Factor VIII chimeric proteins and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of the invention application filed on January 9, 2015, with Chinese application number 201580009942.9 and entitled "Factor VIII Chimeric Protein and Its Use".

[0002] Reference to the electronically submitted sequence list

[0003] The contents of the sequence list, submitted electronically in the form of an ASCII text file (name: 2159_441PC02_SequenceListing_ST25.txt; size: 823,500 bytes; and creation date: January 9, 2015), are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates to factor VIII chimeric proteins and their uses, specifically providing a factor VIII chimeric protein comprising a first polypeptide containing an FVIII protein and a first Ig constant region or a portion thereof; and a second polypeptide containing a VWF protein having a D' domain and a D3 domain, an XTEN sequence having a length of less than 288 amino acids, and a second Ig constant region or a portion thereof, wherein the first polypeptide and the second polypeptide are associated with each other. The invention also includes nucleotides, vectors, host cells, and methods of using the chimeric protein. Background Technology

[0005] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding coagulation factor VIII (FVIII), affecting 1 in 10,000 to 2 in 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients affected by hemophilia A can be treated with infusions of purified or recombinant FVIII. However, the half-life of all commercially available FVIII products is known to be approximately 8–12 hours, requiring frequent intravenous administration to patients. See Weiner MA and Cairo, MS, Pediatric Hematology Secrets, Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Supplement 4:S2-8 (2008). In addition, many methods have been explored to prolong the half-life of FVIII. For example, methods under development for extending the half-life of coagulation factors include PEGylation, glycoglycolation, and conjugation to albumin. See Dumont et al., Blood. 119(13): 3024-3030 (published online January 13, 2012). However, regardless of the protein engineering used, current long-acting FVIII products under development have been reported to have limited half-lives—only about 1.5 to 2 hours in preclinical animal models. See ibid. Consistent results have been demonstrated in humans, for example, with reported improvements of up to about 1.7 times in the half-life of rFVIIIFc compared to ADVATE® in patients with hemophilia A. See ibid. Therefore, despite minor improvements, an increase in half-life may indicate the presence of other T1 / 2 limiting factors. See Liu, T. et al., 2007 ISTH Conference, Abstract #PM-035; Henrik, A. et al., 2011 ISTH Conference, Abstract #P=MO-181; Liu, T. et al., 2011 ISTH Conference Abstract #P-WE-131.

[0006] The half-life of plasma von Willebrand factor (VWF) is approximately 16 hours (ranging from 13 to 18 hours). (Goudemand J et al., J Thromb Haemost 2005;3:2219–27). The VWF half-life can be affected by many factors, including glycosylation patterns, ADAMTS-13 (disintegrin and metalloproteinases with the thrombospondin motif-13), and various mutations in VWF.

[0007] In plasma, 95–98% of FVIII circulates as a tight, non-covalent complex with full-length VWF. The formation of this complex is important for maintaining adequate plasma levels of FVIII in vivo. Lenting et al., Blood. 92(11):3983–96 (1998); Lenting et al., J. Thromb. Haemost. 5(7): 1353–60 (2007). Full-length wild-type FVIII exists primarily 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 in the heavy chain and at position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII. The activated FVIII, along with activating factor IX, calcium, and phospholipids (“factor X activating enzyme complex”), induces factor X activation, resulting in the production of 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, along with calcium, cross-links and stabilizes the soluble fibrin polymers, thereby forming cross-linked (insoluble) fibrin. Activated FVIII is rapidly cleared from circulation via proteolysis.

[0008] Due to the inconvenience caused by frequent dosing and the timing of dosing, there is still a need to develop FVIII products that require a lower dosing frequency, i.e., FVIII products with a half-life 1.5 to 2 times longer than the half-life limit. Summary of the Invention

[0009] This invention provides a chimeric protein comprising (i) a first polypeptide comprising a factor VIII (“FVIII”) protein fused to a constant region of a first immunoglobulin (“Ig”) or a portion thereof, and (ii) a second polypeptide comprising a van Wilbond factor (“VWF”) protein containing a D' domain and a D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via an XTEN sequence therebetween, wherein the XTEN sequence contains fewer than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide. Certain embodiments include chimeric proteins as described herein, wherein the XTEN sequence in the second polypeptide consists of an amino acid sequence of length between 12 and 287 amino acids.

[0010] Chimeric proteins as described herein are also disclosed, wherein, compared to corresponding fusion proteins comprising a first polypeptide and a second polypeptide, wherein the second polypeptide of the fusion protein comprises an XTEN sequence containing at least 288 amino acids, the chimeric protein exhibits a longer half-life. Some embodiments include the XTEN sequence AE288 containing at least 288 amino acids. In some embodiments, AE288 is SEQ ID NO: 8.

[0011] Chimeric proteins as described herein are also disclosed, wherein the XTEN sequence of the second polypeptide contains about 36, about 42, about 72, or about 144 amino acids. In some embodiments, the XTEN sequence of the second polypeptide is selected from AE42, AE72, AE144, AG42, AG72, or AG144.

[0012] Some embodiments include chimeric proteins as described herein, wherein the XTEN sequence of the second polypeptide is selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 63.

[0013] In some embodiments, the first polypeptide further comprises a second XTEN sequence that links the FVIII protein to a first Ig constant region or a portion thereof. Chimeric proteins as described herein are also disclosed, wherein the first polypeptide comprises a third XTEN sequence inserted at one or more insertion sites within the FVIII protein. In some embodiments, the first polypeptide further comprises a second XTEN sequence inserted at one or more insertion sites within the FVIII protein. In some embodiments, the first polypeptide comprises a third XTEN sequence that links the FVIII protein to a first Ig constant region or a portion thereof.

[0014] Chimeric proteins as described herein are also disclosed, wherein the second XTEN sequence, the third XTEN sequence, or the second XTEN sequence and the third XTEN sequence are each independently selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288 and AG144. In some embodiments, the second XTEN sequence, the third XTEN sequence, or both the second and third XTEN sequences are each independently selected from SEQ ID NO: 8; SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17; SEQ ID NO: 54; SEQ ID NO: 19; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 15; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 63. In some embodiments, the second XTEN sequence, the third XTEN sequence, or both the second and third XTEN sequences are each independently AE288 or AG288. In some embodiments, the XTEN sequence in the second polypeptide is fused to a second Ig constant region or a portion thereof via a linker. In some embodiments, the linker is a cleavable linker.

[0015] Some embodiments include chimeric proteins as described herein, wherein the linker may be cleaved by a protease selected from: factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, granzyme-B, TEV, enterokinase, protease 3C, sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. In some embodiments, the linker may be cleaved by factor IIa (thrombin).

[0016] Chimeric proteins as described herein are also disclosed, wherein the linker comprises one or more cleavage sites comprising an amino acid sequence selected from the following: RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: 103), RRRRS (SEQ ID NO: 104), TQSFNDFTR (SEQ ID NO: 1), SVSQTSKLTR (SEQ ID NO: 3), DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), KLTRAET (SEQ ID NO: 121), DFTRVVG (SEQ ID NO: 122), TMTRIVGG (SEQ ID NO: 123), SPFRSTGG (SEQ ID NO: 124), LQVRIVGG (SEQ ID NO: 125), PLGRIVGG (SEQ ID NO: 126), IEGRTVGG (SEQ ID NO: 127), 128, 129, 120 ... 127), LTPRSLLV (SEQ ID NO: 128), LGPVSGVP (SEQ ID NO: 129), VAGDSLEE (SEQ ID NO: 130), GPAGLGGA (SEQ ID NO: 131), GPAGLRGA (SEQ ID NO: 132), APLGLRLR (SEQ ID NO: 133), PALPLVAQ (SEQ ID NO: 134), ENLYFQG (SEQ ID NO: 135), DDDKIVGG (SEQ ID NO: 136), LEVLFQGP (SEQ ID NO: 137), LPKTGSES (SEQ ID NO: 138), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), and IEPRSFS (SEQ ID NO: 194). In some embodiments, the connector comprises TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). In some embodiments, the cleavage site comprises the amino acid sequence LVPRG (SEQ ID NO: 6). In other embodiments, the cleavage site comprises the amino acid sequence IEPRSFS (SEQ ID NO: 194). In still other embodiments, the cleavage site comprises the amino acid sequence IEPRSFS (SEQ ID NO: 194), wherein the cleavage site is not the full-length a2 region of FVIII.In some embodiments, the cleavage site comprises a fragment of the a2 region of FVIII containing at least the sequence IEPR (SEQ ID NO: 200). In other embodiments, the cleavage site comprises a fragment of the a2 region of FVIII containing at least the sequence IEPR (SEQ ID NO: 200), wherein the cleavage site is not the full-length a2 region. In some embodiments, the cleavage site is cleavable in thrombin cleavage assays as provided herein or as known in the art.

[0017] Some embodiments include chimeric proteins as described herein, wherein the first Ig constant region or a portion thereof comprises a first Fc region and / or the second Ig constant region or a portion thereof comprises a second Fc region. In some embodiments, the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof extend the half-life of the chimeric protein. In some embodiments, the first polypeptide and the second polypeptide are fused via a linker. In some embodiments, the first polypeptide and the second polypeptide are fused via a processable linker. In some embodiments, the first Ig constant region or a portion thereof associates with the second Ig constant region or a portion thereof. In some embodiments, the first Ig constant region or a portion thereof associates with the second Ig constant region or a portion thereof via a covalent bond. In some embodiments, the covalent bond is a disulfide bond.

[0018] Chimeric proteins comprising each of the following formulas (a)-(hh) are also disclosed: (a) FVIII-F1:F2-L2-X-L1-V; (b) FVIII-F1:V-L1-X-L2-F2; (c) F1-FVIII:F2-L2-X-L1-V; (d) F1-FVIII:V-L1-X-L2-F2; (e) FVIII-X2-F1:F2-L2-X1-L1-V; (f) FVIII-X2-F1:V-L1-X1-L2-F2; (g) FVIII(X2)-F1:F2-L2-X1-L1-V; (h) FVIII(X2)-F1:V-L1-X1-L2-F2; (i) F1-X2-F1:F2-L2-X1-L1-V; (j) F1-X2-F1:V-L1-X1-L2-F2; (k) V-L1-X-L2-F2-L3-FVIII-L4-F1; (l)V-L1-X-L2-F2-L3-F1-L4-FVIII; (m) F1-L4-FVIII-L3-F2-L2-X-L1-V; (n) FVIII-L4-F1-L3-F2-L2-X-L1-V; (o) FVIII-L4-F1-L3-V-L1-X-L2-F2; (p)FVIII-L4-F1-L3-F2-L2-X-L1-V; (q) F2-L2-X-L1-V-L3-F1-L4-FVIII; (r)F2-L2-X-L1-V-L3-FVIII-L4-F1; (s)V-L1-X1-L2-F2-L3-FVIII(X2)-L4-F1; (t)V-L1-X1-L2-F2-L3-F1-L4-FVIII(X2); (u) F1-L4-FVIII(X2)-L3-F2-L2-X1-L1-V; (v) F-L4-FVIII(X2)-L3-V-L1-X1-L2-F2; (w) FVIII(X2)-L4-F1-L3-V-L1-X1-L2-F2; (x)FVIII(X2)-L4-F1-L3-F2-L2-X1-L1-V; (y) F2-L2-X1-L1-V-L3-F1-L4-FVIII(X2); (z) F2-L2-X1-L1-V-L3-FVIII(X2)-L4-F1; (aa) V-L1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1; (bb) V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII; (cc) F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V; (dd) F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2; (ee) FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1; (ff) FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V; (gg) F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or (hh) F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2;

[0019] Where V is a VWF protein containing a D' domain and a D3 domain, X or X1 is a first XTEN sequence containing less than 288 amino acids, X2 is a second XTEN sequence, FVIII includes an FVIII protein, FVIII(X2) includes an FVIII protein having a second XTEN sequence inserted into one or more insertion sites within the FVIII protein, F1 is a first Ig constant region or a portion thereof, F2 is a second Ig constant region or a portion thereof, L1, L2, L3, L4 or L5 are optional linkers, (-) is a peptide bond; and (:) is a covalent or non-covalent bond.

[0020] Some implementations include chimeric proteins as described herein, wherein X or X1 consists of an amino acid sequence of length between 12 and 287 amino acids.

[0021] In some embodiments, the exception compared to including the formula is that X or X1 is a corresponding chimeric protein of AE288, as chimeric proteins as described herein exhibit a longer half-life. In some embodiments, AE288 is SEQ ID NO:8.

[0022] Some embodiments include chimeric proteins as described herein, wherein X or X1 contains about 36, about 42, about 72, or about 144 amino acids. In some embodiments, X or X1 is selected from AE42, AE72, AE144, AG42, AG72, or AG144. In some embodiments, X or X1 is selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 63. In some embodiments, X2 comprises an amino acid sequence of at least about 36 amino acids, at least about 42 amino acids, at least about 144 amino acids, at least about 288 amino acids, at least about 576 amino acids, or at least about 864 amino acids. In some embodiments, X2 is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. In some embodiments, X2 is selected from SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 17; SEQ ID NO: 54; SEQ ID NO: 19; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 15; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 63. In some embodiments, X2 is AE288 or AG288.

[0023] Chimeric proteins containing X or X1 and / or X2, as described herein, are also disclosed, exhibiting a longer half-life compared to chimeric proteins not containing X or X1 and / or X2. In some embodiments, L1 and / or L2 are cleavable linkers. In some embodiments, L4 and / or L5 are cleavable linkers. In some embodiments, the linker may be cleaved by a protease selected from: factor XIa, factor XIIa, angiotensin, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, granzyme-B, TEV, enterokinase, protease 3C, sorting enzyme A, MMP-12, MMP-13, MMP-17, and MMP-20. In some embodiments, the linker may be cleaved by factor IIa (thrombin).

[0024] Some embodiments include chimeric proteins as described herein, wherein the linker comprises one or more cleavage sites comprising an amino acid sequence selected from the following: RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: 103), RRRRS (SEQ ID NO: 104), TQSFNDFTR (SEQ ID NO: 2), SVSQTSKLTR (SEQ ID NO: 3), DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), KLTRAET (SEQ ID NO: 121), DFTRVVG (SEQ ID NO: 122), TMTRIVGG (SEQ ID NO: 123), SPFRSTGG (SEQ ID NO: 124), LQVRIVGG (SEQ ID NO: 125), PLGRIVGG (SEQ ID NO: 126), IEGRTVGG (SEQ ID NO: 127), 128, 129, 120 ... 127), LTPRSLLV (SEQ ID NO: 128), LGPVSGVP (SEQ ID NO: 129), VAGDSLEE (SEQ ID NO: 130), GPAGLGGA (SEQ ID NO: 131), GPAGLRGA (SEQ ID NO: 132), APLGLRLR (SEQ ID NO: 133), PALPLVAQ (SEQ ID NO: 134), ENLYFQG (SEQ ID NO: 135), DDDKIVGG (SEQ ID NO: 136), LEVLFQGP (SEQ ID NO: 137), and LPKTGSES (SEQ ID NO: 138). In some embodiments, the linker comprises TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). In some embodiments, the linker comprises the amino acid sequence LVPRG (SEQ ID NO: 6). In some embodiments, the connector includes region a1 of FVIII, region a2 of FVIII, region a3 of FVIII, or any combination thereof. In some embodiments, the connector includes a fragment of region a2 of FVIII. In some cases, the fragment of region a2 may contain the sequence DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88).In other embodiments, a smaller fragment of the a2 region of FVIII may be used, including a fragment having the sequence IEPRSFS (SEQ ID NO: 194). In one particular embodiment, the adapter comprises the amino acid sequence IEPRSFS (SEQ ID NO: 194). In another embodiment, the adapter comprises the amino acid sequence IEPRSFS (SEQ ID NO: 194), wherein the adapter is not the full-length a2 region of FVIII.

[0025] Chimeric proteins as described herein are also disclosed, wherein the a2 region of FVIII contains at least about 80%, about 85%, about 90%, about 95%, or 100% of the same amino acid sequence as ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106) or DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88). In some embodiments, the a1 region contains at least about 80%, about 85%, about 90%, about 95%, or 100% of the same amino acid sequence as ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107). In some embodiments, region a3 contains at least about 80%, about 85%, about 90%, about 95%, or 100% of the same amino acid sequence as ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 108). In some embodiments, F1 contains a first Fc region and / or F2 contains a second Fc region.

[0026] Some embodiments include chimeric proteins as described herein, wherein chimeric proteins containing F1 and F2 exhibit a longer half-life compared to chimeric proteins without F1 and F2. In some embodiments, L3 is a processable linker. In some embodiments, the VWF protein associates with the FVIII protein via a non-covalent bond. In some embodiments, the half-life of the chimeric protein is prolonged compared to FVIII proteins without VWF protein and / or XTEN sequence or compared to wild-type FVIII. In some embodiments, the half-life of the chimeric protein is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, 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 FVIII proteins without VWF protein or XTEN sequence or wild-type FVIII.

[0027] Chimeric proteins as described herein are also disclosed, wherein the half-life of the chimeric protein is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In some embodiments, the half-life of the chimeric protein in HemA mice is about 40 hours. In some embodiments, the VWF protein does not substantially bind to the VWF clearance receptor. In some implementations, the VWF protein can protect the FVIII protein from cleavage by one or more proteases, protect the FVIII protein from activation, stabilize the heavy and / or light chains of the FVIII protein, or prevent the FVIII protein from being cleared by one or more scavenger receptors.

[0028] Some embodiments include chimeric proteins as described herein, wherein the VWF protein inhibits or prevents endogenous VWF binding to the FVIII protein by masking or blocking the VWF binding site on the FVIII protein. In some embodiments, the VWF binding site is located in the A3 domain or C2 domain of the FVIII protein, or both the A3 and C2 domains. In some embodiments, the VWF binding site comprises amino acid sequences corresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO: 65. In some embodiments, the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are the same or different. In some embodiments, the FVIII protein is linked to and / or has inserted at least two, at least three, at least four, at least five, or at least six XTEN sequences.

[0029] Chimeric proteins as described herein are also disclosed, wherein the FVIII protein comprises one or more domains of FVIII selected from the following: A1 domain, a1 acidic region, A2 domain, a2 acidic region, B domain, A3 domain, a3 acidic region, C1 domain, C2 domain, one or more of these domains, and any combination thereof.

[0030] Chimeric proteins as described herein are also disclosed, wherein one or more insertion sites in the FVIII protein are located within one or more domains of the FVIII protein selected from the following: A1 domain, α1 acidic region, A2 domain, α2 acidic region, A3 domain, B domain, C1 domain, C2 domain, and any combination thereof, or are located between one or more domains of the FVIII protein selected from the following: A1 domain and α1 acidic region, α1 acidic region and A2 domain, A2 domain, etc. The insertion site in the FVIII protein may be an amino acid domain and an a2 acidic region, an a2 acidic region and a B domain, a B domain and an A3 domain, an A3 domain and a C1 domain, a C1 domain and a C2 domain, or any combination thereof, or located between two domains of the FVIII protein selected from the following: A1 domain and an a1 acidic region, an a1 acidic region and an A2 domain, an A2 domain and an a2 acidic region, an a2 acidic region and a B domain, a B domain and an A3 domain, an A3 domain and a C1 domain, a C1 domain and a C2 domain, or any combination thereof. In some embodiments, one or more insertion sites in the FVIII protein are one or more amino acids selected from the group consisting of amino acid residues in Tables 7, 8, 9, and 10. In some embodiments, the insertion site in the FVIII protein is located immediately downstream of amino acid 745, which corresponds to the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is located immediately downstream of residues 1656 and 1900 of the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is immediately downstream of residues 26, 1656, and 1900 of the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is immediately downstream of residues 403 and 745 of the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is immediately downstream of residues 745 and 1900 of the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the insertion site in the FVIII protein is immediately downstream of residues 18 and 745 of the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the FVIII protein is a double-stranded FVIII isotype. In some embodiments, the FVIII protein is a single-stranded FVIII isotype. In some embodiments, the FVIII protein includes a B domain or a portion thereof. In some embodiments, the FVIII protein is an FVIII lacking the SQ B domain.

[0031] Some embodiments include chimeric proteins as described herein, wherein the single-chain FVIII isoform contains at least one amino acid substitution at residues corresponding to: residue 1648, residue 1645, or two residues corresponding to the full-length maturation factor VIII polypeptide (SEQ ID NO: 65) or residue 754, residue 751, or two residues corresponding to the SQ BDD factor VIII (SEQ ID NO: 67). In some embodiments, the amino acid substitution is an amino acid other than arginine. In some embodiments, the double-chain FVIII isoform comprises a first chain containing an FVIII heavy chain and a second chain containing an FVIII light chain, wherein the heavy chain and the light chain are associated with each other by a metallic bond. In some embodiments, the D' domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 21. In some embodiments, the D3 domain comprises at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence 867 to 1240 of SEQ ID NO: 21. In some embodiments, the VWF protein is a monomer.

[0032] Chimeric proteins as described herein are also disclosed, comprising at least two, at least three, at least four, at least five, or at least six VWF proteins. In some embodiments, the VWF proteins comprise at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence 764 to 1240 of SEQ ID NO: 21. In some embodiments, the VWF proteins are substantially composed of or comprised of amino acids 764 to 1240 of SEQ ID NO: 21. In some embodiments, the VWF proteins contain at least one amino acid substitution at residues corresponding to residues 1099, 1142, or both residues 1099 and 1142 of SEQ ID NO: 21. In some embodiments, the VWF proteins contain amino acids other than cysteine ​​that substitute for residues corresponding to residues 1099, 1142, or both residues 1099 and 1142 of SEQ ID NO: 21. In some embodiments, the VWF protein further includes the D1 domain, D2 domain, or both D1 and D2 domains of VWF.

[0033] Some implementations include chimeric proteins as described herein, wherein the VWF protein further comprises a VWF domain selected from the following: A1 domain, A2 domain, A3 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more of these domains, and any combination thereof.

[0034] Chimeric proteins as described herein are also disclosed, wherein the VWF protein is substantially composed of or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the D1, D' and D3 domains of VWF or fragments thereof; (3) the D2, D' and D3 domains of VWF or fragments thereof; (4) the D1, D2, D' and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3 and A1 domains of VWF or fragments thereof.

[0035] Some implementations include chimeric proteins as described herein, wherein the VWF protein further comprises a signal peptide operatively linked to the VWF protein, either VWF or FVIII.

[0036] Chimeric proteins as described herein are also disclosed, wherein one or more linkers are at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues in length. In some embodiments, one or more linkers are about 1 to about 2000 amino acid residues in length. In some embodiments, one or more linkers comprise a gly / ser peptide. In some implementations, the gly / ser peptide has the formula (Gly4Ser). n (SEQ ID NO: 94) or S(Gly4Ser) n (SEQ ID NO: 164), where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some implementations, (Gly4Ser) n The linker is (Gly4Ser)3 (SEQ ID NO: 100) or (Gly4Ser)4 (SEQ ID NO: 165). In some embodiments, the linker comprises 20, 35, 48, 73, or 95 amino acids. In some embodiments, the cleavable linker is SGGGGSGGGGSGGGGSGGGGSGGGSLVPRGSGG (SEQ ID NO: 166).

[0037] In some implementations, the chimeric protein, as described herein, is polysialylated, polyethylene glycolated, or hydroxyethyl starch-modified.

[0038] Also disclosed are chimeric proteins as described herein, wherein the first polypeptide comprises FVIII161 (SEQ ID NO: 69), FVIII169 (SEQ ID NO: 70), FVIII173 (SEQ ID NO: 72), FVIII195 (SEQ ID NO: 73), FVIII196 (SEQ ID NO: 74), FVIII199 (SEQ ID NO: 75), FVIII201 (SEQ ID NO: 76), FVIII203 (SEQ ID NO: 77), FVIII204 (SEQ ID NO: 78), FVIII205 (SEQ ID NO: 79), FVIII266 (SEQ ID NO: 80), FVIII267 (SEQ ID NO: 81), FVIII268 (SEQ ID NO: 82), FVIII269 (SEQ ID NO: 83), FVIII271 (SEQ ID NO: 84), and FVIII272 (SEQ ID NO: 85). The first polypeptide comprises at least about 80%, 90%, 95%, 99%, or 100% identical to FVIII282 (SEQ ID NO: 85) or FVIII282 (SEQ ID NO: 159), and the second polypeptide comprises at least about 80%, 90%, 95%, 99%, or 100% identical to VWF057 (SEQ ID NO: 152) or VWF059 (SEQ ID NO: 197). In some embodiments, the first polypeptide comprises FVIII169 (SEQ ID NO: 70), and the second polypeptide comprises VWF057 (SEQ ID NO: 152). In other embodiments, the first polypeptide comprises FVIII169 (SEQ ID NO: 70), and the second polypeptide comprises VWF059 (SEQ ID NO: 197). In yet another embodiment, the first polypeptide comprises FVIII169 (SEQ ID NO: 70), and the second polypeptide comprises VWF062 (SEQ ID NO: 199). In some implementations, chimeric proteins effectively prevent and / or stop bleeding from subjects in need.

[0039] Also disclosed is a polynucleotide or a group of polynucleotides encoding the chimeric protein as described herein. In some embodiments, the polynucleotide as described herein further comprises a polynucleotide chain encoding PC5 or PC7.

[0040] Some implementations include a vector comprising a polynucleotide as described herein and one or more promoters operatively linked to said polynucleotide or said group of polynucleotides.

[0041] In some implementations, the vectors described herein further include additional vectors comprising a polynucleotide chain encoding PC5 or PC7.

[0042] Also disclosed is a host cell comprising the polynucleotides or vectors described herein. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is selected from HEK293 cells, CHO cells, and BHK cells.

[0043] Also disclosed is a pharmaceutical composition comprising a chimeric protein, a polynucleotide, a carrier or host cell, and a pharmaceutically acceptable carrier as described herein. In some embodiments, the chimeric protein has an extended half-life compared to the wild-type FVIII protein. In some embodiments, the half-life of the chimeric 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 compared to the wild-type FVIII.

[0044] Some embodiments include the composition as described herein, wherein the half-life of the chimeric protein is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In some embodiments, the half-life of the chimeric protein in HemA mice is about 40 hours. In some embodiments, the composition as described herein is administered via a route selected from the group consisting of: topical application, intraocular application, parenteral application, intrathecal application, subdural application, and oral application. In some implementations, parenteral administration is administered intravenously or subcutaneously.

[0045] In some embodiments, the compositions described herein are used to treat bleeding disorders or conditions in a subject in need. In some embodiments, the bleeding disorder or condition is selected from the group consisting of: bleeding coagulation disorders, joint effusion, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, iliopsoas sheath bleeding, and any combination thereof. In some embodiments, the subject is scheduled to undergo surgery. In some embodiments, the treatment is preventative or as needed.

[0046] Also disclosed is a method for prolonging or increasing the half-life of a chimeric protein, wherein the method comprises adding an effective amount of the chimeric protein, polynucleotide, vector, host cell, or composition as described herein to a subject in need, wherein the VWF protein, XTEN sequence, first Ig constant region or a portion thereof, and second Ig constant region or a portion thereof increase the half-life of the chimeric protein.

[0047] Some implementations include a method of treating a bleeding disorder or condition in a subject in need, comprising administering an effective amount of a chimeric protein, polynucleotide, carrier, host cell, or composition as described herein, wherein the bleeding disorder or condition is selected from the group consisting of: bleeding coagulation disorders, joint effusion, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, and iliopsoas sheath bleeding. In some implementations, the subject is an animal. In some implementations, the animal is a human. In some implementations, the subject is suffering from hemophilia A. In some implementations, the treatment is preventative or as needed. In some implementations, the effective amount is from 0.1 μg / kg to 500 mg / kg.

[0048] Also disclosed is a method as described herein, wherein the chimeric protein, polynucleotide, carrier, host cell, or composition described herein is administered via a route selected from the group consisting of: local administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. In some embodiments, parenteral administration is selected from the group consisting of: intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration.

[0049] Some embodiments include a method for preparing a chimeric protein, comprising transfecting one or more host cells with a polynucleotide or vector as described herein, and expressing the chimeric protein in the host cells. In some embodiments, the method described herein further comprises isolating the chimeric protein. In some embodiments, the chimeric protein effectively stops and / or prevents bleeding in a subject. Attached Figure Description

[0050] Figure 1 A schematic diagram of a chimeric protein is shown, comprising a first polypeptide comprising an FVIII protein (A1-A2-partial or complete B-A3-C1-C2) fused to an Fc region, wherein an XTEN is inserted into the FVIII protein at an insertion site; and a second polypeptide comprising a VWF protein containing a D'D3 domain, an XTEN having less than 288 amino acids, a thrombin-cleavable linker, and a second Fc region. The insertion of the XTEN into the FVIII protein and / or its fusion with the VWF protein prolongs the half-life of the chimeric protein by increasing the hydrodynamic radius and by blocking receptor-mediated clearance. The D'D3 domain of the VWF blocks the interaction between FVIII and endogenous VWF, stabilizing the FVIII protein and prolonging the half-life of the chimeric protein. The Fc domain allows the D'D3 domain to be covalently linked to the FVIII protein and prolongs the half-life of the chimeric protein via an FcRn-mediated recycling pathway. The thrombin-cleavable linker enables the release of the D'D3 domain upon activation of FVIII and ensures proper alignment between the D'D3 domains of FVIII and VWF.

[0051] Figure 2 This demonstrates a three-plasmid expression system for the FVIII-XTEN-Fc:D'D3-XTEN-Fc heterodimer: the first plasmid contains a nucleotide sequence encoding a single-stranded FVIII-XTEN-Fc, with XTEN inserted into the B domain; the second plasmid contains a nucleotide sequence encoding D1D2D'D3-XTEN-Fc, with the XTEN sequence containing fewer than 288 amino acids; and the third plasmid contains a nucleotide sequence encoding PACE (a protopeptide processing enzyme). When the three polypeptides are expressed from the three plasmids, the D1D2 protopeptide domain of VWF can be processed from the D'D3 domain via intracellular processing. The resulting complex contains three products: the first molecule is the FVIII-XTEN / D'D3 heterodimer, the second molecule is a byproduct, namely the homodimer of D'D3-XTEN-Fc, and the third molecule is another byproduct, namely FVIII(XTEN)-Fc.

[0052] Figure 3This demonstrates the cumulative effect of XTEN inserts on the half-life extension of heterodimers. FVIII169 comprises an FVIII protein fused to the Fc region with a B-domain deletion, wherein an XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to the mature full-length FVIII. FVIII205 comprises an FVIII protein fused to the Fc region with a B-domain deletion, wherein an XTEN sequence (e.g., AE144) is inserted at amino acid 18 corresponding to the mature full-length FVIII, and another XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to the mature full-length FVIII. VWF031 comprises the D' and D3 domains of VWF fused to the Fc region via a thrombin-cleavable linker (without XTEN). VWF034 comprises the D' and D3 domains of VWF fused to both AE288 and the Fc region. The half-life of FVIII169 / VWF031 (inverted triangle) in HemA mice is 16.7 hours; the half-life of FVIII205 / VWF031 (circle) in HemA mice is 29.4 hours; and the half-life of FVIII169 / VWF034 (square) in HemA mice is 31.1 hours.

[0053] Figure 4 The results show that AE144 XTEN, when inserted between the D'D3 and Fc domains of VWF, confers a superior half-life compared to AE288 XTEN. For example, while VWF169 / VWF034 (square) has a half-life of 31.1 hours in HemA mice, FVIII169 / VWF057 (circle) has a half-life of 42 hours in HemA mice. VWF057 contains the D'D3 domain of VWF fused to the AE144 and Fc regions.

[0054] Figure 5 This demonstrates that the Fc domain is necessary for the extended half-life of the chimeric protein heterodimer. When the half-life of FVIII205 / VWF031 (circle) in HemA mice was compared to that of FVIII263 / VWF050 (square), which contains a mutation at the FcRn binding site (IHH triple mutant Fc) and therefore cannot recycle via the FcRn pathway, the half-life of FVIII263 / VWF050 (23 hours) was shorter than that of VWF205 / VWF031 (29.4 hours). This indicates that the Fc region is essential for half-life extension.

[0055] Figure 6Inset A shows the similar acute efficacy of the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer compared to the B-domain-deficient FVIII (SQ BDD FVIII) in a HemA mouse tail clamp model. Mice were administered 75 IU / kg, and activity was measured by aPTT assay. SQ BDD FVIII is shown as a circle, while FVIII169 / VWF034 is shown as a square, FVIII169 / VWF057 as a rhombus, and the mediator is shown as an inverted triangle. Construct details of FVIII169, VWF034, and VWF057 are shown elsewhere in this paper. Figure 6 Inset B shows a comparison of the acute efficacy of FVIII169 / VWF034 and B-domain-deficient FVIII (SQ BDD FVIII) in HemA mice at a dose of 37.5 IU / kg, with activity measured by aPTT assay. Median blood loss (uL) in mice in each treatment group is indicated by horizontal lines; blood loss (uL) in C57 / BL6 mice is shown as a hollow triangle; blood loss (uL) after administration of rBDD-FVIII at 37.5 IU / kg is shown as a hollow circle; blood loss (uL) after administration of FVIII169 / VWF034 at 37.5 IU / kg is shown as a hollow square; and blood loss (uL) after administration of the carrier is shown as an inverted triangle.

[0056] Figure 7 Figure AB shows that the rFVIII169 / VWF057 heterodimer provides long-lasting protection to HemA mice in a tail vein transverse bleeding model. Figure 7 Inset A shows rebleeding data in mice that received rFVIII169 / VWF057 72 hours before tail injury (square), SQ BDD-FVIII 48 hours before tail injury (diamond), SQBDD FVIII 24 hours before tail injury (inverted triangle), and the mediator (circle). Activity was measured by aPTT assay. The X-axis shows time in hours, and the Y-axis shows the percentage of mice that did not bleed. Figure 7 Small image B shows Figure 7 The corresponding survival data for the four mouse species are shown in inset A. Mice treated with SQ BDDFVIII 72 hours prior to tail injury showed similar protection against rebleeding and survival compared to mice treated with SQ BDDFVIII 24 hours prior to tail injury.

[0057] Figure 8Inset A shows similar rebleeding data in mice that received rFVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer 96 hours prior to injury, compared to mice that received rBDD-FVIII 24 hours prior to injury. Solid squares represent rebleeding data in mice that received FVIII169 / VWF034 24 hours prior to injury; hollow squares represent rebleeding data in mice that received FVIII169 / VWF034 96 hours prior to injury; solid rhombuses represent rebleeding data in mice that received FVIII169 / VWF057 24 hours prior to injury; hollow rhombuses represent rebleeding data in mice that received FVIII169 / VWF057 96 hours prior to injury; solid circles represent rebleeding data in mice that received rBDD-FVIII 24 hours prior to injury; hollow circles represent rebleeding data in mice that received rBDD-FVIII 48 hours prior to injury; and solid triangles represent rebleeding data in mice that received the medium. The X-axis shows time in hours, and the y-axis shows the percentage of mice that did not bleed.

[0058] Figure 8 Inset B shows the survival curves in mice that received rFVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer 96 hours prior to injury, compared to mice that received rBDD-FVIII 24 hours prior to injury. The X-axis represents time in hours, and the y-axis represents the percentage of survival. (Symbols and symbols are not provided in the original text.) Figure 8 The same applies to small figure A.

[0059] Figure 9Diagrams showing representative FVIII-VWF heterodimers and constructs FVIII169, FVIII286, VWF057, VWF059, and VWF062. For example, the FVIII169 construct comprises an FVIII protein with a B domain deletion having an R1648A substitution fused to the Fc region, wherein the XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to the mature full-length FVIII (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-Fc). The FVIII286 construct comprises an FVIII protein fused to the Fc region with a B domain deletion having an R1648 substitution, wherein an XTEN sequence (e.g., AE288) is inserted at amino acid 745 corresponding to the mature full-length FVIII, and there is an additional a2 region between FVIII and Fc (A1-a1-A2-a2-288XTEN-a3-A3-C1-C2-a2-Fc). VWF057 is a VWF-Fc fusion construct comprising a D'D3 domain of a VWF protein attached to the Fc region via a VWF adapter containing an LVPRG thrombin site (“LVPRG”; SEQ ID NO: 6) and a GS adapter (“GS”). The D'D3 domain has two amino acid substitutions, C336A and C379A, and the XTEN sequence (AE144) is inserted between the D'D3 domain and the VWF adapter (D'D3-144XTEN-GS+LVPRG-Fc). VWF059 is a VWF-Fc fusion construct containing a D'D3 domain of a VWF protein linked to the Fc region via acidic region 2 (a2) of FVIII as a VWF linker (with two amino acid substitutions, C336A and C379A), wherein an XTEN sequence (AE144) is inserted between the D'D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct containing a D'D3 domain of a VWF protein linked to the Fc region (with two amino acid substitutions, C336A and C379A), wherein an XTEN sequence (AE144) is inserted between the D'D3 domain and the Fc region (D'D3-144XTEN-Fc).

[0060] Figure 10The diagram shows FVIII / VWF heterodimer constructs, such as FVIII169 / VWF057, FVIII169 / VWF059, FVIII169 / VWF059A, and FVIII169 / VWF073. Arrows indicate sites where optional adapters are added to introduce thrombin cleavage sites. FVIII169 / VWF057 has an adapter containing an LVPRG (SEQ ID NO: 6). FVIII169 / VWF059 has an adapter containing the FVIII a2 region (i.e., is...). dkntgdyyedsyedisayllskknnaieprsfs The connector is dkth (SEQ ID NO: 106). FVIII169 / VWF059A has a connector including a truncated FVIII a2 region (i.e., dkntgdyyeds yedisayllsknnaieprsfs The connector of dkth (SEQ ID NO: 88). FVIII169 / VWF073 has a connector within the VWF073 construct (SEQ ID NO: 175) consisting of a segment composed of IEPRSFS (SEQ ID NO: 194) containing the FVIII a2 region.

[0061] Figure 11 The small image AC shows SDS-PAGE images after thrombin digestion of FVIII169 / VWF057 and FVIII-Fc controls. Figure 11 Inset A shows staining of an SDS-PAGE gel with anti-D3 antibody (AB 96340). The arrows highlight “LCFc:D'D3-XTEN-Fc”, which is the unly cleaved full-length FVIII169 / VWF057; and “D'D3-144 XTEN”, which is the fragment obtained after cleavage by thrombin. Figure 11 Inset B shows staining of an SDS-PAGE gel with anti-HC antibody (GMA012). The arrows highlight the FVIII heavy chain (“HC”) and the FVIII A2 domain. Figure 11 Inset C shows an overlay of insets A and B. Samples were collected at the time points indicated at the top of each plot. Arrows point to the relevant proteins.

[0062] Figure 12 The small image (AC) shows an SDS-PAGE image after thrombin digestion of FVIII169 / VWF059. Figure 12 Inset A shows staining of an SDS-PAGE gel with anti-D3 antibody (AB 96340). The arrows highlight “LCFc:D'D3-XTEN-Fc”, which is the unlycracked full-length FVIII169 / VWF059; and “D'D3-144 XTEN”, which is the fragment obtained after lysis by thrombin. Figure 12Inset B shows staining of the SDS-PAGE gel with anti-HC antibody (GMA012). Arrows highlight the unly cleaved full-length FVIII169 / VWF059; D'D3-144 XTEN-a3, which is the fragment obtained after cleavage by thrombin; and "A2", which is the A2 domain of FVIII. Figure 12 Inset C shows an overlay of insets A and B. Samples were collected at the time points indicated at the top of each plot.

[0063] Figure 13 This shows acute efficacy data for HemA mice treated with FVIII169 / VWF059 (circles) compared to HemA mice treated with the BDD-FVIII control (squares). Blood loss was measured after tail clamping. p=0.9883. Detailed Implementation

[0064] The present invention relates to a chimeric protein comprising two polypeptides, the first polypeptide comprising an FVIII protein fused to a first Ig constant region, and the second polypeptide comprising a VWF protein fused to a second Ig constant region or a portion thereof via an XTEN sequence, wherein the XTEN sequence contains less than 288 amino acids.

[0065] I. Definition

[0066] It should be noted that the term "a" refers to one or more of that entity; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a," "one or more," and "at least one" are used interchangeably in this document.

[0067] Furthermore, when used herein, “and / or” should be considered as each of the two specified features or components disclosed herein, with or without the other. Thus, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0068] It should be understood that whenever an aspect is described in this document as “comprising”, other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd edition, 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, revised edition, 2000, Oxford University Press provide comprehensive dictionaries for those skilled in the art using many of the terms used in this disclosure.

[0070] Units, prefixes, and symbols are represented in their accepted Système International de Unites (SI) form. Numerical ranges include the numerical values ​​defining the range. Unless otherwise indicated, amino acid sequences are written from left to right with the amino-to-carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which are derived from the entire specification. Therefore, the terms defined below are more fully explained by reference to the entire specification.

[0071] The term “about” is used in this document to mean approximately, roughly, about, or around. When the term “about” is used with a numerical range, it modifies that range by extending the boundaries above and below the stated value. Generally, the term “about” can modify values ​​above and below the stated value by varying upwards or downwards (increasing or decreasing), for example, by 10%.

[0072] The terms "polynucleotide" or "nucleotide" are intended to cover both single and multiple nucleic acids, and refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In some embodiments, polynucleotides contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as seen in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. "Isolated" nucleic acid or polynucleotide means a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, for the purposes of this invention, a recombinant polynucleotide encoding a factor VIII polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or recombinant polynucleotides purified (partially or substantially purified) from other polynucleotides in solution form. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this invention. The isolated polynucleotides or nucleic acids of this invention further include molecules synthesized from them. In addition, polynucleotides or nucleic acids may include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0073] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While “stop codons” (TAG, TGA, or TAA) are not typically translated into amino acids, they can be considered part of a coding region; however, any side-joint sequences (e.g., promoters, ribosome binding sites, transcription terminators, introns, etc.) are not part of a 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 polynucleotide construct, e.g., on a single vector, or in a separate polynucleotide construct, e.g., on a separate (different) vector. Thus, a single vector may contain only a single coding region, or may contain two or more coding regions; for example, a single vector may encode binding domain A and binding domain B separately as described below. Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions fused to or not fused to nucleic acids encoding the binding domains of the present invention. Heterogeneous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains.

[0074] Certain proteins secreted by mammalian cells associate with a secretion signal peptide, which is cleaved from the mature protein once the elongated protein chain has been evicted across the rough endoplasmic reticulum. Those skilled in the art will understand that the signal peptide is typically fused to the N-terminus of a polypeptide and cleaved from the complete or “full-length” polypeptide to produce the secreted or “mature” form of the polypeptide. In some embodiments, a native signal peptide, or a functional derivative thereof, retaining that sequence, is used to guide the secretion of the polypeptide operatively associated with it. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0075] The term "downstream" when referring to a nucleotide sequence means a nucleic acid or nucleotide sequence located at the 3' end of a reference nucleotide sequence. In some embodiments, the downstream nucleotide sequence refers to a sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site. The term "downstream" when referring to a polypeptide sequence means an amino acid or amino acid insertion site located at the C-terminus of a reference amino acid. For example, an insertion site immediately downstream of amino acid 745 corresponding to mature wild-type FVIII protein means an insertion site between amino acids 745 and 746 corresponding to mature wild-type FVIII protein.

[0076] The term "upstream" refers to a nucleotide sequence located 5' of a reference nucleotide sequence. In some implementations, the upstream nucleotide sequence refers to a sequence located 5' to the side of a coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0077] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequence), inside, or downstream (3' non-coding sequence) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, then the polyadenylation signal and transcription termination sequence will typically be located at the 3' end of the coding sequence.

[0078] Polynucleotides encoding gene products (e.g., polypeptides) may include promoters and / or other transcriptional or translational control elements operatively associated with one or more coding regions. In an operative association, the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory regions in such a way that the expression of the gene product is placed under the influence or control of the regulatory region. For example, if inducing promoter function results in the transcription of mRNA encoding a gene product encoded by the coding region, and if the nature of the connection between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or the ability of the DNA template to be transcribed, then the coding region and the promoter are “operatively associated.” In addition to promoters, other transcriptional control elements (e.g., enhancers, operons, repressors, and transcription termination signals) may also be operatively associated with coding regions to direct the expression of the gene product.

[0079] Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, those functioning in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcomavirus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globulin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Also suitable transcriptional control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters induced by interferon or interleukin).

[0080] Similarly, a variety of translation control elements are known to those skilled in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from microRNA viruses (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

[0081] As used herein, the term "expression" refers to the process by which polynucleotides produce gene products (e.g., RNA or polypeptides). This includes, but is not limited to, the transcription of polynucleotides into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into polypeptides. Expression produces "gene products." As used herein, gene products can be nucleic acids (e.g., messenger RNA produced through gene transcription) or polypeptides translated from transcripts. Gene products described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation or splicing) or polypeptides with post-translational modifications (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage).

[0082] "Vector" refers to any medium used to clone and / or transfer nucleic acids into host cells. A vector can be a replicon to which another nucleic acid segment can be linked to cause the linked segment to replicate. "Replicon" refers to any genetic element (e.g., plasmid, bacteriophage, phage, chromosome, virus) that acts as an autonomous replication unit in vivo, i.e., capable of replicating under its own control. The term "vector" includes both viral and non-viral vectors used to introduce nucleic acids into cells in vitro, ex vivo, or in vivo. Many vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be achieved by ligating an appropriate polynucleotide fragment into a selected vector having complementary sticky ends.

[0083] Vectors can be engineered to encode selectable markers or reporter agents that provide selection or identification of cells already containing the vector. Expression of selectable markers or reporter agents allows for the identification and / or selection of host cells that contain and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include genes providing resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, such as anthocyanin regulatory genes, isopentyltransferase genes, etc. Examples of reporter agents known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered as reporter agents.

[0084] The term "plasmid" refers to an extrachromosomal element that often carries a gene that is not part of the cell's central metabolism and is typically in the form of a circular double-stranded DNA molecule. This element can be a linear, circular, or supercoiled autonomously replicating sequence, a genome-integrated sequence, a bacteriophage, or a nucleotide sequence derived from any source, having single-stranded or double-stranded DNA or RNA. Many of these nucleotide sequences have been conjugated or reassembled into a unique structure capable of introducing a promoter fragment and a selected gene product, along with an appropriate 3' untranslated sequence, into the cell.

[0085] Usable eukaryotic viral vectors include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and poxvirus (e.g., vaccinia virus) vectors, baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cell transfection agents), DNA-protein complexes, and biopolymers.

[0086] A cloning vector is a sequentially replicated nucleic acid unit of a certain length, containing a "replicon" (such as a plasmid, bacteriophage, or granulosome) as the origin of replication, to which another nucleic acid segment can be ligated to cause the ligated segment to replicate. Some cloning vectors can replicate in one cell type (e.g., bacteria) and be expressed in another cell type (e.g., eukaryotic cells). Cloning vectors typically contain one or more sequences that can be used to select the cell containing the vector and / or one or more multiple cloning sites for inserting the target nucleic acid sequence.

[0087] The term "expression vector" refers to a medium designed to enable the expression of an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is positioned in a manner operatively associated with the regulatory region as described above.

[0088] The vector is introduced into the host cell by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, liposome transfection (lysosome fusion), using a gene gun, or a DNA vector transporter.

[0089] As used herein, “culture” means the incubation of cells in vitro under conditions that allow cell growth or division, or the maintenance of cells in a viable state. As used herein, “cultured cells” means cells that have multiplied in vitro.

[0090] As used herein, the term "polypeptide" is intended to cover both single "polypeptides" and multiple "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains having two or more amino acids, and does not refer to a product of a specific length. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains having two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" also means a product having post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modifications by non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any manner, including through chemical synthesis.

[0091] "Isolated" polypeptides or fragments, variants, or derivatives thereof refer to polypeptides that are not in their native environment. No specific level of purification is required. For example, isolated polypeptides may simply be removed from their native or natural environment. For the purposes of this invention, recombinant polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique.

[0092] This invention also includes fragments or variants of polypeptides and any combination thereof. The terms "fragment" or "variant," when referring to the polypeptide-binding domain or binding molecule of this invention, include any polypeptide that retains at least some properties of the reference polypeptide (e.g., FcRn binding affinity of the Fc binding domain or Fc variant, coagulation activity of the FVIII variant, or FVIII binding activity of the VWF fragment). In addition to the specific antibody fragments discussed elsewhere herein, polypeptide fragments also include proteolytic fragments and deletion fragments, but not naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide-binding domain or binding molecule of this invention include fragments as described above and polypeptides whose amino acid sequences are altered due to amino acid substitution, deletion, or insertion. Variants may be naturally occurring or non-natural. Non-natural variants may be generated using mutagenesis techniques known in the art. Variant polypeptides may contain conserved or non-conserved amino acid substitutions, deletions, or additions.

[0093] As used herein, the term "VWF protein" or "VWF protein" means any VWF fragment that interacts with FVIII and retains at least one or more properties typically provided by full-length VWF to FVIII, such properties as preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that could lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII rather than VWF-bound FVIII, and / or stabilizing the interaction between the FVIII heavy and light chains.

[0094] "Conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is substituted by another amino acid from the same side chain family, the substitution is considered conserved. In another embodiment, a string of amino acids may be replaced by a structurally similar string of conserved substitutions that differ in the sequence and / or composition of the side chain family members.

[0095] As is known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with that of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package for Unix, 8th Edition, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to obtain the optimal homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is 95% identical to the reference sequence of the present invention, parameters should of course be set to calculate the percentage of identity over the full length of the reference polypeptide sequence and to allow homology vacancies to account for up to 5% of the total number of amino acids in the reference sequence.

[0096] As used herein, the “corresponding amino acid” or “equivalent amino acid” in a VWF or FVIII protein sequence is identified by aligning the first VWF or FVIII sequence to maximize the identity or similarity with the second VWF or FVIII sequence. The numbering used to identify the equivalent amino acid in the second VWF or FVIII sequence is based on the numbering used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0097] As used herein, the term "insertion site" refers to a position in an FVIII polypeptide or a fragment, variant, or derivative thereof immediately upstream of the location of the insertable heterologous portion. An "insertion site" is designated by a number corresponding to the amino acid at the insertion site in mature natural FVIII (SEQ ID NO: 65), immediately adjacent to the N-terminus of the insertion site. For example, the phrase "a3 contains XTEN at the insertion site corresponding to amino acid 1656 of SEQ ID NO: 65" indicates that the heterologous portion is located between the two amino acids corresponding to amino acids 1656 and 1657 of SEQ ID NO: 65.

[0098] As used herein, the phrase "immediately downstream of an amino acid" refers to the position immediately following the terminal carboxyl group of the amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position immediately following the terminal amine group of the 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 synonymously with "inserted at the insertion site."

[0099] As used herein, the terms “insertion,” “inserted,” “inserted into,” or grammatically relevant terms refer to the position of XTEN in the chimeric polypeptide relative to a similar position in native mature human FVIII. As used herein, these terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII and do not indicate, imply, or imply any method or process used to prepare the chimeric polypeptide. For example, with respect to the chimeric polypeptide provided herein, the phrase “insertion of XTEN immediately downstream of residue 745 of the FVIII polypeptide” means that the chimeric polypeptide contains XTEN immediately downstream of amino acid 745, which corresponds to amino acid 745 in native mature human FVIII, defined, for example, by amino acids 745 and 746 corresponding to native mature human FVIII.

[0100] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. Amino acid sequences typically found in individual proteins can be fused together in polypeptide form, or amino acid sequences typically found in the same protein can be newly arranged and placed in a fusion polypeptide, such as the fusion of the factor VIII domain and the Ig Fc domain of this invention. Fusion proteins are produced, for example, by chemical synthesis or by generating and translating polynucleotides in which peptide regions are encoded in the desired relationship. Chimeric proteins may further comprise a second amino acid sequence associated with the first amino acid sequence via covalent non-peptide or non-covalent bonds.

[0101] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life can be expressed as the time required for half the amount administered to a subject to be cleared from circulation and / or other tissues in an animal. When constructing a clearance profile of a given polypeptide over time, the profile is typically biphasic, consisting of a rapid α phase and a longer β phase. The α phase typically represents the equilibrium reached between the intravascular and extravascular spaces of the administered Fc polypeptide and is determined in part by the size of the polypeptide. The β phase typically represents the catabolic metabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and FVIII-containing chimeric proteins are monophasic and therefore do not have an α phase, but only a single β phase. Therefore, in some 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 human antibodies in humans is 21 days.

[0102] As used herein, the term "link" refers to the covalent or non-covalent attachment of a first amino acid sequence or nucleotide sequence to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence may be directly attached to or adjacent to the second amino acid or nucleotide sequence, or an intercalation sequence may covalently attach the first sequence to the second sequence. The term "link" not only means the fusion of the first amino acid sequence at its C-terminus or N-terminus into the second amino acid sequence, but also includes the insertion of the entire first amino acid sequence (or the second amino acid sequence) into any two amino acids of the second amino acid sequence (or correspondingly, the first amino acid sequence). In one embodiment, the first amino acid sequence may be linked to the second amino acid sequence via a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence via a phosphodiester bond or a linker. The linker may be a peptide or polypeptide (for polypeptide chains), a nucleotide or nucleotide chain (for nucleotide chains), or any chemical moiety (for both polypeptide chains and polynucleotide chains). The term "link" is also indicated by a hyphen (-).

[0103] As used herein, the term "association with" refers to the formation of a covalent or non-covalent bond between a first amino acid chain and a second amino acid chain. In one embodiment, the term "association with" means a covalent non-peptide bond or a non-covalent bond. This association may be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or disulfide bridge with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 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, which are numbered using the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metallic bonds, hydrogen bonds, disulfide bonds, σ bonds, π bonds, δ bonds, glycosidic bonds, agnostic bonds, bent bonds, dipole bonds, π-opposite bonds, double bonds, triple bonds, quadruple bonds, pentaple bonds, hexaple bonds, conjugation, hyperconjugation, aromaticity, hapticity, or anti-bonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metallic bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetrical hydrogen bonds), van der Walls forces, London dispersion forces, mechanical bonds, halogen bonds, metalophilicity, insertion, stacking, entropic forces, or chemical polarity.

[0104] As used herein, the term "monomer-dimer hybrid" refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain associated with each other by disulfide bonds, wherein the first chain comprises a coagulation factor (e.g., factor VIII) and a first Fc region, and the second chain comprises, substantially comprises, or comprises a second Fc region without a coagulation factor. Therefore, a monomer-dimer hybrid construct is a hybrid comprising a monomeric aspect having only one coagulation factor and a dimeric aspect having two Fc regions.

[0105] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Some enzymatic cleavage sites include intracellular processing sites. In one embodiment, the polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during a coagulation cascade, such that cleavage at said site occurs at the site of clot formation. Exemplary sites include, for example, those recognized by thrombin, factor XIa, or factor Xa. Exemplary FXIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3). Exemplary thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), and IEPRSFS (SEQ ID NO: 194). Other enzymatic cleavage sites are known in the art and are described elsewhere herein.

[0106] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide that is a target of an enzyme that functions after the polypeptide is translated. In one embodiment, the enzyme functions during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before the protein is secreted from the cell. Examples of processing sites include, for example, those targeted by the PACE / furin family of endopeptidases (where PACE is an acronym for paired basic amino acid cleaving enzyme). These enzymes are localized to the Golgi membrane and cleave proteins on the carboxyl-terminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein, enzymes of the "furin protease" family include, for example, PCSK1 (also known as PC1 / Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin protease 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.

[0107] In constructs that include more than one processing or cleavage site, it should be understood that the sites may be the same or different.

[0108] The term "furin protease" refers to the enzyme corresponding to EC number 3.4.21.75. Furin protease is a subtilis protease-like protonopeptide, also known as PACE (paired basic amino acid lyase). Furin protease causes the deletion of segments of inactive precursor proteins to convert them into biologically active proteins. During its intracellular transport, the propeptide of VWF can be cleaved from the mature VWF molecule by furin protease. In some embodiments, furin protease cleaves D1D2 from D'D3 of VWF. In other embodiments, the nucleotide sequence encoding furin protease can be expressed together with the nucleotide sequence encoding the VWF fragment so that the D1D2 domain can be removed by intracellular cleavage of furin protease.

[0109] In constructs that include more than one processing or cleavage site, it should be understood that the sites may be the same or different.

[0110] As used herein, “processable connector” means a connector that includes at least one intracellular processing site described elsewhere in this document.

[0111] As used herein, a hemostatic disorder refers to a genetically inherited or acquired condition characterized by a tendency to bleed spontaneously or due to trauma, resulting from an impaired or absent ability to form fibrin clots. Examples of such disorders include hemophilia. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease"), and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, van Wilbond disease; factor XI deficiency (PTA deficiency); factor XII deficiency; deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII; and Bernard-Soulier syndrome, which is a GPIb deficiency or impairment. VWF receptor GPIb can be defective, leading to a lack of primary clot formation (primary hemostasis) and an increased tendency to bleed, as well as thrombasthenia of Glanzman and Naegeli. In liver failure (acute and chronic forms), there is an insufficiency of clotting factors produced by the liver; this can increase the risk of bleeding.

[0112] The chimeric molecules of this invention can be used preventively. As used herein, the term "preventive treatment" refers to the administration of the molecule prior to a bleeding episode. In one embodiment, a subject requiring a general hemostatic agent is undergoing or is about to undergo surgery. The chimeric protein of this invention can be administered as a preventative agent before or after surgery. The chimeric protein of this invention can be administered during or after surgery to control acute bleeding episodes. Surgeries may include, but are not limited to, liver transplantation, hepatectomy, dental procedures, or stem cell transplantation.

[0113] The chimeric protein of this invention is also used for on-demand treatment. The term "on-demand treatment" refers to the administration of the chimeric molecule in response to symptoms of bleeding or before an activity that may lead to bleeding. In one aspect, on-demand treatment may be given to a subject at the onset of bleeding (such as after an injury) or when bleeding is expected (such as before surgery). In another aspect, on-demand treatment may be given before an activity that increases the risk of bleeding (such as contact sports).

[0114] As used in this article, the term "acute bleeding" refers to an episode of bleeding regardless of the underlying cause. For example, a subject may have trauma, uremia, a hereditary bleeding disorder (such as factor VII deficiency), a platelet disorder, or be resistant due to the production of antibodies against coagulation factors.

[0115] As used herein, treatment refers to, for example, reducing the severity of a disease or symptom; shortening the duration of a disease; improving one or more symptoms associated with a disease or symptom; providing a beneficial effect to a subject with a disease or symptom, without necessarily curing the disease or symptom; or preventing or treating one or more symptoms associated with a disease or symptom. In one implementation, the term "treatment" means maintaining the FVIII trough level in a subject at at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL by administering the chimeric protein or VWF fragment of the present invention. In another implementation, treatment means maintaining the trough level of FVIII between approximately 1 and approximately 20 IU / dL, approximately 2 and approximately 20 IU / dL, approximately 3 and approximately 20 IU / dL, approximately 4 and approximately 20 IU / dL, approximately 5 and approximately 20 IU / dL, approximately 6 and approximately 20 IU / dL, approximately 7 and approximately 20 IU / dL, approximately 8 and approximately 20 IU / dL, approximately 9 and approximately 20 IU / dL, or approximately 10 and approximately 20 IU / dL. Treatment of the disease or condition may also include maintaining FVIII activity in the subject at a level similar to that in non-hemophilic subjects of at least approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0116] II. Chimeric proteins

[0117] This invention relates to the use of a VWF protein fused to an XTEN sequence to extend the half-life of a chimeric protein by preventing or inhibiting the association of an FVIII half-life limiting factor (i.e., endogenous VWF) with the FVIII protein. Endogenous VWF associates with approximately 95% to approximately 98% of FVIII in a non-covalent complex. Although endogenous VWF is an FVIII half-life limiting factor, it is also known that endogenous VWF binding to the FVIII protein can protect FVIII in various ways. For example, a full-length VWF (in a multimeric form with approximately 250 kDa) can protect FVIII from protease cleavage and FVIII activation, stabilize the FVIII heavy and / or light chains, and prevent FVIII from being cleared by scavenger receptors. However, endogenous VWF also limits the FVIII half-life by preventing endocytosis and by systemically clearing the FVIII-VWF complex via the VWF clearance pathway. Without being bound by theory, it is believed that endogenous VWF is a half-life limiting factor preventing chimeric proteins fused to half-life extensions from having a half-life approximately twice that of wild-type FVIII. Therefore, this invention relates to using VWF proteins (e.g., VWF fragments) containing D' and D3 domains to prevent or inhibit the interaction between endogenous VWF and FVIII proteins, and simultaneously increasing the half-life of the resulting FVIII protein by using an XTEN sequence combined with an Ig constant region or a portion thereof. Specifically, this invention shows that shorter XTEN sequences (i.e., XTENs containing less than 288 amino acids in length, i.e., XTENs shorter than 288 amino acids) are superior in extending the half-life of chimeric proteins.

[0118] In one embodiment, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing a D' domain and a D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via an XTEN sequence therebetween, wherein the XTEN sequence contains less than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide. In another embodiment, the XTEN sequence in the second polypeptide consists of an amino acid sequence of length between 12 and 287 amino acids. In other embodiments, the chimeric protein exhibits a longer half-life compared to a corresponding fusion protein comprising a first polypeptide and a second polypeptide, wherein the second polypeptide comprises an XTEN sequence containing at least 288 amino acids (e.g., AE288, e.g., SEQ ID NO: 8). In other embodiments, the XTEN sequence in the second polypeptide contains at least about 36, at least about 42, at least about 72, or at least about 144 amino acids, but less than 288 amino acids, such as AE42, AE72, AE144 (AE144, AE144_2A, AE144_3B, AE144_4A, AE144_5A, AE144_6B), AG42, AG72, or AG144 (AG144, AG144_A, AG144_B, AG144_C, AG144_F), for example, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 64. NO: 63.

[0119] The chimeric protein of the present invention may further include a second XTEN sequence that links the FVIII protein to the first Ig constant region or a portion thereof.

[0120] In some embodiments, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing a D' domain and a D3 domain of VWF, the VWF protein being fused to a second Ig constant region or a portion thereof via a first XTEN sequence therebetween, wherein the XTEN sequence contains less than 288 amino acid residues, and wherein the first polypeptide is linked or associated with the second polypeptide, and wherein the first polypeptide further comprises a second XTEN sequence inserted at one or more insertion sites within the FVIII protein or fused to the FVIII protein and / or a portion thereof. Thus, in one embodiment, the second XTEN sequence is inserted at one or more insertion sites within the FVIII protein. In another embodiment, the second XTEN sequence is fused to the FVIII protein and / or a portion thereof. In other embodiments, the second XTEN sequence is inserted at one or more insertion sites within the FVIII protein, and a third XTEN sequence is fused to the FVIII protein and / or a portion thereof.

[0121] The second XTEN sequence and / or the third XTEN sequence can be any length of XTEN amino acid. For example, second and / or third XTEN sequences, such as AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144, are disclosed elsewhere in this document, for example, SEQ ID NO: 8; SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17; SEQ ID NO: 54; SEQ ID NO: 19; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 15; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 63. In a particular embodiment, the second XTEN sequence and / or the third XTEN sequence is AE288 or AG288, for example, SEQ ID NO: 8 or 19.

[0122] In some embodiments, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof via an optional linker, wherein an optional XTEN sequence (X2) is inserted into one or more insertion sites within the FVIII protein or fused to the FVIII protein or the first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing a D' domain and a D3 domain of VWF, the VWF protein being fused to the second Ig constant region or a portion thereof via an XTEN sequence (X1) between the VWF protein and the second Ig constant region or a portion thereof, wherein the XTEN sequence (X1) contains less than 288 amino acid residues and is fused to the VWF protein via a linker, and wherein the first polypeptide and the second polypeptide are associated. In some embodiments, the present invention relates to a chimeric protein comprising (i) a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof via an optional linker, wherein an optional XTEN sequence (X2) is inserted into or fused to one or more insertion sites within the FVIII protein or to the FVIII protein or to the first Ig constant region or a portion thereof, and (ii) a second polypeptide comprising a VWF protein containing a D' domain and a D3 domain of VWF, the VWF protein being fused to the second Ig constant region or a portion thereof via an XTEN sequence (X1) between the VWF protein and the second Ig constant region or a portion thereof, wherein the XTEN sequence (X1) contains less than 288 amino acid residues and is fused to the second Ig constant region or a portion thereof via a linker, and wherein the first polypeptide and the second polypeptide are associated. In other embodiments, the linker that fuses the XTEN sequence (X1) to the VWF protein or the second Ig constant region or a portion thereof is a cleavable linker. Non-limiting examples of cleavable linkers are shown elsewhere herein. In a particular embodiment, the linker is a thrombin-cleavable linker.

[0123] In some embodiments, the chimeric protein is two polypeptide chains, a first chain comprising the first polypeptide described above, and a second chain comprising the second polypeptide described above. For example, the two polypeptide chains comprise (i) a first chain comprising a single-chain FVIII protein, a first Ig constant region or a portion thereof, and an optional XTEN sequence inserted into or fused to one or more insertion sites within the FVIII protein or to the FVIII protein or to the first Ig constant region or a portion thereof, and (ii) a second chain comprising a VWF protein fused to the second Ig constant region or a portion thereof via an XTEN sequence (X1) therebetween, wherein the XTEN sequence (X1) contains less than 288 amino acids.

[0124] In some embodiments, the chimeric protein is two polypeptide chains, the first chain containing the heavy chain of the FVIII protein, and the second chain containing, from the N-terminus to the C-terminus, the light chain of the FVIII protein, an optional XTEN sequence inserted into or fused to one or more insertion sites within the FVIII protein or to the FVIII protein or a portion thereof, the first Ig constant region or a portion thereof, an optional adapter (e.g., a processable adapter), the VWF protein, the XTEN sequence (X1), a second optional adapter (e.g., a cleavable adapter), and a second Ig constant region or a portion thereof.

[0125] In other embodiments, the chimeric protein is three polypeptide chains: (i) a first chain comprising the heavy chain of the FVIII protein; (ii) a second chain comprising the light chain of the FVIII protein, a first Ig constant region or a portion thereof, and an optional XTEN sequence inserted into or fused to the heavy chain or the light chain of the FVIII protein at one or more insertion sites or fused to the FVIII protein or the first Ig constant region or a portion thereof; and (iii) a third chain comprising a VWF protein fused to a second Ig constant region or a portion thereof via an XTEN sequence (X1) between the two chains, wherein the first chain and the second chain are associated by non-covalent bonds, such as metal bonds, and the second chain and the third chain are associated by covalent bonds, such as disulfide bonds.

[0126] In other embodiments, the chimeric protein is a single-chain protein comprising, from its N-terminus to its C-terminus, a single-chain FVIII protein, an optional XTEN sequence inserted into or fused to one or more insertion sites within the FVIII protein or to a portion thereof, and a first Ig constant region or a portion thereof, an optional adapter (e.g., a processable adapter), a VWF protein, an XTEN sequence (X1), a second optional adapter (e.g., a cleavable adapter), and a second Ig constant region or a portion thereof.

[0127] In some implementations, the chimeric protein comprises one of the following formulas (a)-(hh): (a) FVIII-F1:F2-L2-X-L1-V; (b) FVIII-F1:V-L1-X-L2-F2; (c) F1-FVIII:F2-L2-X-L1-V; (d) F1-FVIII:V-L1-X-L2-F2; (e) FVIII-X2-F1:F2-L2-X1-L1-V; (f) FVIII-X2-F1:V-L1-X1-L2-F2; (g) FVIII(X2)-F1:F2-L2-X1-L1-V; (h) FVIII(X2)-F1:V-L1-X1-L2-F2; (i) F1-X2-F1:F2-L2-X1-L1-V; (j) F1-X2-F1:V-L1-X1-L2-F2; (k)V-L1-X-L2-F2-L3-FVIII-L4-F1; (l)V-L1-X-L2-F2-L3-F1-L4-FVIII; (m) F1-L4-FVIII-L3-F2-L2-X-L1-V; (n) FVIII-L4-F1-L3-F2-L2-X-L1-V; (o) FVIII-L4-F1-L3-V-L1-X-L2-F2; (p)FVIII-L4-F1-L3-F2-L2-X-L1-V; (q) F2-L2-X-L1-V-L3-F1-L4-FVIII; (r)F2-L2-X-L1-V-L3-FVIII-L4-F1; (s)V-L1-X1-L2-F2-L3-FVIII(X2)-L4-F1; (t)V-L1-X1-L2-F2-L3-F1-L4-FVIII(X2); (u) F1-L4-FVIII(X2)-L3-F2-L2-X1-L1-V; (v) F-L4-FVIII(X2)-L3-V-L1-X1-L2-F2; (w) FVIII(X2)-L4-F1-L3-V-L1-X1-L2-F2; (x)FVIII(X2)-L4-F1-L3-F2-L2-X1-L1-V; (y) F2-L2-X1-L1-V-L3-F1-L4-FVIII(X2); (z) F2-L2-X1-L1-V-L3-FVIII(X2)-L4-F1; (aa) V-L1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1; (bb) V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII; (cc) F1-L5-X2-L4-FVIII-L3-F2-L2-X2-L1-V; (dd) F1-L5-X2-L4-FVIII-L3-V-L1-X2-L2-F2; (ee) FVIII-L5-X2-L4-F2-L3-V-L1-X1-L2-F1; (ff) FVIII-L5-X2-L4-F2-L3-F1-L2-X1-L1-V; (gg) F1-L2-X1-L1-V-L3-F2-L4-X2-L5-FVIII; or (hh) F1-L2-X1-L1-V-L3-FVIII-L5-X2-L4-F2; V represents the VWF protein, which contains a D' domain and a D3 domain. X or X1 is the first XTEN sequence containing fewer than 288 amino acids. X2 is the second XTEN sequence. FVIII includes the FVIII protein.

[0128] FVIII(X2) includes an FVIII protein having a second XTEN sequence inserted at one or more insertion sites within the FVIII protein. F1 is the first Ig constant region or a part thereof. F2 is the second Ig constant region or a part thereof. L1, L2, L3, L4, or L5 are optional connectors. (-) is a peptide bond; and (:) indicates a covalent or non-covalent bond.

[0129] In one embodiment, X or X1 consists of an amino acid sequence of length between 12 and 287 amino acids. In another embodiment, the chimeric protein exhibits a longer half-life compared to a corresponding fusion protein containing the formula AE288 (e.g., SEQ ID NO: 8).

[0130] In other embodiments, X or X1 contains at least about 36, at least about 42, at least about 72, or at least about 144 amino acids, but less than 288 amino acids, such as AE42, AE72, AE144 (AE144, AE144_2A, AE144_3B, AE144_4A, AE144_5A, AE144_6B), AG42, AG72, or AG144 (AG144, AG144_A, AG144_B, AG144_C, AG144_F), for example SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 14; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; or SEQ ID NO: 63.

[0131] In other embodiments, X2 comprises an amino acid sequence of at least about 36 amino acids, at least 42 amino acids, at least 144 amino acids, at least 288 amino acids, at least 576 amino acids, or at least 864 amino acids, such as AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG144, for example, SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 8; SEQ ID NO: 11; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 14. In a particular embodiment, X2 is AE288 or AG288, for example, SEQ ID NO: 8 or 19.

[0132] In some embodiments, chimeric proteins containing X or X1 and / or X2 have an extended half-life compared to chimeric proteins without X or X1 and / or X2. In other embodiments, L1 and / or L2 are cleavable linkers. In other embodiments, L4 and / or L5 are cleavable linkers.

[0133] II.A. Van Wilbond Factor (VWF) Protein

[0134] VWF (also known as F8VWF) is a large polysaccharide protein present in plasma and constitutively produced in the endothelium (in the Weibel-Palade body), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a protein with 2813 amino acids. Each monomer contains several specific domains with specific functions, namely the D' / D3 domain (which binds factor VIII), the A1 domain (which binds platelet GPIb receptor, heparin and / or possibly collagen), the A3 domain (which binds collagen), the C1 domain (where the RGD domain binds platelet integrin αIIbβ3 when activated in this domain), and the "cysteine ​​knot" domain at the C-terminus of the protein (VWF shares this domain with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ), and β-human chorionic gonadotropin (βHCG).

[0135] In one embodiment, the VWF protein is a VWF fragment. As used herein, the term "VWF fragment" includes, but is not limited to, functional VWF fragments comprising D' and D3 domains capable of inhibiting the binding of endogenous VWF to FVIII. In one embodiment, the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment blocks the VWF binding site on the FVIII protein, thereby inhibiting the interaction between the FVIII protein and endogenous VWF. VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0136] The 2813-amino acid sequence of human VWF is reported in GenBank under accession number NP_000543.2. The nucleotide sequence encoding human VWF is reported in GenBank under accession number NM_000552.3. The nucleotide sequence of human VWF is designated as SEQ ID NO: 20. SEQ ID NO: 21 is the amino acid sequence of the full-length VWF. The domains of VWF are listed in Table 1.

[0137] Table 1. VWF sequences

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] The VWF protein used herein may be a VWF fragment containing the D' and D3 domains of a VWF, wherein the VWF fragment binds 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 VWF domains selected from the group consisting of: A1 domain, A2 domain, A3 domain, D1 domain, D2 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more of these domains, and any combination thereof. In one embodiment, the VWF fragment comprises, is substantially composed of, or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the D1, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the D1, D2, D', and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3, and A1 domains of VWF or fragments thereof. The VWF fragment described herein does not contain a site for binding to a VWF scavenging receptor. In another embodiment, the VWF fragment described herein is not amino acids 764 to 1274 of SEQ ID NO: 21. The VWF fragments of the present invention may comprise any other sequence linked to or fused to the VWF fragment. For example, the VWF fragment described herein may further comprise a signal peptide.

[0147] In one embodiment, a VWF fragment containing both a D' and a D3 domain binds to or associates with the FVIII protein. By binding to or associating with the FVIII protein, the VWF fragment of the present invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy and light chains of FVIII, and prevents FVIII from being cleared by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with the FVIII protein and blocks or prevents the FVIII protein from binding to phospholipids and activated protein C. By preventing or inhibiting the binding of the FVIII protein to endogenous full-length VWF, the VWF fragment of the present invention reduces FVIII clearance by VWF scavenger receptors and thus prolongs the half-life of the chimeric protein. Therefore, the prolonged half-life of the chimeric protein is due to the binding to or association of the FVIII protein with a VWF fragment lacking a VWF scavenger receptor binding site and the shielding or protection of the FVIII protein from endogenous VWF containing a VWF scavenger receptor binding site by the VWF fragment. FVIII proteins bound to or protected by VWF fragments can also be recycled. By eliminating the VWF clearance pathway receptor binding sites contained in the full-length VWF molecule, the FVIII / VWF heterodimer of the present invention is shielded from the VWF clearance pathway, thereby further extending the FVIII half-life.

[0148] In one embodiment, the VWF protein suitable for use in this invention comprises a D' domain and a D3 domain of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 21, and wherein the VWF protein prevents or inhibits endogenous VWF binding to FVIII. In another embodiment, the VWF protein comprises a D' domain and a D3 domain of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 21, and wherein the VWF protein prevents or inhibits endogenous VWF binding to FVIII. In some embodiments, the VWF protein described herein comprises, is substantially composed of, or is composed of: a D' and D3 domain of VWF, said domains being at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 21, wherein said VWF protein prevents or inhibits endogenous VWF binding to FVIII. In other embodiments, the VWF protein comprises, is substantially composed of, or is composed of: D1, D2, D', and D3 domains, said domains being at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23 to 1240 of SEQ ID NO: 21, wherein said VWF protein prevents or inhibits endogenous VWF binding to FVIII. In other embodiments, the VWF protein further comprises a signal peptide operatively linked thereto.

[0149] In some embodiments, the VWF protein suitable for use in this invention is substantially composed of or consists of: (1) a D'D3 domain, a D1D'D3 domain, a D2D'D3 domain, or a D1D2D'D3 domain and (2) up to about 10 amino acids (e.g., any sequence of amino acids 764 to 1240 to 764 to 1250 of SEQ ID NO: 21), up to about 15 amino acids (e.g., any sequence of amino acids 764 to 1240 to 764 to 1255 of SEQ ID NO: 21), up to about 20 amino acids (e.g., any sequence of amino acids 764 to 1240 to 764 to 1260 of SEQ ID NO: 21), up to about 25 amino acids (e.g., amino acids 764 to 1240 to 764 to 1260 of SEQ ID NO: 21). The VWF sequence may be any sequence of amino acids 764 to 1265 of SEQ ID NO: 21, or an additional VWF sequence of up to about 30 amino acids (e.g., any sequence of amino acids 764 to 1240 of SEQ ID NO: 21 to amino acids 764 to 1260 of SEQ ID NO: 21). In certain embodiments, the VWF protein comprising or substantially consisting of the D' and D3 domains is neither amino acids 764 to 1274 of SEQ ID NO: 21 nor a full-length mature VWF. In some embodiments, the D1D2 domain is expressed in trans form along with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis form along with the D'D3 domain.

[0150] In other embodiments, VWF proteins comprising a D'D3 domain linked to the D1D2 domain further include an intracellular cleavage site (e.g., a cleavage site for PACE (furin protease) or PC5) that allows the D1D2 domain to be cleaved from the D'D3 domain after expression. Non-limiting examples of intracellular cleavage sites are disclosed elsewhere herein.

[0151] In other embodiments, the VWF protein comprises a D' domain and a D3 domain, but does not contain an amino acid sequence selected from the group consisting of: (1) amino acids 1241 to 2813 corresponding to SEQ ID NO: 21, (2) amino acids 1270 to 2813 corresponding to SEQ ID NO: 21, (3) amino acids 1271 to 2813 corresponding to SEQ ID NO: 21, (4) amino acids 1272 to 2813 corresponding to SEQ ID NO: 21, (5) amino acids 1273 to 2813 corresponding to SEQ ID NO: 21, (6) amino acids 1274 to 2813 corresponding to SEQ ID NO: 21, and any combination thereof.

[0152] In other embodiments, the VWF protein of the present invention comprises, is substantially composed of, or is composed 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 to 1479 of SEQ ID NO: 21, wherein the VWF protein prevents endogenous VWF from binding to FVIII. In a particular embodiment, the VWF protein is not amino acids 764 to 1274 of SEQ ID NO: 21.

[0153] In some embodiments, the VWF protein of the present invention comprises a D' domain and a D3 domain, but does not comprise at least one VWF domain selected from the group consisting of: (1) an A1 domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2 domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CK domain, (11) a CK domain and a C2 domain, (12) a CK domain, a C2 domain and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, and a B3 domain, (14) a CK domain, a C2 domain, a C1 domain, a B3 domain, and a B2 domain, (15) a CK domain, a C... 2. Structural domain, C1 structural domain, B3 structural domain, B2 structural domain and B1 structural domain, (16) CK structural domain, C2 structural domain, C1 structural domain, B3 structural domain, B2 structural domain, B1 structural domain and D4 structural domain, (17) CK structural domain, C2 structural domain, C1 structural domain, B3 structural domain, B2 structural domain, B1 structural domain, D4 structural domain and A3 structural domain, (18) CK structural domain, C2 structural domain, C1 structural domain, B3 structural domain, B2 structural domain, B1 structural domain, D4 structural domain, A3 structural domain and A2 structural domain, (19) CK structural domain, C2 structural domain, C1 structural domain, B3 structural domain, B2 structural domain, B1 structural domain, D4 structural domain, A3 structural domain, A2 structural domain and A1 structural domain, and (20) any combination thereof.

[0154] In other embodiments, the VWF protein comprises a D'D3 domain and one or more domains or modules. Examples of such domains or modules include, but are not limited to, those disclosed in Zhour et al., Blood, published online April 6, 2012: DOI 10.1182 / blood-2012-01-405134, which is incorporated herein by reference in its entirety. For example, the VWF protein may comprise a D'D3 domain and one or more domains or modules selected from the group consisting of: A1 domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL-4 module, C1 module, C2 module, C3 module, C4 module, C5 module, C6 module, and any combination thereof.

[0155] In other embodiments, the VWF protein is linked to a heterologous moiety, wherein the heterologous moiety is inserted downstream of the N-terminus or C-terminus of the VWF protein or immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the VWF protein. For example, the insertion site of the heterologous moiety in the VWF protein may be in a D' domain, a D3 domain, or both. The heterologous moiety may be a half-life extender.

[0156] In some embodiments, the VWF protein suitable for use in this invention forms polymers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, or higher-order polymers. In other embodiments, the VWF protein is a monomer having only one VWF protein. In some embodiments, the VWF protein of this invention may have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF protein may include amino acid substitutions, deletions, additions, or modifications to prevent the VWF protein from forming disulfide bonds or from forming dimers or polymers. In another embodiment, the amino acid substitution is within the D' and D3 domains. In a particular embodiment, the VWF protein suitable for use in this invention contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 21. The at least one amino acid substitution may be any amino acid not naturally present in wild-type VWF. For example, the amino acid substitution can be any amino acid other than cysteine, such as isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, or histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the formation of multimers of VWF protein.

[0157] In some embodiments, the VWF protein applicable herein may be further modified to improve its interaction with FVIII, for example, by improving its binding affinity for FVIII. As a non-limiting example, the VWF protein includes a serine residue at residue 764 corresponding to amino acid SEQ ID NO: 21 and a lysine residue at residue 773 corresponding to amino acid SEQ ID NO: 21. Residues 764 and / or 773 may contribute to the binding affinity of the VWF protein for FVIII. In other embodiments, the VWF protein applicable to the present invention may have other modifications, such as polyethylene glycolation, glycosylation, hydroxyethyl starchization, or polysialylation.

[0158] II. B. XTEN sequence

[0159] As used herein, “XTEN sequence” refers to an extended length polypeptide having a non-naturally occurring, substantially non-repetitive sequence consisting primarily of small hydrophilic amino acids, wherein said sequence has low or no secondary or tertiary structure under physiological conditions. As a chimeric protein partner, XTEN can act as a carrier, thereby conferring certain desired pharmacokinetic, physicochemical, and pharmaceutical properties, for example, when linked to the VWF protein or FVIII sequence of the present invention to generate a chimeric protein. These desired properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics. As used herein, “XTEN” explicitly excludes antibodies or antibody fragments, such as single-chain antibodies or Fc fragments of light or heavy chains.

[0160] This invention provides a shorter XTEN sequence that, when fused to a VWF protein and / or a second Ig constant region or a portion thereof, provides improved half-life extension properties compared to a longer XTEN sequence. Therefore, the XTEN sequence fused to the VWF protein and / or a portion thereof contains less than 288 amino acids in length, i.e., shorter than 288 amino acids. In one embodiment, the XTEN sequence fused to the VWF protein and / or a portion thereof consists of an amino acid sequence of length between 12 and 287 amino acids. In another embodiment, the XTEN sequence fused to the VWF protein and / or a portion thereof contains at least about 36 amino acids, at least about 42 amino acids, at least about 72 amino acids, or at least about 144 amino acids, but less than 288 amino acids. In other embodiments, the XTEN sequence fused to the VWF protein and / or a portion thereof is selected from AE36, AG36, AE42, AG42, AE72, AG72, AE144, or AG144. In one embodiment, the XTEN sequence fused to the VWF protein and / or a portion thereof is an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 14, wherein the chimeric protein exhibits an improved half-life compared to a chimeric protein without the XTEN sequence.

[0161] The chimeric protein of the present invention may further comprise additional (second, third, or more) XTEN sequences. The additional XTEN sequences may be further fused to the FVIII protein or the first Ig constant region or a portion thereof. The additional XTEN sequences may be of any length. For example, the additional XTEN sequence fused to the FVIII protein or the first Ig constant region or a portion thereof is a peptide or polypeptide having more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In some embodiments, the additional XTEN sequence is a peptide or polypeptide having more than about 20 to about 3,000 amino acid residues, more than about 30 to about 2,500 residues, more than about 40 to about 2,000 residues, more than about 50 to about 1,500 residues, more than about 60 to about 1,000 residues, more than about 70 to about 900 residues, more than about 80 to about 800 residues, more than about 90 to about 700 residues, more than about 100 to about 600 residues, more than about 110 to about 500 residues, or more than about 120 to about 400 residues.

[0162] An XTEN sequence (i.e., an XTEN sequence fused to the VWF protein and / or a portion thereof of the second Ig constant region or thereof, or an XTEN sequence fused to the FVIII protein and / or a portion thereof of the first Ig constant region or inserted at one or more insertion sites within the FVIII protein) may comprise one or more sequence motifs having 9 to 14 amino acid residues or 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 comprises, is substantially composed of, or is composed of 4 to 6 types of amino acids selected from the group consisting of: glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P). See US 2010-0239554A1.

[0163] In some embodiments, the XTEN sequence comprises a non-overlapping sequence motif, wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% of the sequence consists of multiple non-overlapping sequence units selected from a single motif family chosen from Table 2A, thereby producing a family sequence. As used herein, “family” means that the XTEN has a motif selected only from a single motif class from Table 2A; namely, AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and any other amino acids in the XTEN not derived from the family motif are selected to achieve desired properties, such as allowing incorporation of the encoding nucleotide into a restriction site, into a cleavage sequence, or achieving better linkage with FVIII or VWF. In some embodiments of the XTEN family, the XTEN sequence comprises multiple non-overlapping motif units from the AD, AE, AF, AG, AM, AQ, BC, or BD motif families, resulting in an XTEN exhibiting the aforementioned homology range. In other embodiments, the XTEN comprises multiple motif sequence units from two or more motif families from Table 2A. These sequences can be selected to achieve desired physical / chemical characteristics conferred by the amino acid composition of the motif, as described more fully below, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repeatability. In the embodiments described above in this paragraph, the methods described herein can be used to select and assemble motifs incorporated into the XTEN to achieve an XTEN having approximately 36 to approximately 3000 amino acid residues.

[0164] Table 2A. XTEN sequence motifs and motif families with 12 amino acids

[0165]

[0166]

[0167] ● Individual motif sequences that, when used together in various permutations, produce a "family sequence".

[0168] In some embodiments, the XTEN sequence used in this invention is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144. AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG 396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF57 6. AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, A E1044, 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.

[0169] In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of: AE42 (SEQ ID NO: 9), AE72 (SEQ ID NO: 10), AE144_2A (SEQ ID NO: 55), AE144_3B (SEQ ID NO: 56), AE144_4A (SEQ ID NO: 57), AE144_5A (SEQ ID NO: 58), AE144_6B (SEQ ID NO: 59), AG144_A (SEQ ID NO: 60), AG144_B (SEQ ID NO: 61), AG144_C (SEQ ID NO: 62), AG144_F (SEQ ID NO: 63), AE864 (SEQ ID NO: 15), AE576 (SEQ ID NO: 16), AE288 (SEQ ID NO: 17), AE864 (SEQ ID NO: 15), AE576 (SEQ ID NO: 16), AE288 (SEQ ID NO: 17), AE864 (SEQ ID NO: 18), AE864 (SEQ ID NO: 19), AE864 (SEQ ID NO: 10 ... 8) AE288_2 (SEQ ID NO: 54), AE144 (SEQ ID NO: 11), AG864 (SEQ ID NO: 17), AG576 (SEQ ID NO: 18), AG288 (SEQ ID NO: 19), AG144 (SEQ ID NO: 14) and any combination thereof.In another embodiment, the XTEN sequence is selected from the group consisting of: AE42 (SEQ ID NO: 9), AE72 (SEQ ID NO: 10), AE144_2A (SEQ ID NO: 55), AE144_3B (SEQ ID NO: 56), AE144_4A (SEQ ID NO: 57), AE144_5A (SEQ ID NO: 58), AE144_6B (SEQ ID NO: 59), AG144_A (SEQ ID NO: 60), AG144_B (SEQ ID NO: 61), AG144_C (SEQ ID NO: 62), AG144_F (SEQ ID NO: 63), AE864 (SEQ ID NO: 15), AE576 (SEQ ID NO: 16), AE288 (SEQ ID NO: 8), AE288_2 (SEQ ID NO: 54), AE144 (SEQ ID NO: 11), AG864 (SEQ ID NO: 17), AG576 (SEQ ID NO: 18), AG288 (SEQ ID NO: 19), AG144 (SEQ ID NO: 14), and any combination thereof. In a particular embodiment, the XTEN sequence is AE288. The amino acid sequences of certain XTEN sequences of the present invention are shown in Table 2B.

[0170] Table 2B. XTEN sequences

[0171]

[0172]

[0173]

[0174] In embodiments in which the XTEN component comprises less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), or less than 100% of its sequence consists of sequence motifs from Table 3 or XTEN sequences from Tables 4 and 13-17, the other amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but preferably from hydrophilic amino acids, such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) are dispersed throughout the XTEN sequence, located within or between sequence motifs, or concentrated in one or more short segments of the XTEN sequence, for example, to create a linker between the XTEN component and the FVIII or VWF component. In the case where the XTEN component comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids are hydrophobic residues so that the resulting sequence generally lacks secondary structure, for example, not having more than 2% α-helix or 2% β-sheet, as determined by the methods disclosed herein. Hydrophobic residues less advantageous in constructing XTENs include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, XTEN sequences can be designed to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or not contain the following amino acids: cysteine ​​(to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine, and glutamine (to avoid deamidation). Therefore, in some embodiments, the XTEN component, which contains other amino acids besides glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), has a sequence of less than 5% (as measured by the Chou-Fasman algorithm) of residues that promote α-helix and β-sheet formation, and has at least 90% or at least about 95% or more of random coil formation, as measured by the GOR algorithm.

[0175] In other embodiments, the XTEN sequence used in this invention affects the physical or chemical properties, such as pharmacokinetics, of the chimeric protein of this invention. The XTEN sequence used in this invention may exhibit one or more of the following advantageous properties: conformational flexibility, enhanced water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius. In a particular embodiment, the XTEN sequence linked to the FVIII protein of this invention increases pharmacokinetic properties, such as a longer terminal half-life or an increased area under the curve (AUC), to increase the in vivo residence time of the chimeric protein described herein compared to wild-type FVIII. In other embodiments, the XTEN sequence used in this invention increases pharmacokinetic properties, such as a longer terminal half-life or an increased area under the curve (AUC), to increase the in vivo residence time of the FVIII protein compared to wild-type FVIII.

[0176] One embodiment of the present invention is an FVIII / VWF fusion protein comprising an FVIII portion fused to an Fc region and a VWF portion fused to an Fc region, wherein an XTEN sequence (e.g., AE288) is inserted within the FVIII portion, and wherein an XTEN sequence having less than 288 amino acids (e.g., AE144) is inserted between the VWF portion and the Fc portion. As described in the examples, the insertion of an XTEN sequence having less than 288 amino acids between the VWF portion and the Fc portion has a greater effect on the pharmacokinetics of the chimeric protein than the insertion of an XTEN sequence having 288 amino acids between the VWF portion and the Fc portion. For example, compared to an XTEN sequence having 288 amino acids, the insertion of an XTEN sequence having less than 288 amino acids between the VWF portion and the Fc portion in the FVIII / VWF fusion protein can increase the terminal half-life of the chimeric protein. In some embodiments, the terminal half-life is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% relative to the insertion of an XTEN sequence having 288 amino acids. In one particular embodiment, the terminal half-life is increased by at least about 35% relative to the insertion of an XTEN sequence having 288 amino acids. Inserting an XTEN sequence having less than 288 amino acids can also increase the AUC value of the chimeric protein. In some embodiments, the AUC is increased by at least about 50%, at least about 100%, or at least about 200% relative to the insertion of an XTEN sequence having 288 amino acids. In one particular embodiment, the AUC is increased by about two-fold. Inserting an XTEN sequence having less than 288 amino acids can also reduce the clearance rate of the chimeric protein. For example, the clearance rate may be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% relative to the insertion of an XTEN sequence having 288 amino acids. Compared to inserting XTEN with 288 amino acids, inserting XTEN sequences with fewer than 288 amino acids can increase the mean retention time (MRT) and / or decrease the steady-state apparent volume of distribution (Vss).

[0177] A variety of methods and assays can be used to determine the physical / chemical properties of proteins containing the XTEN sequence. These methods include, but are not limited to, analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, reversed-phase HPLC, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractive index, and UV / Vis spectroscopy. Other methods are disclosed in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

[0178] Other examples of XTEN sequences that can be used according to the present invention are disclosed in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1 or 2011 / 0172146A1 or International Patent Publication Nos. WO 2010091122A1, WO 2010144502A2, WO 2010144508A1, WO 2011028228A1, WO 2011028229A1, WO 2011028344A2 or WO 20130122617A1.

[0179] II.C. Factor VIII (FVIII) protein

[0180] Unless otherwise specified, as used herein, “FVIII protein” means a functional FVIII polypeptide that performs its normal function in coagulation. The term FVIII protein includes functional fragments, variants, analogs, or derivatives that retain the function of full-length wild-type factor VIII in the coagulation pathway. “FVIII protein” may be used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include, but are not limited to, the ability to activate coagulation, the ability to act as a cofactor of factor IX, or the ability to [follow the coagulation pathway]. 2+ In the presence of phospholipids, it forms a factor X activating enzyme complex with factor IX, which then converts factor X into its activated form Xa. FVIII proteins can be human, porcine, canine, rat, or rodent FVIII proteins. Furthermore, comparisons between human and other species of FVIII have identified potentially conserved residues required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

[0181] Many tests can be used to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, colorimetric assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often via the Clauss method), platelet count, platelet function tests (often via PFA-100), TCT, bleeding time, mixing test (whether abnormality correction is performed if the patient's plasma is mixed with normal plasma), coagulation factor assay, antiphospholipid antibody, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), Russell's viper venom time (dRVVT), miscellaneous platelet function tests, coagulation elastography (TEG or Sonoclot), and coagulation elastography (TEM). ® For example, ROTEM ® ) or euglobulin dissolution time (ELT).

[0182] The aPTT test is a performance indicator that measures the effectiveness of both the "intrinsic" coagulation pathway (also known as the contact activation pathway) and the common coagulation pathway. This test is typically used to measure the coagulation activity of commercially available recombinant coagulation factors (such as FVIII or FIX). It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

[0183] ROTEM analysis provides comprehensive information on the following hemodynamics: clotting time, clot formation, clot stability, and dissolution. The various parameters in coagulation elastography depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many other factors that influence these interactions. This assay provides a complete overview of secondary hemostasis.

[0184] The FVIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants, and modified forms. The following shows an example of a full-length human FVIII sequence.

[0185] Table 3. Amino acid sequence of full-length factor VIII

[0186] (Full-length FVIII (FVIII signal peptide is underlined; FVIII heavy chain is double-underlined; B domain is italicized; and FVIII light chain is in plain text format)

[0187]

[0188]

[0189] Table 4. Nucleotide sequence encoding full-length FVIII (SEQ ID NO: 66)*

[0190]

[0191]

[0192]

[0193] *The underlined nucleic acid encodes a signal peptide.

[0194] FVIII peptides include full-length FVIII, full-length FVIII minus the N-terminal Met, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and / or FVIII with a complete or partial deletion of the B domain. In some embodiments, FVIII variants include a deletion of the B domain, whether partial or complete.

[0195] The sequence of native mature human FVIII is shown as SEQ ID NO: 65. The native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are structure-associated "A domains", B is a "B domain", C1 and C2 are structure-associated "C domains", and a1, a2, and a3 are acidic spacer regions. Regarding the primary amino acid sequence positions in SEQ ID NO:65, the A1 domain of human FVIII extends from Ala1 to approximately Arg336, the a1 spacer region extends from approximately Met337 to approximately Val374, the A2 domain extends from approximately Ala375 to approximately Tyr719, the a2 spacer region extends from approximately Glu720 to approximately Arg740, the B domain extends from approximately Ser741 to approximately Arg1648, the a3 spacer region extends from approximately Glu1649 to approximately Arg1689, the A3 domain extends from approximately Ser1690 to approximately Leu2025, the C1 domain extends from approximately Gly2026 to approximately Asn2072, and the C2 domain extends from approximately Ser2073 to Tyr2332. The designation of the boundaries between the domains and regions of FVIII may vary in different references, except for specific protein hydrolysis cleavage sites. Therefore, the boundaries indicated in this paper are specified as approximate boundaries using the term “approximate”.

[0196] The human FVIII gene was isolated and expressed in mammalian cells (Toole, JJ et al., Nature 312:342-347 (1984); Gitschier, J. et al., Nature 312:326-330 (1984); Wood, WI et al., Nature 312:330-337 (1984); Vehar, GA et al., Nature 312:337-342 (1984); WO 87 / 04187; WO 88 / 08035; WO 88 / 03558; and U.S. Patent No. 4,757,006). The FVIII amino acid sequence was deduced from cDNA as shown in U.S. Patent No. 4,965,199. Furthermore, FVIII with partial or complete deletion of the B domain is shown in U.S. Patent Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B domain is replaced by the human factor VB domain as shown in U.S. Patent No. 5,004,803. The cDNA sequence and amino acid sequence encoding human factor VIII are shown as SEQ ID NO: 1 and 2, respectively, in U.S. Application Publication No. 2005 / 0100990.

[0197] The porcine FVIII sequence was published in Toole, JJ et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). Furthermore, the complete porcine cDNA sequence obtained by PCR amplification of the FVIII sequence from a porcine spleen cDNA library has been reported in Healey, JF et al., Blood 88:4209-4214 (1996). Hybrid human / porcine FVIIIs with substitutions of all domains, all subunits, and specific amino acid sequences are disclosed in U.S. Patent No. 5,364,771 to Lollar and Runge and WO 93 / 20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII and chimeric FVIIIs with porcine A1 and / or A2 domains replacing the corresponding human domains have been reported in WO 94 / 11503. U.S. Patent No. 5,859,204 to Lollar, JS also discloses porcine cDNA and its inferred amino acid sequence. U.S. Patent No. 6,458,563 discloses a porcine FVIII lacking a B domain.

[0198] U.S. Patent No. 5,859,204 to Lollar, JS, reports a functional mutant of FVIII with reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463 to Lollar, JS, also reports an FVIII mutant with reduced immunoreactivity. U.S. Application Publication No. 2005 / 0100990 to Saenko et al. reports a functional mutation in the A2 domain of FVIII.

[0199] In one embodiment, FVIII (or the FVIII portion of a chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an FVIII amino acid sequence having amino acids 1 to 1438 of SEQ ID NO: 67 or amino acids 1 to 2332 of SEQ ID NO: 65 (with signal sequence) or an FVIII amino acid sequence having amino acids 1 to 19 of SEQ ID NO: 64 and amino acids 1 to 1438 of SEQ ID NO: 67 or amino acids 1 to 19 of SEQ ID NO: 64 and amino acids 1 to 2332 of SEQ ID NO: 65 (with signal sequence), wherein said FVIII has coagulation activity, for example as a cofactor activator IX to convert factor X into activator X. FVIII (or the FVIII portion of a chimeric protein) may be identical to the FVIII amino acid sequence (without signal sequence) having amino acids 1 to 1438 of SEQ ID NO: 67 or amino acids 1 to 2332 of SEQ ID NO: 65. FVIII may further include a signal sequence.

[0200] The “B domain” of FVIII used herein is identical to the B domains known in the art as defined by internal amino acid sequence identity and proteolytic cleavage sites, such as residues Ser741-Arg1648 of full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence residues Glu1649-Arg1689 are commonly referred to as the a3 acidic region. The boundary positions of all domains including the B domain in porcine, mouse, and canine FVIII are also known in the art. In one implementation, the B domain of FVIII is missing (“B domain missing factor VIII” or “BDD FVIII”). An instance of BDD FVIII is REFACTO. ® (Recombinant BDD FVIII), which has the same sequence as the factor VIII portion having the sequence in Table 5. (The BDD FVIII heavy chain is double-underlined; the B domain is in italics; and the BDD FVIII light chain is in plain text). The nucleotide sequence encoding Table 6 (SEQ ID NO: 68) is shown in Table 6.

[0201] Table 5. Amino acid sequence of factor VIII with B domain deletion (BDD FVIII)

[0202]

[0203]

[0204] Table 6. Nucleotide sequence encoding BDD FVIII (SEQ ID NO: 68)*

[0205]

[0206]

[0207] *The underlined nucleic acid encodes a signal peptide.

[0208] The “FVIII with missing B domain” 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 FVIII sequence with missing B domain of the present invention includes any of the deletions disclosed in column 4, line 4 through column 5, line 28 of U.S. Patent No. 6,316,226 (also in U.S. 6,346,513) and in Examples 1-5. In another embodiment, the B-domain deletion factor VIII is the S743 / Q1638 B-domain deletion factor VIII (SQ BDD FVIII) (e.g., factor VIII has a deletion from amino acid 744 to amino acid 1637, or factor VIII has amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 65, i.e., SEQ ID NO: 67). In some embodiments, the B-domain deletion FVIII of the present invention has the deletion disclosed in column 2, lines 26-51 of U.S. Patent No. 5,789,203 (and US 6,060,447, US 5,595,886, and US 6,228,620) and in Examples 5-8. In some implementations, the missing B domain factor VIII has the missing information described in the following: column 1, line 25 to column 2, line 40 of U.S. Patent No. 5,972,885; column 6, lines 1-22 and Example 1 of U.S. Patent No. 6,048,720; column 2, lines 17-46 of U.S. Patent No. 5,543,502; column 4, lines 22 to column 5, lines 36 of U.S. Patent No. 5,171,844; column 2, lines 55-68 of U.S. Patent No. 5,112,950. Figure 2And Example 1; column 2, line 2 through column 19, line 21 of U.S. Patent No. 4,868,112 and Table 2; column 2, line 1 through column 3, line 19; column 3, line 40 through column 4, line 67; column 7, line 43 through column 8, line 26; and column 11, line 5 through column 13, line 39 of U.S. Patent No. 7,041,635; or column 4, lines 25-53 of U.S. Patent No. 6,458,563. In some embodiments, the B-domain-deficient FVIII lacks most of the B-domain but still contains the B-domain N-terminal sequence necessary for the hydrolysis of the primary translation product protein into two polypeptide chains in vivo, as disclosed in WO 91 / 09122. In some embodiments, the B-domain-deficient FVIII is constructed with the deletion of amino acids 747-1638, i.e., a virtually complete deletion of the B-domain. Hoeben RC et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). Factor VIII with a B domain deletion may also contain deletions of amino acids 771–1666 or 868–1562 of FVIII. Meulien P. et al. Protein Eng. 2(4): 301-6 (1988). Additional B-domain deletions as part of this invention include the following: deletions of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5939-5942), 797 to 1562 (Eaton et al., Biochemistry (1986) 25:8343-8347), 741 to 1646 (Kaufman (PCT Publication No. WO 87 / 04187)), 747-1560 (Sarver et al., DNA (1987) 6:553-564), 741 to 1648 (Pasek (PCT Application No. 88 / 00831)), or 816 to 1598 or 741 to 1648 (Lagner (Behring Inst. Mitt. (1988)). Issue 82: 16-25); EP 295597). In other embodiments, BDD FVIII comprises an FVIII polypeptide containing a B domain and retaining one or more N-linked glycosylation sites, said sites being, for example, residues 757, 784, 828, 900, 963, or optionally 943 of the amino acid sequence corresponding to the full-length FVIII sequence.Examples of B-domain fragments include 226 or 163 amino acids 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) (i.e., retaining the first 226 or 163 amino acids of the B-domain). In other embodiments, BDD FVIII further includes a point mutation at residue 309 (from Phe to Ser) to improve the expression of the BDD FVIII protein. See Miao, HZ et al., Blood 103(a): 3412-3419 (2004). In other embodiments, BDD FVIII comprises an FVIII polypeptide containing a portion of a B domain but not containing 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 aforementioned deletions may be made in any FVIII sequence.

[0209] In some embodiments, FVIII has a partial B domain. In some embodiments, the FVIII protein having a partial B domain is FVIII198. FVIII198 is a single-chain FVIIIFc molecule-226N6 containing a partial B domain. The number 226 represents the N-terminal 226 amino acids of the FVIII B domain, and N6 represents the six N-glycosylation sites in the B domain.

[0210] In one embodiment, FVIII is cleaved following arginine or the corresponding arginine residue (in other variants) at amino acid 1648 (in full-length factor VIII or SEQ ID NO: 65) or amino acid 754 (in factor VIII with the S743 / Q1638 B domain missing or SEQ ID NO: 67), thereby producing heavy and light chains. In another embodiment, FVIII comprises heavy and light chains linked or associated by non-covalent bonds mediated by metal ions.

[0211] In other embodiments, FVIII is a single-chain FVIII that has not been cleaved after arginine or the corresponding arginine residue (in other variants) at amino acid 1648 (in full-length FVIII or SEQ ID NO: 65), amino acid 754 (in FVIII with the S743 / Q1638 B domain missing or SEQ ID NO: 67). The single-chain FVIII may contain one or more amino acid substitutions. In one embodiment, the amino acid substitution is at residue 1648, residue 1645, or both of the full-length maturation factor VIII polypeptide (SEQ ID NO: 65) or residue 754, residue 751, or both of the SQ BDD factor VIII (SEQ ID NO: 67). The amino acid substitution can be any amino acid other than arginine, such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolidone, or taurine.

[0212] FVIII can be further cleaved by thrombin and then activated to FVIIIa, thereby acting as a cofactor of activating factor IX (FIXa). Activated FIX then forms an X enzyme complex together with activated FVIII, and converts factor X into activating factor X (FXa). For activation, FVIII is cleaved by thrombin after three arginine residues at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the FVIII sequence with a B domain deletion), which yields FVIIIa having a 50 kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chain. In one embodiment, the FVIII protein suitable for use in this invention is an inactive FVIII. In another embodiment, the FVIII protein is an activated FVIII.

[0213] Proteins having an FVIII polypeptide linked or associated with a VWF protein may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to SEQ ID NO: 65 or 67, wherein said sequence has FVIII coagulation activity, for example, activating factor IX as a cofactor to convert factor X into activating factor X (FXa).

[0214] As used herein, “hybrid” or “chimeric” polypeptides and proteins include combinations of a first polypeptide chain (e.g., a VWF protein fused to an XTEN sequence having less than 288 amino acids and a first Ig constant region or a portion thereof) and a second polypeptide chain (e.g., an FVIII protein fused to a second Ig constant region or a portion thereof), thereby forming a heterodimer. In one embodiment, the first polypeptide and the second polypeptide in the hybrid associate with each other through protein-protein interactions (such as charge-charge or hydrophobic interactions). In another embodiment, the first polypeptide comprises a VWF protein-XTEN-Fc fusion protein, and the second polypeptide comprises an FVIII-Fc fusion protein, thereby making the hybrid a heterodimer, wherein the XTEN contains less than 288 amino acids. In other embodiments, the first polypeptide comprises a VWF protein-XTEN-Fc fusion protein, and the second polypeptide comprises an FVIII(X)-Fc fusion protein, thereby making the hybrid a heterodimer, wherein the XTEN contains less than 288 amino acids. The first and second polypeptides can associate via covalent bonds (e.g., disulfide bonds) between the first and second Fc regions. The first and second polypeptides can further associate with each other through the binding of the VWF fragment to the FVIII protein.

[0215] The FVIII protein applicable to this invention may include an FVIII having one or more additional XTEN sequences that do not affect the FVIII coagulation activity. The XTEN sequence may be fused to the C-terminus or N-terminus of the FVIII protein or inserted between one or more groups of two amino acid residues in the FVIII protein, while the insertion does not affect the FVIII coagulation activity or FVIII function. In one embodiment, the insertion improves the pharmacokinetic properties (e.g., half-life) of the FVIII protein. In another embodiment, there may be multiple insertions, such as more than two, three, four, five, six, seven, eight, nine, or ten insertions. Examples of insertion sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15, or any combination thereof.

[0216] An FVIII protein linked to one or more XTEN sequences can be represented as FVIII(X2) or FVIII. (a→b) -X-FVIII (c→d) FVIII (a→b) X2 comprises, is substantially composed of, or consists of the first part of the FVIII protein from amino acid residue "a" to amino acid residue "b"; X2 comprises, is substantially composed of, or consists of one or more XTEN sequences, FVIII (c→d) It comprises, is essentially composed of, or consists of the second part 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 part of the FVIII protein. b is the C-terminal amino acid residue of the first part of the FVIII protein, but it is also the N-terminal amino acid residue of the two amino acids at the insertion site of the XTEN sequence. c is the N-terminal amino acid residue of the second part of the FVIII protein, but it is also the C-terminal amino acid residue of the two amino acids at the insertion site of the XTEN sequence. d is the C-terminal amino acid residue of the FVIII protein, and

[0217] The first part of the FVIII protein and the second part of the FVIII protein are different from each other, and together they have a length sufficient to give the FVIII protein FVIII coagulation activity.

[0218] In one embodiment, the first and second portions of the FVIII protein are fragments of SEQ ID NO:65 [Full-length mature FVIII sequence] or SEQ ID NO:67 [FVIII with B domain deletion], for example, the N-terminal and C-terminal portions, respectively. In some embodiments, the first portion of the FVIII protein includes the A1 and A2 domains of the FVIII protein. The second portion of the FVIII protein includes the A3 domain, the C1 domain, and an optional C2 domain. In other embodiments, the first portion of the FVIII protein includes the A1 and A2 domains, and the second portion of the FVIII protein includes a portion of the B domain, the A3 domain, the C1 domain, and an optional C2 domain. In other embodiments, the first portion of the FVIII protein includes the A1, A2, and a portion of the B domain of the FVIII protein, and the second portion of the FVIII protein includes the A3 domain, the C1 domain, and an optional C2 domain. In other embodiments, the first portion of the FVIII protein comprises the first portions of the A1, A2, and B domains of the FVIII protein. The second portion of the FVIII protein comprises the 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 an amino acid residue immediately upstream of the site where one or more XTEN sequences are inserted or linked, and “c” may be an 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 amino acid sequence of the FVIII protein. For example, FVIII(a→b) It 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: 67 [FVIII amino acid sequence with B domain deletion] or SEQ ID NO: 65 [full-length FVIII], and FVIII (c→d) It may be amino acids 746 to 1438 of SEQ ID NO: 67 or amino acids 1641 to 2332 of SEQ ID NO: 65, respectively.

[0219] In some respects, the insertion site in the FVIII protein is located in one or more domains of the FVIII protein (which is the N-terminus, A1 domain, A2 domain, A3 domain, B domain, C1 domain, C2 domain, C-terminus, or two or more combinations thereof) or between two domains of the FVIII protein (which is the A1 domain and the α1 acidic region, and the α1 acidic region and the A2 domain, the A2 domain and the α2 acidic region, the α2 acidic region and the B domain, the B domain and the A3 domain, and the A3 domain and the C1 domain, the C1 domain and the C2 domain, or any combination thereof). For example, the insertion sites for insertable XTEN sequences are selected from the following groups: N-terminus and A1 domain, N-terminus and A2 domain, N-terminus and A3 domain, N-terminus and B domain, N-terminus and C1 domain, N-terminus and C2 domain, N-terminus and C-terminus, A1 domain and A2 domain, A1 domain and A3 domain, A1 domain and B domain, A1 domain and C1 domain, A1 domain and C2 domain, A1 domain and C-terminus, A2 domain, etc. The domain and A3 structural domain, A2 structural domain and B structural domain, A2 structural domain and C1 structural domain, A2 structural domain and C2 structural domain, A2 structural domain and C terminal, A3 structural domain and B structural domain, A3 structural domain and C1 structural domain, A3 structural domain and C2 structural domain, A3 structural domain and C terminal, B structural domain and C1 structural domain, B structural domain and C2 structural domain, B structural domain and C terminal, C1 structural domain and C2 structural domain, C1 structural domain and C terminal, C2 structural domain and C terminal, and two or more combinations thereof. Non-restrictive examples of insertion sites are listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0220] FVIII proteins with XTEN sequences inserted downstream of one or more amino acids (e.g., one or more XTEN insertion sites) or linked to the C-terminus or N-terminus retain FVIII activity after being linked to or inserted by the XTEN sequence. The XTEN sequence can be inserted into the FVIII protein once, twice, three times, four times, five times, or six times to ensure that the insertion does not affect FVIII activity (i.e., the FVIII protein retains its clotting properties).

[0221] The FVIII protein applicable to this invention can be inserted via an optional adapter at the N-terminus or C-terminus of the FVIII protein to one or more XTEN polypeptides, or via one or more optional adapters immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein. In one embodiment, the two amino acid residues of the inserted XTEN sequence or the amino acid residues to which the XTEN sequence is attached correspond to two or one amino acid residues selected from the group consisting of residues in Tables 7, 8, 9, and 10 and any combination thereof in SEQ ID NO: 65 [Full-length Mature FVIII].

[0222] In other embodiments, at least one XTEN sequence is inserted into any one or more XTEN insertion sites disclosed herein, or any combination thereof. In one aspect, at least one XTEN sequence is inserted into one or more XTEN insertion sites disclosed in one or more amino acids disclosed in Table 7.

[0223] Table 7: Exemplary XTEN Insertion Sites

[0224]

[0225]

[0226]

[0227]

[0228] *Indicates the insertion point of XTEN based on the amino acid number of mature full-length human FVIII, wherein the insertion may be on the N-terminal side or C-terminal side of the indicated amino acid.

[0229] In some embodiments, one or more XTEN sequences are inserted into about six amino acids upstream or downstream of amino acids 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 or 1910 or any combination thereof corresponding to SEQ ID NO: 65.

[0230] Table 8. Exemplary XTEN Insertion Range

[0231]

[0232] * The distance from the insertion residue refers to the relative number of amino acids from the N-terminus (negative) or C-terminus (positive) of the designated insertion residue (residue "0") where the insertion can take place. The label "-x" indicates that the insertion site is x amino acids from the N-terminus of the designated insertion residue. Similarly, the label "+x" indicates that the insertion site is x amino acids from the C-terminus of the designated insertion residue.

[0233] For example, "-1, +2" indicates an insertion at the N-terminus or C-terminus of an amino acid residue represented as -1, 0, +1, or +2.

[0234] In other embodiments, one or more XTEN sequences are inserted immediately downstream of an amino acid group consisting of one or more insertion sites in Table 9 corresponding to full-length mature human FVIII.

[0235] Table 9. Exemplary XTEN insertion sites or ranges

[0236]

[0237] * Indicates the range of insertion sites numbered relative to the amino acid numbering of mature human FVIII.

[0238] In other embodiments, one or more XTENs are inserted into the B domain of FVIII. In one example, the XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 740 and 1640 is optionally absent. In another example, the XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 740 and 1690 is optionally absent. In other examples, the XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 741 and 1648 is optionally absent. In other examples, the XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 743 and 1638 is optionally absent. In other instances, XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 745 and 1656 is optionally absent. In some instances, XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 745 and 1657 is optionally absent. In some instances, XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 745 and 1667 is optionally absent. In other instances, XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 745 and 1686 is optionally absent. In some other instances, XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 747 and 1642 is optionally absent. In other instances, XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO: 65, wherein the FVIII sequence between amino acids 751 and 1667 is optionally absent.

[0239] In some embodiments, one or more XTENs are inserted into one or more amino acids downstream of the insertion site of an amino acid selected from the group of amino acid residues in Table 10.

[0240] Table 10: FVIII XTEN Insertion Sites and Construct Labels

[0241]

[0242]

[0243]

[0244] * Amino acid number indicating mature FVIII protein

[0245] 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 can be inserted into each of the FVIII “A” domains containing at least two “permissive loops” that allow insertion of at least one XTEN polypeptide without eliminating the procoagulant activity of the recombinant protein or the ability of the recombinant protein to be expressed in vivo or in vitro in host cells. A permissive loop is a region that, among other properties, allows insertion of at least one XTEN sequence and also has high surface or solvent exposure and high conformational flexibility. The A1 domain contains the permissive loop-1 (A1-1) region and the permissive loop-2 (A1-2) region, the A2 domain contains the permissive loop-1 (A2-1) region and the permissive loop-2 (A2-2) region, and the A3 domain contains the permissive loop-1 (A3-1) region and the permissive loop-2 (A3-2) region.

[0246] In one aspect, the first permissible ring (A1-1) in the FVIII A1 domain is located between β chain 1 and β chain 2, and the second permissible ring (A1-2) in the FVIII A2 domain is located between β chain 11 and β chain 12. The first permissible ring (A2-1) in the FVIII A2 domain is located between β chain 22 and β chain 23, and the second permissible ring (A2-2) in the FVIII A2 domain is located between β chain 32 and β chain 33. The first permissible ring (A3-1) in the FVIII A3 domain is located between β chain 38 and β chain 39, and the second permissible ring (A3-2) in the FVIII A3 is located between β chain 45 and β chain 46. In some aspects, the surface-exposed flexible ring structure containing A1-1 corresponds to the region in naturally mature human FVIII from about amino acid 15 to about amino acid 45 of SEQ ID NO: 65, for example, from about amino acid 18 to about amino acid 41 of SEQ ID NO: 65. In other aspects, the surface-exposed flexible ring structure comprising A1-2 corresponds to the region in natural mature human FVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO: 65, for example, from about amino acid 218 to about amino acid 229 of SEQ ID NO: 65. In other aspects, the surface-exposed flexible ring structure comprising A2-1 corresponds to the region in natural mature human FVIII from about amino acid 395 to about amino acid 421 of SEQ ID NO: 65, for example, from about amino acid 397 to about amino acid 418 of SEQ ID NO: 65. In other embodiments, the surface-exposed flexible ring structure comprising A2-2 corresponds to the region in natural mature human FVIII from about amino acid 577 to about amino acid 635 of SEQ ID NO: 65, for example, from about amino acid 595 to about amino acid 607 of SEQ ID NO: 65. In some aspects, the surface-exposed flexible ring structure comprising A3-1 corresponds to the region in natural mature human FVIII from about amino acid 1705 to about amino acid 1732 of SEQ ID NO: 65, for example, from about amino acid 1711 to about amino acid 1725 of SEQ ID NO: 65. In other aspects, the surface-exposed flexible ring structure comprising A3-2 corresponds to the region in natural mature human FVIII from about amino acid 1884 to about amino acid 1917 of SEQ ID NO: 65, for example, from about amino acid 1899 to about amino acid 1911 of SEQ ID NO: 65.

[0247] In another embodiment, one or more amino acids into which at least one XTEN sequence is inserted are located within the a3 domain, for example, within amino acids 1649 to 1689 corresponding to the full-length mature FVIII polypeptide. In a particular embodiment, the XTEN sequence is inserted between amino acids 1656 and 1657 of SEQ ID NO: 65 (full-length mature FVIII). In a particular embodiment, an FVIII protein comprising an XTEN sequence inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 65 further comprises a deletion from amino acids 745 to 1656 corresponding to SEQ ID NO: 65.

[0248] In some embodiments, one or more insertion sites for one or more XTEN insertions are immediately downstream of one or more amino acids 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) 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) Two or more of them.

[0249] In one embodiment, the FVIII protein suitable for use in this invention comprises two XTEN sequences: a first XTEN sequence inserted into a first XTEN insertion site and a second XTEN sequence inserted into a second XTEN insertion site. Non-limiting examples of the first and second XTEN insertion sites are listed in Table 11.

[0250] Table 11. Exemplary insertion sites for two XTENs

[0251]

[0252]

[0253] The two XTENs inserted into or linked to the FVIII protein may be the same or different. In some embodiments, the FVIII protein to which this invention is applicable comprises two XTEN sequences inserted into the FVIII protein: a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 65, and a second XTEN sequence inserted immediately downstream of amino acid 2332 (C-terminus) corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 18, 26, 40, 1656, or 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 18, 26, or 40 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 599 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acids 18, 26, 40, 399, 403, 1725, 1720, 1900, 1905, or 2332 corresponding to SEQ ID NO: 65. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acids 18, 26, or 40 corresponding to SEQ ID NO: 65. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 18, 26, or 40 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 399 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 65.In a particular embodiment, the FVIII protein comprises two XTEN sequences: a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 65, and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 65, wherein the FVIII protein further comprises a deletion from amino acid 745 to amino acid 1685 corresponding to SEQ ID NO: 65, a mutation or substitution at amino acid 1680 corresponding to SEQ ID NO: 65 (e.g., Y1680F), a mutation or substitution at amino acid 1648 corresponding to SEQ ID NO: 65 (e.g., R1648A), or at least two mutations or substitutions at amino acid 1648 (e.g., R1648A) and amino acid 1680 (e.g., Y1680F) corresponding to SEQ ID NO: 65. In a particular embodiment, the FVIII protein comprises two XTEN sequences, a first XTEN inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 65, and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 65, wherein the FVIII protein further has a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 65.

[0254] In some embodiments, the FVIII protein comprises three XTEN sequences: a first XTEN sequence inserted at a first XTEN insertion site, a second XTEN sequence inserted at a second XTEN insertion site, and a third XTEN sequence inserted at a third XTEN insertion site. The first, second, or third XTEN sequences may be the same or different. The first, second, and third insertion sites may be selected from any group of insertion sites disclosed herein. In some embodiments, the FVIII protein comprising the three XTEN sequences may further comprise a mutation or substitution, such as amino acid 1648 corresponding to SEQ ID NO: 65, for example, R1648A. For example, non-limiting examples of the first, second, and third XTEN insertion sites are listed in Table 12.

[0255] Table 12. Exemplary insertion sites for three XTENs

[0256]

[0257] In some embodiments, the FVIII protein comprises three XTEN sequences: a first XTEN sequence inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 65, a second XTEN sequence inserted downstream of amino acid 403 corresponding to SEQ ID NO: 65, and a third XTEN sequence inserted downstream of amino acids 1656, 1720, or 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 65, and the third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 65, and the third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 65, and the third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 65, and the third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acids 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or 1910 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted downstream of amino acid 745 corresponding to SEQ ID NO: 65, and the third XTEN sequence is inserted downstream of amino acid 2332 corresponding to SEQ ID NO: 65.

[0258] In other embodiments, the FVIII protein of the present invention comprises four XTEN sequences: a first XTEN sequence inserted at a first insertion site, a second XTEN sequence inserted at a second insertion site, a third XTEN sequence inserted at a third insertion site, and a fourth XTEN sequence inserted at a fourth insertion site. The first, second, third, and fourth XTEN sequences may be identical, different, or in combination thereof. In some embodiments, the FVIII protein comprising the four XTEN sequences may further comprise mutations or substitutions, such as amino acid 1648 corresponding to SEQ ID NO: 65, for example, R1648A. Non-limiting examples of the first, second, third, and fourth XTEN insertion sites are listed in Table 13.

[0259] Table 13. Exemplary insertion sites for four XTENs

[0260]

[0261]

[0262]

[0263] In some embodiments, the FVIII protein comprises five XTEN sequences: a first XTEN sequence inserted at a first insertion site, a second XTEN sequence inserted at a second insertion site, a third XTEN sequence inserted at a third XTEN insertion site, a fourth XTEN sequence inserted at a fourth XTEN insertion site, and a fifth XTEN sequence inserted at a fifth XTEN insertion site. The first, second, third, fourth, or fifth XTEN sequences may be the same, different, or in combination thereof. Non-limiting examples of the first, second, third, fourth, and fifth insertion sites are listed in Table 14.

[0264] Table 14. Exemplary insertion sites for five XTENs

[0265]

[0266] In some embodiments, the FVIII protein comprises six XTEN sequences: a first XTEN sequence inserted at a first XTEN insertion site, a second XTEN sequence inserted at a second XTEN insertion site, a third XTEN sequence inserted at a third XTEN insertion site, a fourth XTEN sequence inserted at a fourth XTEN insertion site, a fifth XTEN sequence inserted at a fifth XTEN insertion site, and a sixth XTEN sequence inserted at a sixth XTEN insertion site. The first, second, third, fourth, fifth, or sixth XTEN sequences may be the same, different, or combinations thereof. Examples of the six XTEN insertion sites include, but are not limited to, the insertion sites listed in Table 15.

[0267] Table 15. Exemplary XTEN insertion sites for six XTENs

[0268]

[0269] In one particular example, the first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 65, and the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65 (full-length mature FVIII). In another example, the first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 65, and the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65. In some examples, the first XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 65, and the second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 65, and the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 65, and the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 65, the second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 65, and the third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65. In some embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 65, a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65, and a third XTEN is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO: 65. In some embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 65, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 65, a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65, and a fourth XTEN is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO: 65.

[0270] In a particular embodiment, the XTEN sequence is inserted between amino acids 745 and 746 of the full-length factor VIII or at the corresponding insertion site of factor VIII with a missing B domain.

[0271] In some embodiments, the chimeric protein of the present invention comprises two polypeptide sequences, the first polypeptide sequence comprising a sequence selected from FVIII-161 (SEQ ID NO: 69), FVIII-169 (SEQ ID NO: 70), FVIII-170 (SEQ ID NO: 71), FVIII-173 (SEQ ID NO: 72), FVIII-195 (SEQ ID NO: 73), FVIII-196 (SEQ ID NO: 74), FVIII-199 (SEQ ID NO: 75), FVIII-201 (SEQ ID NO: 76), FVIII-203 (SEQ ID NO: 77), FVIII-204 (SEQ ID NO: 78), FVIII-205 (SEQ ID NO: 79), FVIII-266 (SEQ ID NO: 80), FVIII-267 (SEQ ID NO: 81), FVIII-268 (SEQ ID NO: 82), FVIII-269. The second polypeptide sequence comprises an amino acid sequence that is at least about 80%, 90%, 95%, or 100% identical to the sequence selected from VWF031 (SEQ ID NO: 86), VWF034 (SEQ ID NO: 87), or VWF-036.

[0272] II.D. Ig constant region or part thereof

[0273] The chimeric protein of the present invention also includes two Ig constant regions or portions thereof, a first Ig constant region or portion thereof fused to the FVIII protein via an optional linker, and a second Ig constant region or portion thereof fused to the VWF protein via an XTEN sequence having less than 288 amino acids. The Ig constant regions or portions thereof can be combined with the XTEN sequence and the VWF protein to improve the pharmacokinetic or pharmacodynamic properties of the chimeric protein. In some embodiments, the Ig constant regions or portions thereof prolong the half-life of the molecule fused to the Ig constant regions or portions thereof.

[0274] The Ig constant region contains domains denoted as CH (constant weight) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may contain three or four CH domains. Some isotypes (e.g., IgG) also contain hinge regions. See Janeway et al. 2001, Immunobiology, Garland Publishing, NY, NY.

[0275] The Ig constant region or a portion thereof used to generate the chimeric protein of the present invention can be obtained from many different sources. In some embodiments, the Ig constant region or a portion thereof is derived from human Ig. However, it should be understood that the Ig constant region or a portion thereof may be derived from the Ig of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, the Ig constant region or a portion thereof may be derived from any Ig class (including IgM, IgG, IgD, IgA, and IgE) and any Ig isotype (including IgG1, IgG2, IgG3, and IgG4). In one embodiment, human isotype IgG1 is used.

[0276] Various Ig constant region gene sequences (e.g., human constant region gene sequences) are available in publicly disclosed, publicly available repository forms. Constant region domain sequences may be selected that possess specific effector functions (or lack specific effector functions) or have specific modifications to reduce immunogenicity. Many antibody and antibody-encoding gene sequences have been disclosed, and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences or portions thereof) can be obtained from these sequences using techniques recognized in the art. Genetic material obtained using any of the foregoing methods can then be modified or synthesized to obtain the polypeptides of the present invention. It should be further understood that the scope of the invention covers alleles, variants, and mutations of constant region DNA sequences.

[0277] The sequence of the Ig constant region or a portion thereof can be cloned, for example, using polymerase chain reaction and primers selected for amplifying the target domain. To clone the sequence of the Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody gene amplified by PCR. PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, in "PCR Protocols: A Guide to Methods and Applications" (Innois et al., eds., Academic Press, San Diego, CA (1990); Ho et al., 1989. Gene 77:51; Horton et al., 1993. Methods Enzymol. 217:270). PCR can be initiated using common constant region primers or using more specific primers based on publicly available heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of antibodies. In this case, the library can be screened using common primers or larger homology probes (such as mouse constant region probes). Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of 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 (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). Cloning of antibody sequences is further described in U.S. Patent No. 5,658,570, filed January 25, 1995 by Newman et al., which is incorporated herein by reference.

[0278] The Ig constant region used herein may include all structural domains and hinge regions or portions thereof. In one embodiment, the Ig constant region or a portion thereof includes the CH2 structural domain, the CH3 structural domain, and the hinge region, namely the Fc region or FcRn binding pair.

[0279] As used herein, the term "Fc region" is defined as the portion of a polypeptide corresponding to the Fc region of native Ig, formed by dimerization of the corresponding Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. Conversely, the terms "genetic fusion Fc region" or "single-chain Fc region" (scFc region) as used herein refer to a synthetic dimer Fc region containing genetically linked Fc domains within a single polypeptide chain (i.e., encoded in a single continuous genetic sequence).

[0280] In one implementation, the "Fc region" refers to a portion of a single Ig heavy chain that begins in a hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, with the first residue of the heavy chain constant region considered as 114) and ends at the C-terminus of the antibody. Therefore, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0281] Depending on the Ig isotype, the Fc region of the Ig constant region may include CH2, CH3, and CH4 domains as well as a hinge region. Chimeric proteins containing the Fc region of Ig impart several desirable properties to the chimeric protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors, such as the neonatal Fc receptor (FcRn) (US Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03 / 077834; US2003-0235536A1), all of which are incorporated herein by reference in their entirety.

[0282] The Ig constant region or a portion thereof may be an FcRn binding pair. FcRn is active in adult epithelial tissue and is expressed in the intestinal lumen, lung airways, nasal surface, vaginal surface, colon, and rectal surface (US Patent No. 6,485,726). The FcRn binding pair is the portion of Ig that binds to FcRn.

[0283] 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 IgG (but not other Ig classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transporting IgG across the cell from the lumen to the serous membrane, followed by release of IgG at relatively high pH in the interstitial fluid. It is expressed in adult epithelial tissues (US Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO 03 / 077834; US2003-0235536A1), including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), proximal renal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358), as well as nasal epithelium; vaginal surface; and biliary surface.

[0284] The FcRn binding conjugates applicable to this invention encompass molecules that can be specifically bound by the FcRn receptor, including intact IgG, the Fc fragment of IgG, and other fragments including the complete binding region of the FcRn receptor. The region of the Fc moiety of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact region between Fc and FcRn is close to the junction of the CH2 and CH3 domains. The Fc-FcRn contacts are all within a single IgG heavy chain. The FcRn binding conjugates include intact IgG, the Fc fragment of IgG, and other fragments of IgG including the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. All references to the amino acid numbering of Ig or Ig fragments are based on Kabat et al., 1991, Sequences of Proteins of Immunological Interest, USDepartment of Public Health, Bethesda, Md.

[0285] Fc regions or FcRn binding partners bound to FcRn can efficiently shuttle across the epithelial barrier via FcRn, thereby providing a non-invasive means of systemic administration of desired therapeutic molecules. Additionally, fusion proteins containing Fc regions or FcRn binding partners are pinocytochemically derived from FcRn-expressing cells. However, these fusion proteins are recycled back into the circulation after being labeled for degradation, thereby increasing the in vivo half-life of these proteins. In some embodiments, a portion of the Ig constant region is an Fc region or FcRn binding partner, which typically associates with another Fc region or another FcRn binding partner via disulfide bonds and other non-specific interactions to form dimers and higher-order multimers.

[0286] Two FcRn receptors can bind to a single Fc molecule. Crystallographic data indicate that each FcRn molecule binds to a single polypeptide of the Fc homodimer. In one embodiment, linking an FcRn binding partner (e.g., the Fc fragment of IgG) to a bioactive molecule provides a means of delivery of the bioactive molecule orally, buccally, sublingually, rectally, vaginally, via aerosol, nasally, or through the lungs, or via the eye. In another embodiment, the chimeric protein can be invasively administered, for example subcutaneously or intravenously.

[0287] The FcRn binding partner region is a molecule or part thereof that can be specifically bound to an FcRn receptor and subsequently actively transported by the FcRn receptor in the Fc region. Specific binding refers to the formation of a relatively stable complex between two molecules under physiological conditions. Specific binding is characterized by high affinity and low to moderate binding capacity, distinguishing it from non-specific binding, which typically has low affinity and moderate to high binding capacity. Generally, when the affinity constant KA is higher than 10... 6 M -1 or higher than 10 8 M -1 In this case, binding is considered specific. If necessary, non-specific binding can be reduced without substantially affecting specific binding by changing the binding conditions. Appropriate binding conditions, such as molecular concentration, ionic strength of the solution, temperature, allowable binding time, and concentration of the blocking agent (e.g., serum albumin, casein), can be optimized by skilled technicians using conventional techniques.

[0288] In some embodiments, the chimeric protein of the present invention comprises one or more truncated Fc regions, however, the Fc regions are still sufficient to impart Fc receptor (FcR) binding properties to the Fc regions. For example, the Fc region containing the FcRn-binding portion (i.e., the FcRn-binding portion) comprises approximately amino acids 282-438 (EU number) of IgG1, wherein the major contact sites are amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Therefore, the Fc region of the present invention may contain or be composed of an FcRn-binding portion. The FcRn-binding portion may be derived from any isotype of heavy chain including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn-binding portion from an antibody having human isotype IgG1 is used. In another embodiment, an FcRn-binding portion from an antibody having human isotype IgG4 is used.

[0289] In another embodiment, the “Fc region” comprises an amino acid sequence of the Fc domain or an amino acid sequence derived from the Fc domain. In some embodiments, the Fc region comprises at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain (approximately amino acids 216-230 of the antibody Fc region, according to EU designation), a CH2 domain (approximately amino acids 231-340 of the antibody Fc region, according to EU designation), a CH3 domain (approximately amino acids 341-438 of the antibody Fc region, according to EU designation), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, the Fc region comprises the complete Fc domain (i.e., the hinge domain, CH2 domain, and CH3 domain). In some embodiments, the Fc region comprises, substantially comprises, or comprises the following: a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof); a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof); a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof); or a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In other embodiments, the Fc region lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). In a particular embodiment, the Fc region comprises or comprises the following: amino acids corresponding to EU numbers 221 to 447.

[0290] The Fc region, referred to herein as F, F1, or F2, can be obtained from many different sources. In one embodiment, the Fc region of the polypeptide is derived from human Ig. However, it should be understood that the Fc region can be derived from the Ig of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, or guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, polypeptides containing the Fc domain or a portion thereof can be derived from any Ig class (including IgM, IgG, IgD, IgA, and IgE) and any Ig isotype (including IgG1, IgG2, IgG3, and IgG4). In another embodiment, human isotype IgG1 is used.

[0291] In some embodiments, the Fc variant confers a change in at least one effector function conferred by the Fc region containing the wild-type Fc domain (e.g., improved or reduced ability of the Fc region to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides engineered cysteine ​​residues.

[0292] The Fc region of this invention may employ Fc variants known in the art to impart effector function and / or alter FcR or FcRn binding (e.g., enhancement or reduction). Specifically, the binding molecule of this invention may include, for example, variations (e.g., substitutions) at one or more amino acid positions disclosed in the following: International PCT publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO0 2 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A 2. WO04 / 099249A2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 12378 0A2, WO06 / 019447A1, WO06 / 047350A2 and WO06 / 085967A2; US Patent Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or US US Patents 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956 and 7,317,091, each of which is incorporated herein by reference. In one embodiment, specific variations may be made at one or more disclosed amino acid positions (e.g., specific substitutions of one or more amino acids disclosed in the art). In another embodiment, different variations may be made at one or more disclosed amino acid positions (e.g., different substitutions of one or more amino acid positions disclosed in the art).

[0293] The Fc region or FcRn binding pair of IgG can be modified according to a well-recognized procedure (such as site-directed mutagenesis) to produce modified IgG or its Fc fragments or portions that will be bound by FcRn. Such modifications include modifications that maintain or even enhance binding to FcRn at sites distant from the FcRn contact site, as well as modifications within the contact site. For example, human IgG1 Fc (Fc... The following single amino acid residues in γ1) can be substituted without significantly reducing the binding affinity of Fc to FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A , N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A , T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N 389A, 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 indicates that wild-type proline is replaced by alanine at position 238. For example, a specific embodiment may include the N297A mutation, thereby removing a highly conserved N-glycosylation site. Other amino acids besides alanine can also be substituted at the wild-type positions specified above. Mutations can be introduced individually into the Fc region, resulting in more than one hundred Fc regions different from the natural Fc. In addition, combinations of two, three or more of these individual mutations can be introduced together, thereby generating hundreds more Fc regions.Furthermore, one Fc region of the construct of the present invention may be mutated, and another Fc region of the construct may not be mutated at all, or both may be mutated, but the mutations are different.

[0294] Some of the above mutations can confer novel functions to the Fc region or FcRn binding partners. For example, one embodiment has N297A, which removes a highly conserved N-glycosylation site. This mutation reduces immunogenicity, thereby enhancing the circulating half-life of the Fc region, and preventing 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). As another example of novel functions resulting from the above mutations, affinity for FcRn can, in some cases, increase beyond that of the wild type. This increased affinity can reflect an increased "association" rate, a decreased "dissociation" rate, or both. Examples of mutations believed 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).

[0295] In addition, at least three human Fcγ receptors appear to recognize binding sites on IgG within the lower hinge region, typically amino acids 234-237. Therefore, another instance of novel functional and potentially reduced immunogenicity can arise from mutations in this region, such as, for example, by replacing amino acids 233-236 "ELLG" of human IgG1 with the corresponding sequence "PVA" from IgG2 (with one amino acid deletion). It has been shown that when said mutations are introduced, FcγRI, FcγRII, and FcγRIII, which mediate the functions of various effectors, will not bind to IgG1. (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613).

[0296] In one embodiment, the Ig constant region or a portion thereof (e.g., the Fc region) is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO: 89 or SEQ ID NO: 3 of U.S. Patent No. 5,739,277), and optionally further comprising a sequence selected from HQSLGTQ (SEQ ID NO: 90), HQNLSDGK (SEQ ID NO: 91), HQNISDGK (SEQ ID NO: 92), or VISSHLGQ (SEQ ID NO: 93) (or SEQ ID NO: 11, 1, 2, and 31 of U.S. Patent No. 5,739,277, respectively).

[0297] In another embodiment, the immunoglobulin constant region or a portion thereof includes an amino acid sequence in the hinge region or a portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or a portion thereof. The disulfide bonds formed by the immunoglobulin constant region or a portion thereof place a first polypeptide containing FVIII and a second polypeptide containing a VWF fragment together such that endogenous VWF does not displace the VWF fragment and does not 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 prevents interaction between endogenous VWF and FVIII protein. This inhibition of interaction between VWF and FVIII protein allows the chimeric protein to have a half-life exceeding twice the limit. The hinge region or a portion thereof may further be linked to one or more of the following domains: CH1, CH2, CH3, fragments thereof, and any combination thereof. In a particular embodiment, the immunoglobulin constant region or a portion thereof is the hinge region and CH2.

[0298] In some embodiments, the Ig constant region or a portion thereof is hemiglycosylated. For example, a chimeric protein comprising two Fc regions or FcRn binding partners may contain a first glycosylated Fc region (e.g., a glycosylated CH2 region) or an FcRn binding partner and a second unglycosylated Fc region (e.g., an unglycosylated CH2 region) or an FcRn binding partner. In one embodiment, a linker may be inserted between the glycosylated Fc region and the unglycosylated Fc region. In another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all Fc regions are glycosylated. In other embodiments, the Fc region may be unglycosylated, i.e., the unglycosylated portion is glycosylated.

[0299] In some embodiments, the chimeric protein of the present invention comprises amino acid substitutions (e.g., Fc variants) of the Ig constant region or a portion thereof, which alter the antigen-independent effector function of the Ig constant region, particularly the protein's cyclic half-life.

[0300] When compared to the absence of these substituted proteins, the proteins exhibit increased or decreased binding to FcRn, and thus increased or decreased serum half-life, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and the molecules are suitable for use in methods of treating mammals (where a prolonged half-life of the administered peptide is desired, for example, for the treatment of chronic diseases or conditions) (see, for example, U.S. Patents 7,348,004, 7,404,956, and 7,862,820). Conversely, Fc variants with decreased affinity for FcRn are expected to have shorter half-lives, and the molecules are also suitable for administration to mammals, for example, when a shortened circulation time is advantageous, such as for in vivo diagnostic imaging or in cases where the starting peptide has toxic side effects when present in circulation for a prolonged period. Fc variants with decreased affinity for FcRn are also less likely to cross the placenta, and are therefore also suitable for treating diseases or conditions in pregnant women. Furthermore, other applications requiring reduced FcRn binding affinity include those requiring localization to the brain, kidney, and / or liver. In one exemplary embodiment, the chimeric protein of the present invention exhibits reduced transport across the glomerular epithelium from vascular structures. In another embodiment, the chimeric protein of the present invention exhibits reduced transport across the blood-brain barrier (BBB) ​​from the brain into the intervascular space. In one embodiment, the FcRn-binding altered protein comprises at least one Fc region or FcRn binding pair (e.g., one or two Fc regions or FcRn binding pairs) having one or more amino acid substitutions within an "FcRn binding loop" in the Ig constant region. The FcRn binding loop comprises amino acid residues 280-299 (according to EU designation) of the wild-type full-length Fc region. In other embodiments, the Ig constant region or a portion thereof in the chimeric protein of the present invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding pair having one or more amino acid substitutions within a 15 μFcRn "contact region". As used herein, the term 15Ǻ FcRn “contact region” includes residues at the following locations in the full-length Fc portion of the wild type: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU number). In other embodiments, the Ig constant region or a portion thereof of the present invention having modified FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions at an amino acid position corresponding to any of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K) and 438.Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication No. WO05 / 047327, which is incorporated herein by reference.

[0301] The Fc region or FcRn binding partner used in this invention may also contain amino acid substitutions that alter the glycosylation of the chimeric protein, as recognized in the art. For example, the Fc region or FcRn binding partner of the chimeric protein linked to the VWF fragment or FVIII protein may include an Fc region with mutations that result in reduced glycosylation (e.g., N-linked or O-linked glycosylation), or may include a wild-type Fc portion with altered sugar form (e.g., low trehalose or no trehalose).

[0302] In one embodiment, the unprocessed chimeric protein of the present invention may comprise a genetically fused Fc region (i.e., a scFc region), wherein two or more of the constituent Ig constant regions or portions thereof are independently selected from the Ig constant regions or portions thereof described herein. In one embodiment, the Fc regions of the dimer Fc regions are identical. In another embodiment, at least two Fc regions are different. For example, the Fc regions or FcRn binding pairs of the proteins of the present invention contain the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In other embodiments, the Fc regions or FcRn binding pairs of the proteins of the present invention may differ in sequence at one or more amino acid positions. For example, at least two Fc regions or FcRn binding partners may differ at approximately 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), approximately 10 positions, approximately 15 positions, approximately 20 positions, approximately 30 positions, approximately 40 positions, or approximately 50 positions.

[0303] II.E. Connector

[0304] The chimeric protein of the present invention further comprises one or more linkers. One type of linker is a cleavable linker, which can be cleaved by various proteases when administered to a subject in vivo, for example, at a coagulation site. In one embodiment, the cleavable linker allows cleavage of a portion of the chimeric protein (e.g., VWF protein) from the XTEN sequence at the coagulation cascade site, thereby imparting its FVIIIa activity to the activated FVIII (FVIIIa). Another type of linker is a processable linker, which contains an intracellular cleavage site and is therefore cleavable by intracellular processing enzymes in the host cell, thereby allowing appropriate expression of the polypeptide and formation of the chimeric protein.

[0305] One or more linkers may be present between any two proteins in the chimeric protein. In one embodiment, the chimeric protein comprises a first polypeptide comprising (i) an FVIII protein and (ii) a first Ig constant region or a portion thereof; and a second polypeptide comprising (iii) a VWF protein, (iv) a linker (e.g., a cleavable linker), (v) an XTEN sequence, and (vi) a second Ig constant region or a portion thereof. In another embodiment, the chimeric protein comprises a first polypeptide comprising (i) an FVIII protein and (ii) a first Ig constant region or a portion thereof; and a second polypeptide comprising (iii) a VWF protein, (iv) an XTEN sequence, (v) a linker (e.g., a cleavable linker), and (vi) a second Ig constant region or a portion thereof. In other embodiments, the chimeric protein comprises a first polypeptide comprising (i) an FVIII protein and (ii) a first Ig constant region or a portion thereof; and a second polypeptide comprising (iii) a VWF protein, (iv) a first linker (e.g., a cleavable linker), (v) an XTEN sequence, (vi) a second linker (e.g., a cleavable linker), and (vii) a second Ig constant region or a portion thereof. In some embodiments, the first polypeptide further comprises a linker, such as a cleavable linker, between the FVIII protein and the first Ig constant region.

[0306] In some embodiments, the chimeric protein comprises a single chain containing (i) an FVIII protein, (ii) a first Ig constant region or a portion thereof, (iii) a linker (e.g., a processable linker), (iv) a VWF protein, (v) an XTEN sequence, and (vi) a second Ig constant region or a portion thereof. In other embodiments, the chimeric protein comprises a single chain containing (i) an FVIII protein, (ii) a first Ig constant region or a portion thereof, (iii) a first linker (e.g., a processable linker), (iv) a VWF protein, (v) a second linker (e.g., a cleavable linker), (vi) an XTEN sequence, and (vii) a second Ig constant region or a portion thereof. Following expression of the chimeric protein in a host cell, the processable linker can be processed; thus, the chimeric protein produced in the host cell can be in a final form comprising two or three polypeptide chains.

[0307] The linker suitable for use in this invention may comprise any organic molecule. In one embodiment, the linker comprises a polymer, such as polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker comprises 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 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids. In one embodiment, the linker includes an XTEN sequence. Other examples of XTEN can be used according to the present invention and are disclosed in U.S. Patent Publications 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1 or 2011 / 0172146 A1 or International Patent Publications WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1 or WO 2011028344 A2. In another embodiment, the connector is a PAS sequence.

[0308] In one embodiment, the linker is a polymer, such as polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker is an amino acid sequence. The linker 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 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids.

[0309] Examples of connectors are well known in the art. In one embodiment, the connector comprises sequence G. n The connector may contain a sequence (GA). n The connector may contain a sequence number (GGS). n In other embodiments, the connector includes (GGGS). n (SEQ ID NO:101). In other embodiments, the connector includes a sequence (GGS). n (GGGGS) n (SEQ ID NO: 95). In these cases, n can be an integer from 1 to 100. In other cases, n can be an integer from 1 to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 96), GGSGGSGGSGGSGGG (SEQ ID NO: 97), GGSGGSGGGGSGGGGS (SEQ ID NO: 98), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 99), or GGGGSGGGGSGGGSGGGS (SEQ ID NO: 100). The linker does not eliminate or attenuate the activity of VWF protein or the coagulation activity of factor VIII. Optionally, the linker enhances the activity of the VWF protein or the coagulation activity of the factor VIII protein, for example by further reducing steric hindrance and making the VWF protein or the factor VIII portion more readily available to its target binding site.

[0310] In one embodiment, the linker length suitable for the chimeric protein is 15-25 amino acids. In another embodiment, the linker length suitable for the chimeric protein is 15-20 amino acids. In some embodiments, the linker length for the chimeric protein is 10-25 amino acids. In other embodiments, the linker length for the chimeric protein is 15 amino acids. In still other embodiments, the linker for the chimeric protein is (GGGGS). n (SEQ ID NO: 94), where G represents glycine, S represents serine, and n is an integer from 1 to 20.

[0311] II. F. Cleavage Site

[0312] Cleavable linkers may have a portion capable of being cleaved chemically (e.g., by hydrolyzing ester bonds), enzymatically (i.e., by having a protease cleavage sequence), or photolytically (e.g., by having a chromophore, such as 3-amino-3-(2-nitrophenyl)propionic acid (ANP)) to release one molecule from another.

[0313] In one embodiment, the cleavable linker includes one or more cleavage sites at the N-terminus, C-terminus, or both. In another embodiment, the cleavable linker consists substantially of one or more cleavable sites or is composed of one or more cleavable sites. In other embodiments, the cleavable linker comprises the heterologous amino acid linker sequence or polymer described herein and one or more cleavable sites.

[0314] In some embodiments, the cleavable adapter includes one or more cleavage sites (i.e., intracellular processing sites) that can be cleaved within the host cell. Non-limiting examples of cleavage sites include RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: 103), and RRRRS (SEQ ID NO: 104).

[0315] In some embodiments, the cleavable linker includes an a1 region from FVIII, an a2 region from FVIII, an a3 region from FVIII, a thrombin-cleavable site comprising an XVPR (SEQ ID NO: 105) and an external binding site interaction motif of PAR1 (where X is an aliphatic amino acid), or any combination thereof. The a2 region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu720 to Arg740 corresponding to full-length FVIII, wherein the a2 region is capable of being cleaved by thrombin. In a particular embodiment, the cleavable linker suitable for use in the present invention comprises an a2 region comprising ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106). In other embodiments, the cleavable linker for the present invention includes an a1 region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Met337 to Arg372 corresponding to full-length FVIII, wherein the a1 region is cleavable by thrombin. In a particular embodiment, the a1 region comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107). In some embodiments, the cleavable linker for the present invention includes an a3 region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to Glu1649 to Arg1689 corresponding to full-length FVIII, wherein the a3 region is cleavable by thrombin. In a particular embodiment, the cleavable linker for the present invention includes an a3 region comprising ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 108).

[0316] In other embodiments, the cleavable linker includes a thrombin cleavage site comprising an XVPR (SEQ ID NO: 105) and a PAR1 external binding site interaction motif, wherein the PAR1 external binding site interaction motif comprises SFLLRN (SEQ ID NO: 109). The PAR1 external binding site interaction motif may further comprise an amino acid sequence selected from the following: P, PN, PND, PNDK (SEQ ID NO: 110), PNDKY (SEQ ID NO: 111), PNDKYE (SEQ ID NO: 112), PNDKYEP (SEQ ID NO: 113), PNDKYEPF (SEQ ID NO: 114), PNDKYEPFW (SEQ ID NO: 115), PNDKYEPFWE (SEQ ID NO: 116), PNDKYEPFWED (SEQ ID NO: 117), PNDKYEPFWEDE (SEQ ID NO: 118), PNDKYEPFWEDEE (SEQ ID NO: 119), PNDKYEPFWEDEES (SEQ ID NO: 120), or any combination thereof. In some embodiments, the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine.

[0317] In other embodiments, the cleavable linker includes one or more cleavage sites that are cleaved by a protease after administration of a chimeric protein containing said cleavable linker to a subject. In one embodiment, the cleavage site is cleaved by a protease selected from the group consisting of: factor XIa, factor XIIa, vasodilator, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase-2, MMP-12, MMP-13, MMP-17, and MMP-20. In another embodiment, the cleavage site is selected from the group consisting of: FXIa cleavage site (e.g., KLTR↓AET (SEQ ID NO: 121)), FXIa cleavage site (e.g., DFTR↓VVG (SEQ ID NO: 122)), FXIa cleavage site (e.g., TMTR↓IVGG (SEQ ID NO: 123)), vasodilator cleavage site (e.g., SPFR↓STGG (SEQ ID NO: 124)), FVIIa cleavage site (e.g., LQVR↓IVGG (SEQ ID NO: 125)), FIXa cleavage site (e.g., PLGR↓IVGG (SEQ ID NO: 126)), FXa cleavage site (e.g., IEGR↓TVGG (SEQ ID NO: 127)), FIIa (thrombin) cleavage site (e.g., LTPR↓SLLV (SEQ ID NO: 128)), elastase-2 cleavage site (e.g., LGPV↓SGVP (SEQ ID NO: 129)), and granzyme-B cleavage site (e.g., VAGD↓SLEE). (SEQ ID NO: 130)), MMP-12 cleavage site (e.g., GPAG↓LGGA (SEQ ID NO: 131)), MMP-13 cleavage site (e.g., GPAG↓LRGA (SEQ ID NO: 132)), MMP-17 cleavage site (e.g., APLG↓LRLR (SEQ ID NO: 133)), MMP-20 cleavage site (e.g., PALP↓LVAQ (SEQ ID NO: 134)), TEV cleavage site (e.g., ENLYFQ↓G (SEQ ID NO: 135)), enterokinase cleavage site (e.g., DDDK↓IVGG (SEQ ID NO: 136)), protease 3C (PRESCISSION™) cleavage site (e.g., LEVLFQ↓GP (SEQ ID NO: 137)), and sorting enzyme A cleavage site (e.g., LPKT↓GSES) (SEQ ID NO: 138). In some embodiments, the FXIa cleavage sites include, but are not limited to, for example, TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3).Non-restrictive exemplary thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88) or IEPRSFS (SEQ ID NO: 194) and sequences containing ALRPR (SEQ ID NO: 7), consisting essentially of ALRPR (SEQ ID NO: 7), or consisting of ALRPR (SEQ ID NO: 7) (e.g., ALRPRVVGGA (SEQ ID NO: 145)).

[0318] In a particular embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). In another embodiment, the cleavage site comprises DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88) or a fragment thereof. In one particular embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 194). In another embodiment, the cleavage site comprises EPRSFS (SEQ ID NO: 195), wherein the cleavage site is not the full-length a2 region of FVIII. In another embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 200). In another embodiment, the cleavage site comprises IEPRSFS (SEQ ID NO: 200), wherein the cleavage site is not the full-length a2 region of FVIII, or does not contain the full-length a2 region of FVIII. In other embodiments,The cleavage sites include DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), KNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 139), NTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 140), TGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 141), GDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 142), DYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 143), YYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 144), YEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 176), EDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 177), DSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 178), SYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 179), YEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 180), EDISAYLLSKNNAIEPRSFS (SEQ ID NO: 181), DISAYLLSKNNAIEPRSFS (SEQ ID NO: 182), ISAYLLSKNNAIEPRSFS (SEQ ID NO: 183), SAYLLSKNNAIEPRSFS (SEQ ID NO: 184), AYLLSKNNAIEPRSFS (SEQ ID NO: 185), YLLSKNNAIEPRSFS (SEQ ID NO: 186), LLSKNNAIEPRSFS (SEQ ID NO: 187), LSKNNAIEPRSFS (SEQ ID NO: 188), SKNNAIEPRSFS (SEQ ID NO: 189), KNNAIEPRSFS (SEQ ID NO: 190), NNAIEPRSFS (SEQ ID NO: 191), NAIEPRSFS (SEQ ID NO: 192), AIEPRSFS (SEQ ID NO: 193), or IEPRSFS (SEQ ID NO: 194). In other embodiments,The cleavage sites include DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), KNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 139), NTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 140), TGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 141), GDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 142), DYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 143), YYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 144), YEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 176), EDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 177), DSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 178), SYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 179), YEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 180), EDISAYLLSKNNAIEPRSFS (SEQ ID NO: 181), DISAYLLSKNNAIEPRSFS (SEQ ID NO: 182), ISAYLLSKNNAIEPRSFS (SEQ ID NO: 183), SAYLLSKNNAIEPRSFS (SEQ ID NO: 184), AYLLSKNNAIEPRSFS (SEQ ID NO: 185), YLLSKNNAIEPRSFS (SEQ ID NO: 186), LLSKNNAIEPRSFS (SEQ ID NO: 187), LSKNNAIEPRSFS (SEQ ID NO: 188), SKNNAIEPRSFS (SEQ ID NO: 189), KNNAIEPRSFS (SEQ ID NO: 190), NNAIEPRSFS (SEQ ID NO: 191), NAIEPRSFS (SEQ ID NO: 192), AIEPRSFS (SEQ ID NO: 193), or IEPRSFS (SEQ ID NO: 194).The cleavage site described herein is not the full-length FVIII a2 region. In some embodiments, the cleavable linker is cleavable in thrombin cleavage assays as provided herein or as known in the art.

[0319] III. Polynucleotides, vectors, and host cells

[0320] This invention also provides a polynucleotide encoding the chimeric protein of the invention. In one embodiment, the first and second polypeptide chains may be encoded by a single polynucleotide chain. In another embodiment, the first and second polypeptide chains are encoded by two different polynucleotides (i.e., a first nucleotide sequence and a second nucleotide sequence). In yet another embodiment, the first and second nucleotide sequences are on two different polynucleotides (e.g., different vectors).

[0321] This invention includes a polynucleotide encoding a single polypeptide chain (e.g., FVIII(X2)-F1-L3-F2-L2-X1-L1-V), wherein FVIII(X2) comprises an FVIII protein having an XTEN sequence inserted at one or more insertion sites, F1 comprises a first Ig constant region or a portion thereof, such as a first Fc region, L1 comprises a first linker, V comprises a VWF protein, X1 comprises an XTEN sequence having a length of less than 288 amino acids, L2 comprises a second linker, L3 comprises a third linker, and F2 comprises a second Ig constant region or a portion thereof, such as a second Fc region. This invention also includes two polynucleotides: a first polynucleotide sequence encoding a first polypeptide comprising an FVIII protein fused to a first Ig constant region or a portion thereof, and a second polynucleotide sequence encoding a second polypeptide comprising a VWF protein, an XTEN sequence having a length of less than 288 amino acids, and a second Ig constant region or a portion thereof. In some embodiments, chimeric proteins comprising two or three polypeptide chains may be encoded by a single polynucleotide chain, which is then processed into two or three (or more) polypeptide chains. In other embodiments, chimeric proteins comprising these polypeptide chains may be encoded by two or three polynucleotide chains.

[0322] In other embodiments, the polynucleotide set further comprises an additional nucleotide chain encoding the protein convertase (e.g., a second nucleotide chain when the chimeric polypeptide is encoded by a single polynucleotide chain, or a third nucleotide chain when the chimeric protein is encoded by two polynucleotide chains). The protein convertase may be selected from the group consisting of: protoprotein convertase subtilisin / kexin type 5 (PCSK5 or PC5), protoprotein convertase subtilisin / kexin type 7 (PCSK7 or PC5), yeast Kex 2, protoprotein convertase subtilisin / kexin type 3 (PACE or PCSK3), and two or more combinations thereof. In some embodiments, the protein convertase is PACE, PC5, or PC7. In a particular embodiment, the protein convertase is PC5 or PC7. See International Application No. PCT / US2011 / 043568.

[0323] As used herein, an expression vector is any nucleic acid construct containing elements necessary for transcription and translation of inserted coding sequences when introduced into a suitable host cell, or, in the case of an RNA viral vector, elements necessary for replication and translation. Expression vectors may include plasmids, phage particles, viruses, and their derivatives.

[0324] The expression vector of the present invention will comprise a polynucleotide encoding the chimeric protein described herein. In one embodiment, one or more of the coding sequences of a first polypeptide comprising an FVIII protein and a first Ig constant region; a second polypeptide comprising a VWF protein, an XTEN sequence having less than 288 amino acids and a second Ig constant region or a portion thereof; or both are operatively linked to an expression control sequence. As used herein, two nucleic acid sequences are operatively linked when they are covalently linked in a manner that allows each constituent nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operatively linked when they are covalently linked in a manner that places the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operatively linked if inducing a promoter in the 5' gene expression sequence results in transcription of the coding sequence, and if the nature of the link between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to guide transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Therefore, if a gene expression sequence can enable transcription of a nucleic acid sequence to translate the resulting transcript into a desired protein or polypeptide, then the gene expression sequence will be operatively linked to that nucleic acid sequence.

[0325] As used herein, gene expression control sequences are any regulatory nucleotide sequences, such as promoter sequences or promoter-enhancer combinations, that facilitate efficient transcription and translation of the encoded nucleic acid to which they are operatively linked. Gene expression control sequences may be, for example, mammalian or viral promoters, such as constitutive or inducible promoters. Constitutive mammalian promoters include, but are not limited to, promoters of genes such as hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoters, and other constitutive promoters. Exemplary viral promoters constitutively functioning in eukaryotic cells include, for example, promoters of long terminal repeats (LTRs) from cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus, and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters suitable for use in gene expression sequences according to this invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducer. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

[0326] Generally, the gene expression control sequence will, where necessary, include 5' non-transcriptional and 5' non-translational sequences, such as TATA boxes, capping sequences, CAAT sequences, etc., which are involved in the initiation of transcription and translation, respectively. In particular, the 5' non-transcriptional sequence will include a promoter region comprising a promoter sequence for transcriptional control of the coding nucleic acid to which it is operably bound. The gene expression sequence may optionally include enhancer sequences or upstream activator sequences, as desired.

[0327] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 viruses; polymorphonuclear viruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia virus; polioviruses; and RNA viruses, such as retroviruses. Other vectors well known in the art can be readily employed. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by target genes. Non-cytopathic viruses include retroviruses, whose life cycle involves the reverse transcription of genomic viral RNA into DNA, followed by provirus integration into the host cell DNA. Retroviruses have been approved for use in human gene therapy trials. Those retroviruses with replication defects (i.e., capable of guiding the synthesis of desired proteins but not producing infectious particles) are most suitable. The genetically altered retroviral expression vectors described above have general utility for efficiently transducing genes in vivo. The standard protocol for generating replication-defective retroviruses (which includes the following steps: incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, generating a recombinant retrovirus from the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) is provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0328] In one implementation, the virus is adeno-associated virus (AAV), a double-stranded DNA virus. AAV can be engineered to be replication-defective and capable of infecting a wide range of cell types and species. It further possesses advantages such as: thermal and lipid solvent stability; high transduction frequency in different cell lineages (including hematopoietic cells); and lack of inhibition against repeated infection, thus allowing for multilevel transduction. AAV has been reported to integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis from retroviral infection and the variability of the inserted gene expression signature. Furthermore, wild-type AAV infection has been successfully carried out in tissue culture for more than 100 passages in the absence of selective pressure, suggesting that AAV genome integration is a relatively stable event. AAV can also function in an extrachromosomal manner.

[0329] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been found to be particularly advantageous for delivering genes into cells in vivo because they cannot replicate within the host genome and integrate into it. However, these plasmids, with promoters compatible with host cells, can extract gene expression peptides operatively encoded within the plasmid. Some commonly used plasmids available from commercial vendors include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Other examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well known to those skilled in the art. Additionally, standard molecular biology techniques can be used to custom-design plasmids to remove and / or add specific DNA fragments.

[0330] In an insect expression system that can be used to produce the protein of the present invention, *Autographa californica* nuclear polyhidrosis virus (AcNPV) is used as a vector for expressing the foreign gene. The virus is grown in *Spodoptera frugiperda* cells. The coding sequence can be cloned into a non-essential region of the virus (e.g., the polyhedral gene) and placed under the control of an ACNPV promoter (e.g., a polyhedral promoter). Successful insertion of the coding sequence results in the inactivation of the polyhedral gene and the production of non-closed recombinant viruses (i.e., viruses lacking the protein coat encoded by the polyhedral gene). These recombinant viruses are then used to infect *Spodoptera frugiperda* cells in which the inserted gene is expressed. (See, for example, Smith et al. (1983) J Virol 46:584; U.S. Patent No. 4,215,051). Other examples of this expression system can be found in Ausubel et al. (1989), Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

[0331] Another system that can be used to express the proteins of this invention is the glutamine synthase gene expression system, also known as the "GS expression system" (Lonza Biologics PLC, Berkshire UK). This expression system is detailed in U.S. Patent No. 5,981,216.

[0332] In mammalian host cells, numerous virus-based expression systems are available. In cases where adenovirus is used as the expression vector, the coding sequence can be linked to the adenoviral transcription / translation control complex, such as the late promoter and triple leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) will produce a recombinant virus that is viable in the infected host and capable of expressing the peptide. (See, for example, Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter can be used. (See, for example, Maskett et al. (1982) Proc Natl Acad Sci USA 79:7415; Maskett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.)

[0333] To increase production efficiency, polynucleotides can be engineered to encode multiple units of the protein of the present invention, separated by enzyme cleavage sites. The resulting polypeptides can be cleaved (e.g., by treatment with a suitable enzyme) to recover the polypeptide units. This increases the yield of polypeptides driven by a single promoter. When used in a suitable viral expression system, the translation of each polypeptide encoded by the mRNA is guided within the transcript; for example, by the internal ribosome entry site IRES. Thus, the polycistronic construct guides the transcription of a single large polycistronic mRNA, which in turn guides the translation of multiple individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and significantly increases the yield of polypeptides driven by a single promoter.

[0334] The vectors used in transformation will typically contain selectable markers for identifying the transformants. In bacterial systems, this may include antibiotic resistance genes, such as ampicillin or kanamycin. Selectable markers used in cultured mammalian cells include genes that confer resistance to drugs such as neomycin, hygromycin, and methotrexate. Selectable markers can be amplifiable selectable markers. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen CC et al. (1983) Proc Natl Acad SciUSA 80:2495-9. Selectable markers were reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the selection of selectable markers is entirely within the realm of ordinary skill.

[0335] The selectable marker can be introduced into the cell simultaneously with the target gene on a separate plasmid, or they can be introduced on the same plasmid. If on the same plasmid, the selectable marker and the target gene can be under the control of different promoters or the same promoter, the latter arrangement of which produces bicistronic information. This type of construct is known in the art (e.g., U.S. Patent No. 4,713,339).

[0336] Expression vectors can encode tags that allow for easy purification of recombinant proteins. Examples include, but are not limited to, the vector pUR278 (Ruther et al. (1983) EMBO J 2:1791), in which the coding sequence of the protein to be expressed can be co-linked with the lac z coding region into the vector to generate a tagged fusion protein; the pGEX vector can be used to express the proteins of this invention tagged with glutathione S-transferase (GST). These proteins are generally soluble and can be readily purified from cells by adsorption onto glutathione-agarose beads followed by elution in the presence of free glutathione. The vector includes cleavage sites (thrombin or factor Xa protease or presentation protein) for easy removal of the tag after purification. TM (Pharmacia, Peapack, NJ)

[0337] One or more expression vectors are then transfected or co-transfected into suitable target cells to which the polypeptide will be expressed. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host expression vector systems can be used to express the proteins described herein, including both prokaryotic and eukaryotic cells. These systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA or plasmid DNA expression vectors containing appropriate coding sequences (e.g., *Escherichia coli*); yeast or filamentous fungi transformed with recombinant yeast or fungal expression vectors containing appropriate coding sequences; insect cell systems infected with recombinant viral expression vectors containing appropriate coding sequences (e.g., baculoviruses); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors containing appropriate coding sequences (e.g., Ti plasmids); or animal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

[0338] In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell with a defined nucleus. Animal eukaryotic cells include cells of vertebrates (e.g., mammals) and cells of invertebrates (e.g., insects). Plant eukaryotic cells may explicitly include, but are not limited to, yeast cells. Eukaryotic cells differ from prokaryotic cells, such as bacteria.

[0339] In some embodiments, the eukaryotic cells are mammalian cells. Mammalian cells are any cells derived from mammals. Mammalian cells explicitly include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is HEK 293 cells, a human embryonic kidney cell line. HEK 293 cells can be obtained from the American Type Culture Collection (Manassas, VA) as CRL-1533 and from Invitrogen (Carlsbad, Calif.) as 293-H cells (catalog number 11631-017) or 293-F cells (catalog number 11625-019). In some embodiments, the mammalian cell is PER.C6. ® Cells, specifically a human cell line derived from the retina. PER.C6 ®Cells can be obtained from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells. CHO cells can be obtained from the American Type Culture Collection (Manassas, VA.) (e.g., CHO-K1; CCL-61). In other embodiments, the mammalian cells are young hamster kidney (BHK) cells. BHK cells can be obtained from the American Type Culture Collection (Manassas, VA.) (e.g., CRL-1632). In some embodiments, the mammalian cells are HKB11 cells, which are hybrid cell lines of HEK293 cells and human B cell lines. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

[0340] In one embodiment, a plasmid including the coding sequence for the FVIII(X2)-Fc fusion, the coding sequence for the VWF protein-L1-X1-L2-Fc, or both, and an optional marker (e.g., zeocin resistance) is transfected into HEK 293 cells to produce a chimeric protein.

[0341] In another embodiment, a plasmid including the coding sequence for the FVIII-Fc fusion, the coding sequence for the VWF protein-L1-X-L2-Fc, or both, and an optional marker (e.g., zeocin resistance) is transfected into HEK 293 cells to produce a chimeric protein.

[0342] In some embodiments, a first plasmid including the coding sequence for the FVIII(X2)-Fc fusion and a first optional marker (e.g., a bleomycin resistance gene), a second plasmid including the coding sequence for the VWF protein-L1-X1-L2-Fc and a second optional marker (e.g., a neomycin resistance gene), and a third plasmid including the coding sequence for a protein convertase and a third optional marker (e.g., a hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The first and second plasmids may be introduced in equal amounts (i.e., a 1:1 molar ratio), or they may be introduced in unequal amounts.

[0343] In other embodiments, a first plasmid including the coding sequence for the FVIII-Fc fusion and a first optional marker (e.g., a bleomycin resistance gene), a second plasmid including the coding sequence for the VWF protein-L1-X-L2-Fc and a second optional marker (e.g., a neomycin resistance gene), and a third plasmid including the coding sequence for a protein convertase and a third optional marker (e.g., a hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The first and second plasmids may be introduced in equal amounts (i.e., a 1:1 molar ratio), or they may be introduced in unequal amounts.

[0344] In other embodiments, a first plasmid including the coding sequence of the FVIII(X2)-Fc fusion and a first optional marker (e.g., a bleomycin resistance gene), a second plasmid including the coding sequence of the VWF protein-L1-X1-L2-Fc fusion and a second optional marker (e.g., a neomycin resistance gene), and a third plasmid including the coding sequence of a protein convertase and a third optional marker (e.g., a hygromycin resistance gene) are co-transfected into HEK 293 cells to produce a chimeric protein. The first and second plasmids may be introduced in equal amounts (i.e., a 1:1 molar ratio), or they may be introduced in unequal amounts.

[0345] In some embodiments, a first plasmid including a chimeric protein encoding the FVIII (with or without XTEN)-F1-L3-F2-L2-X-L1-V coding sequence and a first optional marker (e.g., a bleomycin resistance gene) and a second plasmid including a protein convertase coding sequence and a second optional marker (e.g., a hygromycin resistance gene) are co-transfected into HEK 293 cells to produce the chimeric protein. The promoters for the FVIII(X)-F1 coding sequence and the V-L2-X-L1-F2 coding sequence may be different or they may be the same.

[0346] In other embodiments, the transfected cells are stably transfected. These cells can be selected and maintained as stable cell lines using conventional techniques known to those skilled in the art.

[0347] Host cells containing a protein-containing DNA construct are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" means a medium containing the nutrients required for cell growth. Nutrients required for cell growth may include carbon sources, nitrogen sources, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the medium may contain fetal bovine serum or fetal bovine serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium will typically select cells containing the DNA construct by, for example, drug selection or the absence of essential nutrients, which are supplemented by selectable markers on or co-transfected with the DNA construct. Cultured mammalian cells are typically grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbad, CA.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, CA.). The selection of a medium suitable for the specific cell line used is at the level of a person skilled in the art.

[0348] To co-express two polypeptide chains of a chimeric protein, host cells are cultured under conditions that allow for the expression of both chains. As used herein, culture means maintaining viable cells in vitro for at least a certain period of time. Maintenance may, but does not necessarily, involve an increase in the viable cell population. For example, cells maintained in culture may be static in terms of population size but remain viable and capable of producing the desired product, such as a recombinant protein or a recombinant fusion protein. Conditions suitable for culturing eukaryotic cells are well known in the art and include appropriate selection of culture media, culture supplements, temperature, pH, oxygen saturation, etc. For commercial purposes, culture may include the use of any of a variety of scale-up systems, including shaker flasks, roller flasks, hollow fiber bioreactors, stirred tank bioreactors, airlift bioreactors, wavebag bioreactors, and other systems.

[0349] Cell culture conditions are also selected to allow the VWF fragment to associate with the FVIII protein. Conditions that allow expression of the VWF fragment and / or the FVIII protein may include the presence of a vitamin K source. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 medium (Invitrogen, Carlsbad, CA) or OptiCHO medium (Invitrogen, Carlsbad, CA) supplemented with 4 mM glutamine.

[0350] In one aspect, the present invention relates to a method for expressing, preparing or producing the chimeric protein of the invention, comprising a) transfecting a host cell containing a polynucleotide encoding the chimeric protein and b) culturing the host cell in a culture medium under conditions suitable for expressing the chimeric protein, wherein the chimeric protein is expressed.

[0351] In other embodiments, protein products containing an FVIII protein linked to a first Ig constant region or a portion thereof and / or a VWF protein fused to a second Ig constant region or a portion thereof via an XTEN sequence are secreted into the culture medium. The culture medium is then separated from the cells, concentrated, filtered, and subsequently passed through two or three affinity columns, such as a protein A column and one or two anion exchange columns.

[0352] In some respects, the present invention relates to chimeric proteins produced by the methods described herein.

[0353] In vitro production allows for scale-up to produce large quantities of the desired modified peptides of the present invention. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or a continuous stirred-tank reactor, or, for example, immobilized or embedded cell culture in hollow fibers, microcapsules, agarose microbeads, or ceramic columns. If necessary and / or required, the peptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC), DEAE-cellulose chromatography, or affinity chromatography.

[0354] IV. Pharmaceutical Compositions

[0355] Compositions containing the chimeric proteins of the present invention may contain a pharmaceutically acceptable carrier. For example, they may contain excipients and / or adjuvants that facilitate the formulation of the active compound into a preparation designed for delivery to the site of action.

[0356] Pharmaceutical compositions may be formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular) via rapid concentration. Injectable formulations may be provided, for example, in preservative-added unit dosage forms in ampoules or multi-dose containers. Compositions may be in the form of suspensions, solutions, or emulsions in oily or aqueous media and may contain formulations such as suspensions, stabilizers, and / or dispersants. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable media (e.g., pyrogen-free water).

[0357] Formulations suitable for parenteral administration also include aqueous solutions of the active compound in a water-soluble form (e.g., water-soluble salts). Additionally, suspensions of the active compound in a suitable oily injectable suspension can be administered. Suitable lipophilic solvents or media include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides). Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the molecules of the invention for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delay agents, water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc. In some embodiments, the composition comprises an isotonic agent, such as a sugar, a polyol (e.g., mannitol, sorbitol), or sodium chloride. In other embodiments, the composition includes pharmaceutically acceptable substances that enhance the shelf life or effectiveness of the active ingredient, such as humectants or small amounts of excipients, such as humectants or emulsifiers, preservatives or buffers.

[0358] The compositions of the present invention can be in various forms, including, for example, liquids (e.g., injectable and infusionable solutions), dispersions, suspensions, semi-solids, and solid dosage forms. Preferred forms depend on the administration method and therapeutic application.

[0359] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentrations. A sterile injectable solution can be prepared by incorporating the active ingredient, in the desired amount, with one or a combination of the ingredients listed above into a suitable solvent, followed by filtration sterilization. Dispersions are typically prepared by incorporating the active ingredient into a sterile medium containing a basic dispersion medium and any other desired ingredients from the ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution. Appropriate flowability of the solution can be maintained, for example, by using a coating agent (such as lecithin), in the case of a dispersion by maintaining the desired particle size, and by using a surfactant. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., monostearate and gelatin) in the composition.

[0360] The active ingredient may be formulated with a controlled-release formulation or device. Examples of such formulations and devices include implants, percutaneous patches, and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations and devices are known in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, ed. JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0361] Injectable reservoir formulations can be prepared by forming a drug within a microencapsulation matrix of a biodegradable polymer such as poly(lactide-polyglycolic acid). The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the polymer used. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Reservoir injectable formulations can also be prepared by encapsulating the drug in liposomes or microemulsions.

[0362] Complementary active compounds may be incorporated into the composition. In one embodiment, the chimeric protein of the present invention is formulated with another coagulation factor or a variant, fragment, analog, or derivative thereof. For example, coagulation factors include, but are not limited to, factors V, VII, VIII, IX, X, XI, XII, XIII, prothrombin, fibrinogen, van Wilbond factor, or recombinant soluble tissue factor (rsTF), or any activated form of the foregoing. The coagulation factor of the hemostatic agent may also include an antifibrinolytic agent, such as ε-aminohexanoic acid or tranexamic acid.

[0363] Dosing regimens can be adjusted to provide the optimal required response. For example, a single large pill can be administered, several fractionated doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is advantageous to formulate parenteral compositions in dosage units for ease of administration and to achieve dosage uniformity. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0364] In addition to active compounds, liquid dosage forms may also contain inert ingredients such as water, ethanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oil, glycerol, tetrahydrofuran alcohol, polyethylene glycol, and fatty acid esters of sorbitol.

[0365] Non-limiting examples of suitable drug carriers are also described in Remington's Pharmaceutical Sciences by EW Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition may also contain pH buffers and wetting agents or emulsifiers.

[0366] For oral administration, the pharmaceutical composition may be in tablet or capsule form prepared by conventional means. The composition may also be prepared as a liquid, such as a syrup or suspension. Liquids may include suspensions (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (lecithin or acacia), non-aqueous mediators (e.g., almond oil, oily esters, ethanol, or fractionated vegetable oils), and preservatives (e.g., methylparaben or propylparaben or sorbic acid). Formulations may also include flavoring agents, coloring agents, and sweeteners. Alternatively, the composition may be provided in a dry product form reconstituted with water or another suitable mediator.

[0367] For buccal administration, the composition may be in tablet or lozenge form according to conventional protocols.

[0368] For administration by inhalation, the compound intended for use according to the invention is preferably delivered in the form of an aerosol with or without excipients, or as an aerosol spray from a pressurized package or nebulizer optionally containing a propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for delivering a metered quantity. Capsules and cartridges, such as gelatin, can be formulated for use in inhalers or blowpipes, containing a mixture of the compound with a suitable powder matrix such as lactose or starch.

[0369] The pharmaceutical composition may also be formulated, for example, in the form of a suppository or retention enema containing a conventional suppository base (such as cocoa butter or other glycerides) for rectal administration.

[0370] In one embodiment, the pharmaceutical composition comprises a chimeric protein, a polynucleotide encoding the chimeric protein, a carrier comprising the polynucleotide or a host cell comprising the carrier, and a pharmaceutically acceptable carrier. The FVIII protein in the chimeric protein has an extended half-life compared to wild-type FVIII protein or a corresponding FVIII protein without the VWF fragment. In one embodiment, the half-life of the chimeric protein is extended by at least about 1.5 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 compared to wild-type FVIII. In another implementation, the half-life of factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0371] In some embodiments, the composition is administered via a route selected from the group consisting of: topical application, intraocular application, parenteral application, intrathecal application, subdural application, and oral application. Parenteral application may be intravenous or subcutaneous.

[0372] In other embodiments, the composition is used to treat bleeding disorders or conditions in a subject in need. Bleeding disorders or conditions are selected from the group consisting of: bleeding coagulation disorders, joint effusion, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, iliopsoas sheath bleeding, and any combination thereof. In other embodiments, the subject is scheduled to undergo surgery. In other embodiments, the treatment is preventative or as needed.

[0373] V. Gene therapy

[0374] The chimeric protein of this invention can be produced in vivo in mammals, such as human patients, and the use of gene therapy methods to treat bleeding disorders or conditions selected from the group consisting of: bleeding coagulation disorders, joint effusion, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, and iliopsoas sheath bleeding. In one embodiment, the bleeding disorder or condition is hemophilia. In another embodiment, the bleeding disorder or condition is hemophilia A. This involves administering a suitable chimeric protein-encoding nucleic acid operably linked to a suitable expression control sequence. In one embodiment, these sequences are incorporated into a viral vector. Viral vectors suitable for the gene therapy include adenovirus vectors, lentivirus vectors, baculovirus vectors, Epstein-Barr virus vectors, papovaviral vectors, vaccinia virus vectors, herpes simplex virus vectors, and adeno-associated virus (AAV) vectors. The viral vector may be a replication-defective viral vector. In other embodiments, the adenovirus vector has a deletion in its E1 or E3 gene. When using an adenovirus vector, mammals may not be exposed to nucleic acids encoding selectable marker genes. In other embodiments, the sequence is incorporated into a non-viral vector known to those skilled in the art.

[0375] VI. Using chimeric proteins

[0376] This invention relates to a method for preventing or inhibiting endogenous VWF binding to FVIII protein using the chimeric protein described herein. This invention also relates to a method using a chimeric protein having an FVIII protein linked to or a portion of the XTEN and Ig constant regions.

[0377] One aspect of the invention relates to preventing or inhibiting the interaction between FVIII and endogenous VWF by blocking or masking the VWF binding site on FVIII from endogenous VWF, and simultaneously using an XTEN sequence combined with an Ig constant region or a portion thereof, which may also be a half-life extender, to extend the half-life of the chimeric protein. In one embodiment, the invention relates to a method for constructing an FVIII protein with a half-life longer than wild-type FVIII. Chimeric proteins suitable for the method include any one or more chimeric proteins described herein.

[0378] Another aspect of the invention includes a method of administering a chimeric protein comprising an FVIII protein having a half-life longer than that of wild-type FVIII to a subject in need, wherein the method includes administering the chimeric protein described herein to the subject.

[0379] In one embodiment, the present invention relates to a method for improving the half-life of a chimeric protein comprising FVIII and VWF proteins using an XTEN sequence and an Ig constant region or a portion thereof, which prevents or inhibits the interaction of endogenous VWF with the FVIII protein. FVIII proteins linked to an XTEN sequence (e.g., FVIII(X)) and subsequently bound to or associated with a VWF protein fused to the XTEN and an Ig constant region or a portion thereof are shielded or protected from VWF clearance pathways, and thus have a reduced clearance rate compared to FVIII proteins not bound to a VWF protein. Therefore, the half-life of the shielded FVIII protein is maximized compared to FVIII proteins not bound to or associated with an XTEN sequence and a VWF protein. In some embodiments, FVIII proteins associated with or protected by a VWF protein and linked to an XTEN sequence are not cleared by VWF clearance receptors. In other embodiments, FVIII proteins associated with or protected by VWF proteins and linked to the XTEN sequence are removed from the system more slowly than FVIII proteins not associated with or protected by VWF proteins and linked to the XTEN sequence.

[0380] In one aspect, chimeric proteins comprising FVIII proteins linked to an XTEN sequence or FVIII proteins that bind to or associate with a VWF protein linked to an XTEN sequence have reduced clearance from circulation because the VWF protein does not contain a VWF clearance receptor binding site. The VWF protein prevents or inhibits the systemic clearance of FVIIIs bound to or associated with the VWF protein via the VWF clearance pathway. VWF proteins suitable for use in this invention may also provide at least one or more VWF-like FVIII protective properties provided by endogenous VWFs. In some embodiments, the VWF protein or XTEN sequence may also mask one or more FVIII clearance receptor binding sites, thereby preventing FVIII clearance via its own clearance pathway.

[0381] In some implementations, the prevention or inhibition of FVIII protein binding to endogenous VWF by the VWF protein or the XTEN sequence can occur in vitro or in vivo.

[0382] A method for increasing the half-life of a chimeric protein is also provided, comprising administering the chimeric protein described herein to a subject in need. The half-life of inactive FVIII bound to or associated with full-length VWF in plasma is approximately 12 to 14 hours. In type 3 VWD, where VWF is almost absent in circulation, the half-life of FVIII is only about six hours, resulting in mild to moderate symptoms of hemophilia A in said patients due to reduced FVIII concentrations. The half-life of the chimeric protein of the present invention, which is linked or associated with a VWF fragment or XTEN sequence, may be at least about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 times greater than that of an inactive FVIII that is bound or associated with full-length VWF.

[0383] In one embodiment, a chimeric protein comprising a first polypeptide (which contains an FVIII protein and a first Ig constant region or a portion thereof) and a second polypeptide (which contains a VWF protein, an XTEN having less than 288 amino acids, and an Ig constant region or a portion thereof) exhibits a half-life at least about 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7, 8, 9, or 10 times longer than a corresponding chimeric protein or wild-type FVIII comprising the same first polypeptide and a second polypeptide without the XTEN sequence. In another embodiment, a chimeric protein comprising a first polypeptide (which comprises FVIII protein and a first Ig constant region or a portion thereof) and a second polypeptide (which comprises VWF protein, an XTEN having less than 288 amino acids, and an Ig constant region or a portion thereof) exhibits a half-life that is about 2 to 5 times, about 3 to 10 times, about 5 to 15 times, about 10 to 20 times, about 15 to 25 times, about 20 to 30 times, about 25 to 35 times, about 30 to 40 times, or about 35 to 45 times longer than a corresponding chimeric protein comprising the same first polypeptide and a second polypeptide without the XTEN sequence, or wild-type FVIII. In a particular embodiment, in FVIII and VWF dual knockout mice, the half-life of the chimeric protein of the present invention is at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 times longer than the half-life of wild-type FVIII.

[0384] In some implementations, the chimeric protein exhibits a half-life of approximately 40 hours in mice.

[0385] In some embodiments, the half-life of the chimeric protein is longer than that of FVIII associated with endogenous VWF. In other embodiments, the half-life of the chimeric protein is at least about 1.5, 2, 2.5, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, or 5.0 times that of wild-type FVIII or FVIII protein associated with endogenous VWF.

[0386] In some embodiments, as a result of the invention, the chimeric protein has a prolonged half-life compared to VWF-free FVIII protein or wild-type FVIII. The half-life of the chimeric protein of the present invention is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, 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 the half-life of VWF-free chimeric protein or wild-type FVIII. In one embodiment, the half-life of FVIII is about 1.5 times to about 20 times, about 1.5 times to about 15 times, or about 1.5 times to about 10 times longer than the half-life of wild-type FVIII. In another embodiment, compared to wild-type FVIII or FVIII protein without VWF protein, the half-life of FVIII is extended by about 2 to about 10 times, about 2 to about 9 times, about 2 to about 8 times, about 2 to about 7 times, about 2 to about 6 times, about 2 to about 5 times, about 2 to about 4 times, about 2 to about 3 times, about 2.5 to about 10 times, about 2.5 to about 9 times, about 2.5 to about 8 times, about 2.5 to about 7 times, about 2.5 to about 6 times, about 2.5 to about 5 times, about 2.5 to about 4 times, about 2.5 to about 3 times, about 3 to about 10 times, about 3 to about 9 times, about 3 to about 8 times, about 3 to about 7 times, about 3 to about 6 times, about 3 to about 5 times, about 3 to about 4 times, about 4 to about 6 times, about 5 to about 7 times, or about 6 to about 8 times. In other embodiments, the half-life of the chimeric protein of the present invention is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 40 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In other embodiments, the half-life of the chimeric protein of the present invention is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours to about one week, about 24 hours to about one week, about 36 hours to about one week, about 48 hours to about one week, about 60 hours to about one week, about 24 hours to about six days, about 24 hours to about five days, about 24 hours to about four days, about 24 hours to about three days, or about 24 hours to about two days.

[0387] In some embodiments, the chimeric protein of the present invention has a mean half-life of about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about six days, about seven days (one week), about eight days, about nine days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days, depending on the subject.

[0388] Furthermore, the present invention provides a method for treating or preventing bleeding disorders or conditions, comprising administering an effective amount of chimeric protein. In one embodiment, the bleeding disorder or condition is selected from the group consisting of: bleeding coagulation disorders, joint effusion, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, and iliopsoas muscle sheath bleeding. In a particular embodiment, the bleeding disorder or condition is hemophilia A.

[0389] The chimeric protein prepared by this invention, comprising an XTEN sequence and a portion thereof in combination with the VWF protein described herein (which prevents or inhibits the interaction of FVIII protein with endogenous VWF), has many uses as will be recognized by those skilled in the art, including, but not limited to, methods for treating subjects with hemostatic disorders and methods for treating subjects requiring general hemostatic agents. In one embodiment, the present invention relates to a method for treating a subject with a hemostatic disorder, comprising administering a therapeutically effective amount of the chimeric protein.

[0390] The FVIII protein portion of the chimeric protein treats or prevents blood disorders by acting as a cofactor of factor IX on a negatively charged phospholipid surface, thereby forming the X enzyme complex. Activation of the clotting factor binds to the phospholipid surface, localizing this process to the site of vascular damage. On the phospholipid surface, factor VIIIa increases the maximum rate of factor X activation achieved by factor IXa by approximately 200,000 times, resulting in a massive, instantaneous burst of thrombin production.

[0391] The chimeric protein of this invention can be used to treat any hemostatic disorder. Hemostatic disorders that can be treated by applying the chimeric protein of this invention include, but are not limited to, hemophilia A and deficiencies or structural abnormalities associated with factor VIII. In one embodiment, the hemostatic disorder is hemophilia A.

[0392] The chimeric protein of the present invention can be used preventively to treat subjects suffering from hemostatic disorders. The chimeric protein of the present invention can be used to treat acute bleeding episodes in subjects suffering from hemostatic disorders. In another embodiment, the hemostatic disorder may be the result of a defective coagulation factor, such as van Wilbond factor. In one embodiment, the hemostatic disorder is a genetic disorder. In another embodiment, the hemostatic disorder is an acquired disorder. Acquired disorders may be caused by latent secondary diseases or conditions. Irrelevant conditions may be, for example, but not limited to, cancer, autoimmune diseases, or pregnancy. Acquired disorders may be caused by old age or by drug treatment (e.g., cancer chemotherapy) used to treat latent secondary diseases.

[0393] This invention also relates to a method for treating a subject who does not have a congenital hemostatic disorder but suffers from a secondary disease or symptom that leads to the acquisition of a hemostatic disorder (e.g., due to the production of anti-FVIII antibodies or surgery). Therefore, this invention relates to a method for treating a subject requiring a general hemostatic agent, comprising administering a therapeutically effective amount of a chimeric protein prepared by the method of this invention.

[0394] The present invention also relates to methods for reducing the immunogenicity of FVIII or inducing less immunogenicity against FVIII, comprising administering an effective amount of the chimeric protein described herein or its encoded polynucleotide.

[0395] In one implementation, a subject requiring general hemostatic agents is undergoing or about to undergo surgery. The chimeric protein of the present invention can be administered as a preventative measure before, during, or after surgery. The chimeric protein of the present invention can be administered before, during, or after surgery to control acute bleeding episodes.

[0396] The chimeric protein of this invention can be used to treat subjects with acute bleeding episodes who do not have hemostatic disorders. Acute bleeding episodes can be caused by severe trauma, such as surgery, car accidents, wounds, gunshot lacerations, or any other traumatic event that leads to uncontrolled bleeding. Non-limiting examples of bleeding episodes include bleeding coagulopathy, effusion of joints, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, bleeding of the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, and any combination thereof.

[0397] In preventative applications, a composition containing one or more chimeric proteins or mixtures thereof of the present invention is administered to a patient who is not yet in a disease state to enhance the patient's resistance or alleviate symptoms associated with the disease or condition. This amount is defined as the "preventative effective dose." In therapeutic applications, sometimes relatively high doses (e.g., about 1 to 400 mg / kg of peptide per dose, with doses of 5 to 25 mg more commonly used for radioimmunoconjugates and higher doses for cytotoxic drug-modified peptides) are required at relatively short intervals until disease progression is reduced or terminated, and until the patient shows partial or complete improvement in disease symptoms. Thereafter, a preventative regimen may be administered to the patient.

[0398] In some embodiments, the chimeric proteins or compositions of the present invention are used for on-demand treatment of bleeding episodes, including hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscles, oral bleeding, trauma, head trauma (head injury), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, fractures, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding in the iliopsoas sheath. Subjects may require surgical prevention, perioperative management, or surgical treatment. Such surgeries include, for example, minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hernia incision, synovectomy, total knee replacement, craniotomy, bone suture, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

[0399] In one embodiment, the chimeric protein of the present invention is administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, such as orally, sublingually, buccally, nasally, rectally, vaginally, or via the lungs. The chimeric protein of the present invention, comprising the VWF fragment and FVIII protein, can be implanted within or attached to a biopolymer solid carrier that allows for slow release of the chimeric protein to the bleeding site, or implanted in a bandage / dressing. The dosage of the chimeric protein will vary depending on the subject and the specific route of administration used. The dosage can range from 0.1 to 100,000 μg / kg body weight. In one embodiment, the dosage range is 0.1–1,000 μg / kg. In another embodiment, the dosage range is 0.1–500 μg / kg. The protein can be administered continuously or at specific time intervals. Optimal dosage ranges and / or administration durations can be determined using in vitro assays. In vitro assays measuring coagulation factor activity are known in the art, such as the STA-CLOT VIIa-rTF coagulation assay or the ROTEM coagulation assay. In addition, the effective dose can be extrapolated from the dose-response curves obtained from animal models (e.g., hemophilic dogs) (Mount et al. 2002, Blood 99(8):2670).

[0400] The present invention has now been described in detail, and will become more apparent from the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention. All patents, publications and articles mentioned herein are expressly and specifically incorporated herein by reference.

[0401] Example

[0402] Unless otherwise stated, the following materials and methods are used throughout the embodiments.

[0403] Materials and methods

[0404] Generally, unless otherwise indicated, the present invention is practiced using conventional chemical techniques, biophysical techniques, molecular biological techniques, recombinant DNA techniques, immunological techniques (especially antibody techniques), and standard electrophoresis techniques. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), ed. McCafferty, Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., CS.HL Press, Pub. (1999); and Current Protocols in Molecular Biology, ed. Ausubel et al., John Wiley & Sons (1992).

[0405] Example 1: FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer

[0406] This invention relates to the production of a chimeric FVIII molecule in which the Fc domain of IgG is coupled to the D'D3 domain of a van Wilbond factor (VWF) protein. The linked D'D3 domain prevents FVIII from interacting with endogenous VWF multimers. This molecule serves as a platform for incorporating other half-life extension technologies to improve the pharmacokinetics of chimeric proteins. An XTEN sequence is incorporated into the FVIII B domain and between the D'D3 and Fc regions to increase the half-life of the FVIII / VWF heterodimer.

[0407] The thrombin cleavage site between D'D3 and Fc allows the release of the D'D3 domain after the FVIII molecule is activated by thrombin.

[0408] Example 2: Plasmid construction of FVIII-XTEN-Fc / D'D3-Fc heterodimer

[0409] Cloning VWF050 - VWF031 containing the IHH triple mutation

[0410] The IHH triple mutation in Fc prevents interaction with FcRn, thus eliminating the possibility of Fc-containing molecules being recycled via the FcRn pathway. The three mutations in Fc are I253A, H310A, and H435A.

[0411] VWF050 was generated by exchanging the Fc region of the VWF031 plasmid between the RsRII restriction site and the Not 1 restriction site with an Fc fragment containing the IHH triple mutation.

[0412] Cloning VWF057-cloning VWF-Fc with a 144 AE XTEN+35 aa thrombin-cleavable linker.

[0413] Oligomers

[0414] ESC 155 -Oligomer of 144 AE XTEN in VWF034-Reverse

[0415] CCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGAACCTCCACCGCCGCTCGAGGCACCTTCTTCAGTGCTGGTGGGCGAGCCCGCTGGTGACCCTTCCTC

[0416] ESC 156 -Oligomer for the 144 AE XTEN-GS connector in VWF034-Reverse

[0417] GGGGAAGAGGAAGACTGACGGTCCGCCCAGGAGTTCTGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGCCTCCGCTGCCCCGGGGGACCAGGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCC

[0418] ESC 157 -Oligomer of 144 AE XTEN in VWF031-forward

[0419] GTGAAGCCTGCCAGGAGCCGATATCGGGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGC

[0420] Two PCRs were performed to obtain 144 AE-XTEN+ 35 aa GS adapters with thrombin cleavage sites.

[0421] A first PCR reaction was performed using 144-AE XTEN encoding DNA as a template and the ESC 157 / ESC 155 primer pair. The approximately 550 bp PCR product obtained from this reaction was used as a template for a second PCR reaction and amplified using the ESC157 / 156 primer pair. This reaction produced a product of approximately 700 bp. This 700 bp PCR product and the VWF034 plasmid were then digested with EcoRV-HF and RsRII. The plasmid backbone was then digested.

[0422] Next, the 700 bp PCR product was ligated using VWF034.

[0423] IHH triple mutation in clones VWF058-VWF034

[0424] The IHH triple mutation in Fc prevents interaction with FcRn, thus eliminating the possibility of Fc-containing molecules being recycled via the FcRn pathway. The three mutations in Fc are I253A, H310A, and H435A.

[0425] VWF058 was generated by exchanging the Fc region of the VWF034 plasmid between the RsRII restriction site and the Not 1 restriction site with an Fc fragment containing the IHH triple mutation.

[0426] Cloning FVIII-263-FVIII 205 with the IHH triple mutation

[0427] The IHH triple mutation in Fc prevents interaction with FcRn, thus eliminating the possibility of Fc-containing molecules being recycled via the FcRn pathway. The three mutations in Fc are I253A, H310A, and H435A.

[0428] FVIII-263 was generated by exchanging the Fc region of the FVIII 205 plasmid between the RsRII restriction site and the Not 1 restriction site with an Fc fragment containing the IHH triple mutation.

[0429] Cloning FVIII-282 - FVIII-Fc with 144 AE XTEN in the B domain

[0430] ESC 158 -Oligomers targeting 144 AE XTEN in the B domain-forward

[0431] AAGAAGCTTCTCTCAAAACGGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGCCACATCTGGGTCGGAAACGCCAGGC

[0432] ESC 159 -Oligomers targeting 144 AE XTEN in the B domain-reverse

[0433] GGTATCATCATAATCGATTTCCTTCTGATCTGACTGAAGAGTAGTACGAGTTATTTCAGCTTGATGGCGTTTCAAGACTGGTGGGCTCGAGGCACCTTCTTCAGTGCTGGTGGGCGAGCCCGCTGGTGACCCTTCCTCAGTGGACGTAGG

[0434] The first PCR reaction was performed using 144-AE XTEN encoding DNA as a template and the ESC 158 / ESC 159 primer pair. The approximately 550 bp PCR product and FVIII 169 plasmid obtained from this reaction were then digested with AscI and Clal. The plasmid backbone obtained from the digested FVIII 169 was then used to ligate the 550 bp PCR product to obtain FVIII 282.

[0435] Cloning FVIII-283 - FVIII 169 with the IHH triple mutation

[0436] The IHH triple mutation in Fc prevents interaction with FcRn, thus eliminating the possibility of Fc-containing molecules being recycled via the FcRn pathway. The three mutations in Fc are I253A, H310A, and H435A.

[0437] FVIII-283 was generated by exchanging the Fc region of the FVIII 169 plasmid with an Fc fragment containing the IHH triple mutation between the RsRII restriction site and the Not 1 restriction site.

[0438] Example 3: Production of FVIII-XTEN-Fc / D'D3-XTEN-Fc in HEK293 cells

[0439] Figure 2 A schematic diagram showing the expression of the FVIII-XTEN-Fc / D'D3-XTEN-Fc construct. Triple plasmid co-transfection was performed in HEK293 cells using polyethyleneimine (PEI). The first plasmid expressed FVIII-XTEN-Fc, the second plasmid expressed D1D2D'D3-XTEN-Fc, and the third plasmid expressed PACE / furin protease, required for the enzymatic removal of the propeptide (i.e., the D1D2 domain) from D1D2D'D3-XTEN-Fc. The products of this triple plasmid expression system included FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimers, D'D3-XTEN-Fc homodimers, and trace amounts of FVIII-XTEN-Fc hemizygous-like material.

[0440] Example 4: Purification of FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer

[0441] To purify the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer, a tangential flow filtration (TFF) step was used to first concentrate the conditioned medium 10-fold. The product in the filtrate was then further purified sequentially using affinity chromatography and a desalting column. According to HPLC-SEC, the purity of the molecule was acceptable and was further confirmed by Western blotting. The specific activity of the molecule was similar to that of FVIII lacking the B domain, as measured by FVIII activity assay (Example 5) and OD280 measurement.

[0442] Example 5: Specific activity of FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer

[0443] The activities of the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer were measured by FVIII colorimetric assay and activated partial thromboplastin time (aPTT) assay. The specific colorimetric and specific aPTT activities of SQ BDD-FVIII, rFVIII169 / VWF034, and rFVIII169 / VWF057 are listed in Table 16. Compared to SQ BDD-FVIII, we observed similar specific colorimetric activities and a 60% reduction in specific aPTT activities for rFVIII169 / VWF034 and rFVIII169 / VWF057.

[0444] Table 16: Specific activity of heterodimer variants

[0445]

[0446] FVIII Colorimetric Determination

[0447] FVIII activity was measured using the COATEST SP FVIII kit (product number: K824086) from DiaPharma, and all incubations were performed on a 37°C plate heater with shaking.

[0448] The WHO International Standard No. 8 (concentrate, code 07 / 350) for coagulation factor VIII:C was used as the assay standard, with a range of 100 mIU / mL to 0.78 mIU / mL. Two copies of the combined normal human plasma control and test samples (diluted with 1X Coatest buffer) were added to each well of an Immulon 2HB 96-well plate (25 μL / well). Freshly prepared IXa / FX / phospholipid mixture (50 μL), 25 μL of 25 mM CaCl2, and 50 μL of FXa substrate were added sequentially to each well, with incubation for 5 minutes between each addition. After incubation with the substrate, 25 μL of 20% acetic acid was added to terminate the colorimetric reaction, and the absorbance at OD405 was measured using a SpectraMAX plus (Molecular Devices) instrument. Data were analyzed using SoftMaxPro software (version 5.2). The lowest level of quantification (LLOQ) is 7.8 mIU / mL.

[0449] FVIII aPTT Measurement

[0450] The following procedure was performed on a Sysmex CA-1500 coagulation analyzer to determine FVIII aPTT levels: First, 50 μL of aPTT buffer (50 mM Tris, 100 mM NaCl, 1% HSA, pH 7.4) containing manually diluted sample, standard, and control was added to the reaction vessel. Then, 50 μL of FVIII-deficient plasma (George King Bio-Medical, product number: 0800) was added. After incubation at 37°C for 1 minute, 50 μL of aPTT reagent (Actin® FSL activated hyalin reagent - Dade Behring, reference number B4219-2) was added to the reaction mixture and incubated at 37°C for 4 minutes. Subsequently, 50 μL of 20 mM CaCl2 (Dade Behring, reference number ORFO37) was added, and the reaction vessel was immediately transferred to one of the four spectrophotometer channels to measure the amount of refracted light in the mixture, which was converted into coagulation by the instrument's software algorithm. The reported settling time is the duration from the addition of CaCl2 until clot formation occurs. Assay standards were prepared by diluting WHO International Standard 8 FVIII in aPTT buffer at concentrations ranging from 100 mIU / mL to 0.78 mIU / mL. The standard curve was plotted in MS Excel as settling time (in seconds) relative to the logarithm of FVIII activity (base 10) (mIU / mL) on the X-axis, and the activity of individual samples was calculated using a formula for a linear regression line against this standard curve. Based on assay performance, the lower limit of quantification (LLOQ) was 7.8 mIU / mL.

[0451] Example 6: The cumulative effect of XTEN inserts on the half-life extension of heterodimers

[0452] Incorporating the XTEN insert into the heterodimer resulted in a prolonged half-life. Insertion of a single 288 amino acid (aa) AE-XTEN into the FVIII B domain resulted in a half-life of 16.7 hours for the heterodimer in HemA mice. Figure 3 As demonstrated by rFVIII169 / VWF031. To further improve the half-life of the heterodimer, a second XTEN insert of 144 aa or 288 aa in length was incorporated into FVIII169 / VWF031, either in the FVIII A1 domain or immediately downstream of the D'D3 fragment. The heterodimer variants were named FVIII205 / VWF031 and FVIII169 / VWF034.

[0453] The half-lives of rFVIII169 / VWF031, rFVIII205 / VWF031, and rFVIII169 / VWF034 were evaluated in FVIII-deficient (HemA) mice by a single intravenous administration of the test molecules at a dose of 200 IU / kg. Figure 3 Plasma samples were collected at the specified time points indicated in the instructions, and the FVIII activity of the samples was determined by FVIII colorimetric assay. PK parameters were calculated using the WinNonlin-Phoenix program and are listed in Table 17.

[0454] like Figure 3 As shown in Table 17, the addition of a second XTEN insert at the A1 domain of FVIII or downstream of D'D3 further improved the half-life of the heterodimer to 29.45 or 31.10, respectively. Furthermore, clearance and AUC improvements of more than 2-fold were also observed due to both XTEN inserts.

[0455] Table 17: PK parameters of heterodimers in HemA mice

[0456]

[0457] Example 7: When inserted between the D'D3 and Fc domains, 144 aa AE-XTEN provides a better half-life benefit than 288 aa AE-XTEN.

[0458] Another heterodimer, FVIII169 / VWF057, was constructed to attempt to determine the optimal length of the XTEN insert within the D'D3-XTEN-Fc chain, where the length of the XTEN insert was reduced from 288 aa to 144 aa. Figure 4 As shown, the half-life of rFVIII169 / VWF057 increased from 31 hours to 42 hours compared to rFVIII169 / VWF034. Improvements in scavenging rate and AUC of rFVIII169 / VWF057 were also observed, with data listed in Table 18. Therefore, when inserted between the D'D3 and Fc domains of the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer, the 144 aa AE-XTEN insert is superior to AE-288 aaXTEN.

[0459] Table 18: PK parameters of rFVIII169 / VWF034 and rFVIII169 / VWF057 in HemA mice

[0460]

[0461] Example 8: Fc domains extend the half-life of heterodimers

[0462] The Fc domain prolongs the half-life of its fusion protein via an FcRn-mediated recycling pathway. To confirm the necessity of the Fc domain for extending the half-life of the heterodimer, the wild-type Fc domain in rFVIII205 / VWF031 was replaced with a triple mutant (I253A / H310A / H435A; IHH) to form rFVIII263 / VWF050, and the complete elimination of FcRn binding in rFVIII263 / VWF050 was confirmed by surface plasmon resonance (Biacore) assays. The half-life of FVIII263 / VWF050 was evaluated in HemA mice compared to rFVIII205 / VWF031. Increased clearance of rFVIII263 / VWF050 and decreased half-life and AUC were observed. Figure 5 As shown in Table 19. This result demonstrates that, in addition to ensuring the covalent bonding of FVIII and D'D3, the Fc domain is also necessary for improving the half-life of the heterodimer.

[0463] Table 19: PK parameters of rFVIII205 / VWF031 and rFVIII263 / VWF040 in HemA mice

[0464]

[0465] Example 9: Acute efficacy of FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer in a HemA mouse tail clamp bleeding model

[0466] The acute efficacy of the lead heterodimer candidate was evaluated using a HemA mouse tail clamp bleeding model.

[0467] Male HemA mice aged 8–12 weeks were randomly assigned to four treatment groups and treated with a single intravenous administration of SQBDD-FVIII, rFVIII169 / VWF034, rFVIII169 / VWF057, or a carrier solution, respectively. To mimic paroxysmal treatment with FVIII (to restore 50–100% of normal FVIII plasma levels), the selected FVIII treatment dose was 75 IU / kg as measured by FVIII aPTT activity. At this dose level, all tested FVIII variants restored approximately 70% of normal mouse plasma FVIII activity within 5 minutes of administration.

[0468] The blood loss volume of individual animals in the study was plotted on... Figure 6In contrast to animals treated with the vector, a significant reduction in blood loss volume was observed in all FVIII treatment groups. No statistically significant differences in blood loss reduction were found among the three FVIII treatment groups, suggesting that the heterodimer molecule may potentially be as effective as SQ BDD-FVIII for on-demand treatment.

[0469] The blood loss volume of individual animals in the study was plotted on... Figure 6 In contrast to animals treated with the vector, a significant reduction in blood loss volume was observed in all FVIII treatment groups. No statistically significant differences in blood loss reduction were found among the three FVIII treatment groups, suggesting that the heterodimer molecule may potentially be as effective as SQ BDD-FVIII for on-demand treatment.

[0470] In addition, HemA mice were treated with lower doses (37.5 IU / kg) of rBDD-FVIII or rFVIII169 / VWF034, and the results showed that... Figure 6 In Figure B, at the same dose of 75 IU / kg, rFVIII169 / VWF034 provided similar protection to BDD-FVIII in HemA mice following tail clamp injury, indicating that the molecule remained effective in treating severe bleeding episodes in HemA mice at approximately 35% of normal rodent circulating FVIII levels.

[0471] The tail clamping procedure was performed as follows. Briefly, mice were anesthetized with a mixture of 50 mg / kg ketamine and 0.5 mg / kg dexmedetomidine before tail injury and placed on a 37°C heating plate to help maintain body temperature. The mice's tails were then immersed in 37°C saline for 10 minutes to dilate the lateral veins. After vein dilation, the FVIII variant or the carrier solution was injected via the tail vein, followed by 5 minutes after administration using a straight-edged 11-gauge scalpel to sever the distal 5 mm of the tail. Blood was collected over 30 minutes in 13 ml of 37°C saline, and the blood loss volume was determined by the weight change of the blood collection tube: Blood loss volume = (End weight of collection tube - Start weight + 0.10) ml. Statistical analysis was performed using t-tests (Mann Whitney test) and one-way ANOVA (Kruskal-Wallis test; post-hoc test: Dunns multiple comparison test).

[0472] Example 10: The preventive and therapeutic efficacy of FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer in a HemA mouse tail vein transverse bleeding model

[0473] The efficacy of FVIII169 / VWF057 was tested in a HemA mouse tail vein transect (TVT) model. The TVT model induces bleeding by introducing damage into the lateral vein of the mouse tail, mimicking spontaneous bleeding episodes in patients with hemophilia bleeding disorder.

[0474] Male HemA mice aged 8–10 weeks were randomly assigned to four treatment groups and treated with FVIII169 / VWF057 72 hours prior to tail vein injury, or with SQ BDD-FVIII 24 or 48 hours prior to injury. Animals treated with the vector served as negative controls. Events of rebleeding or euthanasia attributable to excessive blood loss within 24 hours of injury were plotted. Figure 7 middle.

[0475] like Figure 7 As shown, unlike mice treated with SQ BDD-FVIII 48 hours before TVT (where only limited protection was observed after injury), mice treated with rFVIII169 / VWF057 72 hours before tail injury showed similar protection against rebleeding and survival compared to mice treated with SQ BDD-FVIII 24 hours before TVT. This indicates that rFVIII169 / VWF057 provides at least a 3-fold or more (e.g., 4-fold) long-lasting protection in HemA mice in the TVT model. Therefore, rFVIII169 / VWF057 may significantly reduce the current treatment frequency for FVIII prophylaxis.

[0476] Similarly, HemA mice were treated with FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimers: rFVIII169 / VWF034 and rFVIII169 / VWF057 24 or 96 hours prior to tail vein injury. Rebleeding and survival data were compared with those obtained via rBDD-FVIII (24 or 48 hours prior to injury) and the vector. Although rebleeding in mice treated with rBDD-FVIII 24 hours prior to injury was similar to that in mice treated with the vector, rebleeding data in mice treated with the heterodimer 24 hours prior to injury were significantly better than those in the vector-treated group. Furthermore, rebleeding data in mice treated with the heterodimer 96 hours prior to injury were similar to those in mice treated with rBDD-FVIII 24 hours prior to injury. Regarding 24-hour survival after TVT injury, compared to a survival rate of less than 50% in mice treated with rBDD-FVIII, more than 90% of mice treated with FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer survived TVT injury when FVIII molecules were administered 24 hours prior to injury. Furthermore, mice treated with FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer 96 hours prior to tail vein injury had better survival (in the case of rFVIII169 / VWF034) or similar survival (in the case of rFVIII169 / VWF057) compared to mice treated with rBDD-FVIII 24 hours prior to injury. Both rebleeding and survival data indicate that FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer therapy has a 4-fold longer efficacy compared to rBDD-FVIII therapy.

[0477] HemA mouse tail vein transverse section model

[0478] The procedure for transverse tail vein incision was performed as follows. Mice were anesthetized with a mixture containing 50 mg / kg ketamine, 0.125 mg / kg dexmedetomidine, and 0.1 mg / kg buprenex. At sufficient depth of anesthesia, the lateral tail vein of the mouse was transversely incised at a diameter of approximately 2.7 mm using a straight-e...

Claims

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

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

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

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

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

8.

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

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

8. The chimeric protein of claim 7, wherein the XTEN sequence of the second polypeptide is selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:14; SEQ ID NO:60; SEQ ID NO:61; SEQ ID NO:62; SEQ ID NO:

63.

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

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

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