is cleaved with thrombin approximately 70-80 times faster than VWF-031 (LVPR). VWF-51 (ALRPRVV) is cleaved 1.8 times faster than VWF-031 (LVPR). VWF-034, which contains 288XTEN along the LVPR site, showed slower cleavage compared to VWF-031.

[0238] VWF-Fc constructs were also generated by introducing different acidic regions (a1, a2, and a3) of the FVIII protein within the linker region. VWF-055, which contains the a2 region between the D'D3 and Fc regions, showed thrombin cleavage similar to that of the VWF-039 construct. As shown in Figure 4, VWF-054 (a1 region) and VWF-056 (a3 region) showed approximately 5-fold reduced thrombin cleavage.

[0239] Figure 5 shows the slopes of the thrombin cleavage curves of the different VWF constructs. From these results, the acidic region 2 (a2) of FVIII appears to be a highly efficient thrombin cleavage site and was incorporated into the FVIII-VWF heterodimer as described herein.

[0240] Example 2. Evaluation of the hemostatic potential of FVIII / VWFD'D3 heterodimers using a HemA patient whole blood ROTEM assay FVIII / VWFD'D3 heterodimers containing different thrombin cleavable linkers were evaluated in a HemA donor whole blood ROTEM (rotational thromboelastometry) assay for their ability on hemostasis. Whole blood samples were collected from donors with severe hemophilia A bleeding disorder using sodium citrate as an anticoagulant. Forty minutes after blood sample collection, FVIII / VWFD'D3 heterodimer variants containing different thrombin cleavable linkers, FVIII155 / VWF031 (48aa, LVPR site), FVIII155 / VWF039 (26aa, LVPR+PAR1 site), FVIII155 / VWF055 (34aa, a2 from FVIII), were diluted in the whole blood sample to the usual final concentrations of 100%, 30%, 10%, and 3% as measured by FVIII chromogenic assay. Immediately after the addition of FVIII / VWFD'D3 heterodimer, the ROTEM reaction was started by the addition of CaCl2. The clotting time (time from the beginning of the test to reach an amplitude of 2 mm) was recorded by the instrument and plotted against the FVIII concentration in the samples (Figure 6). It was hypothesized that the more potent FVIII / VWFD'D3 heterodimer induces a faster clotting process, thus resulting in shorter clotting times compared to the less potent FVIII / VWFD'D3 heterodimer. As shown in Figure 6, samples spiked with FVIII / VWF039 heterodimer had the shortest clotting times at all concentrations tested, and samples spiked with FVIII / VWF031 heterodimer had the longest clotting times at all concentrations. The clotting times of samples spiked with FVIII155 / VWF055 heterodimer were intermediate. Therefore, the ranking of hemostatic ability is FVIII155 / VWF039>FVIII155 / VWF055>FVIII155 / VWF031. Since the only difference between the three molecules is the thrombin-cleavable linker between the VWF protein and the Fc region, this result indicates that the linker containing the LVPR site and the PAR1 non-active site interaction motif and the a2 region of FVIII works better than the LVPR site alone.

[0241] Example 3. Evaluation of the activity of FVIII / VWF heterodimers The FVIII-XTEN / VWF heterodimer constructs were transfected into HEK293F cells using three plasmids, the first expressing FVIII-XTEN-Fc, the second expressing VWF-XTEN-Fc, and the third expressing PACE. Polyethylenimine (PEI) standard protocols were used for transfection, and tissue culture medium was harvested 5 days after transfection. Various combinations of FVIII-VWF heterodimers were purified from the culture medium. The activity of the purified proteins was tested in both chromogenic (two-step) and aPTT (one-step) clotting assays using standard protocols. The introduction of the acidic region 2 (a2) of FVIII between FVIII and Fc or between D'D3 and Fc (shown in Table 7A and Figure 7) improved the aPTT activity of FVIII-VWF heterodimers, as shown in Table 7C. For example, the FVIII169 / VWF059 heterodimer has an a2 thrombin cleavage site within the D'D3-Fc linker region, as shown in Table 7C. has better aPTT activity than FVIII169 / VWF057, which contains the LVPR thrombin site within the D'D3Fc linker.

[0242] Similarly, incorporation of the a2 region between FVIII and Fc increased the one-stage clotting activity of the heterodimers, as evidenced by the improved chromogenic and aPTT ratios of FVIII286 / VWF059 and FVIII286 / VWF062, as shown in Table 7B. [Table 7A] [Table 7B] [Table 7C]

[0243] Example 4: Acute Effects of FVIII-XTEN-Fc / D'D3-XTEN-Fc Heterodimers in the HemA Mouse Tail Cut Bleeding Model The acute effects of heterodimers containing different thrombin cleavable linkers were evaluated using the HemA mouse tail cut bleeding model.

[0244] Male HemA mice aged 8–12 weeks were randomly assigned to one of five treatment groups, each with a SQ Patients were treated with a single intravenous administration of BDD-FVIII, rFVIII169 / VWF034, rFVIII169 / VWF057, rFVIII169 / VWF059, or vehicle solution. To mimic FVIII crisis treatment (to reconstitute 50-100% of normal FVIII plasma levels), the FVIII treatment doses chosen were FVIII aP At this dose level, all tested FVIII variants will reconstitute approximately 70% of normal mouse plasma FVIII activity 5 min after administration, as measured by TT activity, at 75 IU / kg.

[0245] The tail amputation procedure was performed as follows. Briefly, mice were anesthetized with a mixture of 50 mg / kg ketamine / 0.5 mg / kg dexmedetomidine prior to tail injury and placed on a heating pad at 37° C. to help maintain body temperature. The tails of mice were then immersed in 37° C. saline for 10 minutes to allow for dilation of the tributaries. After vasodilation, FVIII variants or vehicle solution were injected via the tail vein, and then the distal 5 mm of the tail was cut using a straight-edged No. 11 scalpel 5 minutes after administration. Blood was collected in 13 ml of 37° C. saline for 30 minutes, and blood loss was determined by the weight change of the collection tube: blood loss = (final weight of collection tube - starting weight + 0.10) ml. Statistical analysis was performed using t-tests (Kolmogorov-Smirnov test) and one-way ANOVA (Kruskal-Wallis test, post-hoc test: Dunns multiple comparison test).

[0246] Blood loss from each individual animal in this study is plotted in Figure 8. A significant reduction in blood loss was observed in all FVIII treatment groups compared to vehicle treated animals (p<0.05, Table 8). A similar reduction in blood loss was observed in all heterodimer treatment groups compared to BDD-FVIII treatment (p>0.5, Table 8), suggesting that the heterodimer molecule may be as effective as SQ BDD-FVIII for on-demand treatment.

[0247] [Table 8]

[0248] pSYN VWF057 nucleotide sequence (VWF D'D3-Fc with LVPR thrombin site in the linker) (SEQ ID NO:79) [ka] [ka] [ka] [ka] pSYN VWF057 protein sequence (VWF D'D3-Fc with LVPR thrombin site in the linker): The bold underlined portion indicates the thrombin-cleavable LVPR containing linker region (SEQ ID NO:80) [ka] pSYN VWF059 nucleotide sequence (VWF D'D3-Fc with acidic region 2 (a2) thrombin site in the linker) (SEQ ID NO:81) [ka] [ka] [ka] [ka] pSYN VWF059 protein sequence (VWF D'D3-Fc with LVPR thrombin site in the linker) - bold underlined portion indicates a2 region (SEQ ID NO:82) [ka] [ka] pSYN VWF062 nucleotide sequence (VWF D'D3-Fc without a thrombin site in the linker) (SEQ ID NO:83) [ka] [ka] [ka] [ka] [ka] [ka] pSYN VWF062 protein sequence (VWF without thrombin site in the linker) D'D3-Fc) (SEQ ID NO: 84) [ka] [ka] pSYN FVIII 286 nucleotide sequence (FVIII-Fc with an additional a2 region between FVIII and Fc) (SEQ ID NO:85) [ka] [ka] [ka] [ka] [ka] [ka] pSYN FVIII286 protein sequence (FVIII-Fc with an additional a2 region between FVIII and Fc, shown in bold and underlined) (SEQ ID NO:86) [ka] [ka] FVIII169 nucleotide sequence (SEQ ID NO:87) [ka] [ka] [ka] [ka] [ka] [ka] FVIII169 protein sequence (SEQ ID NO:88) [ka] VWF034 nucleotide sequence (SEQ ID NO:91) [ka] [ka] [ka] VWF034 protein sequence (SEQ ID NO:92) [ka] [ka]

[0249] The foregoing description of the specific embodiments fully reveals the general nature of the present invention, so that by applying knowledge within the art, one skilled in the art can easily modify and / or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concept of the present invention. Such applications and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology in this specification is for the purpose of description rather than limitation, and that the terminology or terminology in this specification will be interpreted by one of ordinary skill in the art in light of the teaching and guidance.

[0250] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0251] All patents and publications cited within this specification are hereby incorporated by reference in their entirety.

[0252] This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 840,872, filed June 28, 2013, the contents of which are incorporated herein by reference in their entirety.

Claims

[Claim 1] A composition, chimeric molecule or method as described in any part of this specification.

Images