Factor viii complex comprising XTEN and von willebrand factor protein and uses thereof

Abstract

To provide Factor VIII complex comprising XTEN and von Willebrand factor protein and uses thereof.SOLUTION: The invention discloses a chimeric protein comprising a VWF protein with D' domain and D3 domain of VWF, at least one XTEN sequence and a FVIII protein, where the VWF fragment, the XTEN sequence or the FVIII protein are linked or associated with each other. The chimeric protein may comprise further one or more Ig constant region or a part thereof (e.g., Fc region).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding coagulation factor VIII (FVIII), affecting 1 to 2 in every 10,000 live births. Grawetal., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients with hemophilia A receive purified FVII. Coagulation disorders can be treated with the administration of recombinantly produced FVIII. However, all commercially available FVIII products are known to have a half-life of approximately 8-12 hours and must be administered intravenously to patients frequently. See Weiner, M.A., and Cairo, MS, Pediatric Hematology Secrets; Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122Suppl4:S2-8 (2008). Furthermore, many approaches have been attempted to extend the half-life of FVIII. Methods under development to extend the half-life of coagulation factors include, for example, PEGylation, glycoPEGylation, and albumin conjugation. See Dumont et al., Blood. 119(13): 3024-3030 (Published online January 13, 2012). However, regardless of the protein recombinant method used, long-acting FVIII products currently under development have reportedly short half-lives (approximately 1.5 to 2 hours in preclinical animal models). See supra. The results are similar in humans; for example, rFVIIIFc has been reported to provide an approximately 1.7-fold improvement in half-life extension in patients with hemophilia A compared with ADVATE®. See supra. Therefore, despite minor improvements, other T1 / 2-limiting factors for half-life extension are suggested. See Liu, T. et al., 2007 ISTH meeting, abstract #PM-035; Henrik, A. et al., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131. [Background technology]

[0002] The half-life of plasma von Willerbrand factor (VWF) is approximately 12 hours (range 9-15 hours). http: / / www.nhlbi.nih.gov / guidelines / vwd / 2_scientificoverview.htm (Last confirmed October 22, 2011). The half-life of VWF can be affected by many factors, including glycosylation patterns, ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motif-13), and various mutations in VWF.

[0003] In plasma, 95-98% of FVIII circulates in a tight, non-covalent complex with full-length VWF. The composition of this complex is important for maintaining adequate plasma FVIII in vivo. Lenting et al., Blood. 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost. 5(7):1353-60 (2007). Full-length wild-type FVIII is Most FVIII exists as a heterodimer with a heavy chain (MW 200 kD) and a light chain (MW 73 kD). When FVIII is activated by proteolysis at positions 372 and 740 of the heavy chain and at position 1689 of the light chain, VWF, which binds to FVIII, is removed from the activated FVIII. Activated FVIII (tenase complex) along with activated factor IX, calcium, and phospholipids induces the activation of factor X, generating large amounts of thrombin. Thrombin then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form soluble fibrin polymers. Thrombin also activates factor XIII, which, together with calcium, crosslinks and stabilizes the soluble fibrin polymers to form crosslinked (insoluble) fibrin. Activated FVIII is rapidly removed from the circulation by proteolysis.

[0004] Due to the inconvenience caused by frequent administration and dosing schedules, there is a need to develop FVIII products that require less frequent administration, i.e., FVIII products with half-lives 1.5 to 2 times longer than the half-life limit. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lenting et al., Blood.(1998)92(11):3983-96 [Non-patent document 2] Lenting et al., J. Thromb. Haemost.(2007)5(7):1353-60 Summary of the Invention [Means for solving the problem]

[0006] The present invention is directed to chimeric proteins containing (i) a von Willerbrand factor (VWF) fragment containing the D' and D3 domains of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, and wherein the VWF fragment or the XTEN sequence is linked or associated with the FVIII protein. The chimeric protein may contain a single polypeptide chain containing the VWF fragment, the XTEN sequence, and the FVIII protein, or it may contain two polypeptide chains (one chain containing the VWF fragment and the second chain containing the FVIII protein), wherein the XTEN polypeptide is linked to either the VWF fragment or the FVIII protein.

[0007] In one embodiment, the chimeric protein of the invention comprises: (a) VX-FVIII, (b) FVIII-XV, (c) VX:FVIII, (d) XV:FVIII, (e) FVIII:VX, or (f) FVIII:XV wherein: V contains a VWF fragment, X contains one or more XTEN sequences, and FVIII comprises a FVIII protein. A hyphen (-) can be a peptide bond or a linker (e.g., a cleavable linker), and a colon (:) represents a chemical or physical association (e.g., a covalent or non-covalent bond) between polypeptides.

[0008] In other embodiments, the chimeric protein further contains (iv) an immunoglobulin (Ig) constant region or portion thereof (also referred to as F1 or a first Ig constant region or portion thereof) linked to a VWF fragment, an XTEN sequence, a FVIII protein, or any combination thereof. In other embodiments, the chimeric protein further contains an additional Ig constant region or portion thereof (also referred to as F2 or a second Ig constant region or portion thereof). The first Ig constant region or portion thereof may be linked to a VWF fragment or an XTEN sequence, and the second Ig constant region may be linked to a FVIII protein. The first Ig constant region, the second Ig constant region, or portion thereof, or both, may extend the half-life of the FVIII protein.

[0009] In some embodiments, the second Ig constant region or portion thereof (F2) is linked to the VWF fragment by a linker (e.g., a processable linker). In other embodiments, the second Ig constant region or portion thereof (F2) is linked to the (first) Ig constant region. The second Ig constant region or portion thereof (F2) and the first Ig constant region or portion thereof (F1) are associated with the Ig constant region or portion thereof (F1). The second Ig constant region or portion thereof (F2) and the first Ig constant region or portion thereof (F1) can be the same or different. The second Ig constant region or portion thereof can be associated with the Ig constant region or portion thereof by a covalent bond (e.g., a disulfide bond). The VWF fragment linked to the first Ig constant region or portion thereof can also be associated with the FVIII protein linked to the second Fc region by a non-covalent bond. In certain embodiments, the FVIII protein may further contain one or more additional XTEN sequences linked to the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites). In some embodiments, the half-life of the FVIII protein is extended compared to wild-type FVIII or a FVIII protein without a VWF fragment.

[0010] In some embodiments, the chimeric protein comprises: (g)V-L2-X-L1-F1:FVIII-L3-F2, (h)V-L2-X-L1-F1:F2-L3-FVIII, (i)F1-L1-X-L2-V:FVIII-L3-F2, (j)F1-L1-X-L2-V:F2-L3-FVIII, (k)V-L2-X-L1-F1-L4-FVIII-L3-F2, (l)F2-L3-FVIII-L4-F1-L1-X-L2-V, (m) FVIII-L3-F2-L4-V-L2-X-L1-F1, and (n)F1-L1-X-L2-V-L4-F2-L3-FVIII, wherein: V contains a VWF fragment, each of L1, L2, and L3 contains an optional linker (e.g., a cleavable linker); L4 is an optional linker (e.g., a processable linker), FVIII contains the FVIII protein, X contains one or more XTEN sequences; F1 contains any first Ig constant region or portion thereof; F2 contains any second Ig constant region or portion thereof, and (:) is a covalent or non-covalent bond.

[0011] The present invention also relates to chimeric proteins comprising (i) a FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or portion thereof, wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus of the FVIII protein by an optional linker, or inserted immediately downstream (at one or more insertion sites) of one or more amino acids of the FVIIII protein, and wherein the Ig constant region or portion thereof is linked to or associated with the FVIII protein or the XTEN sequence. In one embodiment, the Ig constant region or portion thereof useful in the chimeric protein comprises a first Fc region. In another embodiment, the chimeric protein further comprises an additional Ig constant region or portion thereof. The additional Ig constant region or portion thereof useful in the present invention comprises a second Fc region (linked to or associated with the first Fc region, e.g., covalently linked, for example, by a processable linker) in another embodiment.

[0012] In other embodiments, the chimeric protein contains (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, and (iv) an Ig constant region or a portion thereof (containing the D' and D3 domains of VWF), wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C-terminus of the FVIII protein. The VWF fragment is linked to or inserted immediately downstream (e.g., at one or more insertion sites) of one or more amino acids in the FVIII protein, the VWF fragment is linked to or associated with the FVIII protein or XTEN sequence, and the Ig constant region or portion thereof is linked to the FVIII protein, XTEN sequence, VWF fragment, or any combination thereof. Non-limiting examples of chimeric proteins include the following: (1)FVIII(X1)-L1-F1:V-L2-X2-L3-F2, (2)FVIII(X1)-L1-F1:F2-L3-X2-L2-V, (3)F1-L1-FVIII(X1):V-L2-X2-L3-F2, (4)F1-L1-FVIII(X1);F2-L3-X2-L2-V, (5)FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2, (6)FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V, (7) F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (8)F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V, wherein: FVIII(X1) comprises a FVIII protein and one or more XTEN sequences, wherein the one or more XTEN sequences are linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., at one or more XTEN insertion sites); each of L1, L2, or L3 contains an optional linker, such as a cleavable linker; L4 is a linker (processible linker), X2 contains one or more XTEN sequences, F1 contains an Ig constant region or a portion thereof, F2 contains any additional Ig constant region or portion thereof, and V contains a VWF fragment, (-) is a peptide bond or one or more amino acids, and (:) contains a covalent or non-covalent bond.

[0013] In one embodiment of the present invention, the VWF fragment useful in the chimeric protein does not bind to a VWF clearance receptor that interferes with or inhibits the interaction of the FVIII protein with endogenous VWF. Thus, the chimeric protein containing the VWF fragment is not cleared via the VWF clearance pathway. In other embodiments of the present invention, the VWF fragment can protect the FVIII protein from one or more protease cleavage, protect the FVIII protein from activation, stabilize the heavy and / or light chains of the FVIII protein, or prevent clearance of the FVIII protein by one or more scavenger receptors.

[0014] The VWF fragment can interfere with or inhibit the interaction between the FVIII protein and endogenous VWF, thereby extending the half-life of the FVIII protein compared to a FVIII protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to wild-type FVIII. In other embodiments, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours, at least about 41 hours, at least about 42 hours, at least about 43 hours, at least about 44 hours, at least about 45 hours, at least about 46 hours, at least about 47 hours, at least about 48 hours, at least about 49 hours, at least about 50 hours, at least about 51 hours, at least about 52 hours, at least about 53 hours, at least about 54 hours, at least about 55 hours, at least about 56 hours, at least about 57 hours, at least about 58 hours, at least about 59 hours, at least about 60 hours, at least about 61 hours, at least about 62 hours, at least about 63 hours, at least about 64 hours, at least about 65 hours, at least about 66 hours, at least about 67 hours, at least about 68 hours, at least about 69 hours, at least about 70 hours, at least about 71 hours, at least about 72 hours, at least about 73 hours, at least about 74 hours, at least about 75 hours, at least about 76 hours, at least about 77 hours, at least about 78 hours, at least about 79 hours, at least about 80 hours, at least about 81 hours, at least about 82 hours, At least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0015] The Ig constant region or portion thereof useful in the present chimeric protein contains a first Fc region linked to a VWF fragment by any linker (e.g., a cleavable linker). The chimeric protein may further contain an additional Ig constant region or portion thereof linked to a FVIII protein or XTEN sequence, an Ig constant region or portion thereof, a VWF fragment, or any combination thereof by any linker. In one embodiment, the additional Ig constant region or portion thereof is linked to the FVIII protein by any linker. The additional Ig constant region or portion thereof may contain a second Fc region.

[0016] The Ig constant region or portion thereof useful in the present invention and the additional Ig constant region or portion thereof useful in the present invention may be the same or different.

[0017] In some embodiments, the FVIII protein is linked to the XTEN sequence at the C-terminus or N-terminus of the FVIII protein, or inserted immediately downstream (e.g., at one or more insertion sites) of one or more amino acids in mature native human FVIII, or any combination thereof. The one or more insertion sites in the FVIII protein may be located within one or more domains of the FVIII protein selected from the group consisting of the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the C1 domain, the C2 domain, and any combination thereof; or may be located between one or more domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof; or may be located between two domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof.

[0018] In one embodiment, the one or more insertion sites are located immediately downstream of one or more amino acids in mature native human FVIII (e.g., SEQ ID NO: 4 [full mature FVIII sequence]) selected from the group consisting of amino acid residues in Tables 7, 8, 9, 10, 11, or any combination thereof.

[0019] In other embodiments, one or more insertion sites are located within a permissive loop of mature native human FVIII. In other embodiments, one or more insertion sites are located within the a3 region of mature native human FVIII. For example, an XTEN sequence may be inserted immediately downstream of amino acid 1656 of SEQ ID NO: 4 (full-length mature FVIII). In other embodiments, the FVIII protein is linked to at least two XTEN sequences (e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2): a first XTEN sequence inserted within the a3 region and a second XTEN sequence inserted within a permissive loop of the FVIII protein. In yet other embodiments, the FVIII protein is linked to at least three XTEN sequences: an XTEN sequence inserted within the a3 region, and a second and third XTEN sequences inserted within one or two permissive loops within the FVIII protein (e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2).

[0020] In certain embodiments, the one or more insertion sites for one or more XTEN insertions are selected from the group consisting of: (1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) amino acid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) 1937 amino acids, (52) 2019 amino acids, (53) amino acid 2068, (54) amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188, (58) amino acid 2227, (59) Amino acid 2277, and (60) a combination of two or more thereof. and immediately downstream of one or more amino acids selected from the group consisting of:

[0021] In some embodiments, one XTEN is inserted into the FVIII protein. In some embodiments, two XTENs are inserted into the FVIII protein. In some embodiments, three XTENs are inserted into the FVIII protein.

[0022] In particular examples, a first XTEN is inserted immediately downstream of amino acid 26 of SEQ ID NO:4, and a second XTEN is inserted immediately downstream of amino acid 1720, which corresponds to SEQ ID NO:4 (full-length mature FVIII). In other examples, a first XTEN is inserted immediately downstream of amino acid 403 of SEQ ID NO:4, and a second XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4. In some examples, a first XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a second XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4. In other examples, a first XTEN is inserted immediately downstream of amino acid 26 of SEQ ID NO:4, a second XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a third XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4. In yet another embodiment, a first XTEN is inserted immediately downstream of amino acid 403 of SEQ ID NO:4, a second XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a third XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4. One XTEN is inserted between amino acids 403 and 404 of SEQ ID NO:4, a second XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a third XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4. In certain embodiments, a first XTEN is inserted immediately downstream of amino acid 26, which corresponds to SEQ ID NO:4 (full-length mature FVIII), a second XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4, and a third XTEN is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 of SEQ ID NO:4, a second XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, a third XTEN is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4, and a fourth XTEN sequence is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In another example, the XTEN is inserted immediately downstream of amino acid 745 of SEQ ID NO:4. In a further example, the first XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and the second XTEN is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In some embodiments, the first XTEN is inserted immediately downstream of amino acid 26 of SEQ ID NO:4, the second XTEN is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and the third XTEN is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In another example, the first XTEN is inserted immediately downstream of amino acid 403 of SEQ ID NO:4, and the second XTEN is inserted immediately downstream of amino acid 745 of SEQ ID NO:4. In some embodiments, the first XTEN is inserted immediately downstream of amino acid 745 of SEQ ID NO:4, and the second XTEN is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 18 of SEQ ID NO:4, and a second XTEN is inserted immediately downstream of amino acid 745 of SEQ ID NO:4.

[0023] In some embodiments, the FVIII protein is a two-chain FVIII isoform. In some embodiments, the FVIII protein is a single-chain FVIII isoform.

[0024] In some embodiments, the inserted XTEN is SEQ ID NO: 39 (AE288). In some examples, the inserted XTEN is SEQ ID NO: 38 and 37 (AG144 and AE144). In some examples, the inserted XTEN is SEQ ID NO: 37, 38, and 37 (AE144, AG144, and AE144). In some embodiments, the inserted XTEN is SEQ ID NO: 37 and 40 (AE144 and AE288). In some embodiments, the inserted XTEN is AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AE144_A (SEQ ID NO: 133), AE144_B (SEQ ID NO: 134), AE14 4_C (SEQ ID NO: 135), AE144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combination thereof.

[0025] FVIII proteins useful in the present invention may contain a B domain or a portion thereof (e.g., SQ B-domain deleted FVIII). In one embodiment, the FVIII protein contains a single-chain FVIII. In another embodiment, the single-chain FVIII contains at least one amino acid substitution at a residue corresponding to residues 1648, 1645, or both, of the full-length mature factor VIII polypeptide (SEQ ID NO: 4), or at residues 754, 751, or both, of the SQ BDD factor VIII (SEQ ID NO: 6). In another embodiment, the amino acid substitution is an amino acid other than arginine. In some embodiments, the FVIII protein comprises a FVIII heavy chain and a FVIII light chain, wherein the heavy chain and the light chain are associated with each other by metal binding.

[0026] The FVIII protein may have low affinity or not bind to low-density lipoprotein receptor-related protein (LRP) (e.g., by containing at least one amino acid substitution that reduces affinity for LRP or by containing at least one amino acid substitution that eliminates binding to LRP). Such at least one amino acid substitution may be at residues 471, 484, 487, 490, 497, 2092, 2093, or a combination of two or more thereof, of full-length mature FVIII. In certain embodiments, the amino acid substitution at residue 471, 484, or 497 is an amino acid other than arginine, the amino acid substitution at residue 487 is an amino acid other than tyrosine, the amino acid substitution at residue 2092 is an amino acid other than lysine, or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine.

[0027] In some embodiments, the FVIII protein contains at least one amino acid substitution that renders the FVIII protein more stable than a FVIII protein that does not have the substitution. Such substitutions may be located in the A2 and A3 domains of the FVIII protein (e.g., at residues corresponding to residues 664, 1826, 662, 1828, or a combination of two or more thereof in full-length mature FVIII).

[0028] VWF fragments useful in the present invention contain the D' domain and the D3 domain, both of which are capable of binding to FVIII. The VWF fragment may contain an amino acid sequence of the D' domain that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-866 of SEQ ID NO: 2 and / or an amino acid sequence of the D3 domain that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO: 2. In one embodiment, the VWF fragment is monomeric. In other embodiments, the VWF fragment contains at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments. The VWF fragment may contain amino acids that are at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1240 of SEQ ID NO: 2. The VWF fragment can consist essentially of or consist of amino acids 764-1240 of SEQ ID NO: 2. In certain embodiments, the VWF fragment may contain at least one amino acid substitution at residues corresponding to 1099, 1142, or both 1099 and 1142 of SEQ ID NO: 2. In other embodiments, the VWF fragment further contains the D1 domain, the D2 domain, or the D1 and D2 domains of VWF.

[0029] The VWF fragment may further contain a VWF domain selected from the group consisting of 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, and any combination thereof. For example, the VWF fragment can consist essentially of, or consist 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 some embodiments, the VWF fragment may comprise a VWF fragment. The F fragment further contains the signal peptide of VWF or FVIII operably linked to the VWF fragment.

[0030] One or more of the linkers useful in the present invention have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. In some embodiments, one or more of the linkers have a length of from about 1 to about 200 amino acids. In one embodiment, one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids. In other embodiments, one or more of the linkers contains a gly / ser peptide, an XTEN sequence, or both. Examples of gly / ser peptides include, but are not limited to, (Gly4Ser) n (SEQ ID NO: 139), or S(Gly4Ser) n(SEQ ID NO: 140), where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. For example, (Gly4Ser) nThe linker may be (Gly4Ser)3 (SEQ ID NO: 63) or (Gly4Ser)4 (SEQ ID NO: 138). In one embodiment, the linker contains at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both. In other embodiments, the linker contains a 20-amino acid, 35-amino acid, 48-amino acid, 73-amino acid, or 95-amino acid thrombin-cleavable linker. The cleavable linker may contain one or more cleavage sites by a protease selected from the group consisting of Factor XIa, Factor XIIa, kallikrein, Factor VIIa, Factor IXa, Factor Xa, Factor IIa (thrombin), elastase 2, granzyme B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20 (e.g., TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8)). Non-limiting examples of the one or more cleavage sites include RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO: 23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28), GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34), and LPKTGSES (SEQ ID NO: 35). In some embodiments, the first cleavage site and the second cleavage site are the same or different.

[0031] XTEN sequences useful in the present invention include AE42 (SEQ ID NO: 36), AE144 (SEQ ID NO: 37), AG144 (SEQ ID NO: 38), AE288 (SEQ ID NO: 39), AG288 (SEQ ID NO: 40), AE576 (SEQ ID NO: 41), AG576 (SEQ ID NO: 42), AE864 (SEQ ID NO: 43), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), ), AG144_F (SEQ ID NO: 136), AE288_2 (SEQ ID NO: 137), or AG864 (SEQ ID NO: 44). In certain embodiments, the XTEN sequence contains AG288 and AG288.

[0032] The chimeric proteins of the present invention may be polysialylated, pegylated, or hesylated.

[0033] The present invention also relates to a polynucleotide or set of polynucleotides encoding the chimeric protein. The polynucleotide may further contain a polynucleotide chain encoding PC5 or PC7. The present invention also relates to a vector containing a polynucleotide or set of polynucleotides and one or more promoters operably linked to the set of polynucleotides. The vector may further contain an additional vector (containing a polynucleotide chain encoding PC5 or PC7). The present invention also relates to a host cell containing the polynucleotide or the vector. The host cell may be a mammalian cell (e.g., a HEK293 cell, a CHO cell, or a BHK cell). In some embodiments, the PC5 or PC7 in the host cell cleaves the D1D2 domain of VWF.

[0034] The present invention also provides pharmaceutical compositions containing the chimeric protein, polynucleotide, vector, or host cell and a pharmaceutically acceptable carrier. The compositions of the present invention therefore have an extended half-life compared to wild-type FVIII protein. The half-life of the FVIII protein is extended by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to wild-type FVIII. The half-life of factor FVIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0035] The compositions of the present invention may be administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, or oral administration. In one embodiment, the compositions are administered parenterally (e.g., intravenously or subcutaneously). The compositions of the present invention are useful for treating bleeding disorders or conditions in patients in need thereof. The bleeding disorders or conditions are selected from the group consisting of bleeding coagulation disorders, joint bleeding, muscle bleeding, oral bleeding, severe bleeding, severe bleeding into muscles, severe bleeding in the oral cavity, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fractures, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, iliopsoas sheath bleeding, and any combination thereof. In one embodiment, the subject treated with the chimeric protein is scheduled to undergo surgery. In other embodiments, the treatment is performed prophylactically or on demand.

[0036] The present invention is also directed to a method for preventing or inhibiting the binding of endogenous VWF to FVIII protein, comprising administering to a subject in need thereof an effective amount of the chimeric protein, polynucleotide, vector, host cell or composition, wherein a VWF fragment binds to FVIII protein, thereby preventing or inhibiting the binding of endogenous VWF. The present invention further relates to a method for extending or increasing the half-life of FVIII protein. The present invention provides a method for inhibiting or suppressing the removal of FVIII protein from a cell, the method comprising administering an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition to a subject in need thereof, wherein a VWF fragment binds to the FVIII protein, thereby extending or increasing the half-life of the FVIII protein. The present invention also provides a method for inhibiting or suppressing the removal of FVIII protein from a cell, the method comprising adding an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition to a cell containing the FVIII protein or a polynucleotide encoding the FVIII protein, wherein a protein having VWF activity binds to the FVIII protein. The subject for which the method of the present invention is useful is an animal, for example, a human (e.g., a patient with hemophilia A).

[0037] The present invention also provides a method of treating a bleeding disorder or condition in a subject in need thereof, the method comprising administering an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition, wherein the bleeding disorder or condition is selected from the group consisting of coagulopathy, joint bleeding, muscle bleeding, oral bleeding, severe bleeding, severe bleeding into muscles, severe bleeding into the oral cavity, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, and iliopsoas sheath bleeding. Treatment may be prophylactic or on-demand. In one embodiment, the effective amount is 0.1 μg / kg to 500 mg / kg.

[0038] The invention also includes methods for producing the chimeric proteins, comprising transfecting one or more host cells with a polynucleotide or vector and expressing the chimeric protein in the host cells. For example, the present invention provides the following: (Item 1) A chimeric protein comprising (i) a von Willebrand factor (VWF) protein containing the D' and D3 domains of VWF, (ii) an XTEN sequence, and (iii) an FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, and wherein the VWF fragment or the XTEN sequence is linked or associated with the FVIII protein. (Item 2) (a) VX-FVIII, (b) FVIII-XV, (c) VX:FVIII, (d) XV:FVIII, (e) FVIII:VX, or (f) FVIII:XV, A chimeric protein comprising the formula: V contains a VWF fragment, X contains one or more XTEN sequences; FVIII contains the FVIII protein; (-) is a peptide bond or one or more amino acids; (:) indicates a chimeric protein, which may be covalently or non-covalently bound. (Item 3) 3. The chimeric protein of any one of items 1 or 2, wherein the XTEN sequence is linked to the FVIII protein by a linker. (Item 4) 4. The chimeric protein according to item 3, wherein the linker is a cleavable linker. (Item 5) 5. The chimeric protein according to any one of items 1 to 4, comprising a single-chain polypeptide comprising the VWF fragment, the XTEN sequence, and the FVIII protein. (Item 6) 5. The chimeric protein according to any one of items 1 to 4, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises FVIII, and the second polypeptide chain comprises the VWF fragment and the XTEN sequence. (Item 7) (iv) The chimeric protein of any one of items 1 to 6, further comprising an Ig constant region or a portion thereof linked to either the VWF fragment, the XTEN sequence, or the FVIII protein, or any combination thereof. (Item 8) (g) V-L2-X-L1-F1:FVIII-L3-F2; (h) V-L2-X-L1-F1:F2-L3-FVIII; (i) F1-L1-X-L2-V:FVIII-L3-F2; (j) F1-L1-X-L2-V:F2-L3-FVIII; (k)V-L2-X-L1-F1-L4-FVIII-L3-F2; (l)F2-L3-FVIII-L4-F1-L1-X-L2-V; (m) FVIII-L3-F2-L4-V-L2-X-L1-F1; and (n)F1-L1-X-L2-V-L4-F2-L3-FVIII, A chimeric protein comprising the formula: V contains a VWF fragment, each of L1, L2, and L3 contains an optional linker; L4 is an optional linker, FVIII contains the FVIII protein, X contains one or more XTEN sequences; F1 contains an optional Ig constant region or portion thereof; F2 optionally contains an additional Ig constant region or portion thereof; (-) is a peptide bond or one or more amino acids; (:) indicates a chimeric protein, which may be covalently or non-covalently bound. (Item 9) 9. The chimeric protein of any one of items 7 or 8, wherein the Ig constant region or portion thereof increases the half-life of the VWF fragment. (Item 10) 10. The chimeric protein of any one of items 7 to 9, wherein the Ig constant region or the portion thereof comprises a first Fc region linked to the XTEN sequence or the VWF fragment. (Item 11) 11. The chimeric protein of item 10, wherein the Ig constant region or portion thereof is linked to the XTEN sequence by a linker. (Item 12) Item 13. The chimeric protein according to item 12, wherein the linker contains a cleavable linker. (Item 13) 13. The chimeric protein according to any one of items 7 to 12, further comprising an additional Ig constant region or part thereof. (Item 14) 14. The chimeric protein according to item 13, wherein the additional Ig constant region or portion thereof contains an additional Fc region. (Item 15) The additional Ig constant region or a portion thereof extends the half-life of the FVIII protein. Item 15. The chimeric protein according to Item 14. (Item 16) 16. The chimeric protein according to any one of items 13 to 15, wherein the additional Ig constant region or part thereof is linked to the FVIII protein. (Item 17) 17. The chimeric protein of item 16, wherein the second Fc region is further linked to the VWF fragment by a linker. (Item 18) Item 18. The chimeric protein according to item 17, wherein the linker is a processable linker. (Item 19) 19. The chimeric protein according to any one of items 8 to 18, wherein L4 is a processible linker. (Item 20) 20. The chimeric protein according to any one of items 7 to 19, wherein the additional Ig constant region or part thereof is associated with the Ig constant region or part thereof. (Item 21) 21. The chimeric protein of item 20, wherein the additional Ig constant region or portion thereof is covalently associated with the Ig constant region or portion thereof. (Item 22) 22. The chimeric protein according to item 21, wherein the covalent bond is a disulfide bond. (Item 23) 23. The chimeric protein according to any one of items 1 to 22, wherein the VWF fragment is non-covalently associated with the FVIII protein. (Item 24) 24. The chimeric protein according to any one of items 1 to 23, wherein the half-life of the FVIII protein is extended compared to a FVIII protein not having the VWF fragment or a wild-type FVIII protein. (Item 25) 25. The chimeric protein of item 24, wherein the half-life of the FVIII is extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein not having the VWF fragment or compared to wild-type FVIII. (Item 26) 25. The chimeric protein of item 24, wherein the half-life of the Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. (Item 27) A chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus of the FVIII protein by an optional linker, or at least A chimeric protein wherein the Ig constant region or portion thereof is inserted between two amino acids of one or more insertion sites, and wherein the Ig constant region or portion thereof is linked or associated with the FVIII protein or the XTEN sequence. (Item 28) 28. The chimeric protein of item 27, wherein the XTEN sequence and the Ig constant region or portion thereof extend the half-life of the FVIII protein. (Item 29) 29. The chimeric protein of any one of items 27 and 28, wherein the VWF binding site is located in the A3 domain, or the C2 domain, or both the A3 domain and the C2 domain of the FVIII protein. (Item 30) 30. The chimeric protein according to item 29, wherein the VWF-binding site contains an amino acid sequence corresponding to amino acids 1669 to 1689 and amino acids 2303 to 2332 of SEQ ID NO: 4. (Item 31) 31. The chimeric protein according to any one of Aspects 27 to 30, wherein the half-life of the FVIII protein is extended compared to a FVIII protein not having the Ig constant region or a portion thereof, or a FVIII protein not having the XTEN sequence. (Item 32) 32. The chimeric protein of item 31, wherein the half-life of the FVIII protein is extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein not comprising the Ig constant region or portion thereof, or a FVIII protein not comprising the XTEN sequence, or wild-type FVIII. (Item 33) 32. The chimeric protein of item 31, wherein the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. (Item 34) 34. The chimeric protein according to any one of items 27 to 33, wherein the Ig constant region or the portion thereof comprises a first Fc region. (Item 35) 35. The chimeric protein of any one of items 27 to 34, further comprising an additional Ig constant region or part thereof. (Item 36) 36. The chimeric protein of item 35, wherein the additional Ig constant region or portion thereof contains a second Fc region linked or associated with the first Fc region. (Item 37) 37. The chimeric protein of item 36, wherein the second Fc region is covalently linked to the first Fc region. (Item 38) Item 3. The first Fc region is linked to the second Fc region by a linker. 6. The chimeric protein according to claim 6. (Item 39) 39. The chimeric protein according to item 38, wherein the linker is a processable linker. (Item 40) 35. The chimeric protein according to any one of items 27 to 34, comprising the FVIII protein, the XTEN sequence, and a single-chain polypeptide containing the Ig constant region or a part thereof. (Item 41) 35. The chimeric protein according to any one of Items 27 to 34, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the FVIII protein, and the second polypeptide chain comprises the light chain of the FVIII protein and an XTEN sequence, and the Ig constant region or a portion thereof. (Item 42) 40. The chimeric protein of any one of items 35 to 39, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the FVIII protein, the XTEN sequence, and the Ig constant region, and the second polypeptide chain comprises the additional Ig constant region or a portion thereof. (Item 43) 40. The chimeric protein of any one of Items 35 to 39, comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the FVIII protein and the XTEN sequence, the second polypeptide chain comprises the light chain of the FVIII protein and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the additional Ig constant region or a portion thereof. (Item 44) 40. The chimeric protein according to any one of Items 35 to 39, comprising a single-chain polypeptide containing the FVIII protein, the XTEN sequence, the Ig constant region or a portion thereof, and the additional Ig constant region or a portion thereof. (Item 45) A chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, and (iv) an Ig constant region or a portion thereof, the chimeric protein containing the D' and D3 domains of VWF, wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus of the FVIII protein by an optional linker, or is inserted immediately downstream of one or more insertion sites in the FVIII protein, the VWF fragment is linked or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combination thereof. (Item 46) (1)FVIII(X1)-L1-F1:V-L2-X2-L3-F2; (2)FVIII(X1)-L1-F1:F2-L3-X2-L2-V; (3)F1-L1-FVIII(X1):V-L2-X2-L3-F2; (4)F1-L1-FVIII(X1);F2-L3-X2-L2-V; (5)FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2; (6)FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V; (7)F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2; or (8)F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V, wherein FVIII(X1) comprises a FVIII protein and an XTEN sequence, wherein said XTEN sequence is a chimeric protein comprising the formula linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids ("insertion site(s)") of said FVIII protein; each of L1, L2, or L3 contains an optional linker; L4 is a linker, X2 contains one or more XTEN sequences, F1 contains an Ig constant region or a portion thereof, F2 optionally contains an additional Ig constant region or portion thereof, and V contains a VWF fragment, (-) is a peptide bond or one or more amino acids; and (:) indicates a chimeric protein, which may be covalently or non-covalently bound. (Item 47) 47. The chimeric protein of any one of items 45 or 46, wherein the VWF fragment does not bind to a VWF clearance receptor. (Item 48) 48. The chimeric protein of any one of items 45 to 47, wherein the VWF fragment is capable of protecting the FVIII protein from cleavage by one or more proteases, protecting the FVIII protein from activation, stabilizing the heavy chain and / or the light chain of the FVIII protein, or preventing removal of the FVIII protein by one or more scavenger receptors. (Item 49) 49. The chimeric protein according to any one of items 45 to 48, wherein the Ig constant region or the portion thereof inhibits or prevents endogenous VWF from binding to the FVIII protein by masking or interfering with the VWF binding site of the FVIII protein. (Item 50) 50. The chimeric protein according to item 49, wherein the VWF binding site is located in the A3 domain, or the C2 domain, or both the A3 domain and the C2 domain of the FVIII protein. (Item 51) 51. The chimeric protein according to any one of items 49 or 50, wherein the VWF-binding site contains an amino acid sequence corresponding to amino acids 1669 to 1689 and amino acids 2303 to 2332 of SEQ ID NO: 4. (Item 52) 52. The chimeric protein according to any one of items 45 to 51, wherein the half-life of the FVIII protein is extended compared to a FVIII protein not having the VWF fragment. (Item 53) 53. The chimeric protein of paragraph 52, wherein the half-life of the FVIII protein is extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild-type FVIII. (Item 54) 53. The chimeric protein of item 52, wherein the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. (Item 55) 55. The chimeric protein according to any one of items 45 to 54, comprising the FVIII protein, the XTEN sequence, the VWF fragment, and a single-chain polypeptide containing the Ig constant region or a part thereof. (Item 56) 55. The chimeric protein according to any one of Items 45 to 54, wherein the Ig constant region or the portion thereof comprises a first Fc region. (Item 57) 55. The chimeric protein of any one of items 45 to 54, wherein the Ig constant region or part thereof is linked to the VWF fragment by an optional linker. (Item 58) 58. The chimeric protein according to item 57, wherein the linker contains a cleavable linker. (Item 59) 51. The chimeric protein of any one of Items 45 to 50, further comprising an additional Ig constant region or portion thereof linked to the FVIII protein, the Ig constant region or portion thereof, the VWF fragment, or any combination thereof, optionally via a linker. (Item 60) 60. The chimeric protein of item 59, wherein the additional Ig constant region or portion thereof is linked to the FVIII protein by an optional linker. (Item 61) 61. The chimeric protein according to item 59 or 60, wherein the Ig constant region or portion thereof is a second Fc region. (Item 62) 62. The chimeric protein of any one of items 8 to 26, 42 to 44, and 46 to 61, wherein the Ig constant region or portion thereof and the additional Ig constant region or portion thereof are the same or different. (Item 63) 63. The chimeric protein of any one of items 46 to 62, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof, and the second polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof. (Item 64) 63. The chimeric protein of any one of items 46 to 62, comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the FVIII protein and the XTEN sequence, the second polypeptide chain comprises the light chain of the FVIII protein and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof. (Item 65) 63. The chimeric protein of any one of items 46 to 62, comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the FVIII protein, the second polypeptide chain comprises the light chain of the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof. (Item 66) a first polypeptide chain and a second polypeptide chain, 63. The chimeric protein of any one of Items 46 to 62, wherein the first polypeptide chain comprises the heavy chain of the FVIII protein and the XTEN sequence, and the second polypeptide chain comprises the light chain of the FVIII protein, the Ig constant region or a portion thereof, the VWF fragment, and the additional Ig constant region or a portion thereof. (Item 67) 67. The chimeric protein of any one of items 63 to 66, further comprising an additional XTEN sequence linked to said VWF fragment or said additional Ig constant region or portion thereof. (Item 68) 27. The chimeric protein of any one of items 1 to 26, wherein the FVIII protein is linked to an XTEN sequence at the C-terminus or N-terminus of the FVIII protein, or inserted immediately downstream of one or more amino acids of the FVIII protein, or any combination thereof. (Item 69) 69. The chimeric protein of any one of items 27 to 68, wherein the FVIII protein is linked to at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five XTEN sequences, or at least six XTEN sequences. (Item 70) 70. The chimeric protein according to any one of items 1 to 69, wherein the FVIII protein contains one or more domains of FVIII selected from the group consisting of the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the B domain, the A3 domain, the a3 acidic region, the C1 domain, the C2 domain, one or more fragments thereof, and any combination thereof. (Item 71) The one or more insertion sites of the FVIII protein are located within one or more domains of the FVIII protein selected from the group consisting of the A1 domain, the a1 acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the C1 domain, the C2 domain, and any combination thereof; or the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and 71. The chimeric protein according to any one of Items 27 to 70, wherein the chimeric protein is positioned between one or more domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof, or between two domains of the FVIII protein selected from the group consisting of the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof. (Item 72) 72. The chimeric protein according to any one of Items 27 to 71, wherein one or more insertion sites in the FVIII protein are one or more amino acids selected from the group consisting of the amino acid residues in Table 7, Table 8, Table 9, and Table 10. (Item 73) 73. The chimeric protein of item 72, wherein the XTEN sequence inserted at the insertion site corresponding to amino acid 3R of SEQ ID NO: 4 further contains the amino acid sequence of ATR. (Item 74) one or more insertion sites of said FVIII protein, (1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) amino acid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) 1937 amino acids, (52) 2019 amino acids, (53) amino acid 2068, (54) amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188, (58) amino acid 2227, (59) amino acid 2277, and (60) a combination of two or more thereof; 72. The chimeric protein according to any one of Items 27 to 71, wherein the chimeric protein is located immediately downstream of one or more amino acids selected from the group consisting of: (Item 75) 75. The chimeric protein according to any one of items 1 to 74, wherein the FVIII protein comprises a B domain or a part thereof. (Item 76) 76. The chimeric protein of item 75, wherein the FVIII protein is an SQ B-domain deleted FVIII. (Item 77) 77. The chimeric protein according to any one of items 1 to 76, wherein the FVIII protein contains a single-chain FVIII. (Item 78) 78. The chimeric protein of item 77, wherein the single-chain FVIII contains at least one amino acid substitution at residues corresponding to 1648, 1645, or both, of the full-length mature factor VIII polypeptide (SEQ ID NO: 4) or at residues corresponding to 754, 751, or both, of the SQ BDD factor VIII (SEQ ID NO: 6). (Item 79) 79. The chimeric protein of item 78, wherein the amino acid substitution is an amino acid other than arginine. (Item 80) The FVIII protein comprises a heavy chain of FVIII and a light chain of factor VIII, wherein the heavy chain and the light chain are associated with each other by metal binding. 76. A chimeric protein according to any one of claims 76 to 78. (Item 81) 81. The chimeric protein according to any one of items 1 to 80, wherein the FVIII protein has low affinity for or does not bind to low density lipoprotein receptor-related protein (LRP). (Item 82) 82. The chimeric protein of item 81, wherein the FVIII protein contains at least one amino acid substitution that reduces affinity for the LRP or abolishes binding to the LRP. (Item 83) 83. The chimeric protein of item 82, wherein the at least one amino acid substitution is at a residue position corresponding to residue 471, residue 484, residue 487, residue 490, residue 497, residue 2092, residue 2093, or a combination of two or more thereof, of full-length mature FVIII. (Item 84) 84. The chimeric protein of item 83, wherein the amino acid substitution at residue 471, 484, or 497 is an amino acid other than arginine; the amino acid substitution at residue 487 is an amino acid other than tyrosine; the amino acid substitution at residue 2092 is an amino acid other than lysine; or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine. (Item 85) 85. The chimeric protein of any one of items 1 to 84, wherein the FVIII protein contains at least one amino acid substitution that makes the FVIII protein more stable than a FVIII protein that does not have the substitution. (Item 86) 86. The chimeric protein of item 85, wherein the A2 and A3 domains of the FVIII protein are covalently associated with each other. (Item 87) 87. The chimeric protein according to item 85 or 86, wherein the at least one amino acid substitution is at a residue position corresponding to residue 664, residue 1826, residue 662, residue 1828, or a combination of two or more thereof, of full-length mature FVIII. (Item 88) 88. The chimeric protein according to item 87, wherein the FVIII protein contains (a) a cysteine ​​at the residue corresponding to residue 664 of full-length mature FVIII and a cysteine ​​at the residue corresponding to residue 1826 of full-length mature FVIII, or (b) a cysteine ​​at the residue corresponding to residue 662 of full-length mature FVIII and a cysteine ​​at the residue corresponding to residue 1828 of full-length mature FVIII. (Item 89) 89. The chimeric protein according to any one of items 1 to 26 and 45 to 88, wherein the VWF fragment is not amino acids 764 to 1274 of SEQ ID NO:2. (Item 90) 89. The chimeric protein according to any one of Items 1 to 26 and 45 to 89, wherein the amino acid sequence of the D' domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2. (Item 91) 91. The chimeric protein according to any one of Items 1 to 26 and 45 to 90, wherein the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2. (Item 92) 92. The chimeric protein according to any one of items 1 to 26 and 45 to 91, wherein the VWF fragment is a monomer. (Item 93) 92. The chimeric protein according to any one of items 1 to 26 and 45 to 91, wherein the VWF fragments contain at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments. (Item 94) 94. The chimeric protein of any one of items 1 to 26 and 45 to 93, wherein the VWF fragment contains amino acids that are at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2. (Item 95) 95. The chimeric protein according to item 94, wherein the VWF fragment consists essentially of or consists of amino acids 764 to 1240 of SEQ ID NO: 2. (Item 96) 96. The chimeric protein according to any one of items 1 to 26 and 45 to 95, wherein the VWF fragment contains at least one amino acid substitution at residues corresponding to 1099, 1142, or both 1099 and 1142 of SEQ ID NO: 2. (Item 97) 97. The chimeric protein of item 96, wherein the VWF fragment contains an amino acid other than cysteine ​​substituted for residues corresponding to 1099, 1142, or both 1099 and 1142 of SEQ ID NO:2. (Item 98) 8. The chimeric protein according to any one of items 1 to 26 and 45 to 97, wherein the VWF fragment further contains the D1 domain, the D2 domain, or the D1 and D2 domains of VWF. (Item 99) 99. The chimeric protein according to any one of items 1 to 27 and 45 to 98, wherein the VWF fragment further contains a VWF domain selected from the group consisting of A1 domain, A2 domain, A3 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, and any combination thereof. (Item 100) 99. The chimeric protein according to any one of items 1 to 27 and 45 to 99, wherein the VWF fragment consists essentially of, or consists of: (1) the D' and D3 domains of VWF, or a fragment thereof; (2) the D1, D' and D3 domains of VWF, or a fragment thereof; (3) the D2, D' and D3 domains of VWF, or a fragment thereof; (4) the D1, D2, D' and D3 domains of VWF, or a fragment thereof; or (5) the D1, D2, D', D3 and A1 domains of VWF, or a fragment thereof. (Item 101) 101. The chimeric protein of any one of items 1 to 27 and 45 to 100, wherein the VWF fragment further comprises a signal peptide of VWF or FVIII operably linked to the VWF fragment. (Item 102) 103. The chimeric protein of any one of Items 1 to 101, wherein one or more of the linkers have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. Item 1 to 3, wherein one or more of the linkers has a length of about 1 to about 2000 amino acids. 101. The chimeric protein of any one of claims 101. (Item 104) 104. The chimeric protein of item 103, wherein one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids. (Item 105) 105. The chimeric protein according to any one of items 1 to 104, wherein one or more of the linkers contains a Gly / Ser peptide. (Item 106) The above Gly / Ser peptide is (Gly4Ser) n (SEQ ID NO: 139), or Ser(Gly4Ser) n (SEQ ID NO: 140), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. (Item 107) Above (Gly4Ser) n 107. The chimeric protein of Item 106, wherein the linker is (Gly4Ser)3 (SEQ ID NO: 63) or (Gly4Ser)4 (SEQ ID NO: 138). (Item 108) 108. The chimeric protein of any one of items 1 to 107, wherein the XTEN sequence is selected from the group consisting of AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. (Item 109) 109. The chimeric protein of item 108, wherein the XTEN sequence is selected from the group consisting of SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 127. (Item 110) 109. The chimeric protein of claim 108, wherein the XTEN sequence is AE288 or AG288. (Item 111) 111. The chimeric protein according to any one of items 1 to 110, wherein the linker contains at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both. (Item 112) 112. The chimeric protein according to any one of items 1 to 111, wherein the linker contains 20 amino acids, 35 amino acids, 48 ​​amino acids, 73 amino acids, or 95 amino acids. (Item 113) 113. The chimeric protein according to item 112, wherein the linker is a 48 amino acid thrombin-cleavable linker. (Item 114) 113. The chimeric protein according to item 112, wherein the linker is a 35 amino acid thrombin-cleavable linker. (Item 115) 112. The chimeric protein of item 111, wherein one or more of the cleavage sites is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8). (Item 116) 112. The chimeric protein of claim 111, wherein one or more of the cleavage sites are cleaved by a protease selected from the group consisting of Factor XIa, Factor XIIa, kallikrein, Factor VIIa, Factor IXa, Factor Xa, Factor IIa (thrombin), elastase 2, granzyme B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. (Item 117) One or more of the cleavage sites is selected from the group consisting of RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO: 23), IE 117. The chimeric protein of either item 111 or 116, containing an amino acid sequence selected from the group consisting of GRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28), GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34), and LPKTGSES (SEQ ID NO: 35). (Item 118) 118. The chimeric protein according to any one of Items 111 to 117, wherein the first cleavage site and the second cleavage site are the same or different. (Item 119) 119. The chimeric protein according to any one of items 1 to 118, which is polysialylated, pegylated, or hesylated. (Item 120) A polynucleotide or a set of polynucleotides encoding the chimeric protein according to any one of items 1 to 120. (Item 121) 121. The polynucleotide according to Item 120, further comprising a polynucleotide chain encoding PC5 or PC7. (Item 122) 122. A vector comprising the polynucleotide according to item 120 or 121 and one or more promoters operably linked to the polynucleotide or set of polynucleotides. (Item 123) Item 123. The vector according to Item 122, further comprising an additional vector containing a polynucleotide chain encoding PC5 or PC7. (Item 124) A host cell containing the polynucleotide of any one of items 120 or 121 or the vector of any one of items 122 or 123. (Item 125) 125. The host cell according to item 124, which is a mammalian cell. (Item 126) 126. The host cell according to item 125, wherein the mammalian cell is selected from the group consisting of HEK293 cells, CHO cells, and BHK cells. (Item 127) A pharmaceutical composition comprising the chimeric protein according to any one of Items 1 to 119, the polynucleotide according to any one of Items 120 or 121, the vector according to any one of Items 122 or 123, or the host cell according to any one of Items 124 to 125, and a pharmaceutically acceptable carrier. (Item 128) 128. The composition of claim 127, wherein the FVIII protein has an extended half-life compared to a wild-type FVIII protein. (Item 129) 129. The composition of any one of paragraphs 127 or 128, wherein the half-life of the FVIII protein is extended by at least about 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. (Item 130) 130. The composition of any one of items 128 or 129, wherein the half-life of the Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. (Item 131) 131. The composition of any one of items 127 to 130, administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. (Item 132) Item 132. The composition according to item 131, wherein the parenteral administration is intravenous or subcutaneous administration. (Item 133) 133. The composition of any one of items 127 to 132, for use in treating a bleeding disease or condition in a subject in need thereof. (Item 134) 134. The composition of claim 133, wherein the bleeding disease or condition is selected from the group consisting of bleeding coagulopathy, joint bleeding, muscle bleeding, oral bleeding, severe bleeding, severe bleeding into muscles, severe bleeding in the oral cavity, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, iliopsoas sheath bleeding, and any combination thereof. (Item 135) 135. The composition of any one of items 133 or 134, wherein the subject is scheduled to undergo surgery. (Item 136) 136. The composition according to any one of items 133 to 135, wherein said treatment is carried out prophylactically or on demand. (Item 137) 136. A method for disrupting or inhibiting binding of endogenous VWF to FVIII protein, comprising administering to a subject in need thereof an effective amount of the chimeric protein of any one of items 1 to 26 and 45 to 119, the polynucleotide of any one of items 120 or 121, the vector of any one of items 122 or 123, the host cell of any one of items 124 to 126, or the composition of any one of items 127 to 136, wherein the VWF fragment binds to the FVIII protein, thereby disrupting or inhibiting the binding of endogenous VWF. (Item 138) A method for extending or increasing the half-life of the FVIII protein, wherein the method comprises administering to a subject in need thereof an effective amount of a chimeric protein according to any one of items 1 to 26 and 45 to 119, a polynucleotide according to any one of items 120 or 121, a vector according to any one of items 122 or 123, a host cell according to any one of items 124 to 126, or a composition according to any one of items 127 to 136. wherein the VWF fragment binds to the FVIII protein, thereby extending or increasing the half-life of the FVIII protein. (Item 139) A method for preventing or suppressing the removal of a FVIII protein from a cell, the method comprising adding an effective amount of the chimeric protein of any one of items 1 to 26 and 45 to 119, the polynucleotide of any one of items 120 or 121, the vector of any one of items 122 or 123, the host cell of any one of items 124 to 126, or the composition of any one of items 127 to 136 to a cell containing a FVIII protein or a polynucleotide encoding the FVIII protein, wherein the protein having VWF activity binds to the FVIII protein. (Item 140) 139. The method according to any one of items 137 to 139, wherein the subject is an animal. (Item 141) Item 141. The method of item 140, wherein the animal is a human. (Item 142) 141. The method of claim 140, wherein the subject has hemophilia A. (Item 143) 136. A method for treating a bleeding disease or disorder in a subject in need thereof, comprising administering an effective amount of the chimeric protein according to any one of items 1 to 26 and 45 to 119, the polynucleotide according to any one of items 120 or 121, the vector according to any one of items 122 or 123, the host cell according to any one of items 124 to 126, or the composition according to any one of items 127 to 136, wherein the bleeding disease or disorder is selected from the group consisting of bleeding coagulation disorders, joint bleeding, muscle bleeding, oral bleeding, severe bleeding, severe bleeding into muscles, severe bleeding in the oral cavity, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, and iliopsoas sheath bleeding. (Item 144) 144. The method of claim 143, wherein the treatment is prophylactic or on demand. (Item 145) Item 145. The method according to any one of items 143 and 144, wherein the effective amount is 0.1 μg / kg to 500 mg / kg. (Item 146) The method according to any one of Items 143 to 145, wherein the chimeric protein according to any one of Items 1 to 119, the polynucleotide according to any one of Items 120 or 121, the vector according to any one of Items 122 or 123, the host cell according to any one of Items 124 to 126, or the composition according to any one of Items 127 to 136 is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. (Item 147) 147. The method of claim 146, wherein the parenteral administration is selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration. (Item 148) 124. A method for producing a chimeric protein comprising transfecting one or more host cells with the polynucleotide of any of items 120 or 121 or the vector of any one of items 122 or 123, and expressing the chimeric protein in the host cells. (Item 149) The XTEN insertion site is located at residue 745 of the mature FVIII protein (SEQ ID NO: 4). 119. The chimeric protein according to any one of items 27 to 119, which is immediately downstream of (Item 150) 119. The chimeric protein of any one of items 27 to 119, wherein the XTEN insertion site is immediately downstream of residues 1656 and 1900 of the FVIII protein. (Item 151) 119. The chimeric protein of any one of items 27 to 119, wherein the XTEN insertion sites are immediately downstream of residues 26, 1656, and 1900 of the FVIII protein. (Item 152) 119. The chimeric protein of any one of items 27 to 119, wherein the XTEN insertion site is immediately downstream of residues 403 and 745 of the FVIII protein. (Item 153) 119. The chimeric protein of any one of items 27 to 119, wherein the XTEN insertion site is immediately downstream of residues 745 and 1900 of the FVIII protein. (Item 154) 119. The chimeric protein of any one of items 27 to 119, wherein the XTEN insertion site is immediately downstream of residues 18 and 745 of the FVIII protein. (Item 155) 155. The chimeric protein according to any one of items 149 to 154, wherein the FVIII protein is a two-chain FVIII isoform. (Item 156) 155. The chimeric protein according to any one of items 149 to 154, wherein the FVIII protein is a single-chain FVIII isoform. (Item 157) 157. The chimeric protein of any one of items 149 to 156, containing one XTEN. (Item 158) 157. The chimeric protein of any one of items 149 to 156, comprising two XTENs. (Item 159) 157. The chimeric protein of any one of items 149 to 156, containing three XTEN inserted therein. (Item 160) 158. The chimeric protein according to any one of Items 149 to 157, wherein the XTEN is SEQ ID NO: 39 (AE288). (Item 161) 159. The chimeric protein according to any one of items 149 to 156 and 158, wherein the XTEN is SEQ ID NO: 38 and 37 (AG144 and AE144). (Item 162) 159. The chimeric protein of any one of items 149 to 156 and 159, wherein the XTEN is SEQ ID NO: 37, 38 and 37 (AE144, AG144, and AE144). (Item 163) 158. The chimeric protein of any one of items 149 to 156 and 157, wherein the XTEN is SEQ ID NO: 38 and 39 (AE144 and AE288). (Item 164) 125. The host cell of item 124, wherein the PC5 or PC7 cleaves the D1D2 domain of VWF. (Item 165) at least two XTEN, at least three XTEN, at least four XTEN, 28. The chimeric protein of any one of items 1 to 27, comprising at least five XTENs, or at least six XTENs. [Brief explanation of the drawings]

[0039] [Figure 1-1] Schematic diagrams of VWF fragments. Figure 1A shows three exemplary VWF fragments (VWF-002, VWF-010, and VWF-013) useful in the present invention. VWF-002 contains amino acids 1-477 of SEQ ID NO: 124 (amino acids 764-1240 of SEQ ID NO: 2) and is synthesized without a pre / propeptide sequence. VWF-010 contains a D1D2 domain in addition to the D'D3 domain. VWF-013 contains alanine residues substituting cysteines at residues 336 and 379 of SEQ ID NO: 123, as well as a D1D2D'D3 domain. Figure 1B shows VWF-031, which contains a D1D2D'D3 domain fused to an Ig constant region or portion thereof (e.g., an Fc region) by a cleavable linker (e.g., a 48-amino acid thrombin-cleavable linker). Figure 1C shows VWF-025, the nucleotide sequence encoding the D1D2D'D3 domain contained in the pLIVE vector, and VWF-029, the nucleotide sequence encoding the D1D2D'D3 domain with two amino acid substitutions, C336A and C379A, in the pLIVE vector. [Figure 1-2] Figure 1D shows a full-length VWF fragment containing the propeptide (D1 and D2 domains) and mature subunits (D', D3, A1, A2, A3, D4, B1-3, and C1-2 domains). The VWF fragment is an approximately 250 kDa protein that forms multimers (>20 MDa) through disulfide bonds. The VWF fragment associates with FVIII (95-98%) in noncovalent complexes, thereby extending the half-life of FVIII by protecting FVIII from protease cleavage / activation, stabilizing the heavy and light chains, and preventing FVIII removal by scavenger receptors. The VWF fragment can also limit the half-life of FVIII by removing FVIII-VWF complexes via the VWF receptor and by preventing pinocytosis and recycling of rFVIIIFc. [Figure 2] Pharmacokinetic profiles of rFVIII-XTEN (rFVIII-AE288 or rFVIII-288AE) in VWF D'D3-expressing mice or FVIII and VWF double knockout (DKO) mice. Figure 2A shows the timeline of hydrodynamic injection of D'D3 domain-encoding plasmid DNA (VWF-025) (day 5), intravenous administration of rFVIII-XTEN AE288 (day 0), and PK sample collection (day 5). Figure 2B shows FVIII activity measured by a FVIII chromogenic assay after IV administration of rFVIII-XTEN288 in D1D2D'D3 mice (inverted triangles) and DKO mice (diamonds). Figure 2C shows plasma levels of D'D3 (ng / mL) after administration of VWF-025. The X-axis represents time in hours. [Figure 3]Schematic diagram of an exemplary VWF:FVIII heterodimer construct. The constructs have a consensus structure represented by the formula FVIII-F1-L1-VX-L2-F2 but contain different exemplary variable linkers. The construct shown (FVIII-161) contains a VWF fragment (the D' and D3 domains of VWF (i.e., amino acids 1-477 of SEQ ID NO: 2, with amino acid substitutions C336A and C379A) ​​linked to an XTEN sequence) and heterodimeric FVIII (heavy and light chains associated by metal binding) linked to a first Fc region, which is further linked to a cleavable linker and a second Fc region. The XTEN sequence contained in FVIII-161 is the XTEN AE288 sequence, and the linker is a thrombin-cleavable linker (having 35 amino acids). In FVIII-161, the FVIII protein linked to the first Fc region is linked to the VWF fragment by a processable linker that, upon expression, can be cleaved by intracellular processing enzymes, thereby generating a construct in which the three polypeptide chains associate with each other. [Figure 4]Schematic diagram of examples of FVIII-VWF heterodimers or monomers: FVIII-168, FVIII-175, FVIII-172, FVIII-174, and FVIII170. Construct FVIII-168 contains a single-chain FVIII sequence (arginine residues are replaced by alanine residues at residues 1645 and 1648) linked to a first Fc region fused to a VWF fragment linked to a second Fc region by a thrombin-cleavable linker (having 48 amino acids). AE288 XTEN is inserted into the B domain of the single-chain FVIII sequence. The link between the first Fc region and the VWF fragment contains a linker that can be cleaved by an intracellular processing enzyme (i.e., a processable linker). The FVIII-175 construct contains a single-chain FVIII (arginine residues are replaced by alanine residues at residues 1645 and 1648) linked to AE288 XTEN and a first Fc region, which is linked to a second Fc region by a linker (e.g., a processable linker). The AE288 XTEN is inserted into the B domain of the single-chain FVIII sequence. The FVIII-172 construct contains two polypeptide chains: the first chain contains a heavy chain FVIII sequence fused to AE288 XTEN, and the second chain contains a light chain FVIII sequence, a first Fc region, a linker (e.g., a processable linker), a VWF fragment, a thrombin-cleavable linker (e.g., 48 amino acids), and a second Fc region. The FVIII-174 construct contains two polypeptide chains: the first chain contains a heavy chain FVIII sequence fused to AE288 XTEN, and the second chain contains a light chain FVIII, a first Fc region, a linker (e.g., a processable linker), and a second Fc region. The FVIII-170 construct contains a VWF fragment, AE288 XTEN, a linker (e.g., a 35-amino acid long thrombin-cleavable linker), and a single-chain FVIII sequence. [Figure 5]Pharmacokinetic profiles of FVIII / VWF heterodimers containing XTEN sequences linked to the Fc domain. The FVIII-161, FVIII-168, and FVIII-172 constructs were administered by hydrodynamic injection (HDI) at a dose of 100 μg / mouse to FVIII:VWF double knockout (DKO) mice. The FVIII-170 construct was administered by HDI at a dose of 50 μg / mouse to FVIII:VWF DKO mice. Plasma FVIII activity after HDI was analyzed 24 hours after HDI using a FVIII chromogenic assay. The FVIII activity of the FVIII:VWF heterodimers containing the XTEN sequence and Fc domain was compared with that of BDD-FVIII, which lacks the VWF fragment, XTEN sequence, and Fc domain. [Figure 6]Figure 6A shows a schematic diagram of an example co-transfection system for FVIII-VWF heterodimers. The FVIII-169 construct contains a full-length FVIII sequence (arginine replaced by alanine residues at residues 1645 and 1648, and an XTEN sequence inserted into the single-chain FVIII sequence) linked to an Fc region. VWF-031 contains a D1D2D'D3 fragment (cysteine ​​replaced by alanine residues at positions 336 and 379) linked to another Fc region via a 48-mer thrombin-cleavable linker. After intracellular processing, the FVIII-169 construct produces a full-length single-chain FVIII (SCFVIII) fused to one Fc fragment and the XTEN sequence, while the VWF-031 construct produces a 477-amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds may be formed between the Fc fragment linked to the SC FVIII or D'D3 fragment, allowing for non-covalent association of FVIII and D'D3. (Figure 6B). The FVIII-173 construct contains a heterodimeric FVIII sequence, a heavy chain FVIII sequence linked to an XTEN sequence, and a light chain FVIII sequence linked to an Fc region. VWF-031 is described above. After intracellular processing, the FVIII-173 construct produces a heterodimeric protein, a heavy chain FVIII fused to an XTEN sequence, a light chain FVIII fused to one Fc fragment, and the VWF-031 construct produces a 477-amino acid D'D3 fragment linked to the other Fc fragment. Two covalent bonds may be formed between the light chain FVIII or the Fc fragment linked to the D'D3 fragment, allowing for non-covalent association of FVIII and D'D3. [Figure 7]Figure 7A shows the binding affinity of an exemplary FVIII:VWF containing an XTEN sequence and an Fc domain to immobilized hVWF in an Octet assay. The binding affinity of FVIII-169 / VWF-031 and FVIII-057 (rFVIIIFc) to immobilized hVWF was verified using biolayer interferometry-based measurements (Octet assay). Figure 7A shows the nanomolar binding response of FVIII169 and FVIIIFc pharmaceutical components (positive controls) to immobilized hVWF. Figure 7B shows the binding response of human IgG1 (negative control) to immobilized human VWF. [Figure 8A] Pharmacokinetic (PK) profiles of FVIII-169 in HemA mice and FVIII:VWF double knockout (DKO) mice. Figure 8A shows the PK profiles of FVIII-169 / VWF-031 and FVIIIFc in HemA mice. HemA mice were treated with a single intravenous dose of FVIII-169 / VWF-031 at 200 IU / kg. Plasma samples collected from the mice were examined by a FVIII chromogenic assay. The half-life of FVIII-169 / VWF-031 was calculated using the WinNonlin program. [Figure 8B] In Figure 8B, the PK profiles of FVIII-169 / VWF-031, FVIII-169 / Fc and FVIIIFc in FVIII / VWF DKO mice are shown. [Figure 9A]Figure 9A shows the PK profiles of FVIII-XTEN mutants in D'D3-expressing FVIII / VWF DKO mice. Figure 9A shows a comparison of the PK profiles of FVIII-XTEN mutants (FVIII with one XTEN, FVIII with two XTEN, and FVIII with three XTEN). One, two, or three XTENs were inserted into various positions in FVIII, including the C-terminus and B domain. CT indicates that the XTEN is linked to the C-terminus of FVIII. The insertion site B / CT indicates that one XTEN is inserted between amino acid residues 745 and 746 of the FVIII protein, and the other XTEN is linked to the C-terminus of the FVIII protein. The numbering of amino acid residues corresponds to the SQ BDD FVIII protein sequence. The insertion site 1900 / B / CT indicates that the first XTEN is inserted between amino acid residues 1900 and 1901 of FVIII, the second XTEN is inserted between amino acid residues 745 and 746 of FVIII, and the third XTEN is linked to the C-terminus of FVIII. The mouse strain used for administration of the FVIII-XTEN mutants is a DKO mouse strain expressing the D'D3 domain. [Figure 9B] Figure 9B shows the PK profiles of FVIII-XTEN with three XTEN insertions. The FVIII-XTEN(1900 / B / CT) mutant was administered to either FVIII / VWF DKO mice or HemA mice. The half-lives of FVIII-XTEN(1900 / B / CT) are compared. [Figure 10]The FVIII activity of FVIIIFc (open triangle), FVIII169:Fc (filled circle), and FVIII169:VWF31 (open triangle) in DKO mouse plasma was measured by chromogenic assay. FVIII:Fc contains two chains of FVIII (heavy and light chains) fused to an Fc dimer (i.e., a monomer-dimer hybrid). FVIII169 is described above (contains AE288 in the B domain, immediately downstream of amino acid 745 of the mature FVIII sequence). FVIII169:Fc contains FVIII169 fused to an Fc dimer. FVIII169:VWF31 contains VWF31 in addition to an Fc dimer, where FVIII169 is fused to a first Fc region and VWF31 is fused to a second Fc region, wherein the first Fc region and the second Fc region form a covalent bond (e.g., one or more disulfide bonds). [Figure 11] Effect of Fc, XTEN, and VWF-D'D3 on FVIII half-life extension. BDD-FVIII (REFACTO®) (squares), FVIIFc (circles), FVIII169 / Fc (triangles), and FVIII169 / VWF031 (inverted triangles) were administered to FVIII and VWF double knockout (DKO) mice. FVIII activity was measured by chromogenic assay, and half-life was calculated using the WinNonlin-Phoenix program. The X-axis represents time, and the Y-axis represents plasma FVIII activity in mU / mL. [Figure 12A]Effect of different XTENs in rFVIII-XTEN / VWF heterodimers in HemA mice. Figure 12A shows the plasma FVIII activity (%), normalized to the 5-minute value, of two XTENs inserted immediately downstream of residues 1900 and 1656 of the mature FVIII sequence compared with FVIII-169, which contains an XTEN immediately downstream of residue 745 of the mature FVIII sequence (i.e., FVIII-195 (two-chain FVIII isoform) and FVIII-199 (single-chain FVIII isoform)). FVIII-169 / VWF-031 (closed circles), FVIII-199 / VWF-031 (closed squares), and FVIII-195 / VWF031 (open squares) were administered to HemA mice, and the plasma FVIII activity was measured. [Figure 12B] Figure 12B shows the effect of half-life extension by inserting a second XTEN immediately downstream of residues 403 (A2 domain) and 745 (B domain) of the mature FVIII sequence (i.e., FVIII-203) and residues 745 (B domain) and 1900 (A3 domain) of the mature FVIII sequence (FVIII-204) compared to FVIII-169 (with an XTEN insertion only in the B domain). FVIII-204 / VWF031 (closed triangles), FVIII-169 / VWF-031 (closed circles), FVIII-203 / VWF-031 (closed squares), and scBDD-FVIII (open diamonds) were administered to HemA mice. The X-axis shows the plasma activity of FVIII (%) normalized to the 5-minute value, and the Y-axis shows time in hours. [Figure 12C]Figure 12C shows the half-life extension effect of two XTEN insertions immediately downstream of residue 18 (A1 domain) and residue 745 (B domain) (i.e., FVIII-205) compared to FVIII-169 (with one XTEN insertion in the B domain) and single-chain FVIII without any Fc region or XTEN (i.e., FVIII-207). Figure 12C also shows the half-life extension effect of three XTEN insertions incorporated immediately downstream of residues 26 (A1 domain), 1656 (A3 domain), and 1900 (A3 domain) (i.e., FVIII-201) compared to FVIII-169 (with one XTEN insertion immediately downstream of residue 745). FVIII-205 / VWF-031 (filled squares), FVIII-201 / VWF-031 (inverted triangles), FVIII-169 / VWF-031 (filled circles), and FVIII-207 (open diamonds) were administered to HemA mice. Plasma activity of FVIII (%) (x-axis), normalized to the 5-minute value, was measured in hours over time (y-axis). [Figure 13] FVIII activity of rFVIII-XTEN / VWF-XTEN heterodimers in FVIII / VWF DKO mice. FVIII activity in plasma samples was analyzed by a FVIII chromogenic assay, and a regression curve of FVIII plasma activity (X-axis) as a function of time (Y-axis) was plotted. FVIII-155 (scFVIIIFc without any XTEN) was coexpressed with VWF-034 (VWF-Fc with AE288 XTEN plus a 35-residue thrombin-cleavable linker). The half-life of FVIII-155 / VWF-034 was compared to that of FVIII-169 / VWF-031 (with AE288XTEN inserted within the FVIII B-domain interface (immediately downstream of residue 745 of the mature FVIII polypeptide)). [Figure 14]Schematic diagrams of various rFVIII-XTEN / VWF constructs. These constructs are also described elsewhere herein. Figure 14A shows a single-chain B-domain-deleted FVIII protein (sometimes referred to herein as scBDD-FVIII). The scBDD-FVIII construct contains two substitutions (Arg to Ala) at residues 1645 and 1648. Figure 14B shows a two-polypeptide chain construct (FVIII155 / VWF031), where the first polypeptide chain contains single-chain FVIII linked to an Fc region without any XTEN, and the second chain contains the VWF D'D3 fragment linked to the Fc region. This construct was used as a control. In Figure 14C, two polypeptide chain constructs (FVIII199 / VWF031) are shown (the first chain contains single-chain FVIII linked to an Fc region with a first XTEN inserted just downstream of residue 1900 of the mature FVIII sequence and a second XTEN inserted just downstream of residue 1656 of the mature FVIII sequence; the second chain contains the VWF D'D3 fragment linked to an Fc region). In Figure 14D, two polypeptide chain constructs (FVIII201 / VWF031) are shown (the first chain contains a single-chain FVIII protein linked to an Fc region with a first XTEN inserted just downstream of residue 26 of the mature FVIII sequence, a second XTEN inserted just downstream of residue 1656 of the mature FVIII sequence, and a third XTEN inserted just downstream of residue 1900 of the mature FVIII sequence; the second chain contains a VWF D'D3 fragment linked to an Fc region). Figure 14E shows two polypeptide chain constructs (FVIII169 / VWF031) (the first chain contains a single-chain FVIII protein linked to an Fc region with an XTEN inserted just downstream (designated B) of residue 745 of the mature FVIII sequence, and the second chain contains a VWF D'D3 fragment linked to an Fc region).Figure 14F shows two polypeptide chain constructs (FVIII203 / VWF031) (the first chain contains a single-chain FVIII protein with a first XTEN inserted at residue 745 of the mature FVIII sequence (B) and a second XTEN inserted at residue 1900 of the mature FVIII sequence, and the second chain contains a VWF D'D3 fragment linked to an Fc region). Figure 14G shows two polypeptide chain constructs (FVIII204 / VWF031) (the first chain contains a single-chain FVIII protein linked to an Fc region with a first XTEN inserted just downstream of residue 403 of the mature FVIII sequence and a second XTEN inserted just downstream of residue 745 of the mature FVIII sequence (B) and the second chain contains a VWF D'D3 fragment linked to an Fc region). Figure 14H shows a two polypeptide chain construct (FVIII205 / VWF031) (the first chain contains single-chain FVIII with a first XTE inserted just downstream of residue 18 of the mature FVIII sequence and a second XTEN inserted just downstream of residue 745 of the mature FVIII sequence (B), and the second chain contains VWF D'D3 linked to the Fc region). [Figure 15] FVIII activity of rFVIII-XTEN / VWF and BDD-FVIII in FVIII / VWF DKO mice. FVIII activity in plasma samples was analyzed by FVIII chromogenic assay, and a regression curve of FVIII plasma activity (X-axis) as a function of time (Y-axis) was plotted. The half-life of rFVIII-XTEN / VWF (FVIII-205 / VWF-031) was compared with BDD-FVIII and rFVIIIFc. [Figure 16]Efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimers in HemA mice using a tail snip bleeding model. The efficacy of FVIII169 / VWF034, FVIII205 / VWF031, and BDD-FVIII was compared using a tail snip bleeding model in HemA mice. Median blood loss in ml for 200 IU / kg FVIII169 / VWF034 and FVIII205 / VWF031 was compared with 200 IU / kg BDD-FVIII, 65 IU / kg BDD-FVIII, 20 IU / kg BDD-FVIII, and vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0040] definition It should be noted that the term "a" or "an" refers to one or more of that entity. For example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Similarly, the terms "a," "one or more," and "at least one" can be used interchangeably herein.

[0041] The term "polynucleotide" or "nucleotide" is meant to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct (e.g., messenger RNA (mRNA) or plasmid DNA (pDNA)). In certain embodiments, a polynucleotide contains conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as present 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 in a polynucleotide. An "isolated" nucleic acid or polynucleotide is intended to be a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a Factor VIII polynucleotide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcription products of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention include molecules produced synthetically. Additionally, the polynucleotide or nucleic acid may include regulatory elements, such as promoters, enhancers, ribosomal binding sites, or transcription termination signals.

[0042] As used herein, a "coding region" or "coding sequence" refers to a portion of a polynucleotide that consists of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not typically translated into an amino acid but may be considered part of the codon region; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, and introns, are not part of the coding region. The boundaries of a coding region are typically determined by a start codon at the 5' end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the carboxyl terminus of the resulting polypeptide). Two or more coding regions of the present invention may be present in a single polypeptide construct (e.g., a single vector) or in separate polynucleotide constructs (e.g., separate (different) vectors). A single vector may contain a single coding region, or two or more coding regions (e.g., a single vector may separately encode binding domain A and binding domain B, as described below). Additionally, the vectors, polynucleotides, or nucleic acids of the invention may encode heterologous coding regions, including but not limited to specialized elements or motifs such as secretory signal peptides or heterologous functional domains, either fused or unfused to the nucleic acid encoding the binding domain of the invention.

[0043] Some proteins secreted from mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein once the nascent protein chain has been transported across the rough endoplasmic reticulum. Those skilled in the art will recognize that signal peptides are often 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, the native signal peptide or a functional derivative thereof that retains the ability to direct secretion of an operably linked polypeptide may be used. Alternatively, a heterologous mammalian signal peptide (e.g., human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide), or a functional derivative thereof, may be used.

[0044] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In some embodiments, a downstream nucleotide sequence refers to a sequence that follows the start of transcription. For example, the translation initiation codon of a gene is the codon that initiates transcription. It is located downstream of the section.

[0045] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In some embodiments, an upstream nucleotide sequence is located 5' to a coding region or at the start of transcription. For example, most promoters are located upstream of the start of transcription.

[0046] 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, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence are usually located 3' to the coding sequence.

[0047] A polynucleotide encoding a gene product (e.g., a polypeptide) may contain a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. In operable association, the coding region for a gene product (e.g., a polypeptide) is associated with one or more regulatory regions in such a manner as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and promoter are "operably associated" if inducing promoter function results in transcription of mRNA encoding the gene product encoded by the coding region, and if the natural association between the promoter and coding region does not interfere with the promoter's ability to induce expression of the gene product or the ability of the DNA template to be transcribed. Other promoter-related transcription control elements (e.g., enhancers, operators, repressors, and transcription termination signals) may also be operably associated with a coding region to induce expression of the gene product.

[0048] A variety of transcription control regions are known to those of skill in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells (e.g., promoter and enhancer segments from cytomegalovirus (the immediate early promoter linked to intron A), simian virus 40 (the early promoter), and retroviral (e.g., Rous sarcoma virus) promoters and enhancer segments). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0049] Similarly, various translation control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

[0050] As used herein, the term "expression" refers to the expression of a polynucleotide into a gene product (e.g., Expression refers to the process of producing a nucleic acid (e.g., a nucleic acid, RNA, or polynucleotide). This includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of mRNA into a polypeptide. Expression results in the production of a "gene product." As used herein, a gene product can be either a nucleic acid (e.g., a messenger RNA produced by transcription of a gene) or a polypeptide translated from a transcript. As used herein, a gene product further includes a nucleic acid that has been post-transcriptionally modified (e.g., polyadenylation or splicing), or a polypeptide that has been post-translationally modified (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage).

[0051] A "vector" refers to any vehicle for cloning and / or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be associated and which may also bring about replication of the associated segment. A "replicon" refers to any genetic element (e.g., a plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo (i.e., capable of replication under its own control). The term "vector" includes both viral and non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Many vectors, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses, are known and used in the art. Insertion of a polynucleotide into an appropriate vector can be accomplished by ligating the appropriate polynucleotide fragment into a selected vector having complementary cohesive termini.

[0052] Vectors may be engineered to encode a selectable marker or reporter that allows 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 have incorporated and expressed other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamides, etc., as well as genes that are also used as phenotypic markers (i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.). Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), galactosidase (LacZ), glucuronidase (Gus), etc.

[0053] The term "plasmid" refers to an extrachromosomal element that is not part of the central metabolism of a cell, but which often carries genes, usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, single- or double-stranded DNA or RNA, from any source, in which many nucleotide sequences, along with appropriate 3' untranslated sequences, are linked or recombined into unique structures capable of introducing promoter fragments and DNA sequences for selected gene products into cells.

[0054] Eukaryotic viral vectors that can be used include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, and poxvirus (e.g., vaccinia virus vectors), baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0055] A "cloning vector" refers to a "replicon," a unit length of continuously replicating nucleic acid that contains an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment may be attached and from which replication of the attached segment may be initiated. A cloning vector is capable of replicating in one cell type (e.g., bacteria) and expressing in others (e.g., eukaryotic cells). Cloning vectors usually contain one or more sequences that can be used to select cells that contain the vector and / or contain multiple cloning sites for insertion of nucleic acid sequences of interest.

[0056] The term "expression vector" refers to a vehicle designed to enable expression of an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is inserted in operably linked relation to the control regions described above.

[0057] Vectors are introduced into host cells by methods known in the art (e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or a DNA vector transporter).

[0058] As used herein, "culture," "culturing," and "culturing" refer to incubating cells under in vitro conditions that allow cell growth or division, or maintaining cells in a living state. As used herein, "cultured cells" refers to cells that have been grown in vitro.

[0059] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "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, but 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 is included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting groups, proteolytic cleavage, or substitution with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but must be translated from a designated nucleic acid sequence. They may be made by any method, including chemical synthesis.

[0060] An "isolated" peptide or 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 may be in its native state or may simply be removed from its natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0061] The present invention also includes fragments or variants of polypeptides, and any combination thereof. The terms "fragment" or "variant" when referring to a polypeptide binding domain or polypeptide binding molecule of the present invention include fragments or variants of at least one polypeptide of the reference polypeptide. The present invention also includes any polypeptide that retains the properties of the FcRn-binding domain or Fc variant (e.g., FcRn-binding affinity for the FcRn-binding domain or Fc variant, clotting activity for the FVIII variant, or FVIII-binding activity for the VWF fragment). Polypeptide fragments include proteolytic fragments, deletion fragments, and specific antibody fragments described elsewhere herein, but do not include native full-length polypeptides (or mature polypeptides). Variants of the polypeptide-binding domain or binding molecule of the present invention include the fragments described above, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants may be produced using known mutagenesis techniques. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0062] As used herein, the term "VWF fragment" or "fragments" refers to any VWF fragment that interacts with FVIII and retains at least one or more properties normally conferred to FVIII by full-length VWF (e.g., preventing premature activation to FVIIIa, preventing premature proteolysis, preventing binding to phospholipid members that result in premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and / or stabilizing the interaction between the FVIII heavy and light chains, etc.).

[0063] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is substituted with another amino acid from the same side chain family, the substitution is considered conservative. In other embodiments, strings of amino acids may be conservatively substituted with structurally similar strings that differ in sequence and / or composition of side chain family members.

[0064] It is recognized in the art that "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. As discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art, including, but not limited to, BESTFIT. The sequence similarity can be determined using the Wisconsin Sequence Analysis Package, Version 8 for Unix (registered trademark), Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. BESTFIT uses the partial homology algorithm of Smithand Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the sequence similarity between 2 and 3 sequences. The best homologous segment between the two sequences is found. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are that the percent identity is calculated over the entire length of the reference polypeptide sequence and the total number of amino acids in the reference sequence. The suffix is ​​set to allow a homology gap of up to 5%.

[0065] As used herein, an "amino acid corresponding to" or "equivalent amino acid" in a VWF or FVIII protein sequence is identified by an alignment that maximizes identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify the equivalent amino acid in the second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0066] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or a fragment, variant, or derivative thereof, immediately upstream of a position at which a heterologous moiety may be inserted. An "insertion site" is identified as a number, which is the number of the amino acid in mature native FVIII (SEQ ID NO: 4) that corresponds to the insertion site and is immediately N-terminal to the insertion position. For example, the phrase "a3 has an XTEN at an insertion site corresponding to amino acid 1656 of SEQ ID NO: 4" indicates that the heterologous moiety is located between the two amino acids corresponding to amino acids 1656 and 1657 of SEQ ID NO: 4.

[0067] As used herein, the phrase "immediately downstream of an amino acid" refers to the position immediately adjacent to the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position immediately adjacent to the terminal amino group of an amino acid. Therefore, as used herein, the phrase "between two amino acids at the insertion site" refers to the position where XTEN or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases "inserted immediately downstream of an amino acid" and "inserted between two amino acids at the insertion site" are used interchangeably with "inserted at the insertion site."

[0068] As used herein, the terms "inserted," "inserted into," "inserted into," or grammatically related terms refer to the location of the XTEN in the chimeric polypeptide relative to the analogous location in mature native human FVIII. As used herein, the terms refer to the characteristics of the recombinant FVIII polypeptide relative to mature native human FVIII, but do not suggest, imply, or infer any method or process by which the chimeric polypeptide was made. For example, in reference to the chimeric polypeptides presented herein, the phrase "XTEN is inserted immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric polypeptide contains an XTEN immediately downstream of the amino acid corresponding to amino acid 745 of mature native human FVIII (e.g., surrounded by amino acids corresponding to amino acids 745 and 746 of mature native human FVIII).

[0069] A "fusion" or "chimeric" protein contains a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. Amino acid sequences that are normally present in separate proteins may be brought together in a fusion polypeptide, or amino acid sequences that are normally present in the same protein may be placed in a new arrangement in a fusion polypeptide (e.g., fusion of an Ig Fc domain with the Factor VIII domain of the present invention). Fusion proteins are created, for example, by chemical synthesis, or by creating and translating a polypeptide in which the peptide regions are encoded in the desired relationship. A chimeric protein may further contain a second amino acid sequence related to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0070] As used herein, the term "half-life" refers to the in vivo biological half-life of a particular polypeptide. Half-life may be expressed as the time required for half of an administered dose to be removed from the circulatory system and / or other tissues in an animal. When the clearance curve of a given polypeptide is plotted as a function of time, the curve typically exhibits a steep α The chimeric polypeptide is biphasic, consisting of a long β-phase and a longer α-phase. The α-phase often represents equilibrium between the intravascular and extravascular spaces of the administered chimeric polypeptide and is determined in part by the size of the polypeptide. The β-phase often represents catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins containing FVIII are monophasic, having only a β-phase and no α-phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

[0071] As used herein, the term "linked" refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence may be directly linked or juxtaposed to the second amino acid sequence or nucleotide sequence, or an intervening sequence may covalently link the first sequence to the second sequence. The term "linked" is intended to include not only C- or N-terminal fusion of the first amino acid sequence to the second amino acid sequence, but also insertion of the entire first amino acid sequence between any two amino acids in the second amino acid sequence (or insertion of the entire second amino acid sequence between any two amino acids in the first amino acid sequence). In one embodiment, the first amino acid sequence may be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence by a phosphodiester bond or a linker. A linker may be a peptide or polypeptide (for a polypeptide chain), or a nucleotide or nucleotide chain (for a nucleotide chain), or any chemical bond (for both polypeptide and polynucleotide chains). The term "linked" may also be indicated by a hyphen (-).

[0072] As used herein, the term "associated" 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" refers to a covalent, non-peptide bond or a non-covalent bond. This association may be indicated by a colon (i.e., (:)). In other embodiments, it refers to a covalent bond excluding a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a thiol group on a second cysteine ​​residue. In most native 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 in the EU numbering system)). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, π backbonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, haptic or antibonds. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilic, intercalation, stacking, entropic forces, or chemical polarity.

[0073] As used herein, the term "monomer-dimer hybrid" refers to a chimeric protein containing a first polypeptide chain and a second polypeptide chain associated with each other by disulfide bonds, where the first chain contains a clotting factor (e.g., Factor VIII) and a first Fc region, and the second chain contains, consists essentially of, or consists of a second Fc region without the clotting factor. Thus, a monomer-dimer hybrid construct combines the characteristics of a monomer having only one clotting factor and the characteristics of a monomer having two Fc regions. It is a hybrid containing dimeric features with regions.

[0074] As used herein, the term "cleavage site" or "enzyme cleavage site" refers to a site recognized by an enzyme. Some enzymatic cleavage sites contain intracellular processing sites. In one embodiment, a polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during the coagulation cascade, with cleavage of such a site occurring at the site of clot formation. Examples of such sites include those recognized by thrombin, factor XIa, or factor Xa. Exemplary FXIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO: 45) and SVSQTSKLTR (SEQ ID NO: 46). Exemplary thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 47), TTKIKPR (SEQ ID NO: 48), LVPRG (SEQ ID NO: 49), and ALRPR (amino acids 1-5 of SEQ ID NO: 50). Other enzymatic cleavage sites are known in the art.

[0075] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide that is targeted by 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 region. Intracellular processing enzymes cleave polypeptides before the protein is secreted from the cell. Examples of such processing sites include those targeted by the endopeptidase family PACE / furin (PACE is an acronym for Paired basic Amino acid Cleaving Enzyme). These enzymes are located in the Golgi membrane and cleave proteins on the carboxy-terminal side of an Arg-[any residue]-(Lys or Arg)-Arg sequence motif. 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.

[0076] It is understood that in constructs containing more than one processing or cleavage site, such sites may be the same or different.

[0077] The term "Furin" refers to an enzyme corresponding to EC 3.4.21.75. Furin is a subtilisin-like proprotein convertase, also known as PACE (Paired basic amino acid cleaving enzyme). Furin removes the inactive portion of a proprotein to convert it into a biologically active protein. During intracellular transport, the Furin enzyme may cleave the VWF propeptide from the mature VWF molecule. In some embodiments, Furin cleaves D1D2 from D'D3 of VWF. In other embodiments, a nucleotide sequence encoding Furin may be expressed together with nucleotides encoding a VWF fragment, such that the D1D2 domain is cleaved intracellularly by Furin.

[0078] It will be understood that in constructs containing more than one processing or cleavage site, such sites may be the same or different.

[0079] As used herein, a "processable linker" refers to a linker that contains at least one intracellular processing site, as described elsewhere herein.

[0080] As used herein, a hemostatic disorder refers to an inherited or acquired condition characterized by a tendency to profuse bleeding, either spontaneously or as a result of trauma, due to an inability or impaired ability to form fibrin clots. Examples of such disorders include hemophilia. The three major forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease"), and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, and Bernard-Soulier syndrome (a defect or defect in GPIb). The receptor for VWF, GPIb, may also be defective, resulting in a defect in primary clot formation (primary hemostasis), an increased tendency to bleed, and Glanzmann and Naegeli thrombasthenia (Glanzmann thrombasthenia).In liver failure (acute and chronic conditions), the liver produces insufficient clotting factors, which may increase the risk of bleeding.

[0081] The chimeric molecules of the present invention may be used prophylactically. As used herein, the term "prophylactic treatment" refers to administering the molecule before a bleeding event. In one embodiment, the subject in need of general hemostatic treatment is undergoing or about to undergo surgery. The chimeric proteins of the present invention may be administered before or after surgery as a prophylactic agent. The chimeric proteins of the present invention may be administered during or after surgery to control serious bleeding events. Surgeries include, but are not limited to, liver transplants, liver resections, dental procedures, or stem cell transplants.

[0082] The chimeric proteins of the present invention may also be used for on-demand treatment. The term "on-demand treatment" refers to administering the chimeric molecule in response to an onset of a bleeding episode or prior to an activity that may result in bleeding. In one embodiment, on-demand treatment may be administered to a subject when bleeding begins (e.g., after trauma) or when bleeding is anticipated (e.g., prior to surgery). In another embodiment, on-demand treatment may be administered prior to an activity that increases the risk of bleeding, such as contact sports.

[0083] As used herein, the term "severe bleeding" refers to a bleeding episode regardless of the context, for example, a subject may have trauma, uremia, an inherited bleeding disorder (e.g., factor VII deficiency), a platelet disorder, or resistance due to the development of antibodies against clotting factors.

[0084] As used herein, treat, treatment, or treating refers to reducing the severity of a disease or condition; shortening the duration of the disease course; ameliorating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject having a disease or condition, or providing prophylaxis of one or more symptoms associated with a disease or condition to a subject not in need of treatment for the disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining a trough level of FVIII in a subject of at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL by administering a chimeric protein or VWF fragment of the present invention. In other embodiments, treating or treatment means increasing FVIII trough levels to 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. IU / dL. Treating or treating a disease or condition also includes maintaining FVIII activity in a subject at a level corresponding 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.

[0085] Chimeric proteins The present invention aims to extend the half-life of factor VIII protein by preventing or inhibiting the association of the FVIII half-life limiting factor (i.e., endogenous VWF) with FVIII protein using VWF fragments and XTEN sequences. Endogenous VWF associates with approximately 95-98% of FVIII in noncovalent complexes. Endogenous VWF is the half-life limiting factor of FVIII, and the binding of endogenous VWF to FVIII protein is known to protect FVIII in various ways. For example, full-length VWF (a multimer having approximately 250 kDa) can protect FVIII from protease cleavage and FVIII activation, stabilize the heavy and / or light chains of FVIII, and prevent clearance of FVIII by scavenger receptors. However, at the same time, endogenous VWF limits the half-life of FVIII by interfering with pinocytosis and removing the FVIII-VWF complex from the body via the VWF clearance pathway. Without wishing to be bound by theory, it is believed that endogenous VWF is the half-life limiting factor, such that the half-life of a FVIII protein fused to a half-life extender is no longer than about twice that of wild-type FVIII. Therefore, the present invention aims to use a VWF fragment to interfere with or inhibit the interaction between endogenous VWF and the FVIII protein, thereby extending the half-life of the FVIII protein by using an XTEN sequence alone or in combination with an Ig constant region or a portion thereof. The XTEN sequence may be linked to the FVIII protein or the VWF fragment. The FVIII protein associated with the VWF fragment is thereby cleared from the circulation more slowly by one or more VWF clearance receptors, resulting in the full half-life extension provided by the XTEN sequence or the XTEN sequence in combination with an Ig constant region compared to a FVIII protein without the VWF fragment or wild-type FVIII.

[0086] In one embodiment, the VWF fragment is associated (or linked) with the FVIII protein by a covalent or non-covalent bond. However, in some cases, the physical blockage or chemical association (e.g., non-covalent bond) between the VWF fragment and the FVIII protein may not be strong enough to stabilize the complex containing FVIII and the VWF fragment in the presence of endogenous VWF. For example, a VWF fragment that forms a non-covalent bond with the FVIII protein (without any other bond) may easily dissociate from the FVIII protein in vivo in the presence of endogenous VWF, and the endogenous VWF may be replaced by the VWF fragment (e.g., recombinant VWF, i.e., rVWF). Therefore, the FVIII protein that is non-covalently bound to endogenous VWF may be easily removed from the body via the VWF clearance pathway. To prevent dissociation of the FVIII protein and the VWF fragment, in some embodiments, the association or linkage between the FVIII protein and the VWF fragment is a covalent bond (e.g., a peptide bond, one or more amino acids, or a disulfide bond). In certain embodiments, the association (i.e., linkage) between the accessory moiety and the FVIII protein is a peptide bond or a linker (FVIII / VWF linker) between the FVIII protein and the VWF fragment. Non-limiting examples of linkers are described elsewhere herein. In some embodiments, the VWF fragment has a length of at least about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, A VWF fragment is a polypeptide containing, consisting essentially of, or consisting of 1,900, 2,000, 2,500, 3,000, or 4,000 amino acids. Non-limiting examples of VWF fragments are described elsewhere herein.

[0087] In certain embodiments, the VWF fragment chemically (e.g., non-covalently) binds to or physically blocks one or more VWF-binding sites of the FVIII protein. The VWF-binding sites of the FVIII protein are located within the A3 domain or the C2 domain of the FVIII protein. In yet other embodiments, the VWF-binding sites of the FVIII protein are located within the A3 domain and the C2 domain. For example, the VWF-binding sites of the FVIII protein correspond to amino acids 1669-1689 and / or 2303-2332 of SEQ ID NO: 4 (full-length mature FVIII).

[0088] The present invention also provides chimeric proteins (comprising a FVIII protein and a VWF fragment) that further contain one or more XTEN sequences, thereby providing additional half-life extension properties. The one or more XTEN sequences may be inserted within the FVIII protein or VWF fragment, or may be linked to the N- or C-terminus of the FVIII protein or VWF fragment. The present invention also includes a FVIII protein linked to an XTEN sequence (first half-life extension) and an Ig constant region or portion thereof (second half-life extension), whereby the two half-life extensions extend the half-life of the FVIII protein via two different mechanisms.

[0089] In some embodiments, the chimeric protein contains a FVIII protein linked to a first Ig constant region or portion thereof (e.g., a first FcRn binding partner), a VWF fragment linked to a second Ig constant region or portion thereof (e.g., a second FcRn binding partner), and one or more XTEN sequences inserted into or linked to the FVIII protein or VWF fragment, wherein the VWF fragment prevents a FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein, wherein the first and second Ig constant regions or portions thereof form a covalent bond (e.g., a disulfide bond), and wherein the one or more XTEN sequences extend the half-life of the FVIII protein.

[0090] In certain embodiments, the chimeric proteins of the present invention contain a FVIII protein linked to a VWF fragment by an optional linker (i.e., an FVIII / VWF linker), and one or more XTEN sequences inserted into or linked to the FVIII protein or the VWF fragment, where the VWF fragment prevents a FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein, and the one or more XTEN sequences extend the half-life of the FVIII protein. In one embodiment, the optional linker (FVIII / VWF linker) contains a sortase recognition motif. In another embodiment, the optional linker (FVIII / VWF linker) contains a cleavable site. Examples of cleavable linkers (i.e., linkers containing one or more cleavage sites) are described elsewhere herein.

[0091] Chimeric proteins of the present invention include, but are not limited to: (1) a VWF fragment containing the D' and D3 domains, an XTEN sequence, and FVIII, wherein the XTEN sequence is linked to the VWF fragment; (2) a FVIII protein, an XTEN sequence, and an Ig constant region or a portion thereof, wherein the FVIII protein is linked to the XTEN sequence and the Ig constant region or a portion thereof; or (3) a FVIII protein, an XTEN sequence, and a VWF fragment, wherein XTEN The N sequence is linked to the FVIII protein at the C-terminus or N-terminus, or is inserted immediately downstream of one or more amino acids of FVIII (e.g., at one or more XTEN insertion sites), and the VWF fragment and the FVIII protein are associated with each other.

[0092] (1) Von Willebrand factor (VWF) fragment and FVIII linked to XTEN The present invention is directed to a chimeric protein comprising (i) a VWF fragment containing the D' and D3 domains of VWF, (ii) an XTEN sequence, and (iii) an FVIII protein, wherein (i), (ii), and (iii) are linked or associated with each other. As part of the chimeric protein of the present invention, the VWF linked to the XTEN sequence associates with the FVIII protein, thereby disrupting or inhibiting the interaction between endogenous VWF and the FVIII protein. In certain embodiments, a VWF fragment capable of disrupting or inhibiting the binding of endogenous VWF to the FVIII protein can simultaneously possess at least one VWF-like FVIII protective property. Examples of VWF-like FVIII protective properties include, but are not limited to, protecting FVIII from protease cleavage and FVIII activation, stabilizing the FVIII heavy and / or light chain, and disrupting the removal of FVIII by scavenger receptors. As a result, the VWF fragment inhibits the removal of FVIII protein via the VWF clearance pathway, thereby reducing the removal of FVIII from the body. In some embodiments, the VWF fragment of the present invention binds to or associates with FVIII protein and / or physically or chemically blocks the VWF-binding site of the FVIII protein. The FVIII protein associated with the VWF fragment is thus removed from the circulation more slowly than wild-type FVIII or FVIII not associated with a VWF fragment.

[0093] In one embodiment, the present invention is directed to a chimeric protein comprising (i) a VWF fragment containing the D' and D3 domains of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the XTEN sequence is linked to the VWF fragment (e.g., (a1) VX or (a2) XV, where V contains the VWF fragment and X contains the XTEN sequence), and the VWF fragment is linked to or associated with the FVIII protein. In other embodiments, the VWF fragment and the XTEN sequence are linked by a linker or peptide bond (e.g., (a3) ​​VLX or (a4) XLV). The linker may be a cleavable linker (e.g., a thrombin-cleavable linker) that can be cleaved at the site of coagulation. In other embodiments, the VWF fragment, the XTEN sequence, and the FVIII protein are arranged in a single polypeptide chain. In yet another embodiment, the chimeric protein contains two polypeptide chains, wherein the first chain contains a VWF fragment and an XTEN sequence, and the second chain contains a FVIII protein. In yet another embodiment, the chimeric protein contains three polypeptide chains, wherein the first chain contains a VWF fragment and an XTEN sequence, the second chain contains a FVIII light chain, and the third chain contains a FVIII heavy chain, wherein the first chain and the second chain are associated with each other (e.g., covalently (e.g., disulfide bond)), and the second chain and the third chain are associated with each other (e.g., metal bond). In yet another embodiment, the XTEN sequence may be linked to the N-terminus or C-terminus of the VWF fragment, or may be inserted immediately downstream of one or more amino acids of the VWF fragment.

[0094] In certain embodiments, the chimeric protein of the invention comprises a formula comprising: (a) VX-FVIII; (b) FVIII-XV, (c) VX:FVIII, (d) XV:FVIII, (e) FVIII:VX, (f) FVIII:XV, or (a5) XV-FVIII, where V contains a VWF fragment, X contains one or more XTEN sequences; FVIII contains the FVIII protein; (-) represents a peptide bond or one or more amino acids; and The (:) represents a chemical or physical association. In one embodiment, the (:) represents a chemical association (e.g., at least one non-peptide bond). In another embodiment, the chemical association, i.e., the (:) represents a covalent bond. In another embodiment, the chemical association, i.e., the (:) represents a non-covalent interaction (e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond). In another embodiment, the (:) represents a non-peptide covalent bond. In yet another embodiment, the (:) represents a peptide bond. In yet another embodiment, the (:) represents a physical association between two sequences, where a portion of the first sequence is in close proximity to the second sequence, thereby shielding or preventing a portion of the second sequence from interacting with other moieties by the first sequence, and further, this physical association is maintained without interaction between the second sequence and other moieties. The orientation of polypeptide formulas herein is listed from the N-terminus (left) to the C-terminus (right). For example, the formula VX-FVIII refers to the formula NH-VX-FVIII-COOH. In one embodiment, the formulas described herein may contain any additional sequence between two moieties. For example, the formula VX-FVIII may further contain any sequence at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII, unless otherwise specified. In other embodiments, a hyphen (-) indicates a peptide bond.

[0095] In other embodiments, the chimeric protein of the invention comprises the formula: (a) V(X1)-X2-FVIII; (b) FVIII-X2-V(X1), (c) V(X1):FVIII, (d) FVIII:V(X1), or (a5)X2-V(X1)-FVIII, wherein V(X1) contains a VWF fragment and a first XTEN sequence (X1), where the XTEN sequence is inserted immediately downstream of one or more amino acids of the VWF fragment, and X2 contains one or more optional XTEN sequences; FVIII contains the FVIII protein, (-) represents a peptide bond or one or more amino acids; and (:) is a chemical or physical association.

[0096] In some embodiments, the chimeric protein is directed to a chimeric protein containing (i) a VWF fragment containing the D' and D3 domains of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) a first optional linker, and (v) a second optional linker, wherein the XTEN sequence is linked to the VWF fragment and / or the FVIII protein by the linker. In certain embodiments, the chimeric protein contains the following formula: (b1) V-L1-X-L2-FVIII, (b2) FVIII-L2-X-L1-V, (b3) V-L1-X:FVIII, (b4) X-L1-V:FVIII, (b5) FVIII:V-L1-X, (b6) FVIII:X-L1-V, (b7) X-L1-V-L2-FVIII, or (b8) FVIII-L2-V-L1-X, where V contains a VWF fragment, X contains one or more XTEN sequences; FVIII contains the FVIII protein; L1 contains a first optional linker (e.g., a first cleavable linker), L2 contains a second optional linker (e.g., a second cleavable linker, or an optional processable linker), (-) is a peptide bond or one or more amino acids; and The (:) represents a chemical or physical association. In one embodiment, the (:) represents a chemical association (e.g., at least one non-peptide bond). In another embodiment, the chemical association, i.e., the (:) represents a covalent bond. In another embodiment, the chemical association, i.e., the (:) represents a non-covalent interaction (e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond). In another embodiment, the (:) represents a non-peptide covalent bond. In yet another embodiment, the (:) represents a peptide bond. In yet another embodiment, the (:) represents a physical association between two sequences, where a portion of the first sequence is in close proximity to the second sequence, thereby shielding or preventing a portion of the second sequence from interacting with other moieties by the first sequence, and further, this physical association is maintained without interaction between the second sequence and other moieties. The orientation of polypeptide formulas herein is listed from the N-terminus (left) to the C-terminus (right). For example, the formula (b1) V-L1-X-L2-FVIII refers to the formula NH2-V-L1-X-L2-FVIII-COOH. In one embodiment, the formulas described herein may contain an optional additional sequence between the two moieties. In another embodiment, a hyphen (-) represents a peptide bond.

[0097] In other embodiments of the present invention, FVIII chimeric proteins are provided that do not interact with FVIII half-life limiting factors (e.g., endogenous VWF) while maximizing the half-life of the FVIII protein using XTEN sequences in combination with a second half-life extender or moiety that provides a covalent bond between the FVIII protein and a VWF fragment (e.g., an Ig constant region or portion thereof). In one embodiment, a chimeric protein of the present invention contains (i) a VWF fragment containing the D' and D3 domains of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, and (iv) an Ig constant region or portion thereof (also referred to herein as F), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker (e.g., a cleavable linker), (2) the VWF fragment is associated with or linked to the FVIII protein by an additional optional linker (e.g., a cleavable linker), and (3) the Ig constant region or portion thereof is linked to the VWF fragment, the XTEN sequence, or the FVIII protein. In another embodiment, the chimeric protein of the present invention contains (i) a VWF fragment containing the D' and D3 domains of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) an Ig constant region or portion thereof (F1 or a first Ig constant region), and (v) an additional Ig constant region or portion thereof (F2 or a second Ig constant region), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker (e.g., a cleavable linker), (2) the XTEN sequence or the VWF fragment is linked to the Ig constant region or portion thereof, (3) the FVIII is linked to the additional Ig constant region or portion thereof, and (4) the Ig constant region or portion thereof is associated with or linked to the additional Ig constant region or portion thereof. In one embodiment, the association or linkage between the two Ig constant regions or portions thereof is a covalent bond (e.g., a disulfide bond). In another embodiment, the two Ig constant regions or portions thereof are linked to each other. The association or linkage between the moieties is a processable linker, where the processable linker is processed by a protease within the cell. For example, the chimeric protein contains the formula: (g) V-L2-X-L1-F1:FVIII-L3-F2; (h) V-L2-X-L1-F1:F2-L3-FVIII; (i) F-L1-X-L2-V:FVIII-L3-F2; (j) F-L1-X-L2-V:F2-L3-FVIII; (k)V-L2-X-L1-F1-L4-FVIII-L3-F2; (l)F2-L3-FVIII-L4-F1-L1-X-L2-V; (m) FVIII-L2-F2-L4-V-L2-X-L1-F1; or (n)F1-L1-X-L2-V-L4-F2-L2-FVIII, where V contains a VWF fragment, each of L1 and L3 contains an optional linker; L2 contains an optional linker (e.g., a cleavable linker), L4 is an optional linker (e.g., a processable linker), FVIII contains the FVIII protein, X contains one or more XTEN sequences; F1 contains an optional Ig constant region or portion thereof; F2 optionally contains an additional Ig constant region or portion thereof; (-) is a peptide bond or one or more amino acids; and (:) is a chemical or physical association.

[0098] In some embodiments, the FVIII protein of any construct or formula disclosed herein further contains at least one, at least two, at least three, at least four, at least five, or at least six XTEN sequences, each of which is inserted immediately downstream of one or more amino acids of the FVIII protein or linked to the N-terminus or C-terminus of the FVIII protein. Non-limiting examples of XTEN insertion sites are described elsewhere herein.

[0099] In one embodiment, the (:) represents a chemical association (e.g., at least one non-peptide bond). In another embodiment, the chemical association, i.e., the (:) is a covalent bond. In another embodiment, the chemical association, i.e., the (:) is a non-covalent interaction (e.g., ionic interaction, hydrophobic interaction, hydrophilic interaction, Van der Waals interaction, or hydrogen bond). In another embodiment, the (:) is a non-peptide covalent bond. In yet another embodiment, the (:) is a peptide bond. In yet another embodiment, the (:) represents a physical association between two sequences, where a portion of the first sequence is in close proximity to the second sequence, thereby shielding or preventing a portion of the second sequence from interacting with other moieties by the first sequence, and further, this physical association is maintained without interaction between the second sequence and other moieties. The orientation of polypeptide formulas herein is listed from the N-terminus (left) to the C-terminus (right). For example, the formula (n) F1-L1-X-L2-V-L4-F2-L2-FVIII means the formula NH2-F1-L1-X-L2-V-L4-F2-L2-FVIII-COOH. In one embodiment, the formulas described herein may contain an optional additional sequence between the two moieties. In another embodiment, a hyphen (-) represents a peptide bond.

[0100] In one embodiment, either or both of the Ig constant region or portion thereof (sometimes designated herein as "F" or "F1") and the additional Ig constant region or portion thereof (sometimes designated herein as "F2") linked to the VWF fragment, FVIII protein or In another embodiment, a pair of an Ig constant region or portion thereof (sometimes referred to herein as "F" or "F1") and an additional Ig constant region or portion thereof (sometimes referred to herein as "F2") are linked to a VWF fragment and a FVIII protein, respectively, to form a stronger than non-covalent bond between the FVIII protein and the VWF fragment (i.e., a covalent bond (e.g., a disulfide bond)), thereby preventing the VWF fragment from displacing endogenous VWF in vivo. F1 or F2 may contain an Fc region or an FcRn-binding partner. In another embodiment, either or both of F1 and F2 linked to the VWF fragment and / or FVIII protein form a covalent bond (e.g., a disulfide bond) between F1 and F2, thereby bringing the VWF fragment and the FVIII protein into close proximity and inhibiting the interaction between the VWF fragment and the FVIII protein. In some embodiments, F1 and F2 are the same or different. Non-limiting examples of F1 and F2 may be selected from the group consisting of a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragment, derivative, or analog thereof, and combinations of two or more thereof. In one embodiment, F1, F2, or both, contain at least one CH1 domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or a functional fragment, derivative, or analog thereof. In other embodiments, F1, F2, or both, comprise at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in a hinge-CH2 orientation). In other embodiments, F1, F2, or both, comprise at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in a CH2-CH3 orientation).Examples of such combinations include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain (also known as Fc regions (or Fc domains)) (e.g., a first Fc region or FcRn binding partner for F1 and a second Fc region or FcRn binding partner for F2). In other embodiments, F1 is linked to the VWF fragment by a linker and / or F2 is linked to FVIII by a linker. In some embodiments, F1 and / or F2 comprise, consist essentially of, or consist of a hinge region. Additional non-limiting examples of Fc regions or FcRn binding partners are described elsewhere herein.

[0101] In some embodiments, the chimeric protein of the present invention contains two polypeptide chains: a first polypeptide chain (containing, consisting essentially of, or consisting of a VWF fragment containing the D' and D3 domains, an XTEN sequence, a first Ig constant region or a portion thereof (e.g., a first Fc region) and an optional linker between the VWF fragment and the XTEN sequence, or an XTEN sequence or a first Ig constant region or a portion thereof) and a second polypeptide chain (containing, consisting essentially of, or consisting of a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region)). The linker between the VWF fragment and the first Ig constant region or a portion thereof may be a cleavable linker (e.g., a cleavable linker) that can be cleaved at the site of coagulation. In some embodiments, the first polypeptide chain and the second polypeptide chain are associated with each other. The association between the first chain and the second chain prevents in vivo displacement of endogenous VWF with the first chain containing the VWF fragment. In one embodiment, the association between the first chain and the second chain can be a covalent bond. In certain embodiments, the covalent bond is a disulfide bond. In some embodiments, the FVIII protein of the second chain further comprises one or more XTEN sequences linked to the C-terminus or N-terminus of the FVIII protein, or inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., at least one insertion site disclosed herein). Non-limiting examples of the present invention are described elsewhere herein.

[0102] In another embodiment, a chimeric protein of the invention comprises three polypeptide chains, wherein a first polypeptide chain comprises, consists essentially of, or consists of a heavy chain of a FVIII protein, a second polypeptide chain comprises, consists essentially of, or consists of a light chain of a FVIII protein fused to a first Ig constant region or portion thereof (e.g., a first Fc region), and a third polypeptide chain comprises, consists essentially of, or consists of a VWF fragment containing a D' domain and a D3 domain, an XTEN sequence, a second Ig constant region or portion thereof (e.g., a second Fc region), and an optional linker between the XTEN sequence and the second Ig constant region or portion thereof or an optional linker between the VWF fragment and the XTEN sequence. The linker of the third chain may be a cleavable linker that is cleaved at a coagulation site (e.g., a thrombin cleavage site). In some embodiments, the FVIII heavy chain or the FVIII light chain is linked to one or more XTEN sequences, which can be linked to the N-terminus or C-terminus, or can be inserted into one or more insertion sites within the FVIII sequence. Non-limiting examples of insertion sites are described elsewhere herein.

[0103] In yet another embodiment, the chimeric protein of the present invention comprises a first chain (comprising, consisting essentially of, or consisting of a heavy chain of a FVIII protein) and a second chain (comprising, consisting essentially of, or consisting of a light chain of a FVIII protein, a first Ig constant region or portion thereof (e.g., a first Fc region), a first linker (e.g., a processible linker containing one or more protease cleavage sites that contain one or more intracellular processing sites), a VWF fragment, a second linker (e.g., a thrombin-cleavable linker), an XTEN sequence, and a second Ig constant region or portion thereof (e.g., a second Fc region), wherein the light chain of the FVIII protein is linked to the first Ig constant region or portion thereof, which is further linked to the VWF fragment by the first linker, and wherein the VWF fragment is linked to the XTEN sequence, which is further linked to the second constant region or portion thereof by the second linker. In one embodiment, the first linker is a processable linker and the second linker is a cleavable linker. Upon expression, the chimeric protein can be cleaved by an intracellular processing enzyme that cleaves the processable linker, thereby allowing the chimeric protein to contain, consist essentially of, or consist of three polypeptide chains. Furthermore, the cleavable linker allows the VWF fragment to be cleaved at the site of coagulation.

[0104] In one embodiment, the chimeric protein of the present invention comprises one polypeptide chain comprising a single-chain FVIII protein, a first Ig constant region or portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker), a VWF fragment, an XTEN sequence, a second linker (e.g., a thrombin-cleavable linker), and a second Ig constant region or portion thereof (e.g., a second Fc region), wherein the single-chain FVIII protein is linked to the first Ig constant region or portion thereof, which is further linked to the VWF fragment by a first linker, and the VWF fragment is linked to the XTEN sequence, which is further linked to a second Ig constant region or portion thereof. In one embodiment, the VWF fragment and the XTEN sequence are linked by a second linker. In another embodiment, the XTEN sequence and the second Ig constant region or portion thereof are linked by a second linker. In another embodiment, the second chain further comprises a third linker. The single-chain polypeptide thereby contains a VWF fragment linked to an XTEN sequence by a second linker, and an XTEN linked to a second Ig constant region or portion thereof by a third linker. The second linker and the third linker can be the same or different. In one embodiment, the first linker is a processable linker. In other embodiments, the second linker or the third linker is a processible linker. The linker is a cleavable linker containing one or two cleavable sites. In certain embodiments, the second linker is a thrombin-cleavable linker. Linkers useful in the present invention are described elsewhere herein.

[0105] (2) FVIII, XTEN, and Fc The chimeric protein of the present invention also contains (i) a FVIII protein, (ii) an XTEN sequence (first half-life extender), and (iii) an Ig constant region or a portion thereof (second half-life extender), in which the XTEN sequence is linked to the FVIII protein by an optional linker and to the Ig constant region or a portion thereof by an additional optional linker. The XTEN sequence and the Ig constant region or a portion thereof can be used together to extend the half-life of the FVIII protein. In one embodiment, the chimeric protein is a monomer. In another embodiment, the chimeric protein is a dimer (homodimer or heterodimer).

[0106] The present invention is also directed to chimeric proteins containing (i) a FVIII protein, (ii) an XTEN sequence, (iii) an Ig constant region or portion thereof (i.e., a first Ig constant region or portion thereof (F or F1)), and (iv) an additional Ig constant region or portion thereof (i.e., a second Ig constant region or portion thereof, F2). In one embodiment, the XTEN sequence is linked to the FVIII protein at the N- or C-terminus or inserted immediately one or more amino acids downstream of the FVIII protein (e.g., at one or more XTEN insertion sites), the FVIII protein is linked to the first Ig constant region or portion thereof, and the first Ig constant region or portion thereof and the second constant region or portion thereof are associated or connected to each other by an optional linker. In some embodiments, the chimeric protein is a monomer-dimer hybrid containing a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains a FVIII protein, an XTEN sequence, and a first Ig constant region or portion thereof, and the second polypeptide chain contains, consists essentially of, or consists of a second Ig constant region or portion thereof without the FVIII protein, and wherein the first chain and the second chain are associated with each other. The association between the Ig constant region or portion thereof (e.g., a first Fc region) and the additional Ig constant region or portion thereof (e.g., a second Fc region) is a chemical association or a physical association. In some embodiments, the chemical association is a covalent bond. In other embodiments, the chemical association is a non-covalent interaction (e.g., ionic interaction, hydrophobic interaction, hydrophilic interaction, Van der Waals interaction, or hydrogen bond). In other embodiments, the association is a non-peptide covalent bond. In still other embodiments, the association is a peptide bond.

[0107] In other embodiments, the chimeric protein is a single-chain polypeptide containing a FVIII protein, an XTEN sequence, a first constant region or portion thereof, a linker (e.g., a processable linker), and a second constant region or portion thereof, wherein the single-chain polypeptide is processed by intracellular enzymes after expression into two polypeptide chains.

[0108] In one embodiment, the Ig constant region or portion thereof (sometimes designated "F" or "F1") linked to the FVIII protein, together with the XTEN sequence, can extend the half-life of the FVIII protein. In other embodiments, the Ig constant region or portion thereof (F or F1) is an Fc region or FcRn binding partner described elsewhere herein.

[0109] In other embodiments, an additional Ig constant region or portion thereof (optionally F2 or a second Ig constant region) associated or linked to the first Ig constant region or portion thereof is provided. In another embodiment, the second Ig constant region or portion thereof (F2), together with the first Ig constant region or portion thereof and an XTEN sequence, can also extend the half-life of the FVIII protein. The additional Ig constant region or portion thereof can be an Fc region or FcRn binding partner, as described elsewhere herein.

[0110] In certain embodiments, the second Ig constant region or portion thereof linked to the first Ig constant region or portion thereof is further linked to a VWF fragment and optional XTEN sequence as described elsewhere herein.

[0111] In some embodiments, either or both of the Ig constant region or portion thereof (F or F1 or first Ig constant region or portion thereof) and the additional Ig constant region or portion thereof (i.e., second Ig constant region or portion thereof or F2) (referred to in this paragraph as an Ig constant region or portion thereof) include, but are not limited to, a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragment, derivative, or analog thereof, or a combination of two or more thereof. In one embodiment, the Ig constant region or portion thereof contains at least one CH1 domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or a functional fragment, derivative, or analog thereof. In other embodiments, the Ig constant region or portion thereof includes at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in a hinge-CH2 orientation). In other embodiments, the Ig constant domain or portion thereof comprises at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (eg, in a CH2-CH3 orientation). Examples of such combinations include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain (also known as an Fc region (or Fc domain)) (e.g., a first Fc region). Additional examples of Ig constant regions or portions thereof are described elsewhere herein.

[0112] The chimeric proteins of the present invention may have a FVIII protein with an extended half-life compared to wild-type FVIII. In one embodiment, the half-life of the FVIII protein is extended by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild-type FVIII. In other embodiments, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0113] (3) FVIII, XTEN, and VWF In one embodiment, the chimeric protein of the present invention contains (i) a FVIII protein, (ii) an XTEN sequence, and (iii) a VWF fragment containing the D' and D3 domains of VWF, wherein the FVIII protein is linked to the XTEN sequence, and wherein the FVIII protein is associated with or linked to the VWF fragment. In certain embodiments, the VWF fragment of the chimeric protein described herein is unable to bind to a VWF clearance receptor. In other embodiments, the VWF fragment can protect the FVIII protein from one or more protease cleavage, protect the FVIII protein from activation, stabilize the heavy and / or light chains of the FVIII protein, or prevent removal of the FVIII protein by one or more scavenger receptors. In other embodiments, the VWF fragment prevents or inhibits endogenous VWF from binding to the VWF-binding site of the FVIII protein. The VWF-binding site may be located in the A3 domain or the C2 domain of the FVIII protein, or may be located in both the A3 and C2 domains. In certain embodiments, the VWF-binding site contains an amino acid sequence corresponding to amino acids 1669-1689 and / or 2303-2332 of SEQ ID NO: 2.

[0114] In another embodiment, the chimeric protein contains (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment containing the D' and D3 domains of VWF, and (iv) an Ig constant region or portion thereof, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or N-terminus or inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., at one or more XTEN insertion sites disclosed herein), the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combination thereof. Ig constant regions or portions thereof useful in the chimeric proteins of the present invention are described elsewhere herein. In one embodiment, the Ig constant region or portion thereof can extend the half-life of the FVIII protein. In another embodiment, the Ig constant region or portion thereof contains a first Fc region or a first FcRn binding partner. In yet another embodiment, the Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker. In yet another embodiment, the linker contains a cleavable linker. The chimeric protein may be a single-chain polypeptide, i.e., a monomer (i.e., single chain) containing (i), (ii), (iii), and (iv), or may be a two-chain polypeptide containing a first chain containing (i) and (ii) and a second chain containing (iii) and (iv). In another embodiment, the chimeric protein is a dimer (e.g., a homodimer or a heterodimer). In one embodiment, the chimeric protein contains two chains, each containing (i), (ii), (iii), and (iv).

[0115] In certain embodiments, the chimeric protein contains (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment containing the D' and D3 domains of VWF, (iv) an Ig constant region or portion thereof (sometimes also referred to as "F," "first Ig constant region or portion thereof," or "F2"), and (v) an additional Ig constant region or portion thereof (sometimes also referred to as "F2" or "second Ig constant region or portion thereof"), wherein (1) the FVIII protein is linked to the XTEN sequence at the C-terminus or N-terminus of the FVIII protein, or inserted at one or more amino acids immediately downstream of the FVIII protein (e.g., at one or more XTEN insertion sites disclosed herein), (2) either the XTEN sequence or the FVIII protein is linked to the Ig constant region or portion thereof, (3) the VWF fragment is linked to a second Ig constant region or portion thereof, and (4) the Ig constant region or portion thereof is associated with the second Ig constant region or portion thereof. In one embodiment, the FVII protein or Ig constant region or portion thereof linked to the XTEN sequence is further linked to the VWF fragment by a linker (e.g., a processable linker). In other embodiments, additional Ig constant regions or portions thereof useful in the chimeric proteins of the present invention are further linked to the FVIII protein or Ig constant region or portion thereof by an optional linker (e.g., a processable linker). may be linked to a portion thereof. In some embodiments, a pair of an Ig constant region or portion thereof and an additional Ig constant region or portion thereof are linked to a VWF fragment and a FVIII protein, respectively, resulting in a stronger bond (i.e., a covalent bond (e.g., a disulfide bond)) than the non-covalent bond between the FVIII protein and the VWF fragment, thereby preventing endogenous VWF from being replaced by the VWF fragment in vivo. In other embodiments, either or both of the Ig constant region or portion thereof and the additional Ig constant region or portion thereof can extend the half-life of the FVIII protein or the VWF fragment. In other embodiments, the additional Ig constant region or portion thereof contains a second Fc region or an FcRn binding partner. The Ig constant region or portion thereof and the additional Ig constant region or portion thereof in the chimeric protein are the same or different.

[0116] In certain embodiments, the Ig constant region, or portion thereof, and the additional Ig constant region, or portion thereof, are associated by a chemical or physical association. In one embodiment, the chemical association, i.e., (:), is at least one non-peptide bond. In certain embodiments, the chemical association, i.e., (:), is a covalent bond. In other embodiments, the chemical association, i.e., (:), is a non-covalent interaction (e.g., ionic interaction, hydrophobic interaction, hydrophilic interaction, Van der Waals interaction, or hydrogen bond). In other embodiments, the (:) is a non-peptide covalent bond. In yet other embodiments, the (:) is a peptide bond. In yet other embodiments, the (:) represents a physical association between two sequences, where a portion of the first sequence is in close proximity to the second sequence, thereby protecting or preventing that portion of the second sequence from interacting with the other portion by the first sequence. In some embodiments, the association between the Ig constant region or portion thereof and the additional Ig constant region or portion thereof can be a covalent bond (e.g., a disulfide bond), which prevents the VWF fragment or polypeptide containing the VWF fragment from displacing endogenous VWF. That is, by preventing the interaction between the FVIII protein and endogenous VWF, the half-life limiting factor for the FVIII protein is removed, thereby extending the half-life of the FVIII protein compared to the FVIII protein without the VWF protein or wild-type FVIII.

[0117] In other embodiments, the chimeric protein comprises a formula comprising: (1)FVIII(X1)-L1-F1:V-L2-X2-L3-F2, (2)FVIII(X1)-L1-F1:F2-L3-X2-L2-V, (3)F1-L1-FVIII(X1):V-L2-X2-L3-F2, (4)F1-L1-FVIII(X1);F2-L3-X2-L2-V, (5)FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2, (6)FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V, (7)F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (8)F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V, wherein FVIII(X1) comprises a FVIII protein and one or more XTEN sequences, wherein the one or more XTENs are linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., at one or more XTEN insertion sites disclosed herein); each of L1, L2, or L3 contains an optional linker (e.g., a cleavable linker); L4 is a linker (e.g., a processable linker); X2 contains one or more optional XTEN sequences; F1 contains an Ig constant region or a portion thereof, F2 optionally contains an additional Ig constant region or portion thereof, and V contains a VWF fragment, (-) is a peptide bond or one or more amino acids, and The (:) represents a covalent or non-covalent bond. In one embodiment, the (:) represents a chemical association (e.g., at least one non-peptide bond). In another embodiment, the chemical association, i.e., the (:) is a covalent bond. In another embodiment, the chemical association, i.e., the (:) is a non-covalent interaction (e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond). In another embodiment, the (:) is a non-peptide covalent bond. In yet another embodiment, the (:) is a peptide bond. In yet another embodiment, the (:) represents a physical association between two sequences, where a portion of the first sequence is in close proximity to the second sequence, thereby shielding or preventing a portion of the second sequence from interacting with other moieties by the first sequence, and further, this physical association is maintained without interaction between the second sequence and other moieties. The orientation of the polypeptide formulas herein is listed from the N-terminus (left) to the C-terminus (right). For example, the formula VX-FVIII refers to the formula NH-VX-FVIII-COOH. In one embodiment, the formulas described herein may contain any additional sequence between two moieties. For example, the formula VX-FVIII may further contain any sequence at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII, unless otherwise specified. In other embodiments, a hyphen (-) indicates a peptide bond.

[0118] In one embodiment, the chimeric protein is two polypeptide chains: (A) a first chain containing (i) a single-chain FVIII protein, (ii) an XTEN sequence, and (iii) a first Ig constant region or portion thereof (e.g., a first Fc region or an FcRn binding partner), wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus, or inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., at one or more XTEN insertion sites disclosed herein), and when the XTEN sequence is linked to the FVIII protein at the C-terminus or C-terminus, the first Ig constant region or portion thereof is linked to the XTEN sequence; and When the XTEN sequence is inserted into the FVIII protein, a first Ig constant region or portion thereof is linked to the FVIII protein, and (B) (iv) a VWF fragment containing the D' domain and the D3 domain, (v) a linker, and (vi) a second chain containing a second Ig constant region or portion thereof (e.g., a second Fc region or a second FcRn binding partner), wherein the VWF fragment is linked to a linker (e.g., a cleavable linker), which is further linked to the second Ig constant region or portion thereof, and wherein the first polypeptide chain and the second polypeptide chain are associated with each other (e.g., covalently linked, such as by a disulfide bond). In one embodiment, the linker is a cleavable linker described elsewhere herein (e.g., a thrombin-cleavable linker). In some embodiments, the second chain contains one or more XTEN sequences between (iv) and (v) or (v) and (vi).

[0119] The chimeric protein contains one polypeptide chain containing (i) a single-chain FVIII protein, (ii) an XTEN sequence, (iii) a first Ig constant region or portion thereof (e.g., a first Fc region or a first FcRn-binding partner), (iv) a first linker, (v) a VWF fragment containing the D' domain and the D3 domain, (vi) a second linker, and (vii) a second Ig constant region or portion thereof (e.g., a second Fc region or a second FcRn-binding partner), where (i) through (vii) are linked in that order, or in any order. In one embodiment, the first linker is a processible linker that can be processed or cleaved intracellularly after expression and separate the single-chain polypeptide into two polypeptide chains. In another embodiment, the second linker is a cleavable linker (e.g., a translinker) described herein. As used herein, the XTEN sequence may be linked to the FVIII protein at the N-terminus or C-terminus of the FVIII protein by an optional linker, or may be inserted one or more amino acids immediately downstream of the FVIII protein (e.g., at one or more XTEN insertion sites).

[0120] In some embodiments, the chimeric protein comprises three polypeptide chains: (A) a first polypeptide chain containing (i) a heavy chain of FVIII and (ii) an XTEN sequence, which are linked to each other; (B) a second polypeptide chain containing (iii) a light chain of FVIII and (iv) a first Ig constant region or a portion thereof (e.g., a first Fc region or a first FcRn binding partner), which are linked to each other; and (C) a third polypeptide chain containing (v) a VWF fragment containing a D' domain and a D3 domain, (vi) a linker, and (vii) a second Ig constant region or a portion thereof (e.g., a second Fc region or a second FcRn binding partner), wherein the second chain is related to the first and third chains. In one embodiment, the association between the first and second chains is a chemical or physical association. For example, the association between the first and second chains can be a metal binding. In other embodiments, the association between the second chain and the third chain is also chemically or physically associated (e.g., covalently or non-covalently). In certain embodiments, the association between the second chain and the third chain is via two Ig constant regions or portions thereof and is a disulfide bond. The bond between the second chain and the third chain prevents or inhibits the FVIII protein from binding to endogenous VWF, thereby preventing the FVIII protein from being removed by the VWF clearance pathway. In some embodiments, the linker is a processible linker that is cleaved intracellularly after expression in a host cell. The XTEN sequence used herein is linked to the FVIII protein at the N-terminus or C-terminus of the FVIII protein by an optional linker, or is inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., at one or more XTEN insertion sites).

[0121] In some embodiments, the VWF fragment is directly linked to the FVIII protein containing one or more XTENs via a peptide bond or a linker. One method for linking the VWF fragment and the FVIII protein (containing one or more XTENs inserted or linked directly (e.g., by a peptide bond) or via a linker) can be enzymatic ligation (e.g., sortase). For example, sortase refers to a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving carboxy-terminal sorting signals. For most of the substrates for the sortase enzyme, the recognition signal consists of an LPXTG (Leu-Pro-any-Thr-Gly (SEQ ID NO: 51) motif, followed by a highly hydrophobic transmembrane sequence, followed by a cluster of basic residues (e.g., arginine). Upon transient attachment of the ligation partner to the active site Cys residue via the Thr residue, cleavage occurs between the Thr and Gly, followed by transpeptidation that covalently attaches the protein to the cell wall. In some embodiments, the ligation partner contains a Gly(n). In other embodiments, the chimeric protein further contains a sortase recognition motif. In some embodiments, a VWF fragment is attached to FVIII containing one or more XTENs inserted or linked thereto using sortase-mediated in vitro protein ligation.

[0122] In one embodiment, a VWF fragment linked to a sortase recognition motif by an optional linker may be fused to FVIII linked to Gly(n) by a sortase, where n may be any integer, and where one or more XTENs may be inserted or linked within the FVIII protein. The ligation construct contains a VWF fragment (the N-terminal portion of the construct) and a FVIII protein (the C-terminal portion of the construct) into which one or more XTENs have been inserted or linked, where the sorter and a sortase recognition motif inserted therebetween. Other ligation constructs contain a VWF fragment (the N-terminal portion of the construct), a linker, a sortase recognition motif, and a FVIII protein (the C-terminal portion of the construct) to which one or more XTENs have been inserted or linked. In other embodiments, a FVIII protein linked to a sortase recognition motif by an optional linker may be fused to a VWF fragment linked to Gly(n) by a sortase, where n is any integer. The resulting ligation construct contains a FVIII protein (the N-terminal portion of the construct) to which one or more XTENs have been inserted or linked, and a VWF fragment (the C-terminal portion of the construct), to which a sortase recognition motif has been inserted. Other resulting ligation constructs contain a FVIII protein (the N-terminal portion of the construct) to which one or more XTENs have been inserted or linked, a linker, a sortase recognition motif, and a VWF fragment (the C-terminal portion of the construct). In other embodiments, a VWF fragment linked to a sortase recognition motif by a first optional linker may be fused to a heterologous moiety (e.g., an immunoglobulin constant region or portion thereof (e.g., Fc region)) linked to a thrombin cleavage site by a second optional linker. The resulting construct may contain the VWF fragment (N-terminal portion), the first linker, the sortase recognition motif, the protease cleavage site, the second optional linker, and the heterologous moiety.

[0123] In some embodiments, the VWF fragment is associated with the FVIII protein. The association between the VWF fragment and the FVIII protein may be a chemical association or a physical association. The chemical association may be a non-covalent interaction (e.g., ionic interaction, hydrophobic interaction, hydrophilic interaction, Van der Waals interaction, or hydrogen bond). In yet other embodiments, the association between the FVIII protein and the VWF fragment is a physical association between two sequences (e.g., by further association between a sequence with the FVIII protein and a sequence with the VWF fragment), where a portion of the first sequence is in close proximity to a second sequence, thereby protecting or preventing a portion of the second sequence from interacting with other portions by the first sequence.

[0124] By disrupting or inhibiting the interaction of endogenous VWF with the FVIII protein by the VWF fragment, the chimeric proteins described herein have an increased half-life compared to wild-type FVIII or a control chimeric protein lacking the VWF fragment. In one embodiment, the half-life of the FVIII protein is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to the FVIII protein lacking the VWF fragment. In other embodiments, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In certain embodiments, the half-life of the FVIII protein is extended by at least 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, or at least about 27 hours in HemA mice.

[0125] A) Von Willebrand factor (VWF) fragment VWF (also known as F8VWF) is a large multimeric glycoprotein present in plasma and is constitutively produced in endothelium (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, including the D´ / D3 domain (which binds factor VIII), the A1 domain (which binds to platelet GPIb receptors, heparin, and / or possibly collagen), the A3 domain (which binds to collagen), the C1 domain (in which the RGD domain binds to platelet integrin αIIbβ3 upon activation), and a "cysteine ​​knot" domain at the C-terminus of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), and β-human chorionic gonadotropin (βHCG)).

[0126] As used herein, the term "VWF fragment" includes, but is not limited to, functional VWF fragments containing the D' and D3 domains that can inhibit the binding of endogenous VWF to FVIII. In one embodiment, the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment interferes with the VWF-binding site on the FVIII protein, thereby inhibiting the interaction of the FVIII protein with endogenous VWF. VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0127] The 2813 monomer amino acid sequence of human VWF is reported in GenBank under accession number NP_000543.2. The nucleotide sequence encoding human VWF is reported in GenBank under accession number NM_000552.3. The nucleotide sequence of human VWF is designated as SEQ ID NO: 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

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

[0128] As used herein, the VWF fragment may be a VWF fragment containing the D' and D3 domains of VWF, wherein the VWF fragment binds to factor VIII (FVIII) and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment containing the D' and D3 domains may further contain a VWF domain selected from the group consisting of 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, and any combination thereof. In one embodiment, the VWF fragment contains, consists essentially of, or consists of (1) the D' and D3 domains of VWF or a fragment thereof, (2) the D1, D', and D3 domains of VWF or a fragment thereof, (3) the D2, D', and D3 domains of VWF or a fragment thereof, (4) the D1, D2, D', and D3 domains of VWF or a fragment thereof, or (5) the D1, D2, D', D3, and A1 domains of VWF or a fragment thereof. The VWF fragments described herein do not contain a site that binds to a VWF clearance receptor. In another embodiment, the VWF fragments described herein are not amino acids 764 to 1274 of SEQ ID NO: 2. The VWF fragments of the present invention may contain any other sequence linked or fused to the VWF fragment. For example, the VWF fragments described herein may further contain a signal peptide.

[0129] In one embodiment, the VWF fragment binds to or associates with the FVIII protein. By binding to or associating with the FVIII protein, the VWF fragment of the present invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy and light chains of FVIII, and prevents FVIII from being removed by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with FVIII and interferes with or prevents FVIII from binding to phospholipids and activated protein C. By interfering with or inhibiting the binding of the FVIII protein to endogenous full-length VWF, the VWF fragment of the present invention reduces the removal of FVIII by the VWF clearance receptor, thereby extending the half-life of the FVIII protein. The prolonged half-life of the FVIII protein is due to the binding or association of the VWF fragment, which lacks the VWF clearance receptor binding site, to the VFVIII protein, and the VWF fragment masks or protects the FVIII protein from endogenous VWF, which contains the VWF clearance receptor binding site. By removing the pathway receptor binding site, the FVIII / VWF heterodimer of the present invention is shielded from the VWF clearance pathway, further extending the half-life of FVIII.

[0130] In one embodiment, the VWF fragment of the present invention contains the D' and D3 domains of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-866 of SEQ ID NO: 2, and wherein the VWF fragment interferes with endogenous VWF binding to FVIII. In another embodiment, the VWF fragment contains the D' and D3 domains of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO: 2, and wherein the VWF fragment interferes with endogenous VWF binding to FVIII. In some embodiments, the VWF fragment described herein contains, consists essentially of, or consists 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, wherein the VWF fragment prevents endogenous VWF from binding to FVIII. In other embodiments, the VWF fragment contains, consists essentially of, or consists of the D1, D2, D', and D3 domains that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23-1240 of SEQ ID NO: 2, wherein the VWF fragment prevents endogenous VWF from binding to FVIII. In yet other embodiments, the VWF fragment further comprises a signal peptide operably linked to the VWF fragment.

[0131] In some embodiments, the VWF fragments of the present invention comprise (1) a D'D3 domain, a D1D'D3 domain, a D2D'D3 domain, or a D1D2D'D3 domain, and (2) an additional VWF fragment of up to about 10 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO: 2 to amino acids 764-1250 of SEQ ID NO: 2), an additional VWF fragment of 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), an additional VWF fragment of up to about 20 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), an additional VWF fragment of up to about 20 amino acids (e.g., any sequence from amino acids 764-1255 of SEQ ID NO: 2), an additional VWF fragment of up to about 20 amino acids (e.g., any sequence from amino acids 764-1250 ... The VWF fragment may consist essentially of, or consist of, an additional VWF fragment of up to about 25 amino acids (e.g., from amino acids 764-1240 of SEQ ID NO:2 to any sequence derived from amino acids 764-1260 of SEQ ID NO:2), an additional VWF fragment of up to about 25 amino acids (e.g., from amino acids 764-1240 of SEQ ID NO:2 to any sequence derived from amino acids 764-1265 of SEQ ID NO:2), or an additional VWF fragment of up to about 30 amino acids (e.g., from amino acids 764-1240 of SEQ ID NO:2 to any sequence derived from amino acids 764-1260 of SEQ ID NO:2). In certain embodiments, the VWF fragment containing or consisting essentially of the D' and D3 domains is not amino acids 764-1274 of SEQ ID NO:2, nor is it full-length mature VWF. In some embodiments, the D1D2 domain is expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis with the D'D3 domain.

[0132] In other embodiments, the VWF fragment containing the D'D3 domain linked to the D1D2 domain further contains an intracellular cleavage site (e.g., a PACE (furin) or PC5 cleavage site) that allows cleavage of the D1D2 domain from the D'D3 domain upon expression. Non-limiting examples of intracellular cleavage sites are described elsewhere herein.

[0133] In still other embodiments, the VWF fragment contains the D' domain and the D3 domain, but is selected from the group consisting of: (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 1271 to 2813 of SEQ ID NO: 2; (5) amino acids 1272 to 2813 of SEQ ID NO: 2, (6) amino acids 1274 to 2813 of SEQ ID NO: 2, and any combination thereof.

[0134] In yet another embodiment, the VWF fragment of the invention contains, consists essentially of, or consists of an amino acid sequence corresponding to the D', D3, and A1 domains, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1479 of SEQ ID NO: 2, and wherein the VWF fragment interferes with the binding of endogenous VWF to FVIII. In a specific embodiment, the VWF fragment is not amino acids 764-1274 of SEQ ID NO: 2.

[0135] In some embodiments, the VWF fragments of the present invention contain the D' and D3 domains, but do not include: (1) the A1 domain, (2) the A2 domain, (3) the A3 domain, (4) the D4 domain, (5) the B1 domain, (6) the B2 domain, (7) the B3 domain, (8) the C1 domain, (9) the C2 domain, (10) the CK domain, (11) the CK domain and the C2 domain, (12) the CK domain, the C2 domain, and the C1 domain, (13) the CK domain, the C2 domain, the C1 domain, and the B3 domain, (14) the CK domain, the C2 domain, the C1 domain, the B3 domain, and the B2 domain, (15) the CK domain, the C2 domain, the C1 domain, the B3 domain, the B2 domain, and the B1 domain, (16) the CK domain, the C2 domain, the C1 domain, the B3 domain, the B2 domain, the B1 domain, and the D4 domain, or (17) the CK domain, the C2 domain, and the C1 domain. (17) the VWF fragment does not contain at least one VWF domain selected from the group consisting of: (18) the B3 domain, the B2 domain, the B1 domain, the D4 domain, and the A3 domain; (19) the CK domain, the C2 domain, the C1 domain, the B3 domain, the B2 domain, the B1 domain, the D4 domain, the A3 domain, and the A2 domain; and (20) any combination thereof.

[0136] In yet another embodiment, the VWF fragment contains the D'D3 domain and one or more domains or molecules. Examples of such domains or molecules include, but are not limited to, the domains and molecules disclosed in Zhour et al., Blood published online April 6, 2012: DOI10.1182 / blood-2012-01-405134. For example, the VWF fragment may contain the D'D3 domain and one or more domains or molecules. It may contain a D3 domain and one or more domains or modules selected from the group consisting of an A1 domain, an A2 domain, an A3 domain, a D4N module, a VWD4 module, a C8-4 module, a TIL-4 module, a C1 module, a C2 module, a C3 module, a C4 module, a C5 module, a C5 module, a C6 module, and any combination thereof.

[0137] In yet another embodiment, the VWF fragment is linked to a heterologous moiety, wherein the heterologous moiety is linked to the N-terminus or C-terminus of the VWF fragment or inserted immediately downstream of one or more amino acids of the FVIII protein of the VWF fragment (e.g., one or more XTEN insertion sites). For example, the insertion site for the heterologous moiety of the VWF fragment may be in the D' domain, the D3 domain, or both. The heterologous moiety may be a half-life extender.

[0138] In certain embodiments, the VWF fragments of the present invention form multimers (e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, or higher multimers). In other embodiments, the VWF fragments are monomers having only one VWF fragment. In some embodiments, the VWF fragments of the present invention have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF fragment may contain amino acid substitutions, deletions, additions, or modifications such that the VWF fragment cannot form disulfide bonds or form dimers or multimers. In another embodiment, the amino acid substitutions are within the D' domain and the D3 domain. In a specific embodiment, the VWF fragment of the present invention 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. The at least one amino acid substitution may be any amino acid that does not naturally occur in wild-type VWF. For example, the amino acid substitution may be any amino acid other than cysteine ​​(e.g., isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, or histidine). In another example, the amino acid substitution comprises one or more amino acids that prevent or inhibit the VWF fragment from forming multimers.

[0139] In certain embodiments, VWF fragments useful in the present invention may be further modified to improve their interaction with FVIII (e.g., to improve binding affinity for FVIII). As a non-limiting example, the VWF fragment contains a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO: 2 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO: 2. Residue 764 and / or residue 773 may contribute to the binding affinity of the VWF fragment for FVIII. In other embodiments, the VWF fragments useful in the present invention may have other modifications (e.g., the protein is pegylated, glycosylated, hesylated, or polysialylated).

[0140] B) XTEN sequence As used herein, "XTEN sequence" refers to an extended-length polypeptide having a non-naturally occurring, substantially non-repetitive sequence composed primarily of small hydrophilic amino acids, and having a sequence that has a lower order structure or no secondary or tertiary structure under physiological conditions. As a partner in a chimeric protein, XTEN can serve as a carrier that, when linked to a VWF fragment or FVIII sequence of the invention to create a chimeric protein, provides certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as, for example, single-chain antibodies or Fc fragments of the light or heavy chains.

[0141] In some embodiments, the XTEN sequences of the invention are peptides or polypeptides having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or polypeptide having more than about 20 to about 3000 amino acid residues, about 30 to about 2500 amino acid residues, about 40 to about 2000 amino acid residues, about 50 to about 1500 amino acid residues, about 60 to about 1000 amino acid residues, about 70 to about 900 amino acid residues, about 80 to about 800 amino acid residues, about 90 to about 700 amino acid residues, about 100 to about 600 amino acid residues, about 110 to about 500 amino acid residues, or about 120 to about 400 amino acid residues.

[0142] The XTEN sequences of the invention may contain one or more motif sequences of 9-14 amino acid residues or amino acid sequences that are at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif. wherein the motif contains, consists essentially of, or consists of 4-6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) (see US2010-0239554 A1).

[0143] In some embodiments, the XTEN contains a non-repetitive sequence motif consisting of multiple units of non-repetitive sequence selected from a single motif family selected from Table 2A, such that about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% of the sequence is a family sequence. As used herein, the term "family" means that the XTEN has motifs selected only from single motif categories from Table 2A (i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN), and that any other amino acids in the XTEN that are not from a family motif are selected to obtain required properties (e.g., to allow for the incorporation of a restriction enzyme site by the coding nucleotides, the incorporation of a cleavage sequence, or to improve linkage to FVIII or VWF). In some embodiments of the XTEN family, the XTEN sequence contains multiple units of a non-repetitive sequence motif from the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, and the resulting XTEN sequence exhibits the ranges of homology described above. In other embodiments, the XTEN contains multiple units of motif sequences from two or more of the motif families in Table 2A. These sequences may be selected to obtain desired physical / chemical properties, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness due to the amino acid composition of the motif (described in more detail below). In the above-described embodiments of this paragraph, the motifs incorporated into the XTEN may be selected and assembled using the methods described herein to obtain XTENs of from about 36 to about 3,000 amino acid residues. [Table 2-1] [Table 2-2]

[0144] XTEN may be of various lengths for insertion or linkage into FVIII or VWF. In one embodiment, the length of the XTEN sequence(s) is selected based on the property or function to be achieved in the fusion protein. Based on the intended property or function, XTEN may be a short or medium-length sequence, or a longer sequence that may serve as a carrier. In certain embodiments, XTEN may contain short segments of about 6 to about 99 amino acid residues, medium-length segments of about 100 to about 399 amino acid residues, and longer segments of about 400 to 1000 and up to about 3000 amino acid residues. Thus, FVIII or XTEN linked to or inserted into FVIII can be about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 in length. In other embodiments, the XTEN is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 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 linked to or inserted into FVIII or VWF can vary without adversely affecting the activity of FVIII or VWF. In one embodiment, one or more XTEN used herein are 36, 42, 72, 144, 288, 576, or 864 amino acids in length and may be selected from one or more of the XTEN family sequences, i.e., AD, AE, AF, AG, AM, AQ, BC, or BD.

[0145] In some embodiments, the XTEN sequences used in the present invention are AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE62 4, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, A 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a sequence selected from the group consisting of E948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004 (see US 2010-0239554 A1).

[0146] In one embodiment, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 137), AE864 (SEQ ID NO: 138), AE864 (SEQ ID NO: 139), AE864 (SEQ ID NO: 140), AE864 (SEQ ID NO: 141), AE864 (SEQ ID NO: 142), AE864 (SEQ ID NO: 143), AE864 (SEQ ID NO: 144). 43), AE576 (SEQ ID NO:41), AE288 (SEQ ID NO:39), AE288_2 (SEQ ID NO:137), AE144 (SEQ ID NO:37), AG864 (SEQ ID NO:44), AG576 (SEQ ID NO:42), AG288 (SEQ ID NO:40), AG144 (SEQ ID NO:38), and any combination thereof. In other embodiments, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), AG144_D (SEQ ID NO: 136), AG144_E (SEQ ID NO: 137), AG144_F (SEQ ID NO: 138), AG144_G (SEQ ID NO: 139), AG144_H (SEQ ID NO: 140), AG144_J (SEQ ID NO: 141), AG144_L (SEQ ID NO: 142), AG144_L (SEQ ID NO: 143), AG144_L (SEQ ID NO: 144), AG144_L (SEQ ID NO: 145), AG144_M (SEQ ID NO: 146), AG144_N (SEQ ID NO: 147), AG144_N (SEQ ID NO: 148), AG144_P (SEQ ID NO: 149), AG144_R (SEQ ID NO: 150), AG144_S (SEQ ID NO: 151), AG144_V (SEQ ID NO: 152), AG144_V (SEQ ID In certain embodiments, X is selected from the group consisting of AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combination thereof. The XTEN sequence is AE288. The amino acid sequence for any XTEN sequence of the invention is shown in Table 2B. [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0147] In further embodiments, the XTEN sequences used in the present invention affect the physical or chemical properties (e.g., pharmacokinetics) of the chimeric proteins of the invention. The XTEN sequences used in the present invention may exhibit one or more of the following beneficial properties: conformational flexibility, enhanced aqueous solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius. In certain embodiments, the XTEN sequences linked to the FVIII proteins of the present invention increase pharmacokinetic properties, such as increased half-life or increased area under the curve (AUC), thereby causing the chimeric proteins described herein to persist in vivo for a longer period of time compared to wild-type FVIII. In further embodiments, the XTEN sequences used in the present invention increase pharmacokinetic properties, such as increased half-life or increased area under the curve (AUC), thereby causing the FVIII proteins to persist in vivo for a longer period of time compared to wild-type FVIII.

[0148] Various methods and assays can be used to measure the physical / chemical properties of the protein that contains XTEN sequence.Such methods include but are not limited to analytical centrifugation, EPR, HPLC ion exchange, HPLC size exclusion, HPLC reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, refractive index measurement and UV / visible light spectroscopy.Further methods are disclosed in Amauet et al., Prot Expr and Purif 48, 1-13 (2006).

[0149] Further examples of XTEN sequences that can be used in accordance with the present invention are described in U.S. Patent Application Publication Nos. 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1, or 2011 / 0172146 A1, or International Patent Application Publication Nos. These are disclosed in published patent applications WO2010091122 A1, WO2010144502 A2, WO2010144508 A1, WO2011028228 A1, WO2011028229 A1, and WO2011028344 A2.

[0150] C) Factor VIII (FVIII) protein As used herein, "FVIII protein" means a FVIII polypeptide that is functional in its normal role in the coagulation system, unless otherwise specified. The term FVIII protein includes functional fragments, variants, analogs, or derivatives thereof that retain the function of full-length wild-type factor VIII in the coagulation system. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include, but are not limited to, the ability to activate the coagulation system, act as a cofactor for factor IX, or bind to Ca. 2+ and the ability to form a tenase complex with factor IX in the presence of phospholipids, thereby converting factor X to the activated form, factor Xa. The FVIII protein may be human, porcine, canine, rat, or mouse FVIII protein. Furthermore, comparison of human FVIII with FVIII from other species has identified conserved residues that may be required for its function (Cameronet al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat. No. 6,251,632).

[0151] Many tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM assay, prothrombin time (PT) test (also used to measure INR), fibrinogen test (often by Clauss method), platelet count, platelet function test (often PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the original plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor X Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time. venom time, dRVVT), mixed platelet function test, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0152] 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 commonly used to measure the clotting activity of commercially available recombinant clotting factors (e.g., FVIII or FIX). It is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0153] ROTEM analysis provides information on global hemostatic dynamics: clotting time, clot formation, clot stability, and lysis. Different parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or the many factors that influence their interactions. This assay provides a global overview of secondary hemostasis.

[0154] The sequences of FVIII polypeptides and polynucleotides are known, and many functional fragments, mutants and variants exist. An example of a human FVIII sequence (full length) is shown below: [Table 3-1] [Table 3-2]

[0155] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0156] FVIII polypeptides include full-length FVIII, full-length FVIII minus the N-terminal Met, mature FVIII (minus the signal peptide), mature FVIII with an N-terminal Met, and / or FVIII with a B domain deleted in whole or in part. In certain embodiments, the FVIII variant includes a deleted (either partial or complete) B domain.

[0157] The sequence of native mature human FVIII is shown as SEQ ID NO: 4. The native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2 and A3 are structurally related "A domains," B is the "B domain," C1 and C2 are structurally related "C domains," and a1, a2 and a3 are acidic spacer regions. With respect to the positions of the primary amino acid sequence of SEQ ID NO: 4, the A1 domain of human FVIII extends from Ala1 to about Arg336, the a1 spacer region extends from about Met337 to about Val374, the A2 domain extends from about Ala375 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg1648, the a3 spacer region extends from about Glu1649 to about Arg1689, the A3 domain extends from about Ser1690 to about Leu2025, the C1 domain extends from about Gly2026 to about Asn2072, and the C2 domain extends from about Ser2073 to about Tyr2332. The designation of the locations of the boundaries between domains and regions of FVIII, other than the specific proteolytic cleavage sites, may vary from reference to reference. The boundaries set forth herein are therefore designated as approximations by use of the term "approximately."

[0158] The human FVIII gene has been isolated and expressed in mammalian cells (Toole, JJ, et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, WI, et al., Nature 312:330-337 (1984); Vehar, GA, et al., Nature312:337-342 (1984);WO87 / 04187;WO88 / 08035;WO88 / 0355 8; and U.S. Pat. No. 4,757,006). The FVIII amino acid sequence has been deduced from cDNA as shown in U.S. Pat. No. 4,965,199. Furthermore, partial or complete B domain-deleted FVIIIs 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 B domain of human factor V (U.S. Pat. No. 5,004,803). The cDNA and amino acid sequences encoding human factor VIII are shown in SEQ ID NOs: 4 and 5, respectively, of U.S. Patent Application Publication No. 2005 / 0100990.

[0159] The sequence of porcine FVIII has been published in Toole, JJ, et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). Additionally, FVIII from a porcine spleen cDNA library was also isolated. The complete porcine cDNA sequence obtained from PCR amplification of the II sequence is reported in Healey, JF, et al., Blood 88:4209-4214 (1996). Hybrid human / porcine FVIIIs with specific amino acid sequence substitutions are disclosed in U.S. Pat. No. 5,364,771 and WO 93 / 2009 by Lollar and Runge. 3. More recently, nucleotide sequences corresponding to the amino acid sequences of the A1 and A2 domains of porcine FVIII, and chimeric FVIIIs in which the porcine A1 and / or A2 domains have been replaced with the corresponding human domains, have been reported in WO 94 / 11503. U.S. Patent No. 5,859,204 (Lollar, JS) also discloses the porcine cDNA and deduced amino acid sequence. US Patent No. 6,458,563 discloses a B domain deleted porcine FVIII.

[0160] US Patent No. 5,859,204 (Lollar, JS) describes a method for producing a compound with reduced antigenicity and immunoreactivity. US Patent No. 6,376,463 (Lollar, JS) also reports FVIII mutants with reduced immunoreactivity. Patent application publication no. 2005 / 0100990 (Saenko et al.) describes the A2 domain of FVIII. We report functional mutations in the main.

[0161] In one embodiment, FVIII (or the FVIII portion of the chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6, or amino acids 1 to 2332 of SEQ ID NO: 4 (without the signal sequence), or to the FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 3 and amino acids 1 to 1438 of SEQ ID NO: 6, or to the FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 3 and amino acids 1 to 2332 of SEQ ID NO: 4 (with the signal sequence), wherein FVIII has coagulation activity (e.g., activating factor IX as a cofactor and converting factor X to activated factor X). FVIII (or the FVIII portion of the chimeric protein) may be identical to amino acids 1 to 1438 of SEQ ID NO: 6 or the FVIII amino acid sequence (without the signal sequence) of amino acids 1 to 2332 of SEQ ID NO: 4. FVIII may further contain a signal sequence.

[0162] As used herein, the "B domain" of FVIII is identical to the B domain known in the art, defined by the identity of the internal amino acid sequence and the site of proteolytic cleavage (e.g., residues Ser741-Arg1648 of full-length human FVIII). Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence contains residues Ser1690-Tyr2332. The remaining sequence, i.e., residues Glu1649-Arg1689, is commonly referred to as the a3 acidic region. The location of boundaries for all domains, including the B domain, for porcine, mouse, and canine FVIII are known in the art. In one embodiment, the B domain of FVIII is deleted (B domain-deleted factor VIII, or BDD FVIII). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequence as the VIII portion of the sequence in Table 5 (the heavy chain of BDD FVIII is double underlined, the B domain is italicized, and the BDD FVIII light chain is in standard text). The nucleotide sequence encoding Table 5 (SEQ ID NO: 7) is shown in Table 6. [Table 5-1] [Table 5-2]

[0163] [Table 6-1] [Table 6-2] [Table 6-3]

[0164] "B domain deleted FVIII" may have a complete or partial deletion as disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In some embodiments, the B-domain deleted FVIII sequences of the invention contain any one of the deletions disclosed in U.S. Pat. No. 6,316,226 (or U.S. Pat. No. 6,346,513) at column 4, lines 4-5, line 28 and in Examples 1-5. In other embodiments, the B-domain deleted factor VIII is S743 / Q1638 B-domain deleted factor VIII (SQ BDD FVIII) (e.g., factor VIII having a deletion of amino acids 744-1637 of SEQ ID NO: 4, e.g., factor VIII having amino acids 1-743 and amino acids 1638-2332, i.e., SEQ ID NO: 6). In some embodiments, the B domain-deleted FVIII of the present invention has a deletion as disclosed in U.S. Pat. No. 5,789,203 (or U.S. Pat. Nos. 6,060,447, 5,595,886, and 6,228,620), paragraph 2, lines 26-51, and Examples 5-8. In some embodiments, the B domain-deleted FVIII has a deletion as disclosed in U.S. Pat. No. 5,972,885, paragraph 1, lines 25-2, line 40; or U.S. Pat. No. 6,048,720, paragraph 6, lines 1-22, and Example 1. Defects: the defect disclosed in U.S. Pat. No. 5,543,502, paragraph 2, lines 17-46; the defect disclosed in U.S. Pat. No. 5,171,844, paragraph 4, lines 22 to 5, line 36; the defect disclosed in U.S. Pat. No. 5,112,950, paragraph 2, lines 55-68, Figure 2, and Example 1; and the defect disclosed in U.S. Pat. No. 4,868,112, paragraph 2, lines 2 to 19, line 21. and Table 2; the deletions disclosed in U.S. Patent No. 7,041,635, paragraph 2, lines 1-3, line 19, paragraph 3, lines 40-4, line 67, paragraph 7, lines 43-8, line 26, and paragraph 11, lines 5-13, line 39; or the deletions disclosed in U.S. Patent No. 6,458,563, paragraph 4, lines 25-53. In some embodiments, the B domain-deleted FVIII lacks most of the B domain but contains the amino-terminal sequence of the B domain, which is essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains, as disclosed in WO 91 / 09122. In some embodiments, the B domain-deleted FVIII is constructed with a deletion of amino acids 747-1638 (i.e., a substantially complete deletion of the B domain). Hoeben R.C., et al. J. Biol. Chem. 265(13): 7318-7323(1990). B domain-deleted factor VIII also has the amino acid sequence of FVIII. The B domain may contain deletions of amino acids 771-1666 or 868-1562. Meulien P., et al. Protein Eng. 2(4):301-6(1988). Additional B domains that are part of the present invention may also contain deletions of amino acids 771-1666 or 868-1562. Deletions of the amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5939-5942) Deletion of 797-1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), deletion of 741-1646 (Kaufman (PCT International Patent Application Publication WO 87 / 04187)), deletion of 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), deletion of 741-1648 (Pasek (PCT International Patent Application 88 / 00831)), or deletion of 816-1598, or 7 Deletion of amino acids 41-1648 (Lagner (Behring Inst. Mitt. (1988), EP 295597)). In other embodiments, BDD FVIII includes FVIII polypeptides containing fragments of the B domain that retain 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 the 226 amino acids or 1 amino acid fragment of the B domain disclosed in Miao, HZ, et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, SW, et al., J. Thromb. Haemost. 9: 2235-2242 (2011). In yet another embodiment, the BDD FVIII further contains 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). ...

[0013] The deletions described above may be used in any FVIII polypeptide, including FVIII polypeptides that contain a portion of the furin cleavage site but do not contain one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, SW, et al., J. Thromb. Haemost. 9: 2235-2242 (2011). Each of the foregoing deletions may be used in any FVIII polypeptide. It can be made in sequence III.

[0165] In some embodiments, the FVIII has a partial B domain. In some embodiments, the FVIII with a partial B domain is FVIII198 (SEQ ID NO: 89). FVIII198 is a partial B domain containing 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 of the B domain.

[0166] In one embodiment, FVIII is provided with an arginine residue immediately following amino acid 1648 (of full-length factor VIII, or SEQ ID NO: 4), immediately following amino acid 754 (of S743 / Q1638 B-domain deleted factor VIII, or SEQ ID NO: 6), or It is cleaved immediately after the corresponding arginine residue (in other variants), thereby yielding the heavy and light chains. In other embodiments, the FVIII contains heavy and light chains that are linked or associated by a metal ion-mediated non-covalent bond.

[0167] In other embodiments, the FVIII is a single-chain FVIII that is not cleaved immediately after the arginine at amino acid 1648 (of full-length FVIII or SEQ ID NO: 4), immediately after the arginine at amino acid 754 (of S743 / Q1638 B-domain deleted FVIII or SEQ ID NO: 6), or immediately after the corresponding arginine residue (of other variants). The single-chain FVIII may contain one or more amino acid substitutions. In one embodiment, the amino acid substitutions are at residues corresponding to residues 1648, 1645, or both, of the full-length mature factor VIII polypeptide (SEQ ID NO: 4), or to residues 754, 751, or both, of the SQ BDD factor VIII (SEQ ID NO: 6). The amino acid substitution may be any amino acid other than arginine (e.g., 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).

[0168] FVIII can also be cleaved by thrombin and subsequently activated as FVIIIa, which serves as a cofactor for activated factor IX (FIXa). Activated FVIII and activated FIX together form an Xase complex, converting factor X to activated factor X (FXa). For activation, FVIII is cleaved by thrombin after three arginine residues (amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain-deleted FVIII sequence)), which produces FVIIIa with a 50 kDa A1, a 43 kDa A2, and a 73 kDa A3-C1-C2 chain. In one embodiment, the FVIII protein useful in the present invention is inactive FVIII. In another embodiment, the FVIII protein is activated FVIII.

[0169] A protein having a FVIII polypeptide linked to or related to a VWF fragment may contain a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 6, wherein the sequence has FVIII clotting activity (e.g., activating factor IX as a cofactor and converting factor X to activated factor X (FXa)).

[0170] "Hybrid" or "chimeric" polypeptides and proteins, as used herein, include combinations of a first polypeptide chain (e.g., a VWF fragment optionally fused to a first Ig constant region or portion thereof) and a second polypeptide chain (e.g., FVIII linked to an XTEN sequence optionally fused to a second Ig constant region or portion thereof, thereby forming a heterodimer). In one embodiment, the first and second polypeptides in the hybrid are associated with each other through protein-protein interactions (e.g., charge-charge or hydrophobic interactions, etc.). In other embodiments, the first and second polypeptides in the hybrid are associated with each other through a disulfide bond or other covalent bond(s). Hybrids are described, for example, in U.S. Patent Application Nos. 2004 / 101740 and 2006 / 074199. The second polypeptide may be an identical copy of the first polypeptide or a non-identical polypeptide. In one embodiment, the first polypeptide is a FVIII protein(X)-Fc fusion protein, and the second polypeptide is a polypeptide containing, consisting essentially of, or consisting of an Fc region, wherein the first polypeptide and the second polypeptide are mutually complementary. In other embodiments, the first polypeptide contains a VWF fragment-XTEN-Fc fusion protein and the second polypeptide contains a FVIII-Fc fusion protein, thereby forming a heterodimer. In other embodiments, the first polypeptide contains a VWF fragment-Fc fusion protein and the second polypeptide contains a FVIII(X)-Fc fusion protein, thereby forming a heterodimer. In yet other embodiments, the first polypeptide contains a VWF fragment-XTEN-Fc fusion protein and the second polypeptide contains a FVIII(X)-Fc fusion protein. The first and second polypeptides may be associated with each other through a covalent bond (e.g., a disulfide bond) between the first and second Fc regions. The first and second polypeptides may further be associated with each other by a bond between the VWF fragment and the FVIII protein.

[0171] The FVIII protein useful in the present invention may contain FVIII with one or more additional XTEN sequences that do not affect the coagulation activity of FVIII. Such XTEN sequences may be fused to the C-terminus or N-terminus of the FVIII protein, or may be inserted between one or more of two amino acid residues of the FVIII protein without affecting the coagulation activity or function of FVIII. In one embodiment, the insertion improves the pharmacokinetic properties (e.g., half-life) of the FVIII protein. In other embodiments, the insertion may be multiple insertions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more insertions). Examples of insertion sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15, or any combination thereof.

[0172] The FVIII protein linked to one or more XTEN sequences may be designated FVIII(X), FVIII(X1), FVIII (a→b) -X-FVIII (c→d) where FVIII(a→b) comprises, consists essentially of, or consists of a first portion of a FVIII protein from amino acid residue "a" to amino acid residue "b"; X or X1 comprises, consists essentially of, or consists of one or more XTEN sequences; (c→d) comprises, consists essentially of, or consists of a second portion of the FVIII protein from amino acid residue "c" to amino acid residue "d"; a is the N-terminal amino acid residue of the first portion of the FVIII protein, b is the C-terminal amino acid residue of the first portion of the FVIII protein and is also the amino acid residue two amino acids N-terminal to the insertion site where the XTEN sequence is inserted; c is the N-terminal amino acid residue of the second portion of the FVIII protein and is also the C-terminal amino acid residue two amino acids from the insertion site into which the XTEN sequence is inserted; d is the C-terminal amino acid residue of the FVIII protein, wherein the first portion of the FVIII protein and the second portion of the FVIII protein are not identical to each other and together are of sufficient length that the FVIII protein has FVIII clotting activity.

[0173] In one embodiment, the first portion of the FVIII protein and the second portion of the FVIII protein are fragments of SEQ ID NO: 4 (full-length mature FVIII sequence) or SEQ ID NO: 6 (B-domain deleted FVIII) (e.g., the N-terminal portion and the C-terminal portion, respectively). In one embodiment, the first portion of the FVIII protein contains the A1 and A2 domains of the FVIII protein. The second portion of the FVIII protein contains the A3 domain, the C1 domain, and optionally the C2 domain. In yet another embodiment, the first portion of the FVIII protein contains the A1 and A2 domains, and the second portion of the FVIII protein contains the B domain, the A3 domain, the C1 domain, and optionally the C2 domain. In yet another embodiment, the first portion of the FVIII protein is a fragment of the FVIII protein. and the second portion of the FVIII protein contains the A1 domain, the A2 domain, and a portion of the B domain of the FVIII protein, and the second portion of the FVIII protein contains the A3 domain, the C1 domain, and optionally the C2 domain. In yet other embodiments, the first portion of the FVIII protein contains the A1 domain, the A2 domain, and a first portion of the B domain of the FVIII protein. The second portion of the FVIII protein contains a second portion of the B domain, the A3 domain, the C1 domain, and optionally the C2 domain. In some embodiments, the two amino acids ("b" and "c") may be any one or more of the amino acid residue insertion sites shown in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15. For example, "b" may be the amino acid residue immediately upstream of the site where one or more XTEN sequences are inserted or linked, and "c" may be the amino acid residue immediately downstream of the site where one or more XTEN sequences are inserted or linked. In some embodiments, "a" is the first mature amino acid sequence of the FVIII protein, and "d" is the last mature amino acid sequence of the FVIII protein. (a→b) may be an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 745 of SEQ ID NO: 6 (the B-domain deleted FVIII amino acid sequence) or SEQ ID NO: 4 (full length FVIII), and (c→d) may be amino acids 746 to 1438 of SEQ ID NO: 6 or amino acids 1641 to 2332 of SEQ ID NO: 4, respectively.

[0174] In some embodiments, the insertion site of the FVIII protein may be located within one or more domains of the FVIII protein, such as the A1 domain, the A2 domain, the A3 domain, the B domain, the C1 domain, the C2 domain, the C-terminus, or any combination thereof, or may be located between two domains of the FVIII protein, such as the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, or any combination thereof. For example, insertion sites into which the XTEN sequence may be inserted are selected from the group consisting of the N-terminus and the A1 domain, the N-terminus and the A2 domain, the N-terminus and the A3 domain, the N-terminus and the B domain, the N-terminus and the C1 domain, the N-terminus and the C2 domain, the N-terminus and the C-terminus, the A1 and A2 domains, the A1 and the A3 domains, the A1 and the B domains, the A1 and the C1 domains, the A1 and the C2 domains, the A1 domain and the C-terminus, the A2 and the A3 domains, the A2 and the B domains, the A2 and the C1 domains, the A2 and the C2 domains, the A2 domain and the C-terminus, the A3 and the B domains, the A3 and the C1 domains, the A3 and the C2 domains, the A3 domain and the C-terminus, the B and the C1 domains, the B and the C2 domains, the B domain and the C-terminus, the C1 and the C2 domains, the C1 domain and the C-terminus, the C2 domain and the C-terminus, and combinations of two or more thereof. Non-limiting examples of insertion sites are shown in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0175] FVIII proteins into which an XTEN sequence has been inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., at one or more XTEN insertion sites), or into which an XTEN sequence has been linked at the C-terminus or N-terminus, retain FVIII activity after linkage to or insertion of the XTEN sequence. XTEN sequences may be inserted into the FVIII protein once, or two or more, three or more, four or more, five or more, six or more, or seven or more times, such that the insertion does not affect FVIII activity (i.e., the FVIII protein still maintains its clotting properties).

[0176] FVIII proteins useful in the present invention may have one or more XTEN polypeptides linked to the N-terminus or C-terminus of the FVIII protein by an optional linker, or may be inserted into the FVIII protein at one or more amino acids immediately downstream (e.g., at one or more XTEN insertion sites) by one or more optional linkers. In this embodiment, the two amino acid residues between which the XTEN is inserted or the amino acid residues between which the XTEN sequence is linked correspond to two or more amino acid residues of SEQ ID NO: 4 (full-length mature FVIII) selected from the group consisting of residues in Table 7, Table 8, Table 9, and Table 10, and any combination thereof.

[0177] In other embodiments, at least one XTEN sequence is inserted into any one or more of the XTEN insertion sites disclosed herein, or any combination thereof. In one embodiment, at least one XTEN sequence is inserted into one or more of the XTEN insertion sites disclosed in one or more amino acids disclosed in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

[0178] In some embodiments, one or more XTEN sequences are inserted within approximately 6 amino acids upstream or downstream from amino acid 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905, or 1910 (corresponding to SEQ ID NO: 4), or any combination thereof. [Table 8]

[0179] In other embodiments, one or more XTEN sequences are inserted immediately downstream of one or more amino acids corresponding to full-length mature human FVIII selected from the group consisting of one or more insertion sites in Table 9. [Table 9]

[0180] In yet other embodiments, one or more XTENs are inserted into the B domain of FVIII. In one example, an XTEN is inserted between amino acids 740 and 1640 of SEQ ID NO: 4, where the FVIII sequence between amino acids 740 and 1640 is optionally absent. In another example, an XTEN is inserted between amino acids 741 and 1690 of SEQ ID NO: 4. wherein the FVIII sequence between amino acids 740 and 1690 is In another example, XTEN is inserted between amino acids 741 and 1648 of SEQ ID NO:4, wherein the FVIII sequence between amino acids 741 and 1648 is optionally absent. In yet another example, XTEN is inserted between amino acids 743 and 1638 of SEQ ID NO:4, wherein the FVIII sequence between amino acids 743 and 1638 is optionally absent. In yet another example, XTEN is inserted between amino acids 745 and 1656 of SEQ ID NO:4, wherein the FVIII sequence between amino acids 745 and 1656 is optionally absent. In yet another example, XTEN is inserted between amino acids 745 and 1657 of SEQ ID NO:4, wherein the FVIII sequence between amino acids 745 and 1657 is optionally absent. In yet other examples, XTEN is inserted between amino acids 745 and 1667 of SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1667 is optionally absent. In yet other examples, XTEN is inserted between amino acids 745 and 1686 of SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1686 is optionally absent. In some other examples, XTEN is inserted between amino acids 747 and 1642 of SEQ ID NO: 4, wherein the FVIII sequence between amino acids 747 and 1642 is optionally absent. In yet other examples, XTEN is inserted between amino acids 751 and 1667 of SEQ ID NO: 4, wherein the FVIII sequence between amino acids 751 and 1667 is optionally absent.

[0181] In some embodiments, one or more XTENs are inserted at one or more amino acids immediately downstream of the insertion site amino acid selected from the group consisting of the amino acid residues in Table 10. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0182] In one embodiment, one or more XTEN insertion sites are located within one or more surface-exposed, flexible loop structures (e.g., permissive loops) of the FVIII protein. For example, at least one XTEN sequence is inserted within each FVIII "A" domain, which contains at least two "permissive loops," into which at least one XTEN polypeptide can be inserted without impairing the procoagulant activity of the recombinant protein or the activity of the recombinant protein expressed in vivo or in vitro in host cells. A permissive loop is a region into which at least one XTEN sequence can be inserted and which allows, among other properties, high surface or solvent exposure and high conformational flexibility. The A1 domain contains permissive loop 1 (A1-1) and permissive loop 2 (A1-2) regions, the A2 domain contains permissive loop 1 (A2-1) and permissive loop 2 (A2-2) regions, and the A3 domain contains permissive loop 1 (A3-1) and permissive loop 2 (A3-2) regions.

[0183] In one embodiment, the first permissive loop (A1-1) of the FVIII A1 domain is positioned between β-strand 1 and β-strand 2, and the second permissive loop (A1-2) of the FVIII A2 domain is positioned between β-strand 11 and β-strand 12. The first permissive loop (A2-1) of the FVIII A2 domain is positioned between β-strand 22 and β-strand 23, and the second permissive loop (A2-2) of the FVIII A2 domain is positioned between β-strand 32 and β-strand 33. The first permissive loop (A3-1) of the FVIII A3 domain is positioned between β-strand 38 and β-strand 39, and the second permissive loop (A3-2) of the FVIII A3 domain is positioned between β-strand 45 and β-strand 46. In one embodiment, the surface-exposed, flexible loop structure containing A1-1 corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of SEQ ID NO: 4 (e.g., from about amino acid 18 to about amino acid 41 of SEQ ID NO: 4). In another embodiment, the surface-exposed, flexible loop structure containing A1-2 corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO: 4. In yet another embodiment, the surface-exposed, flexible loop structure containing A2-1 corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 of SEQ ID NO:4 (e.g., from about amino acid 397 to about amino acid 418 of SEQ ID NO:4). In yet another embodiment, the surface-exposed, flexible loop structure containing A2-2 corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 of SEQ ID NO:4 (e.g., from about amino acid 595 to about amino acid 607 of SEQ ID NO:4). In one embodiment, the surface-exposed, flexible loop structure containing A3-1 corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 of SEQ ID NO:4 (e.g., from about amino acid 1711 to about amino acid 1725 of SEQ ID NO:4). In yet another embodiment, the surface-exposed, flexible loop structure containing A3-2 corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 of SEQ ID NO: 4 (e.g., from about amino acid 1899 to about amino acid 1911 of SEQ ID NO: 4).

[0184] In other embodiments, one or more amino acids into which at least one XTEN sequence is inserted are located within the a3 domain (e.g., amino acids 1649-1689 corresponding to the full-length mature FVIII polypeptide). In certain embodiments, the XTEN sequence is inserted between amino acids 1656-1657 of SEQ ID NO:4 (full-length mature FVIII). In certain embodiments, the FVIII containing an XTEN sequence inserted immediately downstream of amino acid 1656 of SEQ ID NO:4 further contains a deletion of amino acids 745-1656 of SEQ ID NO:4.

[0185] In some embodiments, the one or more insertion sites for the one or more XTEN insertions are: (1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) Amino acid 333, (20) Amino acid 336, (21) Amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) Amino acid 487, (29) Amino acid 490, (30) Amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) amino acid 1937, (52) Amino acid 2019, (53) Amino acid 2068, (54) Amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188, (58) amino acid 2227, (59) amino acid 2277, and (60) A combination of two or more of these; and immediately downstream of one or more amino acids selected from the group consisting of:

[0186] In one embodiment, a FVIII protein useful in the present invention comprises two XTEN sequences (a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN insertion site, and a Non-limiting examples of first and second XTEN insertion sites are listed in Table 11. [Table 11-1] [Table 11-2] [Table 11-3]

[0187] The two XTENs inserted into or linked to a FVIII protein can be the same or different. In some embodiments, a FVIII protein useful in the present invention contains two XTEN sequences inserted into the FVIII protein: a first XTEN sequence inserted immediately downstream of amino acid 745 of SEQ ID NO:4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 (C-terminus) of SEQ ID NO:4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 1656, or 1720 of SEQ ID NO:4, and the second XTEN sequence is inserted immediately downstream of amino acid 403 of SEQ ID NO:4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 of SEQ ID NO:4, and the second XTEN sequence is inserted immediately downstream of amino acid 599 of SEQ ID NO:4. In still other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a second XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1725, 1720, 1900, 1905, or 2332 of SEQ ID NO:4. In certain embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4, and a second XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 of SEQ ID NO:4. In some embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 of SEQ ID NO:4, and a second XTEN sequence is inserted immediately downstream of amino acid 399 of SEQ ID NO:4. In other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4, and a second XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 of SEQ ID NO:4. In yet other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4, and a second XTEN sequence is inserted immediately downstream of amino acid 18 of SEQ ID NO:4.In certain embodiments, the FVIII protein contains two XTEN sequences (a first XTEN inserted just downstream of amino acid 745 of SEQ ID NO:4 and a second XTEN inserted just downstream of amino acid 2332 of SEQ ID NO:4), wherein the FVIII protein further has a deletion of amino acid 745 of SEQ ID NO:4 through amino acid 1685 of SEQ ID NO:4, a mutation or substitution at amino acid 1680 of SEQ ID NO:4 (e.g., Y1680F), a mutation or substitution at amino acid 1648 of SEQ ID NO:4 (R1648A), or at least two mutations or substitutions at amino acid 1648 of SEQ ID NO:4 and amino acid 1680 of SEQ ID NO:4 (e.g., R1648A, Y1680F). In certain embodiments, the FVIII protein contains two XTEN sequences (a first XTEN inserted just downstream of amino acid 1656 of SEQ ID NO:4 and a second XTEN inserted just downstream of amino acid 2332 of SEQ ID NO:4), wherein the FVIII protein further has a deletion of amino acids 745 to 1656 of SEQ ID NO:4.

[0188] In certain embodiments, the FVIII protein contains three XTEN sequences (a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN insertion site, and a third XTEN sequence inserted into a third XTEN insertion site). The first, second, or third XTEN sequence may be the same or different. The first, second, and third insertion sites may be selected from any one of the groups of insertion sites disclosed herein. In some embodiments, a FVIII protein containing three XTEN sequences may further contain a mutation or substitution (e.g., at amino acid 1648 of SEQ ID NO: 4, e.g., R1648A). For example, non-limiting examples of first, second, and third XTEN insertion sites are listed in Table 12. [Table 12-1] [Table 12-2]

[0189] In some embodiments, the FVIII protein contains three XTEN sequences (a first XTEN sequence inserted immediately downstream of amino acid 26 of SEQ ID NO:4, a second XTEN sequence inserted immediately downstream of amino acid 403 of SEQ ID NO:4, and a third XTEN sequence inserted immediately downstream of amino acid 1656, 1720, or 1900 of SEQ ID NO:4). In other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 26 of SEQ ID NO:4, a second XTEN sequence is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a third XTEN sequence is inserted immediately downstream of amino acid 1720 or 1900 of SEQ ID NO:4. In yet other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 26 of SEQ ID NO:4, a second XTEN sequence is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4, and a third XTEN sequence is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In yet other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 403 of SEQ ID NO:4, a second XTEN sequence is inserted immediately downstream of amino acid 1656 of SEQ ID NO:4, and a third XTEN sequence is inserted immediately downstream of amino acid 1720 or 1900 of SEQ ID NO:4. In other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 of SEQ ID NO:4, a second XTEN sequence is inserted immediately downstream of amino acid 1720 of SEQ ID NO:4, and a third XTEN sequence is inserted immediately downstream of amino acid 1900 of SEQ ID NO:4. In other embodiments, a first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or 1910 of SEQ ID NO:4, a second XTEN sequence is inserted immediately downstream of amino acid 745 of SEQ ID NO:4, and a third XTEN sequence is inserted immediately downstream of amino acid 1720 or 2332 of SEQ ID NO:4.

[0190] In other embodiments, the FVIII protein of the invention contains four XTEN sequences (a first XTEN sequence is inserted into the first insertion site, a second XTEN sequence is inserted into the second insertion site, a third XTEN sequence is inserted into the third insertion site, and a fourth XTEN sequence is inserted into the fourth insertion site). The first, second, third, and fourth XTEN sequences can be the same, different, or a combination thereof. In some embodiments, a FVIII protein containing four XTEN sequences can further contain a mutation or substitution (e.g., amino acid 1648 (e.g., R1648A) of SEQ ID NO: 4). Non-limiting examples of first, second, third, and fourth XTEN insertion sites are listed in Table 13. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4]

[0191] In some embodiments, the FVIII protein contains five XTEN sequences (a first XTEN sequence is inserted into the first insertion site, a second XTEN sequence is inserted into the second insertion site, a third XTEN sequence is inserted into the third insertion site, a fourth XTEN sequence is inserted into the fourth insertion site, and a fifth XTEN sequence is inserted into the fifth insertion site). The first, second, third, fourth, and fifth XTEN sequences can be the same, different, or a combination thereof. In some embodiments, non-limiting examples of the first, second, third, fourth, and fifth XTEN insertion sites are listed in Table 14. [Table 14]

[0192] In certain embodiments, the FVIII protein contains six XTEN sequences (a first XTEN sequence is inserted into the first insertion site, a second XTEN sequence is inserted into the second insertion site, a third XTEN sequence is inserted into the third insertion site, a fourth XTEN sequence is inserted into the fourth insertion site, a fifth XTEN sequence is inserted into the fifth insertion site, and a sixth XTEN sequence is inserted into the sixth insertion site). The first, second, third, fourth, fifth, and sixth XTEN sequences can be the same, different, or a combination thereof. In some embodiments, examples of six XTEN insertion sites include, but are not limited to, the insertion sites listed in Table 15. [Table 15]

[0193] In particular examples, a first XTEN is inserted between amino acids 26 and 27 of SEQ ID NO:4, and a second XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4 (full-length mature FVIII). In other examples, a first XTEN is inserted between amino acids 403 and 404 of SEQ ID NO:4, and a second XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4. In some examples, a first XTEN is inserted between amino acids 1656 and 1657 of SEQ ID NO:4, and a second XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4. In other examples, a first XTEN is inserted between amino acids 26 and 27 of SEQ ID NO:4, and a second XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4. In yet another embodiment, a first XTEN is inserted between amino acids 1656 and 1657 of SEQ ID NO:4, and a third XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4. In yet another embodiment, a first XTEN is inserted between amino acids 403 and 404 of SEQ ID NO:4, a second XTEN is inserted between amino acids 1656 and 1657 of SEQ ID NO:4, and a third XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4. In yet another embodiment, a first XTEN is inserted between amino acids 403 and 404 of SEQ ID NO:4, a second XTEN is inserted between amino acids 1656 and 1657 of SEQ ID NO:4, and a third XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4. In certain embodiments, a first XTEN is inserted between amino acids 26 and 27 of SEQ ID NO:4, a second XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4, and a third XTEN is inserted between amino acids 1900 and 1901 of SEQ ID NO:4. In some embodiments, a first XTEN is inserted between amino acids 26 and 27 of SEQ ID NO:4, a second XTEN is inserted between amino acids 1656 and 1657 of SEQ ID NO:4, a third XTEN is inserted between amino acids 1720 and 1721 of SEQ ID NO:4, and a fourth XTEN is inserted between amino acids 1900 and 1901 of SEQ ID NO:4.

[0194] In certain embodiments, the XTEN sequence is inserted between amino acids 745 and 746 of full-length factor VIII or at the corresponding insertion site in B-domain deleted factor VIII.

[0195] In some embodiments, the chimeric protein of the present invention contains two polypeptide sequences, and the first polypeptide sequence is selected from the group consisting of FVIII-161 (SEQ ID NO: 101), FVIII-169 (SEQ ID NO: 103), FVIII-170 (SEQ ID NO: 102), FVIII-173 (SEQ ID NO: 104); FVIII-195 (SEQ ID NO: 105); FVIII-196 (SEQ ID NO: 106), FVIII199 (SEQ ID NO: 107), FVIII-201 (SEQ ID NO: 108); FVIII-203 (SEQ ID NO: 109), FVIII-204 (SEQ ID NO: 110), FVIII-205 (SEQ ID NO: 111), FVIII-266 (SEQ ID NO: 112), FVIII-267 (SEQ ID NO: 113), FVIII-268 (SEQ ID NO: 114), FVIII-269 (SEQ ID NO: 115), FVIII-271 (SEQ ID NO: 116), or FVIII and the second polypeptide sequence contains an amino acid sequence that is at least about 80%, 90%, 95%, or 100% identical to a sequence selected from VWF031 (SEQ ID NO: 118), VWF034 (SEQ ID NO: 119), or VWF-036 (SEQ ID NO: 120).

[0196] D) Ig constant region or part thereof The VWF fragment or FVIII protein linked to the XTEN sequence of the present invention may further contain an Ig constant region or a portion thereof. The Ig constant region or a portion thereof may improve the pharmacokinetic or pharmacodynamic properties of the VWF fragment or FVIII protein combined with the XTEN sequence. In certain embodiments, the Ig constant region or a portion thereof extends the half-life of the molecule fused to the Ig constant region or a portion thereof.

[0197] The Ig constant region is composed of domains designated CH (constant heavy chain) 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. The constant region of some isotypes (e.g., IgG) also contains a hinge region. See Janeway et al. 2001, Immunobiology, Garl and Publishing, NY, NY.

[0198] The Ig constant region or a portion thereof for the production of the chimeric proteins of the present invention can be prepared from many different Ig constant regions. The Ig constant region or a portion thereof may be obtained from any source. In some embodiments, the Ig constant region or a portion thereof is derived from human Ig. However, it should be understood that the Ig constant region or a portion thereof may also be derived from Ig of other mammalian species, including, for example, rodents (e.g., mouse, rat, rabbit, guinea pig, etc.), or non-human primate species (e.g., chimpanzee, macaque). Furthermore, the Ig constant region or a portion thereof may be derived from any Ig class, including IgG, IgM, IgA, IgD, and IgE, and any Ig isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgG1 is used.

[0199] Various Ig constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly available deposits. Constant region domain sequences with specific effector functions (or lacking specific effector functions) or with specific modifications to reduce immunogenicity can be selected. Many antibody sequences and sequences of antibody-encoding genes have been published, and appropriate Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be derived from these sequences using techniques known in the art. The resulting genetic material may then be modified or synthesized using any of the aforementioned methods to obtain the polypeptides of the present invention. Furthermore, it should be recognized that the scope of the present invention encompasses alleles, variants, and mutations of constant region DNA sequences.

[0200] The sequence of an Ig constant region or a portion thereof may be cloned, for example, using the polymerase chain reaction and primers selected to amplify the domain of interest. To clone the sequence of an Ig constant region or a portion thereof from an antibody, mRNA may be isolated from hybridoma, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody gene may be amplified by PCR. PCR amplification methods are described in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188, and, for example, "PCR Protocols: A Guide to Methods and Applications" by Innis et al., eds., Academic Press, San Diego, CA (1990); Ho et al., 1989. Gene 77:51; Horton et al., 1993. The consensus constant region primers are described in detail in Methods Enzymol. 217:270. PCR may be initiated by using the nucleotide sequences of the heavy and light chains or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As described above, PCR may be used to isolate DNA clones encoding the heavy and light chains of an antibody. In this case, the library may be screened with consensus primers or larger homologous probes (e.g., mouse constant region probes). Many primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509)); Rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larricket et al. 1989 Biochem. Biophys. Res. Commun. 160:1250), etc. Cloning of antibody sequences is further detailed in US Patent No. 5,658,570 to Newman et al., filed January 25, 1995, which is incorporated herein by reference.

[0201] As used herein, an Ig constant region can include all domains and hinge regions, or portions thereof. In one embodiment, an Ig constant region or portion thereof contains the CH2 domain, CH3 domain, and hinge region (i.e., the Fc domain or FcRn binding partner).

[0202] As used herein, the term "Fc region" is defined as the polypeptide portion corresponding to the Fc region of a native immunoglobulin, i.e., the portion formed by the dimeric association of the Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, a "genetically fused Fc region" or a "single-chain Fc region" The term (scFc region) as used herein refers to a synthetic dimeric Fc region composed of genetically linked Fc domains in a single polypeptide chain (i.e., encoded by a single contiguous gene sequence).

[0203] In one embodiment, "Fc region" refers to the portion of a single Ig heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of the heavy chain constant region, which is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain contains at least the hinge, CH2, and CH3 domains.

[0204] The Fc region of an Ig constant region may contain CH2, CH3, and CH4 domains, as well as a hinge region, depending on the Ig isotype. The inclusion of the Fc region of an Ig in a chimeric protein can provide increased stability, increased serum half-life (see Caponet et al., 1989, Nature 337:525), and enhanced Fc receptor activity, such as the fetal Fc receptor (FcRn). Several desirable properties are conferred to the chimeric proteins, including increased binding to receptors (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1, which are incorporated by reference in their entireties).

[0205] The Ig constant region or a portion thereof may be an FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the intestinal lumen, respiratory tract, nasal cavity surface, vaginal surface, colonic surface, and rectal surface (U.S. Patent No. 6,485,726). An FcRn binding partner is a portion of an Ig that binds to FcRn.

[0206] 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). FcRn receptors bind to IgG at relatively low pH. It binds to IgA (but not to other Ig classes such as IgA, IgM, IgD, and IgE), actively transports IgG transcellularly within the lumen toward the serosal membrane, and then releases IgG at the relatively high pH of intestinal fluid. It is expressed in adult epithelial tissues, including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), proximal tubular epithelial cells (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. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1).

[0207] 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 has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The main contact region of the Fc with FcRn is 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, the Fc fragment of IgG, and other fragments of IgG that contain the complete FcRn-binding region. Major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All amino acid numbering standards for Ig or Ig fragments are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, US Department of Public Health, Bethesda, MD.

[0208] Fc regions bound to FcRn, or FcRn binding partners, are expressed by FcRn. These fusion proteins can efficiently cross epithelial barriers, thereby providing a non-invasive means for systemic administration of desired therapeutic molecules. Furthermore, fusion proteins containing Fc regions or FcRn binding partners are endocytosed by cells expressing FcRn. However, instead of being degraded, these fusion proteins are recycled back into the circulatory system, thereby extending the in vivo half-life of these proteins. In certain embodiments, portions of Ig constant regions are Fc regions or FcRn binding partners, which often associate with other Fc regions or other FcRn binding partners through disulfide bonds and other non-specific interactions to form dimers and higher-order multimers.

[0209] Two FcRn receptors can bind to one Fc molecule. Crystal structure data suggest that each FcRn molecule binds to one polypeptide of an Fc homodimer. In one embodiment, linking an FcRn binding partner (e.g., an Fc fragment of IgG) to a biologically active molecule provides a means for delivering the biologically active molecule via aerosol administration via oral, buccal, sublingual, rectal, vaginal, nasal, or pulmonary routes, or via the intraocular route. In other embodiments, the chimeric protein may be administered invasively (e.g., subcutaneously, intravenously).

[0210] An FcRn binding partner region is a molecule or a portion thereof that can be specifically bound by the FcRn receptor and, as a result, actively transported by the Fc receptor in the Fc region. Specific binding refers to two molecules forming a relatively stable complex under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, which clearly distinguishes it from nonspecific binding (which usually has low affinity and moderate to high capacity). Typically, the affinity constant KA is greater than or equal to 10. 6 M -1 , or 10 8 M -1 If the binding is higher than 0.05, the binding is considered specific. If necessary, the binding conditions can be changed to reduce non-specific binding without substantially affecting specific binding. Appropriate binding conditions (e.g., molecule concentration, ionic strength of the solution, temperature, binding time, concentration of blocking agent (e.g., serum albumin, milk casein, etc.), etc.) can be optimized by one skilled in the art using routine techniques.

[0211] In certain embodiments, the chimeric proteins of the present invention contain one or more fragmented Fc regions sufficient to confer binding properties to the Fc region of an Fc receptor (FcR). For example, the portion of the Fc region that binds to FcRn (i.e., the FcRn-binding portion) contains approximately amino acids 282 to 438 of IgG1 (EU numbering system) (with major contact sites at amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain). Thus, the Fc regions of the present invention may contain or consist of an FcRn-binding portion. The FcRn-binding portion may be derived from a heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn-binding portion derived from a human antibody of isotype IgG1 is used. In other embodiments, an FcRn-binding portion derived from an antibody of human isotype IgG4 is used.

[0212] In other embodiments, an "Fc region" includes an amino acid sequence of an Fc domain or an amino acid sequence derived from an Fc domain. In certain embodiments, an Fc region contains at least one of a hinge (e.g., upper hinge region, middle hinge region, and / or lower hinge region) domain (approximately amino acids 216-230 of an antibody Fc region, EU numbering), a CH2 domain (approximately amino acids 231-340 of an antibody Fc region, EU numbering), a CH3 domain (approximately amino acids 341-438 of an antibody Fc region, EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, the Fc region contains a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc region contains, consists essentially of, or consists of a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), a hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), or a CH2 domain (or portion thereof) fused to both a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In yet other embodiments, the Fc region lacks at least a portion of the CH2 domain (e.g., part or all of the CH2 domain). In certain embodiments, the Fc region contains or consists of amino acids corresponding to 221 to 447 according to EU numbering.

[0213] The Fc region, designated herein as F, F1, or F2, may be obtained from many different sources. In one embodiment, the Fc region of the polypeptide is derived from human Ig. However, it should be understood that the Fc region may also be derived from Ig of other mammalian species, including, for example, rodents (e.g., mouse, rat, rabbit, or guinea pig) or non-human primate species (e.g., chimpanzee, macaque). Furthermore, the Fc domain or portion thereof may be derived from any Ig class, including IgG, IgM, IgA, IgD, and IgE, and any Ig isotype, including IgG1, IgG2, IgG3, and IgG4. In another embodiment, the human isotype IgG1 is used.

[0214] In certain embodiments, the Fc variants provide an alteration in at least one effector function provided by an Fc region containing the wild-type Fc domain (e.g., an improvement or decrease in the activity of the Fc region to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or an improvement or decrease in the activity of the Fc region to cause antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variants provide an engineered cysteine ​​residue.

[0215] The Fc region of the present invention may employ Fc variants known in the art that are known to alter effector function and / or FcR or FcRn binding. Specifically, the binding molecules of the present invention may employ Fc variants described in, for example, International Patent Applications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, and WO03 / 07456. 9A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO 05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447 A1, WO06 / 047350A2, and WO06 / 085967A2; U.S. Patent Publications US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or U.S. Patents No. 5,648,260; No. 5,739,277; No. 5,834,250; No. 5,869,046; No. 6,096,871; No. 6,121,022; No. 6,194,551; No. 6,242, No. 195; No. 6,277,375; No. 6,528,624; No. 6,538,124; No. 6,737,056; No. 6,821,505; No. 6,998,253; No. 7,083,784; No. 7 Nos. 4,404,956 and 7,317,091 (each of which is incorporated herein by reference). In one embodiment, there may be a specific change (e.g., a specific substitution of one or more amino acids known in the art) at one or more of the disclosed amino acid positions. In other embodiments, there may be a different change (e.g., a different substitution of one or more amino acids known in the art) at one or more of the disclosed amino acid positions.

[0216] The Fc region of IgG or FcRn binding partners may be modified, for example, by well-known methods (e.g., site-directed mutagenesis, etc.) to obtain modified IgG or Fc fragments or portions thereof that are bound by FcRn. Such modifications include modifications at sites distant from the FcRn contact site, as well as modifications within the contact site that do not abolish or even enhance binding to FcRn. For example, the FcRn binding domain of human IgG1 Fc (Fc The following single amino acid residues of γ1) can be substituted without significantly impairing the Fc binding affinity to FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, V284A, V285A, V286A, V287A, V288A, V289A, V290A, V291A, V292A, V293A, V294A, V295A, V296A, V297A, V298A, V299A, V300A, V301A, V302A, V303A, V304A, V305A, V310A, V311A, V312A, V313A, V314A, V315A, V316A, V317A, V318A, V319A, V320A, V321A, V322A, V323A, V324A, V325A, V326A, V327A, V328A, V329A, V330A, V331B, V332B, V333A, V334A, V335A, V336A, V337A, V338A, V339A, V340A, V341A, V342A, V343A, V344A, V345A, V346A 83A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V 305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P 331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A (where, for example, P238A represents a substitution of alanine for the wild-type proline at position 238). By way of example, in certain embodiments, an N297A mutation is incorporated to remove a highly conserved N-glycosylation site.In addition to alanine, other amino acids may be substituted for the wild-type amino acid at these specific sites. Mutations may be introduced individually into the Fc, resulting in over 200 Fc regions that differ from the native Fc. Furthermore, combinations of two, three, or more of these individual mutations may be introduced together to generate over hundreds of Fc regions. Furthermore, one of the Fc regions of a construct of the invention may be mutated, while the other Fc region of the construct may not be mutated at all, or both may be mutated with different mutations.

[0217] Some of the above-mentioned mutations may confer new functionalities to the Fc region or FcRn binding partner. For example, in one embodiment, N297A is introduced to remove a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby extending the circulating half-life of the Fc region, and to prevent the Fc region from binding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA without impairing affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). Another example of new functionalities resulting from the above-mentioned mutations is that, in some cases, affinity for FcRn may be increased beyond that of the wild-type. This increase in affinity may reflect an increase in the "on" ratio, a decrease in the "off" ratio, or both an increase in the "on" ratio and a decrease in the "off" ratio. Examples of mutations that are thought to confer increased affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0218] Furthermore, at least three human Fcγ receptors are thought to recognize binding sites on IgG within the lower hinge region (usually amino acids 234-237). Therefore, other novel functionalities and potentially reduced immunogenicity may be provided by mutations in this region, such as substituting amino acids 233-236 (ELLG) of human IgG1 with the corresponding sequence (PVA) of IgG2 (with a single amino acid deletion). FcγRI, FcγRII, and FcγRIII, which mediate various effector functions, have been shown to abolish IgG1 binding when such mutations are introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77, and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0219] In one embodiment, the Ig constant region or portion thereof (e.g., Fc region) is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO: 52) and, optionally, further comprising a sequence selected from HQSLGTQ (SEQ ID NO: 53), HQNLSDGK (SEQ ID NO: 54), HQNISDGK (SEQ ID NO: 55), or VISSHLGQ (SEQ ID NO: 56) (U.S. Patent No. 5,739,277).

[0220] In another embodiment, the immunoglobulin constant region or a portion thereof contains the amino acid sequence of a hinge region or a portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or a portion thereof. The disulfide bonds formed by the immunoglobulin constant region or a portion thereof are located between a first polypeptide containing FVIII and a second polypeptide containing a VWF fragment, so that endogenous VWF does not displace the VWF fragment and bind to FVIII. Therefore, the disulfide bond between the first immunoglobulin constant region or a portion thereof and the second immunoglobulin constant region or a portion thereof inhibits the interaction between endogenous VWF and FVIII protein. This inhibition of the interaction between VWF and FVIII protein extends the half-life of the FVIII protein beyond the two-fold limit. The hinge region or a portion thereof may be further linked to one or more domains selected from CH1, CH2, CH3, fragments thereof, and any combination thereof. In certain embodiments, the immunoglobulin constant region or portion thereof is the hinge region and CH2.

[0221] In some embodiments, the Ig constant region, or a portion thereof, is semi-glycosylated. For example, a chimeric protein containing two Fc regions or FcRn binding partners may contain a first glycosylated Fc region (e.g., a glycosylated CH2 region) or FcRn binding partner and a second non-glycosylated Fc region (e.g., an non-glycosylated CH2 region) or FcRn binding partner. In one embodiment, a linker may be placed between the glycosylated Fc region and the non-glycosylated Fc region. In other embodiments, the Fc region or FcRn binding partner is fully glycosylated (i.e., the entire Fc region is glycosylated). In other embodiments, the Fc region may be non-glycosylated (i.e., none of the Fc moieties is glycosylated).

[0222] In certain embodiments, the chimeric proteins of the present invention contain amino acid substitutions to the Ig constant region or portions thereof (e.g., Fc variants), which alter the antigen-independent effector functions of the Ig constant region (particularly the circulating half-life of the protein).

[0223] Such proteins exhibit increased binding to FcRn compared to proteins without the substitutions. Fc variants with improved affinity for FcRn are expected to have extended serum half-lives, and such molecules are useful for applications in mammalian therapeutic methods where an administered polypeptide with a long half-life is desired (e.g., chronic diseases or disorders) (see, e.g., U.S. Patent Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with reduced FcRn binding affinity are expected to have short half-lives, and such molecules are also useful for administration to mammals where, for example, a short circulation period is advantageous (e.g., in vivo diagnostic imaging, or where the starting polypeptide has toxic side effects if present in the circulation for a long period). Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore useful for treating diseases or disorders in pregnant women. Further, other applications in which reduced FcRn binding affinity is desirable include those in which localization to the brain, kidney, and / or liver is desired. In one exemplary embodiment, the chimeric proteins of the present invention exhibit reduced transport across the kidney glomerular epithelium from the vasculature. In another embodiment, the chimeric proteins of the present invention exhibit reduced transport across the blood-brain barrier (BBB) ​​from the brain into the vascular space. In one embodiment, a protein with altered FcRn binding contains at least one Fc region or FcRn binding partner (e.g., one or two Fc regions or FcRn binding partners) with one or more amino acid substitutions within the "FcRn-binding loop" of an Ig constant region. The FcRn-binding loop is composed of amino acid residues 280 to 299 (according to EU numbering) of a wild-type, full-length Fc region. In other embodiments, the Ig constant region or portion thereof of the chimeric protein of the present invention with altered FcRn binding affinity contains at least one Fc region or FcRn binding partner with one or more amino acid substitutions within the 15 Å FcRn "contact region."As used herein, the term 15 Å FcRn "contact region" includes residues at the following positions of the wild-type, full-length Fc portion: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (according to EU numbering). In other embodiments, the Ig constant region or portion thereof of the present invention with altered FcRn-binding affinity contains at least one Fc region or FcRn-binding partner with one or more amino acid substitutions at amino acid positions corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in International Patent Application Publication No. WO 05 / 047327, which is incorporated herein by reference.

[0224] The Fc region or FcRn-binding partner used in the present invention may also contain amino acid substitutions known in the art that alter the glycosylation of the chimeric protein. For example, the Fc region or FcRn-binding partner of a chimeric protein linked to a VWF fragment or FVIII protein may contain mutations that result in reduced glycosylation (e.g., N-linked or O-linked glycosylation) or may contain an altered glycoform of the wild-type Fc moiety (e.g., a glycan with reduced or no fucose).

[0225] In one embodiment, the unprocessed chimeric proteins of the invention may contain genetically fused Fc regions (i.e., scFc regions) having two or more of their constituent Ig constant regions or portions thereof independently selected from the Ig constant regions or portions thereof described herein. In one embodiment, the Fc regions of the dimeric Fc region are identical. In other embodiments, at least two of the Fc regions are different. For example, the Fc regions of the proteins of the invention or FcRn binding partners may contain the same number of amino acid residues, or they may differ in length by one or more amino acid residues. In yet other embodiments, the Fc regions or FcRn binding partners of the proteins of the invention may differ in sequence at one or more amino acid positions (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc regions or FcRn binding partners of the proteins of the invention may differ in sequence at one or more amino acid positions. For example, at least two of the Fc regions or FcRn binding partners may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions.

[0226] E) Linker The chimeric proteins of the present invention further contain one or more linkers. One type of linker is a cleavable linker that can be cleaved by various proteases (e.g., at the site of coagulation) when administered to a subject in vivo. In one embodiment, the cleavable linker allows a portion (e.g., a VWF fragment) to be cleaved from the chimeric protein at the site where the coagulation cascade occurs, thereby allowing activated FVIII (FVIIIa) to retain its FVIIIa activity. Another type of linker is a processible linker that contains an intracellular cleavage site, which can be cleaved by intracellular processing enzymes in host cells, allowing for convenient expression of the polypeptide and formation of the chimeric protein.

[0227] One or more linkers may be present between any two proteins in the chimeric protein. In one embodiment, the chimeric protein contains (i) a VWF fragment, (ii) an XTEN sequence, and (iii) an FVIII protein, wherein the VWF fragment is linked to the XTEN sequence by a linker (e.g., a cleavable linker), and the XTEN sequence is further linked to the FVIII protein (i.e., VLX-FVIII). In another embodiment, the chimeric protein contains (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence, and the XTEN sequence is linked to the FVIII by a linker (e.g., a cleavable linker) (i.e., VXL-FVIII).

[0228] In certain embodiments, the chimeric protein contains (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a first Ig constant region or portion thereof (e.g., a first Fc region), (iv) an FVIII protein, and (v) a second Ig constant region or portion thereof (e.g., a second Fc region), wherein the VWF fragment is linked to the XTEN sequence by an optional linker (e.g., a cleavable linker). The XTEN sequence may further be linked to the first Ig constant region or portion thereof by a linker (e.g., a cleavable linker). The FVIII protein (with or without the XTEN sequence) may also be linked to the second Ig constant region or portion thereof by an optional linker (e.g., a cleavable linker). In certain embodiments, the chimeric protein further contains one or more linkers (e.g., processable linkers) between the first Ig constant region or portion thereof (e.g., the first Fc region) and the second Ig constant region or portion thereof (e.g., the second Fc region), between the VWF fragment and the second Ig constant region or portion thereof, or between the FVIII protein and the first Ig constant region or portion thereof (e.g., the first Fc region).

[0229] In some embodiments, the present invention provides a method for producing an antibody comprising: (i) a FVIII protein; (ii) an XTEN sequence; (iii) a first Ig constant region or portion thereof (e.g., a first Fc region); and (iv) a second Ig constant region or portion thereof (e.g., a second Fc region), wherein the first Ig constant region or portion thereof and the second Ig constant region or portion thereof Some are linked by processable linkers.

[0230] Linkers useful in the present invention may contain any organic molecule. In one embodiment, the linker contains a polymer (e.g., polyethylene glycol (PEG)) or hydroxyethyl starch (HES). In other embodiments, the linker contains an amino acid sequence. The linker may contain at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker may contain 1 to 5 amino acids, 1 to 10 amino acids, 1 to 20 amino acids, 10 to 50 amino acids, 50 to 100 amino acids, 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids. In one embodiment, the linker contains an XTEN sequence. Additional example XTENs may be used in accordance with the present invention and are described in U.S. Patent Application Publication Nos. 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1, or 2011 / 0172146 A1, or International Patent Application Publication No. WO2010091122 A1, WO2010144502 A2, WO2010144508 A1, WO2011028228 A1, WO2011028229 A1, or WO2011028344 A2. In other embodiments, the linker is a PAS sequence.

[0231] Linkers useful in the present invention may contain any organic molecule. In one embodiment, the linker is a polymer (e.g., polyethylene glycol (PEG)) or hydroxyethyl starch (HES). In other embodiments, the linker is an amino acid sequence. The linker may contain at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker may contain 1 to 5 amino acids, 1 to 10 amino acids, 1 to 20 amino acids, 10 to 50 amino acids, 50 to 100 amino acids, 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids.

[0232] Examples of linkers are known in the art. In one embodiment, the linker has the sequence G n The linker contains the sequence (GA) n The linker may contain the sequence (GGS) n In other embodiments, the linker may contain the sequence (GGGS) n (SEQ ID NO: 57). In yet another embodiment, the linker contains the sequence (GGS) n (GGGGS) n(SEQ ID NO: 58). In these examples, n can be an integer between 1 and 100. In other examples, n can be an integer between 1 and 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 59), GGSGGSGGSGGSGGG (SEQ ID NO: 60), GGSGGSGGGGSGGGGS (SEQ ID NO: 61), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 62), or GGGSGGGGSGGGGGS (SEQ ID NO: 63). The linker does not eliminate or reduce the activity of the VWF fragment or the clotting activity of Factor VIII. Optionally, the linker can further reduce the effects of steric hindrance, for example, making the VWF fragment or Factor VIII protein more accessible to its target binding site. This enhances the activity of the VWF fragment or the coagulation activity of the factor VIII protein.

[0233] In one embodiment, a linker useful for a chimeric protein is 15 to 25 amino acids in length. In another embodiment, a linker useful for a chimeric protein is 15 to 20 amino acids in length. In some embodiments, a linker for a chimeric protein is 10 to 25 amino acids in length. In other embodiments, a linker for a chimeric protein is 15 amino acids in length. In yet another embodiment, a linker for a chimeric protein is (GGGGS) n (SEQ ID NO: 64), where G represents glycine, S represents serine, and n is an integer from 1 to 20.

[0234] F) Cleavage site Linkers may also incorporate moieties that can be cleaved either chemically (e.g., by hydrolysis of an ester bond), enzymatically (i.e., by incorporation of a protease cleavage sequence), or photolytically (e.g., 3-amino-3-(2-nitrophenyl)propionic acid (ANP)) to release one molecule from the other.

[0235] In one embodiment, the linker is a cleavable linker. The cleavable linker may contain one or more cleavage sites at the N-terminus, C-terminus, or both. In other embodiments, the cleavable linker consists essentially of, or consists of, one or more cleavage sites. In other embodiments, the cleavable linker contains a heterologous amino acid linker sequence or polymer described herein and one or more cleavage sites.

[0236] In some embodiments, the cleavable linker contains one or more cleavage sites (i.e., intracellular processing sites) that can be cleaved in a host cell. Non-limiting examples of cleavage sites include RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), and RRRRS (SEQ ID NO: 11).

[0237] In other embodiments, the cleavable linker contains one or more cleavage sites that are cleaved by a protease after the chimeric protein containing the cleavable linker is administered to a subject. In one embodiment, the cleavage site is cleaved by a protease selected from the group consisting of Factor XIa, Factor XIIa, kallikrein, Factor VIIa, Factor IXa, Factor Xa, Factor IIa (thrombin), elastase 2, MMP-12, MMP-13, MMP-17, and MMP-20. In other embodiments, the cleavage site is a XIa cleavage site (e.g., KLTR↓AET (SEQ ID NO: 65)), a FXIa cleavage site (e.g., DFTR↓VVG (SEQ ID NO: 66)), a FXIIa cleavage site (e.g., TMTR↓IVGG (SEQ ID NO: 67)), a kallikrein cleavage site (e.g., SPFR↓STGG (SEQ ID NO: 68)), a FVIIa cleavage site (e.g., LQVR↓IVGG (SEQ ID NO: 69)), a FIXa cleavage site (e.g., PLGR↓IVGG (SEQ ID NO: 70)), a FXa cleavage site (e.g., IEGR↓TVGG (SEQ ID NO: 71)), a FIIa (thrombin) cleavage site (e.g., LTPR↓SLLV (SEQ ID NO: 72)), an elastase-2 cleavage site (e.g., L GPV↓SGVP (SEQ ID NO: 73)), granzyme-B cleavage site (e.g., VAGD↓SLEE (SEQ ID NO: 74)), MMP-12 cleavage site (e.g., GPAG↓LGGA (SEQ ID NO: 75)), MMP-13 cleavage site (e.g., GPAG↓LRGA (SEQ ID NO: 76)), MMP-17 cleavage site (e.g., APLG↓LRLR (SEQ ID NO: 77)), MMP-20 cleavage site (e.g., PALP↓LVAQ (SEQ ID NO: 78)), TEV cleavage site (e.g., ENLYFQ↓G (SEQ ID NO: 79)), enterokinase cleavage site (e.g., DDDK↓IVGG (SEQ ID NO: 80)), protease 3C (PRESCISSION®) cleavage site (e.g., LEVLFQ↓GP (SEQ ID NO: 81), and a sortase A cleavage site (e.g., LPKT↓GSES) (SEQ ID NO: 82). In certain embodiments, FXIa cleavage sites include, but are not limited to, e.g., TQSFNDFTR (SEQ ID NO: 83) and SVSQTSKLTR (SEQ ID NO: 84). Non-limiting examples of thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 85), TTKIKPR (SEQ ID NO: 86), or LVPRG (SEQ ID NO: 87), and a sequence containing, consisting essentially of, or consisting of ALRPR (SEQ ID NO: 17), (e.g., ALRPRVVGGA (SEQ ID NO: 88)).

[0238] In a particular embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).

[0239] Polynucleotides, Vectors, and Host Cells ...

Claims

[Claim 1] A chimeric protein, polynucleotide, composition, or method as described herein.

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