EA044115B1 - CHIMERIC FACTOR VIII PROTEINS AND THEIR APPLICATION - Google Patents

Description

Link to sequence listing submitted electronically

Full contents of the sequence list submitted electronically in an ASCII text file (name: 2159_441PC02_SequenceListing_ST25.txt; size: 823500 bytes; creation date: 9

January, 2015, incorporated herein by reference.

State of the art

Hemophilia A is a bleeding disorder caused by defects in the gene encoding clotting factor VIII (FVIII) and affects 1-2 in 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients with hemophilia A can be treated with infusions of purified or recombinant FVIII. All commercially available FVIII products, however, are known to have a half-life of approximately 8-12 hours, requiring frequent intravenous administration to patients. See Weiner M.A. and Cairo, M.S., Pediatric Hematology Secrets, Lee, M.T., 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried to extend the half-life of FVIII. For example, approaches under development to prolong the half-life of coagulation factors include PEGylation, glycopegylation, and albumin conjugation. See Dumont et al., Blood. 119(13): 3024-3030 (published online January 13, 2012). However, according to the literature, long-acting FVIII drugs currently in development, regardless of the protein engineering method used, have limited half-lives - only up to about 1.5-2 hours in preclinical studies in animal models. See also there. Consistent results have been obtained in humans; for example, use of rFVIIIFc has been reported to increase the half-life to a maximum of about 1.7 times that of ADVATE® in patients with hemophilia A. See ibid. Thus, 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 meeting, abstract #P-M-035; Henrik, A. et al., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131.

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 half-life of VWF can be influenced by a number of factors: glycosylation profile, ADAMTS-13 (disintegrin and metalloprotease with thrombospondin motif-13), and various mutations in the VWF gene.

In plasma, 95-98% of FVIII circulates in a close non-covalent complex with full-length VWF. Formation of this complex is important for maintaining proper 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 is mainly present as a heterodimer consisting of a heavy chain (molecular weight - 200 kDa) and a light chain (molecular weight - 73 kDa). When FVIII is activated by proteolysis at positions 372 and 740 of the heavy chain and position 1689 of the light chain, VWF bound to FVIII is cleaved from activated FVIII. Activated FVIII, together with activated factor IX, calcium and phospholipid (tenase complex), induces activation of factor X, generating thrombin in large quantities. Thrombin, in turn, cleaves fibrinogen to form soluble monomers, which then spontaneously polymerize to form soluble fibrin polymer. Thrombin also activates factor XIII, which, together with calcium, cross-links and stabilizes the soluble fibrin polymer, forming cross-linked (insoluble) fibrin. Activated FVIII is rapidly cleared from the circulation by proteolysis.

Due to the need for frequent administration and the inconvenience of the administration schedule, there is still a need to develop FVIII preparations that require less frequent administration, i.e. FVIII drug, the half-life of which exceeds the above-mentioned 1.5-2-fold limit.

Summary of the invention

The present invention provides a chimeric protein comprising (i) a first polypeptide consisting of a factor VIII (FVIII) protein fused to a first immunoglobulin (IG) constant region or portion thereof, and (ii) a second polypeptide consisting of a von Willebrand factor protein ( VWF), which includes domain D' and domain D3 of VWF, fused to the second constant region of the IG or part thereof through the XTEN sequence located between them, characterized in that the XTEN sequence contains less than 288 amino acid residues, and characterized in that the first polypeptide is linked or connected to a second polypeptide. Certain embodiments of the invention include a chimeric protein as described herein, characterized in that the XTEN sequence in the second polypeptide consists of an amino acid sequence ranging from 12 to 287 amino acids in length.

Also disclosed is a chimeric protein as described herein, wherein 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 of the fusion protein includes an XTEN sequence containing at least at least 288 amino acids. Some embodiments of the invention include an XTEN AE288 sequence containing at least 288 amino acids. In some embodiments, AE288 is the sequence of SEQ ID NO: 8.

- 1 044115

Also disclosed is a chimeric protein as described herein, 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.

Some embodiments of the invention include a chimeric protein as described herein, characterized in that the XTEN sequence of the second polypeptide is selected from

SEQ GO NO: 9, SEQ GO NO: 10, SEQ GO NO: AND,

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.

In certain embodiments of the invention, the first polypeptide further comprises a second XTEN sequence that binds the FVIII protein to the first IG constant region or portion thereof. Also disclosed is a chimeric protein as described herein, wherein the first polypeptide contains a third XTEN sequence inserted into 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 certain embodiments of the invention, the first polypeptide contains a third XTEN sequence that binds the FVIII protein to the first IG constant region or portion thereof.

Also disclosed is a chimeric protein as described herein, wherein the second XTEN sequence, the third XTEN sequence, or the second and third XTEN sequences 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 the second and third XTEN sequences are each independently selected from

SEQ GO NO: 8; SEQ GO NO: 9, SEQ GO NO: 10, SEQ GO

NO: 11, SEQ ID NO: 17; SEQ ID NO: 54; SEQ ID NO: 19; SEQ ID NO: 16; SEQ GO 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 certain embodiments of the invention, 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 portion thereof via a linker. In certain embodiments of the invention, the linker is a cleavable linker.

Some embodiments of the invention include a chimeric protein as described herein, characterized in that the linker is cleavable by a protease selected from factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase 2, granzyme B, tobacco etch virus (TEV) protease, enterokinase, protease 3C, sortase A, matrix metalloprotease-12 (MMP-12), MMP-13, MMP-17 and MMP-20. In some embodiments, the linker is cleavable by factor IIa (thrombin).

Also disclosed is a chimeric protein as described herein, characterized in that the linker contains one or more cleavage sites containing an amino acid sequence selected from

- 2 044115

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), 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 linker comprises the sequence TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO. 146). in some embodiments, the cleavage sites comprise 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 the FVIII protein. In some embodiments, the cleavage site comprises a fragment of the a2 region of the FVIII protein containing at least the sequence IEPR (SEQ ID NO. 200). in other embodiments, the cleavage site comprises a fragment of the a2 region of the FVIII protein containing at least an IEPR sequence (SEQ ID NO. 200), wherein the cleavage site is not the full length a2 region. In certain embodiments of the invention, the cleavage site is amenable to cleavage in a thrombin hydrolysis test, as proposed herein or known in the art.

Some embodiments of the invention include a chimeric protein as described herein, wherein the first IG constant region or a portion thereof contains a first Fc fragment and/or the second IG constant region or a portion thereof contains a second Fc fragment. In some embodiments, the first IG constant region or portion thereof and the second IG constant region or portion thereof prolong the half-life of the chimeric protein. In some embodiments, the first polypeptide and the second polypeptide are fused through a linker. In certain embodiments of the invention, the first polypeptide and the second polypeptide are fused through a processable linker. In some embodiments of the invention, a first IG constant region or a portion thereof is connected to a second IG constant region or a portion thereof. In certain embodiments of the invention, the first IG constant region or portion thereof is connected to the second IG constant region or portion thereof by a covalent bond. In some embodiments, the covalent bond is a disulfide bond.

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Also described is a chimeric protein comprising each of the following formulas (a)-(hh):

(a) FVin-Fl:F2-L2-X-Ll-V;

(b) FVin-Fl:V-Ll-X-L2-F2;

(c) Fl-FVni:F2-L2-X-Ll-V;

(d) Fl-FVni:V-Ll-X-L2-F2;

(e) FVin-X2-Fl:F2-L2-Xl-Ll-V;

(f) FVin-X2-Fl:V-Ll-Xl-L2-F2;

(g) FVin(X2)-Fl:F2-L2-Xl-Ll-V;

(h) FVin(X2)-Fl:V-Ll-Xl-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;

(1) V-L1-X-L2-F2-L3-F1-L4-FVIII;

(m) F1-L4-FVDI-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-Ll-V-L3-Fl-L4-FVin;

(r) F2-L2-X-L1-V-L3-FVIII-L4-F1;

(s) V-Ll-Xl-L2-F2-L3-FVni(X2)-L4-Fl;

(t) V-Ll-Xl-L2-F2-L3-Fl-L4-FVni(X2);

(u) Fl -L4-FVIII(X2)-L3 -F2-L2-X1 -L1 - V;

(v) F-L4-FVIII(X2)-L3 - V-L1 -X1-L2-F2;

(w) FVIII(X2)-L4-F 1-L3 - V-L 1 -X1-L2-F2;

(x) FVIII(X2)-L4-F 1-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-Ll-X2-L2-F2-L3-FVni-L4-X2-L5-Fl;

(bb) V-L 1-X2-L2-F2-L3-F1-L5-X2-L4-F VIII;

(cc) Fl -L5-X2-L4-FVni-L3 -F2-L2-X2-L1 -V;

(dd) F1-L5-X2-L4-FVIII-L3 - V-L 1-X2-L2-F2;

(ee) FVID-L5-X2-L4-F2-L3 - V-L 1 -X1-L2-F1;

(ff) FVID-L5-X2-L4-F2-L3 -Fl -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;

where V is the VWF protein containing domain D' and domain D3, X or X1 is the first XTEN sequence containing less than 288 amino acids, X2 is the second XTEN sequence, FVIII contains the FVIII protein, FVIII(X2) is the FVIII protein with the second an 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 is an optional linker , (-) represents a peptide bond and (:) represents a covalent or non-covalent bond.

Some embodiments of the invention include a chimeric protein as described herein, wherein X or X1 consists of an amino acid sequence ranging from 12 to 287 amino acids in length.

In certain embodiments of the invention, the chimeric protein as described herein exhibits a longer half-life compared to the corresponding chimeric protein comprising the above formula, except that X or X1 is AE288. In some embodiments, AE288 is SEQ ID NO: 8.

Some embodiments of the invention include a chimeric protein as described herein, wherein X or X1 in the formula contains about 36, about 42, about 72, or about 144 amino acids. In certain embodiments, X or X1 in the formula is selected from AE42, AE72, AE144, AG42, AG72, or AG144. In some embodiments, X or X1 in the formula is selected from

SEQ ID NO: 9, SEQ Ш 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 certain 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, at least about 864 amino acids. amino acids. In certain embodiments, X2 is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144. In some embodiments, X2 is selected from

SEQ Ш NO: 8; SEQ Ш NO: 9; SEQ GO 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 certain embodiments of the invention, X2 is AE288 or AG288.

Also disclosed is a chimeric protein as described herein, containing X or X1 and/or X2, which exhibits a longer half-life compared to a chimeric protein not containing X or X1 and/or X2. In some embodiments, L1 and/or L2 is a cleavable linker. In certain embodiments of the invention, L4 and/or L5 is a cleavable linker. In certain embodiments, the linker is cleavable by a protease selected from factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase 2, granzyme B, TEV protease, enterokinase, 3C protease, sortase A, MMP-12, MMP-13, MMP-17 and MMP-20. In some embodiments, the linker is cleavable by factor IIa (thrombin).

Some embodiments of the invention include a chimeric protein as described herein, characterized in that the linker contains one or more cleavage sites containing an amino acid sequence selected from

RRRR (SEQ GO NO: 102), RKRRKR (SEQ GO NO: 103), RRRRS (SEQ GO 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), 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 contains the sequence TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). in certain embodiments, the linker comprises the amino acid sequence LVPRG (SEQ ID NO: 6). in some embodiments, the linker comprises a FVIII a1 region, a FVIII a2 region, a FVIII a3 region, or any combination thereof. In certain embodiments, the linker comprises a fragment of the a2 region of FVIII. In some cases, a fragment of the a2 region may contain the sequence DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88). In yet other embodiments, a smaller fragment of the FVIII a2 region may be used, including a fragment with the sequence IEPRSFS (SEQ ID NO: 194). in one particular embodiment, the linker comprises the amino acid sequence IEPRSFS (SEQ ID NO. 194). in another embodiment of the invention, the linker contains the amino acid sequence

IEPRSFS (SEQ ID NO. 194), and the linker is not the full-length a2 region of FVIII.

Also disclosed is a chimeric protein as described herein, wherein the a2 region of FVIII contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to either the sequence ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (seq id no: 106) or the sequence DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88). in some embodiments, the a1 region contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the sequence ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107).

In certain embodiments, the a3 region contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the sequence

ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ Ш NO: 108).

In some embodiments, F1 contains a first Fc fragment and/or F2 contains a second Fc fragment.

Some embodiments of the invention include a chimeric protein as described herein, wherein the chimeric protein containing F1 and F2 exhibits a longer half-life compared to a chimeric protein not containing F1 or F2. In certain embodiments of the invention, L3 is a cleavable linker. In some embodiments, the VWF protein is linked to the FVIII protein by a non-covalent bond. In some embodiments, the half-life of the chimeric protein is prolonged compared to FVIII protein without the 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-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times , is at least about 11 times or at least about 12 times longer lasting than that of an FVIII protein without the VWF protein or XTEN sequence or than that of a wild-type FVIII protein.

Also disclosed is a chimeric protein 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 is about 40 hours in mice with hemophilia A. In some embodiments, the VWF protein does not substantially bind to the VWF clearance receptor. In some embodiments, the VWF protein is capable of protecting the FVIII protein from degradation by one or more proteases by protecting the FVIII protein from activation, stabilizing the heavy chain and/or light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more phagocytic receptors.

Some embodiments of the invention include a chimeric protein as described herein, wherein the VWF protein inhibits or prevents the binding of endogenous VWF to the FVIII protein by shielding or blocking the VWF binding site on the FVIII protein. In some embodiments, the VWF binding site is located in the A3 domain or the C2 domain of the FVIII protein, or both the A3 domain and the C2 domain. In some embodiments, the VWF binding site comprises an amino acid sequence corresponding to amino acids 1669-1689 and 2303-2332 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 are different. In certain embodiments, at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five XTEN sequences, or at least six XTEN sequences are attached to the FVIII protein and/or inserted into the FVIII protein .

Also disclosed is a chimeric protein as described herein, characterized in that

- 6 044115 that the FVIII protein contains one or more FVIII domains selected from the A1 domain, the al acidic region, the A2 domain, the a2 acidic region, the B domain, the A3 domain, the a3 acidic region, the C1 domain, the C2 domain, one or more fragments thereof and any combinations thereof.

Also disclosed is a chimeric protein as described herein, wherein one or more insertion sites in the FVIII protein are located within one or more domains of the FVIII protein selected from A1 domain, a1 acidic region, A2 domain, a2 acidic region, A3 domain , B domain, C1 domain, C2 domain, and any combinations thereof, or between one or more domains of the FVIII protein selected from the group consisting of A1 domain and a1 acidic region, a1 acidic region and A2 domain, A2 domain and a2 acidic region, acidic the a2 region and the B domain, the B domain and the A3 domain, the A3 domain and the C1 domain, the C1 domain and the C2 domain, and any combination thereof, or between two domains of the FVIII protein selected from the A1 domain and the a1 acidic region, the a1 acidic region and the A2 domain , domain A2 and acidic region a2, acidic region a2 and domain B, domain B and domain A3, domain A3 and domain C1, domain C1 and domain C2 and any combinations 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 table. 7, table. 8, table. 9 and table. 10. In certain embodiments, the insertion sites in the FVIII protein are located immediately downstream of an amino acid corresponding to amino acid 745 in the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion sites in the FVIII protein are located immediately downstream of amino acid residues corresponding to residues 1656 and 1900 in the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion sites in the FVIII protein are located immediately downstream of amino acid residues corresponding to residues 26, 1656, and 1900 in the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion sites in the FVIII protein are located immediately downstream of amino acid residues corresponding to residues 403 and 745 in the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion sites in the FVIII protein are located immediately downstream of amino acid residues corresponding to residues 745 and 1900 in the mature FVIII protein (SEQ ID NO: 65). In certain embodiments, the insertion sites in the FVIII protein are located immediately downstream of amino acid residues corresponding to residues 18 and 745 in the mature FVIII protein (SEQ ID NO: 65). In some embodiments, the FVIII protein is a double-chain isoform of FVIII. In some embodiments, the FVIII protein is a single chain isoform of FVIII. In certain embodiments, the FVIII protein comprises a B domain or a portion thereof. In some embodiments, the FVIII protein is FVIII with the SQ B domain removed.

Some embodiments of the invention include a chimeric protein as described herein, wherein the single chain FVIII isoform contains at least one amino acid substitution at a position corresponding to residue 1648, residue 1645, or both residues in the full-length mature factor FVIII polypeptide (SEQ ID NO: 65) or residue 754, residue 751, or both residues in factor VIII with the SQ B domain deleted (SEQ ID NO: 67). In certain embodiments, the amino acid substitution is an amino acid other than arginine. In some embodiments, the double-chain FVIII isoform consists of a first chain comprising a FVIII heavy chain and a second chain comprising a FVIII light chain, the heavy chain and the light chain being linked together by a metal bond. In certain embodiments, domain D' contains an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-866 of SEQ ID NO: 21. In some In embodiments, the D3 domain contains an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO: 21. In some embodiments, VWF protein is a monomer.

Also disclosed is a chimeric protein as described herein, comprising at least two VWF proteins, at least three VWF proteins, at least four VWF proteins, at least five VWF proteins, or at least six VWF proteins. In certain embodiments, the VWF protein contains an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1240 of SEQ ID NO: 21. In some embodiments implementation of the invention, the VWF protein consists essentially of or consists of amino acids 764-1240 of the sequence SEQ ID NO: 21. In certain embodiments of the invention, the VWF protein contains at least one amino acid substitution at the position corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 sequence SEQ ID NO: 21. In some embodiments, the VWF protein contains an amino acid other than cysteine that replaces the residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 21. In certain embodiments of the invention, the VWF protein additionally contains a D1 domain, a D2 domain, or D1 and D2 domains of the VWF protein.

Some embodiments of the invention include a chimeric protein as described herein, characterized in that the VWF protein further comprises a VWF domain selected from an A1 domain, an A2 domain, an A3 domain, a D4 domain, a B1 domain, a B2 domain, a B3 domain. , domain C1, domain

C2, the CK domain, one or more fragments thereof, and any combination thereof.

Also disclosed is a chimeric protein as described herein, characterized in that the VWF protein consists or essentially consists of: (1) VWF D' and D3 domains or fragments thereof; (2) domains D1, D' and D3 of VWF or fragments thereof; (3) domains D2, D' and D3 of VWF or fragments thereof; (4) domains D1, D2, D' and D3 of VWF or fragments thereof; (5) domains D1, D2, D', D3 and A1 of VWF or fragments thereof.

Some embodiments of the invention include a chimeric protein as described herein, wherein the VWF protein further comprises a VWF or FVIII signal peptide operably linked to the VWF protein.

Also disclosed is a chimeric protein as described herein, wherein one or more linkers have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 9 00, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acid residues. In some embodiments, the one or more linkers are from about 1 to about 2000 amino acid residues in length. In certain embodiments, one or more linkers comprise a gly/ser peptide. In some embodiments, the gly/ser peptide has the formula (Gly ₄ Ser) _n (SEQ ID NO: 94) or S(Gly ₄ Ser) _n (SEQ ID NO: 164), where n is a positive integer selected from group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In certain embodiments, the linker (Gly ₄ Ser) _n is (Gly ₄ Ser) ₃ (SEQ ID NO: 100 ) or (Gly4Ser)4 (SEQ ID NO: 165). In some embodiments, the linker contains 20 amino acids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acids. In certain embodiments, the cleavable linker sequence is SGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGG (SEQ ID NO: 166).

In some embodiments, the chimeric protein as described herein is polysialylated, PEGylated, or HESylated.

Also disclosed is a chimeric protein as described herein, wherein the first polypeptide contains a sequence that is at least about 80%, 90%, 95%, 99%, or 100% identical to

FVIII161 (SEQ Ш 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), FVIII272 (SEQ ID NO: 85) or FVIII282 (SEQ ID NO: 159), and the second polypeptide contains the sequence at least about 80%, 90%, 95%, 99% or 100% identical to either FV057 (SEQ ID NO: 152) or FV059 (SEQ ID NO: 197). In some embodiments, the first polypeptide contains FVIII169 (SEQ ID NO: 70) and the second polypeptide contains FV057 (SEQ ID NO: 152). In other embodiments, the first polypeptide contains FVIII169 (SEQ ID NO: 70) and the second polypeptide contains FV059 (SEQ ID NO: 197). In yet another embodiment, the first polypeptide contains FVIII169 (SEQ ID NO: 70) and the second polypeptide contains FV062 (SEQ ID NO: 199). In some embodiments, the chimeric protein is effective in preventing and/or stopping bleeding in an eligible subject.

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

Some embodiments of the invention include a vector containing a polynucleotide as described herein and one or more promoters operably linked to the polynucleotide or set of polynucleotides.

In some embodiments, the vector as described herein further comprises an additional vector comprising a polynucleotide chain encoding PC5 or PC7.

Also disclosed is a host cell containing a polynucleotide or vector as described herein. In some embodiments, the host cell is a mammalian cell. In certain embodiments of the invention, the mammalian cell is selected from a HEK293 cell (human embryonic kidney 293; human embryonic kidney cell line), a CHO cell (Chinese hamster ovary; Chinese hamster ovary cell line), and a BHK cell (baby ham-8 044115 ster kidney line). kidney cells of a newborn Syrian hamster).

Also disclosed is a pharmaceutical composition comprising a chimeric protein, polynucleotide, vector or host cell as described herein and a pharmaceutically acceptable carrier. In some embodiments, the chimeric protein has a longer half-life than the wild-type FVIII protein. In certain embodiments, the half-life of the chimeric protein is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times , is at least about 11 times or at least about 12 times longer lasting than that of wild-type FVIII protein.

Some embodiments of the invention include a composition as described herein, characterized in that 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 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 certain embodiments, the half-life of the chimeric protein is about 40 hours in mice with hemophilia A. In some embodiments, the composition as described herein is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration , intrathecal administration, subdural administration and oral administration. In certain embodiments of the invention, parenteral administration may be intravenous or subcutaneous administration.

In some embodiments, a composition as described herein is used to treat a hemorrhagic disease or condition in a subject in need thereof. In certain embodiments of the invention, the hemorrhagic disease or condition is selected from the group consisting of a bleeding disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, head injury, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, hemorrhage in the chest cavity, bone fracture, bleeding of the central nervous system, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space, hemorrhage in the iliopsoas muscle sheath and any combinations thereof. In some embodiments, the subject is scheduled for surgery. In certain embodiments of the invention, treatment is prophylactic or on demand.

Also disclosed is a method of prolonging or increasing the half-life of a chimeric protein, which comprises administering an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition as described herein to a subject having an indication therefor, wherein the VWF protein, the XTEN sequence, the first IG constant region, or part thereof and the second IG constant region or part thereof increases the half-life of the chimeric protein.

Some embodiments of the invention include a method of treating a hemorrhagic disease or disorder in a subject having an indication therefor, which includes administering an effective amount of a chimeric protein, polynucleotide, vector, host cell, or composition as described herein, wherein the hemorrhagic disease or disorder is selected from the group consisting of bleeding disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, head injury, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, chest hemorrhage, bone fracture , bleeding of the organs of the central nervous system, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space and hemorrhage in the sheath of the iliopsoas muscle. In some embodiments, the subject is an animal. In some embodiments, the animal is a human. In some embodiments, the subject has hemophilia A. In certain embodiments, the treatment is prophylactic or on-demand. In some embodiments, the effective amount is from 0.1 μg/kg to 500 mg/kg.

Also disclosed is a method as described herein, wherein the chimeric protein, polynucleotide, vector, host cell, or composition as described herein is administered by a route selected from the group consisting of topical application, intraocular

- 9 044115 administration, parenteral administration, intrathecal administration, subdural administration and oral administration. In certain embodiments, parenteral administration is selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration.

Some embodiments of the invention include a method of making 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 cell. In some embodiments, the method as described herein further comprises isolating the chimeric protein. In certain embodiments, the chimeric protein is effective in stopping and/or preventing bleeding in a subject.

Brief description of graphics/figures

In fig. 1 is a schematic representation of a chimeric protein consisting of a first polypeptide that contains the FVIII protein (A1-A2-partial or full B-A3-C1-C2 domain) fused to an Fc fragment, with the sequence XTEN inserted into the insertion site within the FVIII protein , and a second polypeptide that contains a VWF protein containing D'D3 domains, an XTEN sequence of less than 288 amino acids, a thrombin-cleavable linker, and a second Fc fragment. Insertion of an XTEN sequence into the FVIII protein and/or fusion of an XTEN sequence to the VWF protein prolongs the half-life of the chimeric protein by increasing the hydrodynamic radius and blocking receptor-mediated clearance. The D'D3 domains of the VWF protein block the interaction of FVIII with endogenous VWF, stabilize the FVIII protein and prolong the half-life of the chimeric protein. Fc domains can covalently link D'D3 domains to FVIII protein and prolong the half-life of the chimeric protein through the FcRn-mediated metabolic pathway. The thrombin-cleavable linker allows the release of D'D3 domains upon FVIII activation and ensures proper alignment between FVIII and VWF D'D3 domains.

In fig. 2 shows a three-plasmid expression system for FVIII-XTENFc:D'D3-XTEN-Fc heterodimers: the first plasmid containing the nucleotide sequence encoding the single-chain protein FVIII-XTEN-Fc, in which the XTEN sequence is inserted into the B domain; a second plasmid containing a nucleotide sequence encoding a D1D2D'D3-XTEN-Fc protein, in which the XTEN sequence contains less than 288 amino acids; and a third plasmid containing a nucleotide sequence encoding PACE, a propeptide processing enzyme. When three polypeptides are expressed from three plasmids, the D1D2 polypeptide domains of the VWF protein can be cleaved from the D'D3 domains by intracellular processing. The resulting complex contains three products: the first molecule is the FVnI-XTEN/D'D3 heterodimers, the second molecule is a by-product, the D'D3-XTEN-Fc homodimer, and the third molecule is another by-product, i.e. FVIII (XTEN)-Fc.

In fig. Figure 3 shows the additive effects of XTEN insertions on prolonging the half-life of heterodimers. FVIII169 contains an Fc fragment-fused FVIII protein with the B domain removed, in which the XTEN sequence (eg, AE288) is inserted at the position corresponding to amino acid 745 of mature full-length FVIII. FVIII205 contains an Fc fusion of an FVIII protein with the B domain removed, in which an XTEN sequence (e.g., AE144) is inserted at amino acid 18 of mature full-length FVIII, and another XTEN sequence (e.g., AE288) is inserted at amino acid 745 mature full-length FVIII. VWF031 contains the VWF D' domain and D3 domain fused to the Fc moiety via a thrombin-cleavable linker (without XTEN). VWF034 contains the VWF D' domain and D3 domain fused to AE288 and an Fc fragment. The half-life of FVIIII9/FV031 (inverted triangle) is 16.7 hours in hemophilia A mice; the half-life of FVIII205/FV031 (circle) is 29.4 hours in mice with hemophilia A; and the half-life of FVIIII9/FV034 (square) is 31.1 hours in hemophilia A mice.

In fig. Figure 4 shows that when inserted between the D'D3 domains of VWF and the Fc domains, the AE144 XTEN sequence provides greater half-life extension than AE288 XTEN. For example, while the half-life of FVIIII69/FV034 (square) in hemophilia A mice is 31.1 hours, the half-life of FVIIII69//FV057 (circle) in hemophilia A mice is 42 hours. FV057 contains VWF D'D3 domains fused to AE144 and an Fc fragment.

In fig. Figure 5 shows that Fc domains are required to prolong the half-life of chimeric protein heterodimers. Comparing the half-life of FVIII205/FB031 (circle) in hemophilia A mice with that of the FVIII263/FB050 protein (square), which contains mutations in the FcRn binding sites (triple IHH (Indian Hedgehog Homolog) mutation in Fc) and therefore cannot be recycled in the FcRn pathway, the half-life of FVIII263/VWF050 (23 hours) is shorter than the half-life of FVIII205/VWF031 (29.4 hours). This suggests that Fc fragments are required to prolong the half-life.

In fig. Figure 6A shows the similar immediate efficacy of FVIII-XTENFc/D'D3-XTEN-Fc heterodimers compared to B domain deleted FVIII protein (SQ BDD FVIII) in a tail-cut hemorrhage model in hemophilia A mice. Mice were dosed with 75 IU /kg and

- 10 044115 activity was measured in the APTT assay. SQ BDD FVIII is shown in the shape of a circle, FVШ169/ФВ034 - in the shape of a square, FVШ169/ФВ057 - in the shape of a diamond, and the carrier is in the shape of an inverted triangle. Detailed descriptions of the FVIII169, FV034 and FV057 designs are provided elsewhere in this document. In fig. 6B shows a comparison of the immediate efficacy of FVIII169/FV034 and B domain deleted FVIII (SQ BDD FVIII) in hemophilia A mice using a dose of 37.5 IU/kg and measuring activity in the APTT assay. Median blood loss (μL) in mice in each experimental group is indicated by horizontal lines; blood loss (μL) in C57/BL6 mice is indicated by open triangles; blood loss (μl) after administration of rBDD-FVIII at a dose of 37.5 IU/kg is indicated by open circles; blood loss (μL) after administration of FVIII169/FV034 at a dose of 37.5 IU/kg is indicated by open squares and blood loss (μL) after vehicle administration is indicated by inverted triangles.

In fig. 7A, B show that the rFVIII169/FV057 heterodimer provides longer-lasting protection in hemophilia A mice in a model of hemorrhage induced by tail vein transection. In fig. Figure 7A shows data on rebleeding in mice treated with rFVIII169/FV057 72 hours before tail injury (square), SQ BDD-FVIII 48 hours before tail injury (diamond), SQ BDD-FVIII 24 hours before tail injury (inverted triangle ) and carrier (circle). Activity was measured using aPTT assay. The x-axis shows time in hours, and the y-axis shows the percentage of individuals who did not experience bleeding. In fig. 7B shows the corresponding survival data for the four categories of mice shown in FIG. 7A. Mice treated with rFVIIII69/FV057 at a dose of 12 IU/kg 72 hours before tail injury showed similar protection from rebleeding and survival compared to mice treated with SQ BDD-FVIII 24 hours before tail injury.

In fig. Figure 8A shows comparable rates of rebleeding between mice treated with FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers 96 hours before injury and mice treated with rBDD-FVIII 24 hours before injury. Filled squares illustrate data on rebleeding in mice treated with FVIIII69/FV034 24 hours before injury; open squares illustrate data on rebleeding in mice treated with FVIIII69/FV034 96 hours before injury; filled diamonds illustrate data on rebleeding in mice treated with FVIIII69/FV057 24 hours before injury; open diamonds illustrate data on rebleeding in mice treated with FVIIII69/FV057 96 hours before injury; filled circles illustrate rebleeding data in mice treated with rBDD-FVIII 24 h before injury; open circles illustrate rebleeding data in mice treated with rBDD-FVIII 48 h before injury; and filled triangles illustrate rebleeding data in vehicle-treated mice. The x-axis shows time in hours, and the y-axis shows the percentage of individuals who did not experience bleeding.

In fig. 8B shows the survival curves of mice treated with FVIII-XTENFc/D'D3-XTEN-Fc heterodimers 96 hours before injury compared to mice treated with rBDD-FVIII 24 hours before injury. The x-axis shows time in hours, and the y-axis shows survival rate in percentage. The symbols are the same as in Fig. 8A.

In fig. Figure 9 shows schematic representations of typical FVIII-VWF heterodimers and the FVIII169, FVIII286, FVIII286, FV057, FV059, and FV062 constructs. For example, the FVIII169 construct contains a FVIII protein fused to an Fc fragment with the B domain removed and the R1648A substitution in which the XTEN sequence (e.g., AE288) is inserted at the position corresponding to amino acid 745 of mature full-length FVIII (A1-a1-A2-a2-288XTEN-a3 -A3-C1-C2-Fc). The FVIII286 construct contains a FVIII protein fused to an Fc fragment with the B domain removed and the R1648 substitution in which the XTEN sequence (e.g., AE288) is inserted at the position corresponding to amino acid 745 of mature full-length FVIII, with an additional a2 region between FVIII and Fc (A1-a1- A2-a2-288XTEN-a3-A3-C1-C2-a2-Fc). VWF057 is a VWF-Fc fusion construct containing the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e., C336A and C379A), linked to the Fc fragment via a VWF linker containing the thrombin cleavage site LVPRG ( LVPRG; SEQ ID NO: 6) and a glycine-serine linker (GS), and in this construct the XTEN sequence (for example, AE144) is inserted between the D'D3 domain and the VWF linker (D'D3-144XTEN-GS+LVPRG-Fc) . VWF059 is a VWF-Fc fusion construct containing the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e. C336A and C379A), linked to the Fc fragment via the acidic region 2 (a2) of the FVIII protein as a VWF linker, with the XTEN sequence (i.e. AE144) inserted between the D'D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct containing the D'D3 domain of the VWF protein (with two amino acid substitutions in the D'D3 domain, i.e. C336A and C379A) linked to an Fc fragment, the sequence being XTEN (i.e. AE144 ) is inserted between the D'D3 domain and the Fc fragment (D'D3-144XTEN-Fc).

In fig. 10 is a schematic representation of heterodimeric FVIII/VWF constructs, for example FVIII169/FV057, FVIIII69/FV059, FVIIII69/FV059A and FVIII169/FV073. The arrow indicates the site at which an optional linker is added to introduce a thrombin cleavage site. FVIIII69/FV057 has a linker containing LVPRG (SEQ ID NO: 6). FVIIII69/FV059 has a linker containing the a2 region of the FVIII protein (i.e.

ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSDKTH (SEQ ID NO: 106),

FVIII169/FV059A has a linker containing a truncated a2 region of the FVIII protein (ie DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSDKTH (SEQ ID NO: 88)). Fv _ΠΠ 69/φbo73 has a linker within the construct FV073 (SEQ ID NO: 175), which contains a fragment of the a2 region of the FVIII protein, consisting of IEPRSFS (SEQ ID NO: 194).

In fig. 11A-C show SDS-PAGE electropherograms obtained after digestion of FVIIII69/FV057 with thrombin, using FVIII-Fc as a control. In fig. 11A shows staining of an SDS-PAGE gel with anti-D3 antibody (AB 96340). Arrows highlight LCFc:D'D3-XTEN-Fc, which is an uncleaved, full-length FVIIII69/FV057, and D'D3-144 XTEN, which is a fragment obtained as a result of cleavage with thrombin. In fig. 11B shows staining of an SDS-PAGE gel with a heavy chain antibody (HC) (GMA012). Arrows highlight the FVIII heavy chain (HC) and the A2 domain of FVIII. In fig. 11C is a superimposed image of test panels A and B. Samples were collected at the time points indicated at the top of each test panel. Arrows point to the corresponding proteins.

In fig. 12A-C show SDS-PAGE electropherograms obtained after cleavage of FVIIII69/FV059 with thrombin. In fig. 12A shows staining of an SDS-PAGE gel with anti-D3 antibody (AB 96340). Arrows highlight LCFc:D'D3-XTEN-Fc, which is an uncleaved, full-length FVIIII69/FV059, and DD3-144 XTEN, which is a fragment obtained as a result of cleavage with thrombin. In fig. 12B shows SDS-PAGE gel staining with anti-HC antibody (GMA012). The arrows highlight the uncleaved, full-length FVIIII69/FV059, D'D3-144 XTEN-a3, which is a fragment obtained as a result of cleavage by thrombin, and A2, which is the A2 domain of the FVIII protein. In fig. 12C is a composite image of test panels A and B. Samples were collected at the time points indicated at the top of each test panel.

In fig. Figure 13 shows immediate efficacy data in hemophilia A mice treated with FVIIII69/FV059 (circle) compared to hemophilia A mice treated with BDD-FVIII control (square). Blood loss was measured after tail cutting, p=0.9883.

Detailed description of the invention

The present invention relates to a chimeric protein containing two polypeptides, the first of which contains the FVIII protein fused to a first IG constant region, and the second of which contains the 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 acid residues.

I. Definitions.

It should be noted that a singular noun also implies a plural; for example, by the definition of a nucleotide sequence is meant one or more nucleotide sequences. Thus, the terms singular, one or more, and at least one may be used interchangeably herein.

Moreover, the use of the term and/or herein should be taken to specifically describe either of the two specified properties or components separately from or together with the second property or component. Therefore, the term and/or, as used herein in an expression such as A and/or B, is intended to include A and B, A or B, A only and B only.

Likewise, the term and/or, when used in an expression such as A, B and/or C, is intended to cover each of the following aspects: 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; only A; only in; and only S.

It should be understood that wherever the phrase contains is used to describe aspects herein, it is also intended to include otherwise similar aspects described by the terms consists of and/or substantially consists of.

Unless otherwise specified, 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 invention relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one skilled in the art with a general vocabulary for many of the terms used herein.

Units, prefixes and symbols are indicated in their accepted form according to the International System of Units (Systeme International de Unites, SI). Numeric ranges include numbers that define a range. Unless otherwise indicated, amino acid sequences are written from left to right in the direction from amino to carboxyl terminus. The headings set forth herein are not intended to limit the various aspects of the invention that may be apparent by reference to the description as a whole. Accordingly, the terms whose definitions are given immediately below are more

- 12 044115 are fully defined by reference to the description in its entirety.

The term about as used herein means approximately, roughly speaking, about or of the order of magnitude. When the term about is used in conjunction with a range of numerical values, it modifies that range by extending the boundaries above and below the following numerical values. In general, the term about can deviate a numerical value above or below a specified value by an amount, such as 10%, upward or downward (above or below).

The term polynucleotide or nucleotide is intended to encompass a single nucleic acid and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, the polynucleotide contains a conventional phosphodiester linkage or a non-conventional linkage (eg, an amide linkage, as in peptide nucleic acids (PNAs)). The term nucleic acid refers to one or more segments of nucleic acid, such as DNA or RNA fragments, present in a polynucleotide. The term isolated nucleic acid or isolated polynucleotide refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment. For example, a recombinant polynucleotide encoding a factor VIII polypeptide and contained in a vector is considered to be isolated for purposes of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. The isolated polynucleotides or nucleic acids of the present invention further include those molecules obtained by synthesis. In addition, the polynucleotide or nucleic acid may include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

As used herein, the term coding region or coding sequence means the portion of a polynucleotide consisting of codons that can be translated into an amino acid sequence. Although a stop codon (TAG, TGA, or TAA) is not typically translated into an amino acid, it may be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, and the like, are not part of the coding region. coding region. The boundaries of the coding region are typically defined by a start codon at the 5' end, encoding the amino terminus of the polypeptide being synthesized, and a translation stop codon at the 3' end, encoding the carboxyl terminus of the polypeptide being synthesized. Two or more coding regions of the present invention may be present in a single polynucleotide construct, eg on a single vector, or in separate polynucleotide constructs, eg on separate (different) vectors. It follows that one vector may contain just one coding region or two or more coding regions, for example, one vector may separately encode binding domain A and binding domain B, as described below. In addition, a vector, polynucleotide or nucleic acid of the present invention may contain heterologous coding regions, whether fused or not fused to a nucleic acid encoding a binding domain of the present invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

Certain proteins secreted by mammalian cells are associated with a secretory signal peptide, which is cleaved from the mature protein after the growing polypeptide chain has begun to be transferred through the rough endoplasmic reticulum. Those of ordinary skill in the art will recognize that signal peptides are typically coupled to the N-terminus of a polypeptide and cleaved from the finished or full-length polypeptide to produce the secreted or mature form of the polypeptide. In certain embodiments, the native signal peptide or functional derivative of the same sequence retains the ability to direct secretion of a polypeptide operably linked to the peptide or derivative. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (tPA) or murine β-glucuronidase signal peptide, or a functional derivative thereof, may be used.

The term downstream of a nucleotide sequence means that the nucleic acid or nucleotide sequence is located in the 3' direction relative to the reference nucleotide sequence. In certain embodiments of the invention, downstream nucleotide sequences refer to sequences downstream of the transcription start point. For example, the translation initiation codon in a gene lies downstream of the transcription start site. The term downstream, when applied to a polypeptide sequence, means that the amino acid or amino acid insertion site is located at the C-terminus of the reference amino acid sequence. For example, an insertion site immediately downstream of an amino acid corresponding to amino acid 745 in the mature wild-type FVIII protein means that the insertion site is located between amino acid residues corresponding to residues 745 and 746 in the mature wild-type FVIII protein.

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

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

A polynucleotide encoding a gene product, such as a polypeptide, may include a promoter and/or other transcriptional or translational control elements operably linked to one or more coding regions. In the case of a functional connection, the coding region for a gene product, such as a polypeptide, is connected to one or more regulatory regions such that expression of the gene product is influenced or controlled by the regulatory region(s). For example, a coding region and a promoter are operably linked if induction of promoter function results in the transcription of mRNA encoding the 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 expression of that gene product or does not interfere with the ability of DNA -templates are transcribed into RNA. Other transcriptional control elements in addition to the promoter, such as enhancers, repressors, and transcription termination signals, can also be operably linked to the coding region to direct expression of the gene product.

Various areas of transcription control are known to those skilled in the art. These include transcriptional control regions that function in vertebrate cells, such as promoter and enhancer segments from cytomegaloviruses (immediate early promoter combined with intron A), simian virus 40 (early promoter), and retroviruses (eg, Rous sarcoma virus), but not are limited to the above. Other transcriptional control regions include regions derived from vertebrate genes, such as the actin, heat shock protein, bovine growth hormone, and rabbit β-globin genes, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional useful transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters inducible by interferons or interleukins).

Likewise, those of ordinary skill in the art are aware of various translation control elements. These include ribosome binding sites, transcription initiation and termination codons, and elements derived from picornaviruses (particularly the internal ribosome entry site, or IRES, also called the cap independent transcription enhancer [CITE] sequence ]), but are not limited to the above.

As used herein, the term expression refers to the process by which a polynucleotide generates a gene product, such as RNA or a polypeptide. It includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product and translation of mRNA into a polypeptide. Expression results in the formation of a gene product. As used herein, the gene product may be either a nucleic acid, such as messenger RNA, synthesized by transcription of the gene, or a polypeptide translated from the transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

Vector means any carrier for cloning and/or transferring a nucleic acid into a host cell. The vector may be a replicon to which another nucleic acid segment may be attached to effect replication of the attached segment. The term replicon refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions 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 vehicles for introducing a nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be achieved by ligating the appropriate polynucleotide fragments to the selected vector having complementary overhangs.

The vector can be designed to encode selectable markers or reporters that allow selection or identification of cells containing the vector. Expression of selectable markers or reporters allows the identification and/or selection of host cells that contain and express other coding regions located on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide bialaphos, sulfonamides and the like; and genes used as phenotypic markers, e.g. regulatory genes for anthocyanin biosynthesis, isopentanyl transferase gene and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable markers can also be considered reporters.

The term plasmid refers to an extrachromosomal element, often carrying a gene that is not part of the cell's general metabolism, and usually in the form of circular, double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genomic insertion sequences, phage or nucleotide sequences, linear, circular or supercoiled single- or double-stranded DNA or RNA, obtained from any source, in which a number of nucleotide sequences are joined or recombined into a unique construct that is capable of introducing into a cell a promoter fragment and DNA sequence for a selected gene product along with a suitable 3' untranslated sequence.

Eukaryotic virus vectors that can be used include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated virus vectors, and poxviral vectors, such as vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors. Nonviral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

The term cloning vector refers to a replicon that is a unit-length sequentially replicating nucleic acid segment that contains an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment can be attached to effect replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, such as bacteria, and expression in another cell type, such as eukaryotic cells. Cloning vectors typically contain one or more sequences that can be used to select cells containing the vector and/or one or more multiple cloning sites for insertion of target nucleic acid sequences.

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

Methods well known in the art are used to introduce vectors into host cells, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran treatment, calcium phosphate precipitation, lipofection (fusion with liposomes), gene gun or vector DNA vectors.

As used herein, the terms culture, culture, and culture mean the incubation of cells under in vitro conditions to allow them to grow and divide, or to maintain cell viability. As used herein, the term cultured cells means cells that are propagated in vitro.

As used herein, the term polypeptide is intended to include the singular polypeptide and the plural polypeptides and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide means any chain or chains of two or more amino acids, without regard to the specific length of the product. Thus, the definition of polypeptide includes peptides, dipeptides, tripeptides, oligopeptides, proteins, amino acid chains or any other term used to designate a chain or chains of two or more amino acids, and the term polypeptide can be used instead of or interchangeably with any of these terms . The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide may be obtained from a natural biological source or synthesized using recombinant technologies, but is not necessarily translated from its defining nucleic acid sequence. It can be obtained by any method, including chemical synthesis.

An isolated polypeptide or fragment, variant or derivative thereof refers to a polypeptide that is outside its natural environment. No specific level of cleaning is required. For example, the isolated polypeptide may simply be recovered from its native or natural environment. Recombinant polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable method.

The present invention also includes fragments or variants of polypeptides and any combinations thereof. The term fragment or variant as applied to polypeptide binding domains or binding molecules of the present invention includes any polypeptides retaining at least some of the properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for a FVIII or FVIII variant binding activity for the VWF fragment) of the reference polypeptide. Polypeptide fragments include proteolytic fragments, deletion fragments, and specific antibody fragments discussed elsewhere herein, but do not include a naturally occurring full-length polypeptide (or mature polypeptide). Variants of the polypeptide binding domains or binding molecules of the present invention include fragments as described above, as well as polypeptides with amino acid sequences altered due to amino acid substitutions, deletions or insertions. Variants may or may not occur in nature. Non-naturally occurring variants can be obtained using mutagenesis techniques known in the art. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions or additions.

As used herein, the term VWF protein or VWF proteins means any VWF fragments that interact with FVIII and retain at least one or more of the properties typically exhibited by full-length VWF with respect to FVIII, e.g., preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes, which may lead to premature clearance, prevention of binding to FVIII clearance receptors, which can bind unprotected FVIII but not VWF-bound FVIII, and/or stabilization of interactions between the heavy chain and the light chain of FVIII.

A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. The art defines families of amino acid residues with similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine , glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid with a side chain of the same family, the substitution is considered conservative. In another embodiment, the amino acid chain may be conservatively replaced by a structurally similar chain that differs in the order and/or composition of members of the same side chain family.

As is known in the art, the sequence identity of two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. In the cases discussed herein, a particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide. using methods and software/computer programs known in the art, including the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711), but not limiting himself to it. The BESTFIT program uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the segment with the highest degree of similarity between two sequences. When using BESTFIT or other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the present invention, parameters are, of course, set such that the percent identity is calculated over the entire length of the reference polypeptide sequence and that breaks in homology are tolerated up to 5% of the total number of amino acids in the comparison sequence.

As used herein, an amino acid corresponding to an amino acid or an amino acid equivalent to an amino acid in a VWF or FVIII protein sequence is determined by alignment to ensure maximum identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify the equivalent amino acid in the second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

As used herein, the term insertion site refers to a position within a FVIII polypeptide or fragment, variant, or derivative thereof immediately upstream of a position into which a heterologous fragment may be inserted. The insertion site is indicated as a number representing the number of the amino acid in the mature native FVIII protein (SEQ ID NO: 65) to which the insertion site corresponds, that is, the amino acid located immediately N-terminal to the insertion position. For example, expression a3 contains the sequence XTEN at the insertion site corresponding to amino acid 1656 of SEQ ID NO: 65, indicating that the heterologous fragment is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 65.

The expression immediately downstream of an amino acid, as used herein, refers to the position immediately adjacent to the terminal carboxyl group of the amino acid. Likewise, the expression immediately upstream of an amino acid refers to the position immediately next to the terminal amino group of an amino acid. Therefore, the expression between two amino acids of an insertion site as used herein refers to the position between two adjacent amino acids into which the XTEN sequence or any other polypeptide is inserted. Thus, the expressions inserted immediately downstream of an amino acid and inserted between two amino acids of an insertion site are used synonymously with the expression inserted into an insertion site.

The terms inserted, inserted, inserted into, or grammatically related terms as used herein refer to the position of the XTEN sequence in the chimeric polypeptide relative to the same position in the native mature human FVIII protein. As used herein, these terms refer to the comparative characteristics of a recombinant FVIII polypeptide and native mature human FVIII and do not indicate, imply or suggest any method or process by which the chimeric polypeptide was created. For example, with respect to the chimeric polypeptide of the present invention, the expression XTEN sequence inserted immediately downstream of amino acid residue 745 of the FVIII polypeptide means that the chimeric polypeptide contains the XTEN sequence immediately below the amino acid corresponding to amino acid 745 in native mature human FVIII, for example, the XTEN sequence limited amino acids corresponding to amino acids 745 and 746 of native mature human FVIII.

The term fusion or chimeric protein includes a first amino acid sequence linked to a second amino acid sequence to which it does not naturally bind in nature. Amino acid sequences typically present in different proteins may be combined in a fusion polypeptide, or amino acid sequences typically present in a single protein may be arranged in a new configuration within a fusion polypeptide, such as in the case of the factor VIII domain fusion of the present invention with an immunoglobulin Fc domain. The fusion protein is created, for example, by chemical synthesis or by creating and translating a polynucleotide encoding peptide regions in the desired relative arrangement. The chimeric protein may further comprise a second amino acid sequence linked to the first amino acid sequence by a covalent non-peptide bond or non-covalent bond.

As used herein, the term half-life refers to the biological half-life of a particular polypeptide in vivo. The half-life can be thought of as the time required for half of the dose administered to a subject to be eliminated from the bloodstream and/or other tissues of the animal. When plotting the clearance of a given polypeptide as a function of time, a biphasic process is usually noted, with a rapid α phase and a longer β phase. In general, α phase reflects the equilibration of the administered Fc polypeptide concentration between the intravascular and extravascular spaces and is determined in part by the size of the polypeptide. The β phase typically reflects the catabolism of the polypeptide in the intravascular space. In some embodiments, the clearance of the FVIII protein and the FVIII-containing fusion protein follows a single-phase pattern, therefore, they do not have an alpha phase, but only a beta phase. Thus, in certain embodiments of the invention, the term half-life as used herein refers to the half-life of the polypeptide in the β phase. The typical β-phase half-life of a human antibody in humans is 21 days.

The term linked as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence may be directly linked to or placed adjacent to the second amino acid sequence or nucleotide sequence, or alternatively, the first sequence may be covalently linked to the second sequence by an insertion sequence therebetween. The term linked not only means the fusion of a first amino acid sequence with a second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or second amino acid sequence) between any two amino acids of the second amino acid sequence (or first amino acid sequence, respectively) . In one embodiment of the invention, the first amino acid sequence may be linked to the second amino acid sequence via a peptide bond or linker. The first nucleotide sequence may be linked to the second nucleotide sequence via a phosphodiester bond or linker. The linker can be a peptide or polypeptide (for polypeptide chains), or a nucleotide or nucleotide chain (for nucleotide chains), or any chemical structure (for both polypeptides and polynucleotide chains). The term related is also indicated by a hyphen (-).

As used herein, the term connected to refers to a covalent or non-covalent bond between a first amino acid chain and a second amino acid chain. In one embodiment of the invention, the term connected to means a covalent non-peptide bond or non-covalent bond. This connection can be indicated by a colon, i.e. (:). In another embodiment, the term refers to 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 bridge with the thiol group of a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are joined by a disulfide bond and the two heavy chains are joined by two disulfide bonds at positions corresponding to positions 239 and 242 according to the Kabat numbering system (positions 226 or 229 according to the European numbering system). Examples of covalent bond include peptide bond, metal bond, hydrogen bond, disulfide bond, sigma bond, pi bond, delta bond, glycosidic bond, agostic bond, banana bond, donor-acceptor bond, pi-dative bond, double bond, triple bond, quadruple bond, quinary bond, six bond, conjugation, hyperconjugation, aromaticity, hapticity or anti-bonding, but are not limited to the above. Non-limiting examples of non-covalent bonding include ionic bonding (e.g., cation-pi interaction or salt bond), metal bonding, hydrogen bonding (e.g., dihydrogen bond, dihydrogen complex, low-barrier hydrogen bond, or symmetric hydrogen bond), van der Waals force, London dispersion forces, mechanical bonding, halogen bonding, aurophilicity, intercalation, stacking, entropic force or chemical polarity.

As used herein, the term monomer-dimer hybrid refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain linked by a disulfide bond, the first chain containing a blood clotting factor, such as factor VIII, and a first Fc fragment, and the second chain containing , consists essentially of or consists of a second Fc fragment without the coagulation factor. Thus, a monomer-dimer hybrid construct is a hybrid containing a monomer portion with just one coagulation factor and a dimer portion with two Fc moieties.

As used herein, the term cleavage site or enzymatic cleavage site refers to a site recognized by an enzyme. Some enzymatic cleavage sites contain an intracellular processing site. In one embodiment of the invention, the polypeptide contains an enzymatic cleavage site cleaved by an enzyme that is activated during a reaction of the coagulation cascade such that cleavage of such sites occurs at the site of clot formation. Examples of such sites include, for example, sites recognized by thrombin, factor XIa or factor Xa. Examples of factor XIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3).

Examples of thrombin cleavage sites include, e.g.

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.

As used herein, the term processing site or intracellular processing site refers to a type of enzymatic cleavage site in a polypeptide that is targeted by enzymes that perform their function after translation of the polypeptide. In one embodiment of the invention, such enzymes perform their function during the transfer of a polypeptide from the cavity of the Golgi apparatus to the trans-golgi apparatus. Intracellular processing enzymes are more common

- 18 044115 polypeptides are released before protein secretion from the cell. Examples of such processing sites include, for example, target sites of the PACE/furin family of endopeptidases. These enzymes are concentrated on the Golgi membrane and cleave proteins at the carboxyl terminus of the Arg-[any residue]-(Lys or Arg)-Arg motif. As used herein, the furin family of enzymes includes, for example, PCSK1 (also known as PC1/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4) or PCSK7 (also known as PC7/LPC, PC8 or SPC7). Other processing sites are known in the art.

In designs that include more than one processing or cleavage site, it should be understood that these sites may be different or the same.

The term furin refers to enzymes corresponding to EC code 3.4.21.75. Furin is a subtilisin-like proprotein convertase, also known as PACE (Paired basic Amino acid Cleaving Enzyme). Furin removes fragments of inactive precursor proteins to convert them into biologically active proteins. Furin can cleave VWF propeptide from the mature VWF molecule during the intracellular transport of VWF propeptide. In some embodiments, furin cleaves the D1D2 domain from the D'D3 domain of the VWF protein. In other embodiments, the nucleotide sequence encoding furin may be expressed together with the nucleotide sequence encoding the VWF fragment, resulting in the D1D2 domains being cleaved by furin within the cell.

In designs that include more than one processing or cleavage site, it should be understood that these sites may be different or the same.

As used herein, the term processable linker refers to a linker containing at least one intracellular processing site described elsewhere herein.

As used herein, disordered hemostasis means a genetically inherited or acquired condition that is characterized by a tendency to bleed, whether spontaneous or caused by trauma, due to an impaired or absent ability to form a fibrin clot. Examples of such disorders include hemophilia. The three main forms of hemophilia are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency, or Christmas disease), and hemophilia C (factor XI deficiency, a mild bleeding tendency). Other hemostasis disorders include, for example, von Willebrand disease, factor XI deficiency (deficiency of the plasma precursor thromboplastin), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X or factor XIII, Bernard-Soulier syndrome caused by defect or deficiency of platelet glycoprotein 1b (GPIb). GPIb - the VWF receptor - can be defective and lead to impaired formation of the primary clot (impaired primary hemostasis and increased tendency to bleeding) and to Glanzmann-Nageli thrombasthenia (Glanzmann thrombasthenia). In liver failure (acute and chronic), the liver synthesizes clotting factors in insufficient quantities, which may increase the risk of bleeding.

The chimeric molecules of the present invention can be used for prophylactic purposes. As used herein, the term prophylactic treatment refers to the administration of the molecule before a bleeding episode. In one embodiment of the invention, the subject in need of a general hemostatic agent is undergoing surgery or is about to undergo surgery. The chimeric protein of this invention can be administered before or after surgery as a prophylactic agent. The chimeric protein of this invention can be administered before or after surgery to control an acute bleeding episode. Surgery may include, but is not limited to, a liver transplant, liver resection, dental procedures, or stem cell transplant.

The chimeric protein of the present invention can also be used for on-demand treatment. The term on-demand treatment refers to the administration of a chimeric molecule in response to symptoms of a bleeding episode or before engaging in an activity that may result in bleeding. In one aspect of the invention, on-demand treatment may be provided to a subject after bleeding has begun, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect of the invention, on-demand treatment may be provided before an activity that increases the risk of bleeding, such as before playing a contact sport.

As used herein, the term acute bleeding refers to an episode of bleeding, regardless of the underlying cause. For example, the subject may have trauma, uremia,

- 19 044115 hereditary bleeding disorder (eg factor VII deficiency), platelet disease or resistance due to the production of antibodies to clotting factors.

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

II. Chimeric proteins.

The present invention relates to prolonging the half-life of a chimeric protein that uses a VWF protein fused to an XTEN sequence by preventing or inhibiting the association of the FVIII half-life limiting factor, i.e. endogenous VWF, with the FVIII protein. Endogenous VWF is associated in non-covalent complexes with about 95-98% of FVIII. Although endogenous VWF is a factor that limits the half-life of FVIII, endogenous VWF bound to the FVIII protein is also known to protect the FVIII protein in various ways. For example, full-length VWF (as a multimer of approximately 250 kDa) can protect FVIII from protease degradation and FVIII activation, stabilize the FVIII heavy chain and/or light chain, and prevent clearance of FVIII by phagocytic receptors. However, at the same time, endogenous VWF limits the half-life of FVIII by preventing pinocytosis and clearing the FVIII-VWF complex from the system through the VWF clearance pathway. Without regard to any theory, it is believed that endogenous VWF is a half-life limiting factor that prevents the half-life of a chimeric protein fused to a half-life prolonging component from extending the half-life of a chimeric protein fused to more than about twice that of wild-type FVIII. Therefore, the present invention is directed to preventing or inhibiting the interaction between endogenous VWF and FVIII protein by using a VWF protein containing a D' domain and a D3 domain (eg, a VWF fragment), and at the same time increasing the half-life of the resulting resulting from the FVIII protein(s) through the use of the XTEN sequence in combination with the IG constant region or part thereof. In particular, the present invention shows that a shorter XTEN sequence (ie, an XTEN sequence less than 288 amino acids in length, ie, an XTEN sequence shorter than 288 amino acids) provides better extension of the half-life of the chimeric protein.

In one embodiment, the invention provides a chimeric protein comprising (i) a first polypeptide consisting of an FVIII protein fused to a first IG constant region or portion thereof, and (ii) a second polypeptide consisting of a VWF protein that includes a D' domain and a D3 VWF fused to a second IG constant region or a portion thereof by an intervening XTEN sequence, wherein the XTEN sequence contains less than 288 amino acid residues and the first polypeptide is linked or linked to the second polypeptide. In another embodiment, the XTEN sequence in the second polypeptide consists of an amino acid sequence ranging from 12 amino acids to 287 amino acids in length. 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 includes an XTEN sequence containing at least 288 amino acids, e.g., AE288, e.g., SEQ ID NO: 8. In yet other embodiments, the XTEN sequence of 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, e.g.

- 20 044115

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.

The chimeric protein of the present invention may further comprise a second XTEN sequence that binds the FVIII protein to the first IG constant region or portion thereof.

In certain embodiments, the invention provides a chimeric protein comprising (i) a first polypeptide consisting of an FVIII protein fused to a first IG constant region or portion thereof, and (ii) a second polypeptide consisting of a VWF protein that includes a D' domain and a D3 VWF fused to a second IG constant region or a portion thereof by a first XTEN sequence located therebetween, wherein the XTEN sequence contains less than 288 amino acid residues, the first polypeptide is linked or linked to a second polypeptide, and the first polypeptide further contains a second XTEN sequence embedded in one or more insertion sites within the FVIII protein or fused to the FVIII protein and/or the first IG constant region or part thereof. Thus, in one embodiment of the invention, the second XTEN sequence is inserted into one or more insertion sites within the FVIII protein. In another embodiment, the second XTEN sequence is fused to the FVIII protein and/or to the first IG constant region or portion thereof. In other embodiments, a second XTEN sequence is inserted into one or more insertion sites within the FVIII protein, and a third XTEN sequence is fused to the FVIII protein and/or to a first IG constant region or portion thereof.

The second and/or third XTEN sequences may be XTEN amino acid sequences of any length. For example, the second and/or third XTEN sequences may be sequences described elsewhere herein, e.g.

AE42, AE72, AE864, AE576,

AE288, AE144, AG864, AG576, AG288 and AG144, for example, SEQ Ш NO: 8; SEQ Ш 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 specific embodiment, the second and/or third XTEN sequences are AE288 or AG288, for example, SEQ ID NO: 8 or 19.

In certain embodiments, the invention provides a chimeric protein comprising (i) a first polypeptide consisting of an FVIII protein fused to a first IG constant region or portion thereof via an optional linker, wherein the optional XTEN (X2) sequence is inserted into one or more insertion sites within FVIII protein or fused to FVIII protein or a first IG constant region or a portion thereof, and (ii) a second polypeptide consisting of a VWF protein that includes a VWF D' domain and a VWF D3 domain fused to a second IG constant region or a portion thereof through the sequence XTEN (X1) located between the VWF protein and the second IG constant region or part thereof, wherein the sequence XTEN (X1) contains less than 288 amino acid residues and is fused to the VWF protein via a linker, and wherein the first polypeptide is connected to the second polypeptide. In some embodiments, the invention provides a chimeric protein comprising (i) a first polypeptide consisting of an FVIII protein fused to a first IG constant region or portion thereof via an optional linker, wherein the optional XTEN (X2) sequence is inserted into one or more insertion sites within FVIII protein or fused to FVIII protein or a first IG constant region or a portion thereof, and (ii) a second polypeptide consisting of a VWF protein that includes a VWF D' domain and a VWF D3 domain fused to a second IG constant region or a portion thereof through the sequence XTEN (X1), located between the VWF protein and the second IG constant region or part thereof, wherein the sequence XTEN (X1) contains less than 288 amino acid residues and is fused to the second IG constant region or part thereof via a linker, and wherein the first polypeptide is connected to the second polypeptide. In other embodiments, the linker by which the XTEN (X1) sequence is fused to the VWF protein or to the second IG constant region or portion thereof is a cleavable linker. Non-limiting examples of cleavable linkers are described elsewhere herein. In a particular embodiment of the invention, the linker is a thrombin-cleavable linker.

In some embodiments, the chimeric protein is two polypeptide chains, the first of which contains the first polypeptide described above, and the second of which contains opi

- 21 044115 the second polypeptide above. For example, the two polypeptide chains include (i) a first chain comprising a single-chain FVIII protein, a first IG constant region or portion thereof, and an optional XTEN sequence inserted into one or more insertion sites within the FVIII protein or fused to the FVIII protein or the first constant region Ig or a portion thereof, and (ii) a second chain comprising a VWF protein fused to a second constant region of Ig or a portion thereof via an intervening sequence XTEN (X1), wherein the sequence XTEN (X1) contains less than 288 amino acids.

In certain embodiments of the invention, the chimeric protein is two polypeptide chains, the first of which contains the heavy chain of the FVIII protein, and the second of which contains, in the N-terminal to C-terminal direction, the light chain of the FVIII protein, an optional XTEN sequence inserted into one or more than insertion sites within the FVIII protein or fused to the FVIII protein or a first IG constant region or a portion thereof, a first IG constant region or a portion thereof, an optional linker (e.g., a processable linker), a VWF protein, an XTEN (X1) sequence, a second optional linker (eg, a cleavable linker) and a second IG constant region or portion thereof.

In other embodiments, the chimeric protein is three polypeptide chains: (i) a first chain comprising a FVIII protein heavy chain, (ii) a second chain comprising a FVIII protein light chain, a first IG constant region or portion thereof, and an optional XTEN sequence inserted at one or more insertion sites within the heavy chain or light chain of the FVIII protein or fused to the FVIII protein or to the first IG constant region or part thereof, and (iii) a third chain containing the VWF protein fused to the second IG constant region or part thereof via an intervening sequence XTEN (X1), the first chain and the second chain being connected by a non-covalent bond, such as a metal bond, and the second chain and the third chain being connected by a covalent bond, such as a disulfide bond.

In yet other embodiments, the chimeric protein is a single chain protein comprising, from N-terminus to C-terminus, a single chain FVIII protein, an optional XTEN sequence inserted into one or more insertion sites within the FVIII protein or fused to the FVIII protein or with a first IG constant region or a portion thereof, an optional linker (eg, a processable linker), a VWF protein, an XTEN (X1) sequence, a second optional linker (for example, a cleavable linker), and a second IG constant region or a portion thereof.

In certain embodiments of the invention, the chimeric protein contains one of the following formulas (a)-(hh):

(a) FVin-Fl:F2-L2-X-Ll-V;

(b) FVin-Fl:V-Ll-X-L2-F2;

(c) Fl-FVni:F2-L2-X-Ll-V;

(d) Fl-FVni:V-Ll-X-L2-F2;

- 22 044115 (e) FVin-X2-Fl:F2-L2-Xl-Ll-V;

(f) FVin-X2-Fl:V-Ll-Xl-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;

(1) 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-Ll-Xl-L2-F2-L3-FVni(X2)-L4-Fl;

(t) V-Ll-Xl-L2-F2-L3-Fl-L4-FVni(X2);

(u) Fl -L4-FVni(X2)-L3-F2-L2-X1 -L1 - V;

(v) F-L4-FVni(X2)-L3-V-Ll-Xl-L2-F2;

(w) FVIII(X2)-L4-F 1-L3 - V-L1 -X1-L2-F2;

(x) FVIII(X2)-L4-F 1-L3 -F2-L2-X1 -L1 - V;

(y) F2-L2-Xl-Ll-V-L3-Fl-L4-FVni(X2);

(z) F2-L2-Xl-Ll-V-L3-FVni(X2)-L4-Fl;

(aa) V-L 1-X2-L2-F2-L3-FVIII-L4-X2-L5-F1;

(bb) V-L1-X2-L2-F2-L3-F1-L5-X2-L4-FVIII;

(cc) Fl -L5-X2-L4-FVIII-L3 -F2-L2-X2-L1 -V;

(dd) F1-L5-X2-L4-FVIII-L3 - V-L 1-X2-L2-F2;

(ee) FVIII-L5-X2-L4-F2-L3 - V-L 1 -X1-L2-F1;

(ff) FVIII-L5-X2-L4-F2-L3 -Fl -L2-X1 -L1 - V;

(gg) Fl-L2-Xl-Ll-V-L3-F2-L4-X2-L5-FVin; or (hh) F1-L2-X1 -L1 - V-L3 -FVIII-L5-X2-L4-F2;

where V is the VWF protein, which contains a D' domain and a D3 domain,

X or X1 is the first XTEN sequence having less than 288 amino acids,

X2 is the second XTEN sequence, FVIII includes the 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 represents the first constant region of the IG or part thereof,

F2 represents the second constant region of the IG or part thereof,

L1, L2, L3, L4, or L5 represents an optional linker; (-) represents a peptide bond; and (:) represents a covalent bond or a non-covalent bond.

In one embodiment of the invention, the sequence X or

X1 consists of an amino acid sequence ranging in length from 12 amino acids to 287 amino acids. In another embodiment, the chimeric protein exhibits a longer half-life compared to the corresponding chimeric protein of the formula wherein X or X1 is AE288, for example, SEQ ID NO: 8.

In other embodiments, X or X1 in the formula contains at least about

- 23 044115

36, at least about 42, at least about 72, or at least about 144 amino acids, but less than 288 amino acids, e.g.

AE42, AE72, AE144 (AE144, AE144_2A, AE144_ZV, 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.

In yet 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 in length, for example

AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576,

AG288 or AG144, for example, SEQ Ш NO: 9; SEQ Ш NO: 10; SEQ Ш NO: 15; SEQ Ш NO:

16; SEQ Ш NO: 8; SEQ Ш NO: И; SEQ Ш NO: 17; SEQ Ш NO: 18; SEQ ID NO: 19; or

SEQ ID NO: 14.

In a specific embodiment, X2 is AE288 or AG288, for example, SEQ ID NO: 8 or 19.

In certain embodiments, a chimeric protein containing X or X1 and/or X2 exhibits a longer half-life compared to a chimeric protein not containing X or X1 and/or X2. In other embodiments, L1 and/or L2 is a cleavable linker. In yet other embodiments, L4 and/or L5 is a cleavable linker.

I.A. Von Willebrand factor (VWF) proteins.

VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and continuously produced in the endothelium (Weibel-Palade bodies), megakaryocytes (platelet alpha granules), and connective tissue subendothelium. The main monomer of VWF is a protein consisting of 2813 amino acids. Each monomer contains a number of specific domains with a specific function, the D'/D3 domain (which binds to factor VIII), the A1 domain (which binds to the platelet receptor GPIb, heparin, and/or possibly collagen), the A3 domain (which binds to collagen), the C1 domain (in which the RGD domain binds to the platelet-derived integrin αΠΕβ3 when activated), and the cysteine knot domain at the C terminal end of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β ) and beta human chorionic gonadotropin (PHCG)).

In one embodiment of the invention, the VWF protein is a fragment of VWF. The term VWF fragment as used herein includes, but is not limited to, functional VWF fragments containing a D' domain and a D3 domain that have the ability to inhibit the binding of endogenous VWF to FVIII. In one embodiment of the invention, the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment blocks the binding site of the FVIII protein, thereby inhibiting the interaction of the FVIII protein with endogenous VWF. VWF fragments include derivatives, variants, mutants or analogs that retain this VWF activity.

The amino acid sequence of the human VWF monomer (2813 amino acids) is described under accession number _NP_000543.2_ in Genbank. The nucleotide sequence encoding VWF is described under accession number _NM_000552.3_ in Genbank. The nucleotide sequence of human VWF is indicated as SEQ ID NO: 20. SEQ ID NO: 21 is the amino acid sequence of full-length VWF. Each VWF domain is presented in Table. 1.

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Table 1

VWF sequences

FV domains Amino acid sequence Signal peptide VWF (Amino Acids 1-22 from SEQ ID NO: 21) 1 MIPARFAGVL LALALILPGT LC 22 VWF D1D2 Region (Amino Acids 23763 of SEQ ID NO: 21) 23 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 AEGTRGRS STARCSLFGS DFVNTFDGSM YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG LHNSLVKLKH GAGVAMDGQDIQLPLLKGDL RIQHTVTASV RLSYGEDLQM DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD AVLSSPLSHR SKR 763 Domain D' VF 764 801 SLSCRPP MVKLVCPADN LRAEGLECTK TCONYDLECM SMGCVSGCLC PPGMVRHENR CVALERCPCF HOGKEYAPGE TVKIGCNTCV

- 25 044115

851 CRDRKWNCTD NUSRAT 866 Domain D3 FV 867 CSTI GM AHY_LTFDG_LK Y LFPGECQ YVLVQPYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE YNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE kvcglcgnfd 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ.CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCP^SC EERNLRENGY ECEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC YPPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP 1240 Domain A1 FV 1241 GGLVVPPTDA 1251 PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV 1301 VDMMERLRIS QKWVRVAVVE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA 1351 GSQVASTSEV LKYTLFQIFS KIDRPEASRI ALLLMASQEP QRMSRNFVRY 1401 VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1451 DEIVSYLCDL APEAPPPTLP PDMAQVTVG 1479 1480 P GLLGVSTLGP KRNSMVLDVA 1501 FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY 1551 PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA 1600 1601 PNLVYMVTGN PASDEIKRLP GDIQVVPIGV GPNANVQELE RIGWPNAPIL 1651 IQDFETLPRE APDLVLQRCC SGEGLQIPTL SPAPDCSQPL DVILLLDGSS 1701 SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNVVPE 1751 KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV 1801 TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA

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GPAGDSNVVK

1851 LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH 1901 TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC 1951 TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG 2001 CMKSIEVKHS ALSVEXHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV 2051 RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD 2101 GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSHCQ VLLLPFAEC 2151 HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTGNGVC VDWRTPDFCA 2201 MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP 2251 EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK 2301 APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN 2351 PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN 2401 STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV 2451 CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE 2501 VVTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP 2551 QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC 2601 TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC CGRCLPTACT 2651 IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2701 CLAEGGKIMKIPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC 2751 QGKCASKAMY SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSVVYHEVLN 2801 AMECKCSPRK CSK Nucleotide sequence (SEQ ID NO: 20) Full size 1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG

- 27 044115

FV SSSTSATTTTT 51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCА ACACCTTTGА TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTASSTS CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTTC TCG ATT ATTG GGG ACTTCC A G A ATGGC A AG AGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCSATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451 TCAGAC AGAT ASTTSAASAA GACCTGCGGG CTGTGTGGCA ASTTTAASAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATTSST CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGASTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTSSSSTST

- 28 044115

CTCGAGACTG

1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT

1151 GTCC AGGGG A GTGCCTTGTC ACTGGTC A AT CCC ACTTC A A GAGCTTTGAC

1201 A AC AG AT ACT TC ACCTTC AG TGGG ATCTGC CAGT ACCTGC TGGCCCGGGA

1251 TTGCCAGGAC СACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG

1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC

1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA

1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC

1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG

1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC

1551 CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG

1601 ACTTCCTTAC CCCCTCTGGG CTGGCRGAGC CCCGGGTGGA GGACTTCGGG

1651 A ACGCCTGG A AGCTGC ACGG GG ACTGCC AG G ACCTGC AG A AGCAGCACAG

1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT

1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC

1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA

1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG

1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG

1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT

2001 G ACCTGCCGC TCTCTCTCTT ACCCGG ATG A GG A ATGC A AT GAGGCCTGCC

2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC

2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG

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AGATCTTCCA

2151 GCC AG A AG AC ATCTTCTC AG ACC ATC AC AC C ATGTGCT AC TGTGAGGATG

2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC

2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG

2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG

2351 AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG

2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA

2451 TGAGAAC AG A TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA

2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC

2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG

2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAAATACC

2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT

2701 A ACCCTGGG A CCTTTCGG AT CCT AGTGGGG A AT A AGGG AT GCAGCCACCC

2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA

2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCC AT GAAGGATGAG

2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG

2901 C A A AGCCCTC TCCGTGGTCT GGG ACCGCC A CCTG AGC ATC TCCGTGGTCC

2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT

3001 GGC ATCC AG A AC AATG ACCT C ACC AGC AGC AACCTCC AAG TGGAGGAAGA

3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA

3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC

3151 ATG A AGC AG A CG ATGGTGG A TTCCTCCTGT AG A ATCCTT A

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CCAGTGACGT

3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT

3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCTGCTTC

3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT

3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA

3401 ATCTCCGGGA GAACGGGTAT GAGTGTGAGT GGCGCTATAA CAGCTGTGCA

3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCTGT

3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG

3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG

3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG

3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT

3701 GTGAAGCCTG CCAGGAGCCG GGAGGCCTGG TGGTGCCTCC CACAGATGCC

3751 CCGGTGAGCC CCACCACTCT GTATGTGGAG GACATCTCGG AACCGCCGTT

3801 GCACGATTTC TACTGCAGCA GGCTACTGGA CCTGGTCTTC CTGCTGGATG

3851 GCTCCTCCAG GCTGTCCGAG GCTGAGTTTG AAGTGCTGAA GGCCTTTGTG

3901 GTGGACATGA TGGAGCGGCT GCGCATCTCC CAGAAGTGGG TCCGCGTGGC

3951 CGTGGTGGAG TACCACGACG GCTCCCACGC CTACATCGGG CTCAAGGACC

4001 GGAAGCGACC GTCAGAGCTG CGGCGCATTG CCAGCAGGT GAAGTATGCG

4051 GGCAGCCAGG TGGCCTCCAC CAGCGAGGTC TTGAAATACA CACTGTTCCA

4101 A ATCTTC AGC A AG ATCG ACC GCCCTG A AGC CTCCCGC ATC GCCCTGCTCC

4151 TGATGGCCAG CCAGGAGCCC CAACGGATGT CCCGGAACTT TGTCCGCTAC

4201 GTCC AGGGCC TGA AG A AG A A G A AGGTC ATT GTG ATCCCGG

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TGGGCATTGG

4251 GCCCCATGCC AACCTCAAGC AGATCCGCCT CATCGAGAAG CAGGCCCCTG

4301 AGAACAAGGC CTTCGTGCTG AGCAGTGTGG ATGAGCTGGA GCAGCAAAGG

4351 GACGAGATCG TTAGCTACCT CTGTGACCTT GCCCCTGAAG CCCCTCCTCC

4401 TACTCTGCCC CCCGACATGG CACAAGTCAC TGTGGGCCCG GGGCTCTTGG

4451 GGGTTTCGAC CCTGGGGCCC AAGAGGAACT CCATGGTTCT GGATGTGGCG

4501 TTCGTCCTGG AAGGATCGGA CAAAATTGGT GAAGCCGACT TCAACAGGAG

4551 CAAGGAGTTC ATGGAGGAGG TGATTCAGCG GATGGATGTG GGCCAGGACA

4601 GCATCCACGT CACGGTGCTG CAGTACTCCT AC ATGGTGAC CGTGGAGTAC

4651 CCCTTCAGCG AGGCACAGTC CAAAGGGGAC ATCCTGCAGC GGGTGCGAGA

4701 GATCCGCTAC CAGGGCGGCA ACAGGACCAA CACTGGGCTG GCCCTGCGGT

4751 ACCTCTCTGA CCACAGCTTC TTGGTCAGCC AGGGTGACCG GGAGCAGGCG

4801 CCCAACCTGG TCTACATGGT CACCGGAAAT CCTGCCTCTG ATGAGATCAA

4851 GAGGCTGCCT GGAGACATCC AGGTGGTGCC CATTGGAGTG GGCCTAATG

4901 CCAACGTGCA GGAGCTGGAG AGGATTGGCT GGCCCAATGC CCCTATCCTC

4951 ATCC AGG ACT TTGAG ACGCT CCCCCGAGAG GCTCCTGACC TGGTGCTGCA

5001 GAGGTGCTGC TCCGGAGAGG GGCTGCAGAT CCCCACCCTC TCCCCTGCAC

5051 CTGACTGCAG CCAGCCCCTG GACGTGATCC TTCTCCTGGA TGGCTCCTCC

5101 AGTTTCCC AG CTTCTT ATTT TG ATGA A ATG A AG AGTTTCG CCAAGGCTTT

5151 C ATTTCAAAA GCC AAT AT AG GGCCTCGTCT C ACTC AGGTG TCAGTGCTGC

5201 AGTATGGAAG CATCACCACC ATTGACGTGC CATGGAACGT GGTCCCGGAG

5251 AAAGCCCATT TGCTGAGCCT TGTGGACGTC ATGCAGCGGG

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AGGGAGGCCC

5301 CAGCCAAATC GGGGATGCCT TGGGCTTTGC TGTGCGATAC TTGACTTCAG

5351 AAATGCATGG TGCCAGGCCG GGAGCCTCAA AGGCGGTGGT CATCCTGGTC

5401 ACGGACGTCT CTGTGGATTC AGTGGATGCA GCAGCTGATG CCGCCAGGTC

5451 CAACAGAGTG ACAGTGTTCC CTATTGGAAT TGGAGATCGC TACGATGCAG

5501 CCCAGCTACG GATCTTGGCA GGCCCAGCAG GCGACTCCAA CGTGGTGAAG

5551 CTCCAGCGAA TCGAAGACCT CCCTACCATG GTCACCTTGG GCAATTCCTT

5601 CCTCC AC AAA CTGTGCTCTG GATTTGTTAG GATTTGC ATG GATGAGGATG

5651 GGAATGAGAA GAGGCCCGGG GACGTCTGGA CCTTGCCAGA CCAGTGCCAC

5701 ACCGTGACTT GCCAGCCAGA TGGCCAGACC TTGCTGAAGA GTCATCGGGT

5751 CAACTGTGAC CGGGGGCTGA GGCCTTCGTG CCCTAACAGC CAGTCCCCTG

5801 TTAAAGTGGA AGAGACCTGT GGCTGCCGCT GGACCTGCCC CTGYGTGTGC

5851 ACAGGCAGCT CCACTCGGCA CATCGTGACC TTTGATGGGC AGAATTTCAA

5901 GCTGACTGGC AGCTGTTCTT ATGTCCTATT TCAAAACAAG GAGCAGGACC

5951 TGGAGGTGAT TCTCCATAAT GGTGCCTGCA GCCCTGGAGC AAGGCAGGGC

6001 TGC ATG A A AT CC ATCG AGGT GA AGC AC AGT GCCCTCTCCG TCGAGSTGCA

6051 CAGTGACATG GAGGTGACGG TGAATGGGAG ACTGGTCTCT GTTCCTTACG

6101 TGGGTGGG AA C ATGGA AGTC A ACGTTT ATG GTGCC ATC AT GCATGAGGTC

6151 AG ATTC AATC ACCTTGGTC A C ATCTTC AC A TTC ACTCC AC AAAACAATGA

6201 GTTCC A ACTG C AGCTC AGCC CC A AG ACTTT TGCTTC A A AG ACGTATGGTC

6251 TGTGTGGGAT CTGTGATGAG AACGGAGCCA ATGACTTCAT GCTGAGGGAT

6301 GGC AC AGTC A CC AC AG ACTG GAAAAC ACTT GTTCAGGAAT

- 33 044115

GGACTGTGCA

6351 GCGGCCAGGG CAGACGTGCC AGCCCATCCT GGAGGAGCAG TGTCTTGTCC

6401 CCGACAGCTC CCACTGCCAG GTCCTCCTCT TACCACTGTT TGCTGAATGC

6451 C AC AAGGTCC TGGCTCC AGC C AC ATTCTAT GCCATCTGCC AGCAGGACAG

6501 TTGCCACC AG G AGC AAGTGT GTGAGGTGAT CGCCTCTTAT GCCCACCTCT

6551 GTCGGACCAA CGGGGTCTGC GTTGACTGGA GGACACCTGA TTTCTGTGCT

6601 ATGTCATGCC CACCATCTCT GGTCTACAAC CACTGTGAGC ATGGCTGTCC

6651 CCGGCACTGT GATGGCAACG TGAGCTCCTG TGGGGACCAT CCCTCCGAAG

6701 GCTGTTTCTG CCCTCC AG AT A A AGTC ATGT TGG A AGGC AG CTGTGTCCCT

6751 GAAGAGGCCT GCACTCAGTG CATTGGTGAG GATGGAGTCC AGCACCAGTT

6801 CCTGGAAGCC TGGGTCCCGG ACCACCAGCC CTGTCAGATC TGCACATGCC

6851 TCAGCGGGCG GAAGGTCAAC TGCACAACGC AGCCCTGCCC CACGGCCAAA

6901 GCTCCCACGT GTGGCCTGTG TGAAGTAGCC CGCCTCCGCC AGAATGCAGA

6951 CCAGTGCTGC CCCGAGTATG AGTGTGTGTG TGACCCAGTG AGCTGTGACC

7001 TGCCCCCAGT GCCTCACTGT GAACGTGGCC TCCAGCCCAC ACTGACCAAC

7051 CCTGGCGAGT GCAGACCCAA CTTCACCTGC GCCTGCAGGA AGGAGGAGTG

7101 C A A A AG AGTG TCCCC ACCCT CCTGCCCCCC GC ACCGTTTG CCCACCCTTC

7151 GG A AG ACCC A GTGCTGTG AT G AGT ATG AGT GTGCCTGC A A CTGTGTCAAC

7201 TCCACAGTGA GCTGTCCCCT TGGGTACTTG GCCTCAACCG CCACCAATGA

7251 CTGTGGCTGT ACC AC AACC A CCTGCCTTCC CG AC AAGGTG TGTGTCCACC

7301 GAAGC ACC AT CT ACCCTGTG GGCC AGTTCT GGG AGG AGGG CTGCGATGTG

7351 TGCACCTGCA CCGACATGGA GGATGCCGTG ATGGGCCTCC

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GCGTGGCCCA

7401 GTGCTCCCAG AAGCCCTGTG AGGACAGCTG TCGGTCGGGC

TTCACTTACG

7451 TTCTGCATGA AGGCGAGTGC TGTGGAAGGT GCCTGCCATC

TGCCTGTGAG

7501 GTGGTGACTG GCTCACCGCG GGGGGACTCC CAGTCTTCCT

GGAAGAGTGT

7551 CGGCTCCCAG TGGGCCTCCC CGGAGAACCC CTGCCTCATC

AATGAGTGTG

7601 TCCG AGTG A A GG AGG AGGTC TTT AT AC A AC A A AGG A ACGT

CTCCTGCCCC

7651 CAGCTGGAGG TCCCTGTCTG CCCCTCGGGC TTTCAGCTGA

GCTGTAAGAC

7701 CTCAGCGTGC TGCCCAAGCT GTCGCTGTGA GCGCATGGAG

GCCTGCATGC

7751 TCAATGGAC TGTCATTGGG CCCGGGAAGA CTGTGATGAT

CGATGTGTGC

7801 ACGACCTGCC GCTGCATGGT GCAGGTGGGG GTCATCTCTG

GATTCAAGCT

7851 GGAGTGCAGG AAGACCACCT GCAACCCCTG CCCCCTGGGT

TACAAGGAAG

7901 A A A AT A AC AC AGGTG A ATGT TGTGGG AG AT GTTTGCCT AC

GGCTTGCACC

7951 ATTCAGCTAA GAGGAGGACA GATCATGACA CTGAAGCGTG

ATGAGACGCT

8001 CCAGGATGGC TGTGATACTC ACTTCTGCAA GGTCAATGAG

AGAGGAGAGT

8051 ACTTCTGGGA GAAGAGGGTC ACAGGCTGCC CACCCTTTGA

TGAACACAAG

8101 TGTCTTGCTG AGGG AGGT A A A ATT ATG A A A ATTCC AGGC A

CCTGCTGTGA

8151 C AC ATGTG AG G AGCCTG AGT GCA ACG AC AT C ACTGCC AGG

CTGCAGTATG

8201 TCAAGGTGGG AAGCTGTAAG TCTGAAGTAG AGGTGGATAT

CCACTACTGC

8251 CAGGGCAAAT GTGCCAGCAA AGCCATGTAC TCCATTGACA

TCAACGATGT

8301 GCAGGACCAG TGCTCCTGCT GCTCTCCGAC ACGGACGGAG

CCCATGCAGG

8351 TGGCCCTGCA CTGCACCAAT GGCTCTGTTG TGTACCATGA

GGTTCTCAAT

8401 GCCATGGAGT GCAAATGCTC CCCCAGGAAG TGCAGCAAGT GA

In this context, the VWF protein may be a VWF fragment containing the D' domain and the D3 domain of VWF, in which the VWF fragment binds to factor VIII (FVIII) and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment containing domain D' and domain D3 may further comprise a VWF domain 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, C2 domain, CK domain, one or more fragments thereof, and any combination thereof. In one embodiment of the invention, the VWF fragment comprises, consists essentially of, or consists of: (1) VWF D' and D3 domains or fragments thereof; (2)

- 35 044115 domains D1, D' and D3 of VWF or their fragments; (3) domains D2, D' and D3 of VWF or fragments thereof; (4) domains D1, D2, D' and D3 of VWF or fragments thereof; (5) domains D1, D2, D', D3 and A1 of VWF or fragments thereof. The VWF fragment described herein does not contain a binding site for the VWF clearance receptor. In another embodiment, the VWF fragment described herein is not amino acids 764-1274 of SEQ ID NO: 21. The VWF fragment of the present invention may include any other sequences linked to or fused to the VWF fragment. For example, the VWF fragment described herein may further comprise a signal peptide.

In one embodiment of the invention, a VWF fragment containing a D' domain and a D3 domain binds or binds to the FVIII protein. By binding or binding to the FVIII protein, the VWF fragment of the present invention protects FVIII from protease degradation and activation of FVIII, stabilizes the heavy chain and light chain of FVIII, and prevents clearance of FVIII by phagocytic receptors. In another embodiment of the invention, the VWF fragment binds or binds to the FVIII protein and blocks or prevents the binding of the FVIII protein to phospholipid 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 the clearance of FVIII by the VWF clearance receptors , thus prolonging the half-life of the chimeric protein. Thus, the increase in the half-life of the chimeric protein is caused by binding or combining with the VWF fragment, with the missing VWF clearance receptor binding site, with the FVIII protein and shielding or protection of the FVIII protein by the VWF fragment from endogenous VWF containing the VWF clearance receptor binding site. By binding or protecting the FVIII protein by the VWF fragment, recycling of the FVIII protein can also be achieved. By eliminating the VWF clearance receptor binding sites contained in the full-length VWF molecule, the FVIII/VWF heterodimers of the present invention are protected from VWF clearance with a further increase in the half-life of FVIII.

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

In some embodiments, a VWF protein useful for use in the present 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) additional VWF sequence , up to about 10 amino acids in size (e.g., any sequence from amino acids 764-1240 of SEQ ID NO: 21, to amino acids 764-1250 of SEQ ID NO: 21), up to about 15 amino acids (for example, any sequence from amino acids 764-1240 of SEQ ID NO: 21 to amino acids 764-1255 of SEQ ID NO: 21), up to about 20 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO: 21 to amino acids 764-1260 of SEQ ID NO: 21 ), up to about 25 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO: 21 to amino acids 764-1265 from SEQ ID NO: 21) or up to about 30 amino acids (for example, any sequence from amino acids 764-1240 from SEQ ID NO: 21 to amino acids 764-1260 from SEQ ID NO: 21). In a particular embodiment, the VWF protein containing or substantially consisting of a D' domain and a D3 domain is neither amino acids 7,641,274 of SEQ ID NO: 21 nor full-length mature VWF. In some embodiments, the D1D2 domain is expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis with the D'D3 domain.

In other embodiments, the VWF protein containing D'D3 domains associated with D1D2 domains further contains an intracellular cleavage site, for example (PACE (furin) or PC5 cleavage site), allowing cleavage of D1D2 domains from D'D3 domains upon expression. Non-limiting examples of intracellular cleavage sites are described elsewhere herein.

In yet other embodiments, the VWF protein contains a D' domain and a D3 domain, but does not contain an amino acid sequence selected from the group consisting of (1) amino acids 12412813 corresponding to SEQ ID NO: 21, (2) amino acids 1270-2813 corresponding to SEQ ID NO: 21, (3)

- 36 044115 amino acids 1271-2813 corresponding to SEQ ID NO: 21, (4) amino acids 1272-2813 corresponding

SEQ ID NO: 21, (5) amino acids 1273-2813 corresponding to SEQ ID NO: 21, (6) amino acids 12742813 corresponding to SEQ ID NO: 21 and any combinations thereof.

In yet other embodiments, the VWF protein of the present invention comprises, consists essentially of, or consists of an amino acid sequence corresponding to a D' domain, a D3 domain, and an A1 domain, wherein the amino acid sequence is at least 60%, 70%, 75%, 80% , 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1479 of SEQ ID NO: 21, wherein the VWF protein prevents or inhibits the binding of endogenous VWF to FVIII. In a specific embodiment, the VWF protein is not amino acids 764-1274 of SEQ ID NO: 21.

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

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

In yet other embodiments, the VWF protein is linked to a heterologous fragment, wherein the heterologous fragment is attached to the N-terminus or C-terminus of the VWF protein or is inserted immediately downstream of one or more amino acids (eg, one or more XTEN insertion sites) in the VWF protein. For example, insertion sites for a heterologous fragment into the VWF protein may be in the D' domain, the D3 domain, or both domains. The heterologous fragment may prolong the half-life.

In some embodiments, the VWF protein useful in the present invention forms a multimer, such as a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or higher order multimers. In other embodiments, the VWF protein is a monomer containing only one VWF protein. In some embodiments, the VWF protein of the present invention may have one or more amino acid substitutions, deletions, insertions, or modifications. In one embodiment, the VWF protein may include amino acid substitutions, deletions, insertions, or modifications such that the VWF protein loses the ability to form a disulfide bond or form a dimer or multimer. In another embodiment, the amino acid substitution is within the D' domain and the D3 domain. In a specific embodiment, a VWF protein suitable for use in the present invention contains at least one amino acid substitution at a position corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 21. At least one amino acid substitution may be any amino acids that are not naturally occurring in wild-type VWF. For example, the amino acid substitution can be any amino acid other than cysteine, for example, 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 in VWF proteins.

In some embodiments, the VWF protein useful in the present invention may be further modified to improve its interaction with FVIII, for example, to increase its binding affinity to FVIII. As a non-limiting example, the VWF protein contains a serine residue at the position corresponding to amino acid 764 of SEQ ID NO: 21 and a lysine residue at the position corresponding to amino acid 773 of SEQ ID NO: 21. Residues 764 and/or 773 may contribute to protein binding affinity FV with FVIII. In other embodiments, VWF proteins suitable for use in the present invention may have other modifications, for example, the protein may be PEGylated, glycosylated, HESylated, or polysialylated.

- 37 044115

II. B. XTEN sequences.

In this context, the sequence XTEN means extended polypeptides with non-repetitive sequences of non-natural origin, which are mainly composed of small hydrophilic amino acids with a sequence having low or no secondary or tertiary structure under physiological conditions. As a chimeric protein partner, XTEN sequences can serve as a carrier to impart certain desired pharmacokinetic, physicochemical and pharmaceutical properties when combined with the VWF protein or FVIII sequence of the present invention to create a chimeric protein. Such desirable properties include, but are not limited to, improved pharmacokinetic parameters and solubility characteristics. As used herein, the term XTEN specifically excludes antibodies and antibody fragments, such as single chain antibodies or light chain or heavy chain Fc fragments.

The present invention provides a shorter XTEN sequence that provides improved half-life increasing properties compared to a longer XTEN sequence when the XTEN sequence is fused to the VWF protein and/or a second IG constant region or a portion thereof. Therefore, the XTEN sequence fused to the VWF protein and/or the second IG constant region or part thereof is less than 288 amino acids in length, that is, shorter than 288 amino acids. In one embodiment of the invention, the XTEN sequence fused to the VWF protein and/or the second IG constant region or a portion thereof consists of an amino acid sequence having a length of from 12 to 287 amino acids. In another embodiment, the XTEN sequence fused to the VWF protein and/or the second IG constant region or a portion thereof comprises 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 the second IG constant region or 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 the second IG constant region or a portion thereof is an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97% identical, 98%, 99%, or 100% of the sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 14, in which the chimeric protein exhibits an improved half-life compared to the chimeric protein without the XTEN sequence.

The chimeric protein of the present invention may also contain an additional (second, third or more) XTEN sequence. The additional XTEN sequence may also be fused to the FVIII protein or to the first IG constant region or part thereof. Additional XTEN sequences can be of any length. For example, the additional XTEN sequence fused to the FVIII protein or the first IG constant region is a peptide or polypeptide having an approximate number of amino acid residues greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 , 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800 or 2000. In some embodiments, additional sequence XTEN is a peptide or polypeptide with an approximate number of amino acid residues from more than about 20 to about 3000 amino acid residues, from more than about 30 to about 2500 residues, from more than about 40 to about 2000 residues, from more than about 50 to about 1500 residues, from more about 60 to about 1000 residues, from more than about 70 to about 900 residues, from more than about 80 to about 800 residues, from more than about 90 to about 700 residues, from more than about 100 to about 600 residues, from more than about 110 to about 500 residues or more from about 120 to about 400 residues.

XTEN sequences (i.e., an XTEN sequence fused to the VWF protein and/or the second IG constant region or part thereof, or an XTEN sequence fused to the FVIII protein and/or the first IG constant region or part thereof, or inserted into one or more insertion site in the FVIII protein) may contain one or more sequence motifs of 9 to 14 amino acid residues or an amino acid sequence identical to at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to a sequence motif, wherein the motif includes, consists essentially of or consists of 4-6 amino acid species selected from the group consisting of glycine (G), alanine (A), serine ( S), threonine (T), glutamate (E) and proline (P). See US 2010-0239554 A1.

In some embodiments, the XTEN sequence contains non-overlapping sequence motifs in which 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 a plurality of non-overlapping sequence units selected from the single motif family of Table 1. 2A, resulting in the family sequence. In this context, the term family means that XTEN has motifs selected from only one category of motifs from Table. 2A; those. AD, AE, AF, AG, AM, AQ, BC or BD XTEN, and that any other amino acids in XTEN are not from the motif family

- 38 044115 were selected to achieve a desired property, for example, to provide inclusion of a restriction site via coding nucleotides, inclusion of a cleavage sequence, or to achieve more efficient binding to FVIII or VWF. In some embodiments of the XTEN families, the XTEN sequence includes multiple units of non-overlapping sequence motifs from the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC motif family, or a family of BD motifs, wherein the selected XTEN exhibits the range of homology described above. In other embodiments, XTEN includes a plurality of sequence motif units from two or more of the motif families in Table 1. 2A. These sequences can be selected to achieve desired physical/chemical properties, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repeatability, which are provided according to the amino acid composition of the motifs, which is described in more detail below. In the embodiments described above in this paragraph, the motifs embedded in XTEN can be selected and assembled using methods described herein to achieve an XTEN size of from about 36 to about 3000 amino acid residues.

Table 2A

XTEN 12 amino acid sequence motifs and motif families

Seed of your motive* MOTIVE SEQUENCES AD GESPGGSSGSES (SEQ ID NO: 24) AD GSEGSSGPGESS (SEQ ID NO: 25) AD GSSESGSSEGGP (SEQ ID NO: 26) AD GSGGEPSESGSS (SEQ ID NO: 27) AE, AM GSPAGSPTSTEE (SEQ ID NO: 28) AE, AM, AQ GSEPATSGSETP (SEQ ID NO: 29) AE, AM, AQ GTSESATPESGP (SEQ ID NO: 30) AE, AM, AQ GTSTEPSEGSAP (SEQ ID NO: 31) AF, AM GSTSESPSGTAP (SEQ ID

- 39 044115

NO: 32) AF, AM GTSTPESGSASP (SEQ ID NO: 33) AF, AM GTSPSGESSTAP (SEQ ID NO: 34) AF, AM GSTSSTAESPGP (SEQ ID NO: 35) A.G., A.M. GTPGSGTASSSP (SEQ ID NO: 36) A.G., A.M. GSSTPSGATGSP (SEQ ID NO: 37) A.G., A.M. GSSPSASTGTGP (SEQ ID NO: 38) A.G., A.M. GASPGTSSTGSP (SEQ ID NO: 39) AQ GEPAGSPTSTSE (SEQ ID NO: 40) AQ GTGEPSSTPASE (SEQ ID NO: 41) AQ GSGPSTESAPTE (SEQ ID NO: 42) AQ GSETPSGPSETA (SEQ ID NO: 43) AQ GPSETSTSEPGA (SEQ ID NO: 44) AQ GSPSEPTEGTSA (SEQ ID NO: 45) B.C. GSGASEPTSTEP (SEQ ID NO: 46) B.C. GSEPATSGTEPS (SEQ ID NO: 47) B.C. GTSEPSTSEPGA (SEQ ID

- 40 044115

NO: 48) Sun GTSTEPSEPGSA (SEQ ID NO: 49) BD GSTAGSETSTEA (SEQ ID NO: 50) BD GSETATSGSETA (SEQ ID NO: 51) BD GTSESATSESGA (SEQ ID NO: 52) BD GTSTEASEGSAS (SEQ ID NO: 53)

Denotes individual sequences of motifs which, when applied in conjunction with various permutations, yield a family sequence.

In some embodiments, the XTEN sequence used in the present 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, AG3 96, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE 792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE18 12, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908 and AG2004. See US 20100239554 A1.

In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of

AE42 (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 IDNO: 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 another embodiment of the invention, the XTEN sequence is selected from the group consisting of

AE42 (SEQ Ш NO: 9), AE72 (SEQ Ш NO: 10), AE144_2А (SEQ IDNO: 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 IDNO: 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 specific embodiment, the XTEN sequence is AE288. The amino acid sequences for certain XTEN sequences of the present invention are shown in table. 2B.

- 41 044115

Table 2B

XTEN sequences

XTEN Amino acid sequence AE42 SEQ ID NO: 9 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS AE72 SEQ ID NO: 10 GAP TSESATPESG PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG TSTEPSEGSA PGASS AE144 SEQ ID NO: 11 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEG SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSA P.G.T.S.S.A. PESGPGSEPATSGSETPGTSTEPSEGSAP AE144_2A (SEQ ID NO: 55) TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG AE144_3B (SEQ ID NO: 56) SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_4A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAG

- 42 044115

(SEQ ID NO: 57) SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_5A (SEQ ID NO: 58) TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG AE144_6B (SEQ ID NO: 59) TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AG144 SEQ ID NO: 14 GTPGSGTASSSPGSSTPSGATTGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS PGSSPSA STGTGPGTPGSGTASSSPGSSTPSGATGSP AG144_A (SEQ ID NO: 60) GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGS STPSGATTGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP AG144_B (SEQ ID NO: 61) GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGS STPSGATGSAPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSST PSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP AG144_C (SEQ ID NO: 62) GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGS SPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AG144_F (SEQ ID NO: 63) GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGS STPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP AE288 SEQ ID NO: 8 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSTEPS GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS

- 43 044115

AP AE288_2 (SEQ ID NO: 54) GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG288 SEQ ID NO: 19 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GSSPSASTG TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATTGS PGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT GSPGSSPSAS TGTGPGGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA TGS AE576 SEQ ID NO: 16 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG576 SEQ ID NO: 18 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG SSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG PGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG SPGSSTPSG ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT ASSSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT ASSSPGSST

- 44 044115

PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS GATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGS STPSGATTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AE864 SEQ ID NO: 15 AG864 SEQ ID NO: 17 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGS STPSGATGS PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GASPGTSST GSPGTPGSGTASSSPGSSTPSGATTGSPGSSPSASTGTGPGSSPSASTGTG PGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGT ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTG TGPGTPGSG TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA TGSPGSSTP SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG ATGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGS STPSGAATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST PSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP GSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG ASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG TPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

In those embodiments in which the XTEN component(s) are composed of less than 100% amino acid types 4, 5, or 6 selected from glycine (G), alanine (A), serine (S), threonine (T),

- 45 044115 glutamate (E) and proline (P), or less than 100% of the sequence consists of sequence motifs from table. 3 or XTEN sequences from table. 4 and 13-17, other amino acid residues in XTEN may be selected from any of the remaining 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), glutamate (E), and proline (P) may be distributed throughout the XTEN sequence, may be located within or between motifs sequences or may be concentrated into one or more regions of the XTEN sequence, for example, to create a linker between XTEN and FVIII or VWF components. Where the XTEN component contains amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is preferable that less than about 2% or less than about 1% of the amino acid residues are hydrophobic such that the resulting sequences generally lack secondary structure, e.g., having no more than 2% alpha helices or 2% beta sheets, as determined by the methods described herein. Hydrophobic residues less favored in the XTEN structure include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, XTEN sequences can be designed to contain less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid deamidation). Thus, in some embodiments, an XTEN component containing other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) has the sequence less than 5% of the residues contributing to the formation of alpha helices and beta sheets according to the Chow-Fasman algorithm, and has at least 90%, or at least about 95% or more random coil structures, according to the GOR algorithm.

In additional embodiments, the XTEN sequence used in the present invention affects the physical or chemical property, for example, the pharmacokinetics of the chimeric protein of the present invention. The XTEN sequence used in this invention may exhibit one or more of the following beneficial properties: conformational flexibility, increased aqueous solubility, high protease resistance, low immunogenicity, low affinity for mammalian cell receptors, and increased hydrodynamic (or Stokes) radii . In a specific embodiment, the XTEN sequence linked to the FVIII protein of the present invention improves pharmacokinetic properties such as increased terminal half-life and increased area under the curve (AUC) such that the chimeric protein described herein remains increased in vivo time period compared to wild type FVIII. In additional embodiments, the XTEN sequence used in the present invention improves pharmacokinetic properties, such as increasing the terminal half-life or increasing the area under the curve (AUC) such that the FVIII protein remains in vivo for an increased period of time compared to wild type FVIII.

One embodiment of the present invention is a FVIII/VWF fusion protein comprising a FVIII portion fused to an Fc fragment and a VWF portion fused to an Fc fragment, in which an XTEN sequence (e.g., AE288) is inserted into the FVIII portion, and in which the sequence XTEN contains less than 288 amino acids (eg AE144) and is inserted between the VWF portion and the Fc fragment. As described in the Examples, an XTEN insertion of less than 288 amino acids between the VWF portion and the Fc portion has a greater impact on the pharmacokinetics of the chimeric protein than an XTEN insertion of 288 amino acids between the VWF portion and the Fc portion. For example, insertion of an XTEN sequence of less than 288 amino acids between the VWF region and the Fc fragment in an FVIII/VWF fusion protein may increase the terminal half-life of the chimeric protein compared to XTEN having 288 amino acids. 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 by 30% compared to the insertion of the XTEN sequence, which has 288 amino acids. In one particular embodiment, the terminal half-life is increased by at least about 35% compared to the XTEN insert having 288 amino acids. Insertion of an XTEN sequence of less than 288 amino acids may also increase the AUC value of the chimeric protein. In some embodiments, the AUC value is increased by at least about 50%, at least about 100%, or at least about 200% compared to an XTEN insert having 288 amino acids. In a specific embodiment of the invention, the AUC value is increased by approximately 2 times. An insertion of an XTEN sequence of less than 288 amino acids may also reduce the clearance of the chimeric protein. For example, clearance may be reduced by at least about 5% at least about 10% at least about 15% at least about 20% at least

- 46 044115 at least about 25%, or at least about 30%, compared to the insertion sequence

XTEN, which has 288 amino acids. An XTEN sequence insert having less than 288 amino acids may increase the drug's mean retention time (MRT) and/or decrease the apparent volume of distribution at steady state (Vss) relative to an XTEN insert having 288 amino acids.

Various methods and assays can be used to determine the physical/chemical properties of proteins containing the XTEN sequence. Such methods include them, but are not limited by them, analytical centrifugation, EPR, ion-exchange EVE, exclusive EHH, faceted-phase VEZH, lighting scraping, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, ion-exchanging VEZH, ex-exclusive veh JH, ik, yamr , Raman spectroscopy, refractometry and UV/visible spectroscopy. Additional methods are described in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

Additional examples of XTEN sequences, which can be used in accordance with this invention, and disclosed in the patent publications of the USA No. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1 or 2011/0172146 A1, or international patent publications No. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2 or WO 20130122617 A1 .

II.C. Factor VIII (FVIII) protein.

As used herein, FVIII protein means functional FVIII polypeptide in its normal role in the coagulation process unless otherwise indicated. The term FVIII protein includes a functional fragment, variant, analog or derivative thereof that retains the function of full-length wild-type FVIII in the coagulation cascade. FVIII protein is used interchangeably with FVIII or FVIII polypeptide (or protein). Examples of FVIII functions include, but are not limited to, the ability to activate coagulation, the ability to act as a cofactor for factor GC, or the ability to form a tenase complex with factor IX in the presence of Ca ²⁺ and phospholipids, which factor X then converts to the activated form Xa. The FVIII protein can be human, porcine, canine, rat or mouse FVIII protein. In addition, comparison of human FVIII with FVIII from other species has revealed the presence of conserved residues that may be essential for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

A number of tests are available to assess the function of the blood coagulation system: activated partial thromboplastin time (aPTT), chromogenic analysis, rotational thromboelastometry, prothrombin time (PTT) (also used to determine MHO), fibrinogen determination (often by the Claus method), platelet count determination , platelet function test (often by PFA-100), thrombin time test, bleeding time test, mixed test (whether the abnormality is corrected if the patient's plasma is mixed with normal plasma), clotting factor test, antiphospholipid antibody test, D- test dimers, genetic tests (eg, factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), various platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, eg, ROTEM®) or determination Euglobulin lysis time (ELT).

The APTT test is a performance indicator measuring the efficiency of both intrinsic (also known as the intrinsic pathway of coagulation activation) and common coagulation cascades. This test is commonly used to measure the coagulating activity of commercially available recombinant coagulation factors such as FVIII or FIX. It is used in conjunction with the prothrombin time (PT) test, which measures the extrinsic pathway of activation of blood clotting.

Rotational thromboelastometry provides information on the entire kinetics of hemostasis: clotting time, clot formation, clot stability and lysis. Various parameters of thromboelastometry depend on the activity of the plasmatic coagulation system, platelet function, fibrinolysis, or many factors that influence these interactions. This analysis can provide a complete picture of secondary hemostasis.

The FVIII polypeptide and polynucleotide sequences are known, as are many other functional fragments, mutants and modified variants thereof. Examples of human FVIII sequences (full length) are given below.

Table 3. Amino acid sequence of full-length factor VIII (Full-length FVIII (FVIII signal peptide is underlined; heavy chain FVIII is double underlined; Domain B is in italics; and light chain FVIII is presented in plain text).

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Table 3

Signal peptide: (SEQ Ш NO: 64)

MQIELSTCFFLCLLRFCFS

Mature factor VIII (SEQ Ш NO: 65)*

ATRRYYLGAVELSWDYMOSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEF TDHLFNIAKPRPPWMGLLGPTIOAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGA EYDDOTSOREKEDDKVFPGGSHTYVWOVLKENGPMASDPLCLTYSYLSHVDLVKDLN SGLIGALLVCREGSLAKEKTOTLHKFILLF AVFDEGKSWHSETKNSLMODRDAASARA WPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS LEISPITFLTAOTLLMDLGOFLLFCHISSHOHDGMEAYVKVDSCPEEPOLRMKNNEEAED YDDDLTDSEMDVVRFDDDNSPSFIOIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPD DRSYKSO YLNNNGPORIGRKYKKVRFMAYTDETFKTREAIOHESGILGPLLYGEVGDTLLI IFKNOASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKS DPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDORGNOIMSDKRNVILFSVFDENRS WYLTENIORFLPNPAGVOLEDPEFOASNIMHS INGYVFDSLOLSVCLHEVAYWYILSIGA OTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMT ALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTTPEN DIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPS PGaids SLLIENSPSVWQNILESDTEFKKVrPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQK KEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQN FLSEKNKVWGKGEFTKDVGLKEMVFPSSRNLFLINLDNLHENNTHNQEKKIQEEIEKKETLI QENW PSIR PIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVG SLGTSATNSVrYKKVENrVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGS LLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQ EKS PEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVrWAKQGRTERLCSQNPPVLKRHQREI TRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDY GMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMV TFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPT KDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE TKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWY LLSMGSNENIHSIHFSGHVFTVRKKEEY KMALYNLYPGVFETVEMLPSKAGIWRVECLI GEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSIN AWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNST GTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMEL MGCDLNSSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMK VTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSL DPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

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Table 4

Nucleotide sequence encoding full-length FVIII (SEQ ID NO: 66)*

661 ATG CAAATAGAGC TCTCCACCTG

721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC

781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG

841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAGAC

901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT

961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA

1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC

1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT

1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC

1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA

1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC

1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA

1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC

1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA

1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT

- 49 044115

1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC

CAGGCGTCCT TGGAAATCTC

1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT

1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG

1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA

1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT

1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA

1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG

1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT

2041 GGC AT AC AC A GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT

2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC

2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG

2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT

2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT

2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG

2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA

2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA

2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA

- 50 044115

2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT

GATAGTTTGC AGTTGTCAGT

2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT

2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC

2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT

2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC

2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA

2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC

3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC

3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA

3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA

3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA

3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT

3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC

3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT

3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT

3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT

3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA

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3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA

AATGTATCGT CAACAGAGAG

3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC

3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC

3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG

3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG

3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC

3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC

4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT

4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT

4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAACAA

4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC

4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAAA

4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT

4381 AGTTTTGCCT C AGAT AC AT A CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTTCTT

4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA

4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC

4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA

- 52 044115

4621 GAAATATGCA TGC ACC AC AA GGATATCTCC TAATACAAGC

CAGCAGAATT TTGTCACGCA

4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA

4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC

4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC

4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC

4921 CATTGCAAAG GT ATS ATS AT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT

4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA

5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC

5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC

5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC

5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA

5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC

5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT

5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA

5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA

5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC

5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA

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5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA

5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG AT ASSAT ATS

5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG

5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA

5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA

5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG

6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA

6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT

6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC

6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA

6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC

6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG

6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG

6421 GTACTTCACT GAAAATTGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA

6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT

6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG

6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA

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6661 GG AGT AT AAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT

TTTGAGACAG TGGAAATGTT

6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT

GGCGAGCATC TACATGCTGG

6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC

6841 TGGACACATT AGAGATTTTC AG ATT AC AGC TTCAGGACAA TATGGACAGT GGGCCCCAAA

6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC

6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC

7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG

7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTTGGCAA

7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT

7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG

7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA

7321 GATTACTGCT TCATCCTACT TTACCATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG

7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG

7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA

7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA

7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC

7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA

7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA

7741 GGACCTCTAC ^The underlined nucleic acids encode a signal peptide

FVIII polypeptides include full-length FVIII, full-length FVIII without the methionine at the N-terminus, mature FVIII (without the signal sequence), mature FVIII with an additional methionine at the N-terminus, and/or FVIII with complete or partial removal of the B domain. In certain embodiments, FVIII variants include B domain deletions, whether partial or complete deletions.

The sequence of native mature human FVIII is shown in SEQ ID NO: 65. Native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are structurally related domains A, B are domains B, C1 and C2 are structurally related to domains C, and a1, a2 and a3 are acidic spacer regions. Referring to the primary amino acid position in SEQ ID NO:65, human FVIII domain A1 extends from Ala1 to about Arg336, spacer region a1 extends from about Met337 to about Val374, domain A2 extends from about Ala375 to about Tyr719, spacer domain a2 extends from about Glu720 to about Arg740, domain B extends from about Ser741 to about

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Arg1648, spacer domain a3 extends from about Glu1649 to about Arg1689, domain A3 extends from about Ser1690 to about Leu2025, domain C1 extends from about Gly2026 to about Asn2072, and domain C2 extends from about Ser2073 to Tyr2332. In addition to specific proteolytic cleavage sites, the designation of the location of the boundaries between FVIII domains and regions may differ in different literature sources. The boundaries presented herein are indicated as approximate by the use of the term approximate.

The human FVIII gene has been isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I, et al., Nature 312:330-337 (1984); Vehar, G A, et al., Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; and US Patent No. 4,757,006). The amino acid sequence of FVIII was predicted from cDNA, as shown in US patent No. 4,965,199. In addition, FVIII with the B domain partially or completely deleted is shown in US Patent Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B domain is replaced with the human Factor V B domain, as shown in US Pat. No. 5,004,803. The cDNA sequence encoding human factor VIII and the amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively, from US Application Publication No. 2005/0100990.

The sequence of porcine FVIII is published in Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). In addition, the complete porcine cDNA sequence obtained by PCR amplification of FVIII sequences from a porcine spleen cDNA library is described in Healey, J. F., et al., Blood 88:4209-4214 (1996). Hybrid FVIII (human/porcine) having substitutions of all domains, all subunits and specific amino acid sequences is described in US 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 a chimeric FVIII having porcine A1 and/or A2 domains replacing the corresponding human domains were described in WO 94/11503. US Patent No. 5,859,204, Lollar, J.S., also describes porcine cDNA and predicted amino acid sequences. US Patent No. 6,458,563 describes porcine FVIII with the B domain deleted.

US Patent No. 5,859,204, Lollar, J. S. describes functional FVIII mutants with reduced antigenicity and reduced immunoreactivity. US Patent No. 6,376,463, Lollar, J. S. describes FVIII mutants with reduced immunoreactivity. US Application Publication No. 2005/0100990 Saenko et al. describe functional mutations in the A2 domain of FVIII.

In one embodiment, the FVIII (or portion of the FVIII chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% be identical to the amino acid sequence of FVIII being amino acids 1-1438 of SEQ ID NO: 67 or amino acids 1-2332 of SEQ ID NO: 65 (without signal sequence), or the amino acid sequence of FVIII being amino acids 1-19 of SEQ ID NO: : 64, and amino acids 1-1438 of SEQ ID NO: 67, or amino acids 1-19 of SEQ ID NO: 64, and amino acids 1-2332 of SEQ ID NO: 65 (with a signal sequence) in which FVIII has clotting activity , for example, activates factor IX as a cofactor to convert factor X to activated factor X. FVIII (or the FVIII portion of the chimeric protein) may be identical to the amino acid sequence of FVIII being amino acids 1-1438 of SEQ ID NO: 67 or amino acids 12332 of SEQ ID NO: 65 (no signal sequence). FVIII may further include a signal sequence.

Domain B of FVIII in this context is the same domain B known in the art, which is determined by the intrinsic amino acid sequence identity and proteolytic cleavage sites, for example, 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 sequence A3-C1-C2 includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is commonly referred to as the a3 acidic region. The locations of the boundaries of all domains, including the B domains of porcine, mouse and canine FVIII are also known in the art. In one embodiment, the B domain is removed from FVIII (factor VIII with B domain deleted or FVIII BDD). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequence as the factor VIII portion of the sequence in table. 5. (The heavy chain FVIII BDD is double underlined; the B domain is italicized; and the light chain FVIII BDD is presented in plain text). Nucleotide sequence encoding table. 6 (SEQ ID NO: 68) is presented in table. 6.

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Table 5

Amino acid sequence of factor VIII with B domain deleted (BDD FVIII).

BDD FVIII (SEQ ID NO: 67)

ATRRYYLGAVELSWDYMOSDLGEL·PVDARFPPRVPKSFPFNTSVVYKKTLFVEF TDHLFNIAKPRPPWMGLLGPTIOAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGA EYDDOTSOREKEDDKVFPGGSHTYVWOVLKENGPMASDPLCLTYSYLSHVDLVKDLN SGLIGALLVCREGSLAKEKTOTLHKFILL FAVFDEGKSWHSETKNSLMODRDAASARA WPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHROAS LEISPITFLTAOTLLMDLGOFLLFCHISSHOHDGMEAYVKVDSCPEEPOLRMKNNEEAED YDDDLTDSEMDVVRFDDDNSPSFIOIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPD DRSYK SOYLNNNGPORIGRKYKKVRFMAYTDETFKTREAIOHESGILGPLLYGEVGDTLIJ IFKNOASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKS DPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDORGNOIMSDKRNVILFSVFDENRS WYLTENIORFLPNPAGVOLEDPEFOASNIM HSINGYVFDSLOLSVCLHEVAYWYILSIGA OTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMT ALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSONPPVLKRHOREITRTTLOSD QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS SSPHV LRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFR NQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKA WAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE NMERNCRAPCNIQ MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGM STLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQ FIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSSMPLGMESKAISDAQIT ASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPV VNSLDPPLLTRYL RIHPQSWVHQIALRMEVLGCEAQDLY

Table 6 Nucleotide sequence coding

BDD FVIII (SEQ ID NO: 68)*

661 A TGCAAATAGA GCTCTCCACC TGCTTCTTTC

721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC

781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC

841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG

901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC

961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG

- 57 044115

1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG

1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG

1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC

1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC

1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT

1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA

1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC

1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT

1501 CAGTCTATTG GCATGTGATT GGAATGGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG

1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA

1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA

1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG

1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG

1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC

1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG

1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT

1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA

- 58 044115

2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA

ATCAGGAATC TTGGGACCTT

2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC

2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC

2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA

2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT

2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC

2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA

2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC

2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA

2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTTGC

2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT

2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC

2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT

2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG

2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA

2941 GTAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC

3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG

- 59 044115

3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT

TTATGATGAG GATGAAAATC

3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT

TATTGCTGCA GTGGAGAGGC

3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA

3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC

3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG

3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCTTCT

3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT

3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA

3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG

3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG

3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA

3721 CCAAAAGCTG GTACTTCACT GAAAATTGG AAAGAAACTG CAGGGCTCCC TGCAATATCC

3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA

3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA

3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC

3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG

4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC

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4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA

TAAGTGTCAG ACTCCCCTGG

4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC

TTCAGGACAA TATGGACAGT

4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG

4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA

4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA

4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT

4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG

4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT

4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT

4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT

4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC

4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC

4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC

4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA

4921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC

4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT

5041 GCGAGGCACA GGACCTCTAC *Underlined nucleic acids encode a signal peptide

FVIII with the B domain deleted may have complete or partial deletions, as disclosed in US 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, 6,048.7 20, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112 and 6,458,563. In some embodiments, the B domain deleted FVIII sequence of the present invention comprises any of the deletions described in col. 4, line 4 to count. 5, line 28 and examples 1-5 of US Patent No. 6,316,226 (also in US Patent 6,346,513). In another embodiment, Factor VIII with B domain deleted is S743/Q1638 Factor VIII with B domain deleted (SQ BDD FVIII) (e.g., Factor VIII with a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII having amino acids 1-743 and amino acids 1638-2332 from SEQ ID NO: 65, i.e. SEQ ID NO: 67). In some embodiments, the B domain deleted FVIII of the present invention contains the deletion described in col. 2, lines 26-51 and examples 5-8 of US Patent No. 5,789,203 (also US 6,060,447, US 5,595,886 and US 6,228,620). In some embodiments, the B domain deleted FVIII of the present invention contains the deletion described in col. 1, lines 25 to count. 2, line 40 of US Pat. No. 5,972,885; Col. 6, lines 1-22 and example 1 of US Patent No. 6,048,720; count 2, lines 17-46 of US Patent No. 5,543,502; count 4, line 22 to count. 5, line 36 of US Patent No. 5,171,844; count 2, lines 55-68, figure 2 and example 1 of US patent No. 5,112,950; count 2, line 2 to count. 19, line 21 and table 2 of US Patent No. 4,868,112; count 2, line 1 to count. 3, line 19, count. 3, line 40 to count. 4,

- 61 044115 line 67, count. 7, line 43 to count. 8, line 26 and count. 11, line 5 to count. 13, line 39 of US Pat. No. 7,041,635; or count. 4, lines 25-53, US Patent No. 6,458,563. In some embodiments, the B domain deleted FVIII has a large portion of the B domain deleted but still contains the amino terminal B domain sequences that are required for proteolytic processing of the primary translation product into two polypeptide chains in vivo, as described in WO 91/09122. In some embodiments, the B domain deleted FVIII is created with amino acids 747-1638 deleted, i.e. almost complete removal of the B domain. Hoeben R.C., et al., J. Biol. Chem. 265 (13): 7318-7323 (1990). FVIII with the B domain deleted may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 from FVIII. Meulien P., et al., Protein Eng. 2(4): 301-6 (1988). Additional B domain deletions that are part of the present invention include: deletions of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A (1986) 83, 5939-5942) ), 797 to 1562 (Eaton, et al., Biochemistry (1986) 25:8343-8347)), 741 to 1646 (Kaufman (PCT Application Published 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) No. 82:16-25, EP 295597)). In another embodiment, the FVIII BDD includes an FVIII polypeptide containing B domain fragments that retain one or more N-linked glycosylation sites, such as residues 757, 784, 828, 900, 963, or, in some cases, 943, which correspond to amino acid sequence of the full-length FVIII sequence. Examples of B domain fragments include 226 amino acids or 163 amino acids of B domain, as described in Miao, H. Z., et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, S.W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011) (ie, the first 226 amino acids or 163 amino acids of the B domain are retained). In yet other embodiments, BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve BDD FVIII protein expression. See Miao, H. Z., et al., Blood 103(a): 3412-3419 (2004). In yet other embodiments, the FVIII BDD includes a FVIII polypeptide containing a portion of the B domain but lacking one or more furin cleavage sites (eg, Arg1313 and Arg 1648). See Pipe, S. W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011). Each of the above deletions can be made in any FVIII sequence.

In some embodiments, FVIII has a partial B domain. In some embodiments, the FVIII protein with a partial B domain is FVIII 198. FVIII 198 is a partial B domain containing a single-chain FVIIIFc molecule-226N6. The number 226 denotes the N-terminal 226 amino acids of the FVIII B domain, and N6 denotes the six Nglycosylation sites in the B domain.

In one embodiment, FVIII is cleaved immediately after arginine at amino acid 1648 (in full-length Factor VIII or SEQ ID NO: 65), amino acid 754 (in S743/Q1638 domain B deleted Factor VIII or SEQ ID NO: 67), or a corresponding residue arginine (in other variants), which leads to the formation of a heavy chain and a light chain. In other embodiments, FVIII comprises a heavy chain and a light chain that are linked or joined by a metal ion-mediated non-covalent bond.

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

FVIII can additionally be cleaved by thrombin and then activated as FVIIIa, serving as a cofactor for activated factor IX (FIXa). And activated FIX, together with activated FVIII, forms the Xase complex and converts factor X into activated factor X (FXa). To activate, FVIII is cleaved by thrombin after three arginine residues - amino acids 372, 740 and 1689 (corresponding to amino acids 372, 740 and 795 in the FVIII sequence with the B domain removed) as a result of the cleavage, FVIIIa is formed, having chains A1 - 50 kDa, A2 - 43 kDa and A3-C1-C2 - 73 kDa. In one embodiment, the FVIII protein useful in the present invention is inactive FVIII. In another embodiment of the invention, the FVIII protein is activated FVIII.

The protein having the FVIII polypeptide linked or linked to the VWF protein may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100 of the sequence % identical to the sequence of SEQ ID NO: 65 or 67, in which the sequence has FVIII coagulation activity, for example, activating factor IX as a cofactor for converting factor X to activated factor X (FXa).

As used herein, hybrid or chimeric polypeptides and proteins include the combination of a first polypeptide chain, e.g., VWF protein fused to an XTEN sequence of less than 288 amino acids and a first IG constant region or portion thereof, and a second polypeptide chain, e.g., FVIII protein fused to the second IG constant region or part thereof, thus forming a heterodimer. In one embodiment of the invention, the first polypeptide and the second polypeptide in the hybrid are connected to each other through protein-protein interactions, such as charge-charge or hydrophobic interactions. In another embodiment, the first polypeptide contains a VWF-XTEN-Fc fusion protein and the second polypeptide contains a FVIII-Fc fusion protein, forming a hybrid heterodimer in which XTEN contains less than 288 amino acids. In other embodiments, the first polypeptide contains a VWF-XTEN-Fc fusion protein and the second polypeptide contains a FVIII(X)-Fc fusion protein, forming a hybrid heterodimer in which XTEN contains less than 288 amino acids. The first polypeptide and the second polypeptide may be joined by a covalent bond, such as a disulfide bond, between the first Fc moiety and the second Fc moiety. The first polypeptide and the second polypeptide may further be linked to each other through binding between the VWF moiety and the FVIII protein.

A FVIII protein useful in the present invention may include FVIII having one or more additional XTEN sequences that do not affect the clotting activity of FVIII. Such XTEN sequences can be fused to the C-terminus or N-terminus of the FVIII protein or inserted between one or more of two amino acid residues in the FVIII protein, the insertions not affecting the folding activity or function of FVIII. In one embodiment, the inserts improve the pharmacokinetic properties of the FVIII protein (eg, half-life). In another embodiment of the invention, the inserts may be multiple, for example, more than two, three, four, five, six, seven, eight, nine or ten inserts. Examples of embedding sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, 15, or any combination thereof.

The FVIII protein associated with one or more XTEN sequences may be represented as FVIII(X2) or FVIII( _a ^ _b )-X-FVIII( _c ^ _d ), wherein FVIII( _a ^ _b ) contains, consists essentially of, or consists of the first part of the FVIII protein from amino acid residue a to amino acid residue b; X2 contains, essentially consists of, or consists of one or more XTEN sequences, FVIII(c^d) contains, essentially consists of, or consists of a second portion of the FVIII protein from amino acid residue c to amino acid residue d;

a is the N-terminal amino acid residue of the first part of the FVIII protein, b is the C-terminal amino acid residue of the first part of the FVIII protein, but is also the N-terminal amino acid residue of the two amino acid residues of the insertion site into which the XTEN sequence is inserted, c is the N-terminal amino acid residue of the second part of the FVIII protein, but is also the C-terminal amino acid residue of the two amino acids of the insertion site into which the XTEN sequence is inserted, and d is the C-terminal amino acid residue of the FVIII protein, and wherein the first portion of the FVIII protein and the second portion of the FVIII protein are not identical to each other and together are of sufficient length for the FVIII protein to have the clotting activity of FVIII.

In one embodiment, the first portion of the FVIII protein and the second portion 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 deleted), for example, the N-terminal portion and C- end part accordingly. In some embodiments, the first portion of the FVIII protein comprises an A1 domain and an A2 domain of the FVIII protein. The second portion of the FVIII protein contains an A3 domain, a C1 domain, and, in some cases, a C2 domain. In still other embodiments, the first portion of the FVIII protein comprises an A1 domain and an A2 domain, and the second portion of the FVIII protein comprises a portion of a B domain, an A3 domain, a C1 domain, and, in some cases, a C2 domain. In still other embodiments, the first portion of the FVIII protein includes an A1 domain, an A2 domain, and a portion of the B domain of the FVIII protein, and the second portion of the FVIII protein comprises an A3 domain, a C1 domain, and, in some cases, a C2 domain. In still other embodiments, the first portion of the FVIII protein comprises an A1 domain, an A2 domain, and a first portion of the B domain of the FVIII protein. The second part of the FVIII protein contains the second part of the B domain, the A3 domain, the C1 domain and, in some cases, the C2 domain. In some embodiments, the two amino acids (b and c) may represent one or more insertion sites for the amino acid residues listed in Table 1. 7, 8, 9, 10, 11, 12, 13, 14 and 15. For example, b may be an amino acid residue located immediately upstream of the site into which one or more XTEN sequences are inserted or linked, and c may be an amino acid residue located immediately below the site at which one or more XTEN sequences are inserted or linked. In some embodiments of the invention, a is the first

- 63 044115 is the amino acid sequence of the FVIII protein, and d is the last amino acid sequence of the FVIII protein. For example, FVIII,( _a ^b) may be an amino acid sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1-745 of SEQ ID NO: 67 (amino acid sequence of FVIII with B domain deleted) or SEQ ID NO: 65 (full-length FVIII), and FVIII( _C ^ _d ) may be amino acids 746-1438 of SEQ ID NO: 67 or amino acids 1641-2332 of SEQ ID NO: 65, respectively.

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

An FVIII protein in which an XTEN sequence is inserted immediately downstream of one or more amino acids (eg, one or more XTEN insertion sites) in the FVIII protein or linked to the C-terminus or N-terminus retains FVIII activity after binding or insertion of the XTEN sequence. The XTEN sequence may be inserted into the FVIII protein one or more times once, twice, three times, four times, five times, or six times such that the insertions do not affect FVIII activity (i.e., the FVIII protein still retains coagulation property).

An FVIII protein useful in the present invention may be linked to one or more XTEN polypeptides at the N-terminus or C-terminus of the FVIII protein via an optional linker or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites ) in the FVIII protein via one or more optional linkers. In one embodiment, the two amino acid residues into which the XTEN sequence is inserted, or the amino acid residue to which the XTEN sequence binds, correspond to two or one amino acid residue from SEQ ID NO: 65 (full-length mature FVIII), selected from the group consisting of residues , presented in table. 7, table. 8, table. 9 and in table. 10, and any combinations thereof.

In other embodiments, at least one XTEN sequence is inserted into any one or more XTEN insertion sites described herein, or any combination thereof. In one aspect, at least one XTEN sequence is inserted into any one or more XTEN insertion sites described in table. 7.

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Table 7

Typical XTEN embed sites

No. Embedding point XTEN* Remaining embedding FVIII BDD underlying sequence b Domain FVIII 1 0 (N-end) ATR Al 2 3 R RYY Al 3 17 M QSD Al 4 18 Q SDL Al 5 22 G ELP Al 6 24 L PVD Al 7 26 V DAR Al 8 28 A RFP Al 9 32 R RVP Al 10 38 F PFN Al eleven 40 F NTS Al 12 41 N TSV Al 13 60 N I.A.K. Al 14 61 I ACRE Al 15 65 R PPW Al 16 81 Y DTV Al 17 111 G AEY Al 18 116 D QTS Al 19 119 S QRE Al 20 120 Q REK Al

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21 128 V FPG Al 22 129 F PGG Al 23 130 P G.G.S. Al 24 182 G SLA Al 25 185 A KEK Al 26 188 TO TQT Al 27 205 G KSW Al 28 210 S ETK Al 29 211 E TKN Al thirty 216 L MQD Al 31 220 R DAA Al 32 222 A A.S.A. Al 33 223 A SAR Al 34 224 S ARA Al 35 230 TO MHT Al 36 243 P GLI Al 37 244 G LIG Al 38 250 R KSV Al 39 318 D G.M.E. Al 40 333 P QLR Al 42 334 Q LRM Al 43 336 R MKN al 44 339 N NEE al 45 345 D YDD al 46 357 V VRF al 47 367 s FIQ al 48 370 s RPY al 49 375 A KKH A2 50 376 TO China A2 51 378 H PKT A2

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52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2 55 409 S QYL A2 56 416 P QRI A2 57 434 E TFK A2 58 438 T REA A2 59 441 A IQH A2 60 442 I QHE A2 61 463 I IFK A2 62 487 Y SRR A2 63 490 R LPK A2 64 492 P KGV A2 65 493 TO GVK A2 66 494 G VKH A2 67 500 D FPI A2 68 506 G EIF A2 69 518 E DGP A2 70 556 TO ESV A2 71 565 Q IMS A2 72 566 I MSD A2 73 598 P AGV A2 74 599 A G.V.Q. A2 75 603 L EDP A2 76 616 S ING A2 77 686 G L.W.I. A2 78 713 TO NTG A2 79 719 Y EDS A2 80 730 L LSK A2 81 733 TO N.N.A. A2

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82 745 Ν PPV** IN 83 1640 Ρ PVL IN 84 1652 R TTL IN 85 1656 Q SDQ AZ 86 1685 Ν QSP AZ 87 1711 M SSS AZ 88 1713 S SPH AZ 89 1720 Ν R.A.Q. AZ 90 1724 S GSV AZ 91 1725 G SVP AZ 92 1726 S V.P.Q. AZ 93 1741 G S.F.T. AZ 94 1744 Τ QPL AZ 95 1749 R GEL AZ 96 1773 V TFR AZ 97 1792 Υ EED AZ 98 1793 Ε EDQ AZ 99 1796 Q RQG AZ 100 1798 Q G.A.E. AZ 101 1799 G ΑΕΡ AZ 102 1802 Ρ RKN AZ 103 1803 R KNF AZ 104 1807 V ΚΡΝ AZ 105 1808 To ΡΝΕ AZ 106 1827 To DEF AZ 107 1844 Ε KDV AZ 108 1861 Ν TLN AZ 109 1863 L ΝΡΑ AZ BY 1896 Ε R.N.C. AZ 111 1900 R APC AZ

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112 1904 N IQM A3 113 1905 I QME A3 114 1910 R TFK A3 115 1920 A ING A3 116 1937 D QRI A3 117 1981 G VFE A3 118 2019 N KCQ A3 119 2020 TO C.Q.T. Cl 120 2044 G QWA Cl 121 2068 F SWI Cl 122 2073 V DLL Cl 123 2090 R QKF Cl 124 2092 TO FSS Cl 125 2093 F SSL Cl 126 2111 TO W.Q.T. Cl 127 2115 Y RGN Cl 128 2120 T GTL Cl 129 2125 V FFG Cl 130 2171 L N.S.C. Cl 131 2173 S CSM C2 132 2188 A QIT C2 133 2223 V NNP C2 134 2224 N NPK C2 135 2227 TO EWL C2 136 2268 G HQW C2 137 2277 N GKV C2 138 2278 G KVK C2 139 2290 F TPV C2 140 2332 Y C-terminus FVIII C.T.

^Indicates the XTEN insertion point based on the number of amino acids of mature full-length human FVIII, in which the insertion may be on either the N- or C-terminal side of the indicated amino acid

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

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Table 8

Typical XTEN installation ranges

No. XTEN installation point Remaining embedding FVIII BDD underlying sequence Domain FVIII Distance from rest of embedding * 9 32 R RVP Al -3,+6 31 220 R DAA Al - 34 224 S ARA Al +5 43 336 R MKN al -1,+6 44 339 N ΝΕΕ al -4, +5 52 399 V LAP A2 -6, +3 56 416 R QRI A2 +6 75 603 L EDP A2 _6, +6 85 1656 Q SDQ IN -3,+6 87 1711 m sss A3 -6,+1 91 1725 G SVP A3 +6 113 1905 I QME A3 +6 114 1910 R TFK A3 -5,+6

^Distance from insertion residue means the relative number of amino acids distant from the N-terminus (negative numbers) or C-terminus (positive numbers) of the specified insertion residue (residue 0) at which insertion can occur. The designation -x refers to an insertion site that is x amino acids distant from the N-terminal side of the indicated insertion residue. Likewise, the designation +x refers to an insertion site that is x amino acids distant from the C-terminal side of the specified insertion residue.

For example, -1, +2 indicates that the insertion is made at the N-terminus or C-terminus of amino acid residues designated -1, 0, +1 or +2.

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

Table 9

Typical embedding sites or XTEN ranges

No. Embedding point range XTEN* Remaining embedding Domain FVIII 3 18-32 Q Al 8 40 F Al 18 211-224 E Al 27 336-403 R Al, A2 43 599 A A2 47 745-1640 N IN 50 1656-1728 Q B, aZ, AZ 57 1796-1804 R AZ 65 1900-1912 R AZ 81 2171-2332 L C1, C2

*denotes the range of insertion sites numbered according to the number of amino acids of mature human FVIII

In yet other embodiments, one or more XTENs are inserted into the B domains of FVIII. In one example, XTEN is inserted between amino acids 740-1640 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 740-1640 is in some cases absent. In another example, XTEN is inserted between amino acids 741-1690, corresponding to

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SEQ ID NO: 65, in which the FVIII sequence between amino acids 740-1690 is in some cases missing. In other examples, XTEN is inserted between amino acids 741-1648 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 741-1648 is in some cases absent. In yet other examples, XTEN is inserted between amino acids 743-1638 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 743-1638 is in some cases absent. In still other examples, XTEN is inserted between amino acids 7451656 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 7451656 is in some cases absent. In some examples, XTEN is inserted between amino acids 745-1657 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 745-1657 is in some cases absent. In certain examples, XTEN is inserted between amino acids 745-1667 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 745-1667 is in some cases absent. In still other examples, XTEN is inserted between amino acids 745-1686 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 745-1686 is in some cases absent. In some other examples, XTEN is inserted between amino acids 747-1642 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 747-1642 is in some cases absent. In still other examples, XTEN is inserted between amino acids 751-1667 corresponding to SEQ ID NO: 65, in which the FVIII sequence between amino acids 751-1667 is in some cases absent.

In some embodiments, one or more XTENs are inserted into one or more amino acids located immediately downstream of the amino acid of the insertion site from the group consisting of amino acid residues in table. 10.

Table 10

FVIII XTEN embedding sites and design notations

Design number Domain No. of the overlying residue.* No. the underlying remainder. * Overlying sequence Below sequence F8X-1 A1 3 4 ATR RYY F8X-2 A1 18 19 YMQ SDL F8X-3 A1 22 23 DLG ELP

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F8X-4 Al 26 27 LPV DAR F8X-5 Al 40 41 FPF NTS F8X-6 Al 60 61 LFN I.A.K. F8X-7 Al 116 117 YDD QTS F8X-8 Al 130 131 VFP G.G.S. F8X-9 Al 188 189 KEK TQT F8X-10 Al 216 217 NSL MQD F8X-11 Al 230 231 W.P.K. MHT F8X-12 Al 333 334 EEP QLR F8X-13 A2 375 376 SVA KKH F8X-14 A2 403 404 APD DRS F8X-15 A2 442 443 EAI QHE F8X-16 A2 490 491 RRL PKG F8X-17 A2 518 519 TVE DGP F8X-18 A2 599 600 NPA G.V.Q. F8X-19 A2 713 714 CDK NTG F8X-20 BD 745 746 SQN PPV F8X-21 BD 745 746 SQN PPV F8X-22 BD** 745 746 SQN PPV F8X-23 A3 1720 1721 APT KDE F8X-24 A3 1796 1797 EDQ RQG F8X-25 A3 1802 1803 AEP RKN F8X-26 A3 1827 1828 PTK DEF F8X-27 A3 1861 1862 HTN TLN F8X-28 A3 1896 1897 NME R.N.C. F8X-29 A3 1900 1901 NCR APC F8X-3O A3 1904 1905 PCN IQM F8X-31 A3 1937 1938 A.Q.D. QRI F8X-32 Cl 2019 2020 YSN KCQ F8X-33 Cl 2068 2069 EPF SWI F8X-34 Cl 2111 2112 GKK W.Q.T. F8X-35 Cl 2120 2121 NST GTL F8X-36 C2 2171 2172 C.D.L. N.S.C. F8X-37 C2 2188 2189 S.D.A. QIT

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F8X-38 C2 2227 2228 NPK EWL F8X-39 C2 2277 2278 FQN GKV F8X-40 C.T. 2332 N.A. DLY N.A. F8X-41 C.T. 2332 N.A. DLY N.A. F8X-42 Al 3 4 ATR ATR pSDOOOl A2 403 404 pSD0002 A2 599 600 pSD0021 N-terminus 0 1 pSD0022 Al 32 33 pSD0023 Al 65 66 pSD0024 Al 81 82 pSD0025 Al 119 120 pSD0026 Al 211 212 pSD0027 Al 220 221 pSD0028 Al 224 225 pSD0029 Al 336 337 pSD0030 Al 339 340 pSD0031 A2 378 379 pSD0032 A2 399 400 pSD0033 A2 409 410 pSD0034 A2 416 417 pSD0035 A2 487 488 pSD0036 A2 494 495 pSD0037 A2 500 501 pSD0038 A2 603 604 pSD0039 A3 1656 1657 pSD0040 A3 1711 1712 pSD0041 A3 1725 1726 pSD0042 A3 1749 1750 pSD0043 A3 1905 1906 pSD0044 A3 1910 1911 pDS0062 A3 1900 1901

*Indicates the number of amino acids of the mature FVIII protein

In one embodiment, one or more XTEN insertion sites are located within one or more surface-exposed, flexible loop structures of the FVIII protein (eg, an insertion-accessible loop). For example, at least one XTEN sequence may be inserted into each FVIII domain A containing at least two insertable loops into which at least one XTEN polypeptide may be inserted without eliminating the procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to manifest in vivo or in vitro in a host cell. Insertion-accessible loops are regions that allow the insertion of at least one XTEN sequence, characterized by, among other properties, high surface or solvent accessibility and conformational flexibility. Domain A1 contains a region accessible for embedding loop-1 (A1-1) and a region accessible for embedding loop-2 (A1-2); domain A2 contains a region accessible for embedding loop-1 (A2-1) and a region accessible for embedding loop-1 (A2-1). 2 (A2-2) region, domain A3 contains a loop-1 (A3-1) embeddable region and a loop-2 (A3-2) embeddable region.

In one aspect, the first insertion-accessible loop in the FVIII A1 domain (A1-1) is located

- 73 044115 between beta strand 1 and beta strand 2, and the second insertable loop in the FVIII A2 domain (A1-2) is located between beta strand 11 and beta strand 12. The first insertable loop in the A2 FVIII domain (A2-1) is located between beta strand 22 and beta strand 23, and the second insertable loop in the A2 domain of FVIII (A2-2) is located between beta strand 32 and beta strand 33. The first insertable loop in the FVIII A3 domain (A3-1) is located between beta strand 38 and beta strand 39, and the second insertable loop in the FVIII A3 domain (A3-2) is located between beta strand 45 and beta strand 46. In some aspects a flexible, movable surface structure containing A1-1 corresponds to the region in native mature human FVIII from about amino acid 15 to about amino acid 45 in SEQ ID NO: 65, for example, from about amino acid 18 to about amino acid 41 in SEQ ID NO: 65. In other aspects, the flexible structure containing A1-2 corresponds to the region in native mature human FVIII from about amino acid 201 to about amino acid 232 in SEQ ID NO: 65, for example, from about amino acid 218 to about amino acid 229 in SEQ ID NO: 65. In yet other aspects, the flexible, movable surface structure containing A2-1 corresponds to the region in native mature human FVIII from about amino acid 395 to about amino acid 421 in SEQ ID NO: 65, for example, from about amino acid 397 to about amino acid 418 in SEQ ID NO: : 65. In yet other embodiments, the flexible, movable surface structure containing A2-2 corresponds to the region in native mature human FVIII from about amino acid 577 to about amino acid 635 in SEQ ID NO: 65, for example, from about amino acid 595 to about amino acid 607 in SEQ ID NO: 65. In some aspects, the flexible surface-movable structure containing A3-1 corresponds to the region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 in SEQ ID NO: 65, for example, from about amino acid 1711 to about amino acid 1725 in SEQ ID NO: 65. In yet other aspects, the flexible surface-movable structure containing A3-2 corresponds to the region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 in SEQ ID NO: 65, for example , from about amino acid 1899 to about amino acid 1911 in SEQ ID NO: 65.

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

In some embodiments, one or more insertion sites for one or more XTEN insertions are located immediately downstream of one or more amino acids corresponding to full-length mature human FVIII 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 combinations thereof .

In one embodiment, a FVIII protein useful in the present invention contains two XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, and a second XTEN inserted into a second XTEN insertion site. Non-limiting examples of the first XTEN insertion site and the second XTEN insertion site are listed in Table. eleven.

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Table 11

Typical embedding sites for two XTENs

Embedding Embedding site 745 26 40 1 Domain IN A1 A1 Embedding Embedding site 2332 403 403 2 Domain ST A2 A2 18 A1 403 A2 26 A1 599 A2 40 A1 599 A2 18 A1 599 A2 1720 AZ 1900 AZ 1725 AZ 1900 AZ 1711 AZ 1905 AZ 1720 AZ 1905 AZ 1725 AZ 1905 AZ 1656 AZ 26 A1 1656 AZ 18 A1 1656 AZ 40 A1 1656 AZ 399 A2 1656 AZ 403 A2 1656 AZ 1725 AZ 1656 AZ 1720 AZ 1900 AZ 18 A1 1900 AZ 26 A1 1900 AZ 40 A1 1905 AZ 18 A1 1905 AZ 40 A1 1905 AZ 26 A1 1910 AZ 26 A1 18 A1 399 A2 26 A1 399 A2 40 A1 399 A2 18 A1 403 A2 1656 AZ 1900 AZ 1656 AZ 1905 AZ 1711 AZ 40 A1 1711 AZ 26 A1 1720 AZ 26 A1

- 75 044115

1720 AZ 40 A1 1720 AZ 18 A1 1725 AZ 26 A1 1725 AZ 40 A1 1725 AZ 18 A1 1720 AZ 403 A2 1720 AZ 399 A2 1711 AZ 403 A2 1720 AZ 403 A2 1725 AZ 403 A2 1725 AZ 399 A2 1711 AZ 403 A2 1900 AZ 399 A2 1900 AZ 403 A2 1905 AZ 403 A2 1905 AZ 399 A2 1910 AZ 403 A2

The two XTENs embedded in or associated with the FVIII protein may be the same or different. In some embodiments, a FVIII protein useful in the present invention contains two XTEN sequences inserted into the FVIII protein, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 65, and a second XTEN sequence inserted immediately downstream of the amino acid 2332 corresponding to SEQ ID NO: 65 (C-terminus). In another embodiment, the first XTEN sequence is inserted immediately below amino acid 18, 26, 40, 1656, or 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately below amino acid 403 corresponding to SEQ ID NO: 65. In yet other embodiments implementation of the invention, the first XTEN sequence is inserted immediately below amino acid 18, 26 or 40 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately below amino acid 599 corresponding to SEQ ID NO: 65. In yet other embodiments of the invention, the first XTEN sequence is inserted directly below amino acid 1656 corresponding to SEQ ID NO: 65, and a second XTEN sequence is inserted immediately below amino acid 18, 26, 40, 399, 403, 1725, 1720, 1900, 1905, or 2332 corresponding to SEQ ID NO: 65. In certain embodiments of the invention, a first XTEN sequence is inserted immediately below amino acid 1900 corresponding to SEQ ID NO: 65, and a second XTEN sequence is inserted immediately below amino acid 18, 26, or 40 corresponding to SEQ ID NO: 65. In some embodiments of the invention, the first XTEN sequence is inserted immediately below the amino acid 18, 26, or 40, corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately below amino acid 399, corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately below amino acid 1720, corresponding to SEQ ID NO: 65 and the second XTEN sequence is inserted immediately below amino acid 18, 26, or 40 corresponding to SEQ ID NO: 65. In yet other embodiments of the invention, the first XTEN sequence is inserted immediately below amino acid 1720 corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted directly below amino acid 18, corresponding to SEQ ID NO: 65. In a particular embodiment of the invention, the FVIII protein containing the XTEN sequence, the first XTEN sequence is inserted immediately below amino acid 745, corresponding to SEQ ID NO: 65, and the second XTEN sequence is inserted immediately below amino acid 2332, corresponding to SEQ ID NO: 65. SEQ ID NO: 65, wherein the FVIII protein further comprises a deletion from amino acid 745 corresponding to SEQ ID NO: 65 to amino acid 1685 corresponding to SEQ ID NO: 65, a mutation or substitution of amino acid 1680 corresponding to SEQ ID NO: 65, for example, Y1680F a mutation or substitution of amino acid 1648 corresponding to SEQ ID NO: 65, for example, R1648A, or at least two mutations or substitutions of amino acid 1648 corresponding to SEQ ID NO: 65, for example, R1648A, and amino acid 1680 corresponding to SEQ ID NO: 65, for example , Y1680F. In a particular embodiment of the invention, the FVIII protein contains two XTEN sequences, a first XTEN sequence inserted immediately below amino acid 1656 corresponding to SEQ ID NO: 65, and a second XTEN sequence inserted immediately below amino acid 2332 corresponding to SEQ ID NO: 65, in which the FVIII protein is additionally contains a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 65.

-76044115

In certain embodiments, the FVIII protein contains three XTEN sequences, a first XTEN sequence is inserted into a first XTEN insertion site, a second XTEN sequence is inserted into a second XTEN insertion site, and a third XTEN sequence is inserted into a third XTEN insertion site. The first, second or third XTEN sequences may be the same or different. The first, second, and third embedding sites may be selected from the group including any of the embedding sites disclosed herein. In some embodiments, the FVIII protein containing three XTEN sequences may additionally carry a mutation or substitution, for example, amino acid 1648 corresponding to SEQ ID NO: 65, for example, R1648A. For example, non-limiting examples of first, second and third XTEN embedding sites are listed in Table. 12.

Table 12

Typical embedding sites for three XTENs

Embedding 1 Embedding 2 Embedding 3 Embed site Domain Embed site Domain Embed site Domain 26 A1 403 A2 1656 AZ 26 A1 403 A2 1720 AZ 26 A1 403 A2 1900 AZ 26 A1 1656 AZ 1720 AZ 26 A1 1656 AZ 1900 AZ 26 A1 1720 AZ 1900 AZ 403 A2 1656 AZ 1720 AZ 403 A2 1656 AZ 1900 AZ 403 A2 1720 AZ 1900 AZ 1656 AZ 1720 AZ 1900 AZ 745 IN 1900 2332 18 A1 745 IN 2332 ST 26 A1 745 IN 2332 st 40 A1 745 V 2332 st 18 A1 745 V 2332 st 40 A1 745 V 2332 st 403 A2 745 V 2332 st 399 A2 745 V 2332 st 1725 AZ 745 V 2332 st 1720 AZ 745 V 2332 st 1711 AZ 745 V 2332 st 1900 AZ 745 V 2332 st 1905 AZ 745 V 2332 st 1910 AZ 745 V 2332 st

In some embodiments, the FVIII protein contains three XTEN sequences, the first XTEN sequence is inserted immediately below amino acid 26 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted immediately below amino acid 403 corresponding to SEQ ID NO: 65, and the third XTEN sequence is inserted directly below amino acid 1656, 1720, or 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately below amino acid 26 corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted immediately below amino acid 1656 corresponding to SEQ ID NO: 65, and a third XTEN sequence is inserted immediately downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 65. In still other variants

- 77 044115 an embodiment of the invention, a first XTEN sequence is inserted immediately below amino acid 26 corresponding to SEQ ID NO: 65, a second XTEN sequence is inserted immediately below amino acid 1720 corresponding to SEQ ID NO: 65, and a third XTEN sequence is inserted immediately below amino acid 1900 corresponding to SEQ ID NO: 65. In yet other embodiments, a first XTEN sequence is inserted immediately below amino acid 403 corresponding to SEQ ID NO: 65, a second XTEN sequence is inserted immediately below amino acid 1656 corresponding to SEQ ID NO: 65, and a third XTEN sequence is inserted immediately below amino acid 1720 or 1900, corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately below amino acid 403 or 1656, corresponding to SEQ ID NO: 65, the second XTEN sequence is inserted immediately below amino acid 1720, corresponding to SEQ ID NO: 65 and the third XTEN sequence is inserted immediately below amino acid 1900 corresponding to SEQ ID NO: 65. In other embodiments, the first XTEN sequence is inserted immediately below amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or 1910, corresponding to SEQ ID NO: 65, a second XTEN sequence is inserted immediately below amino acid 745, corresponding to SEQ ID NO: 65, and a third XTEN sequence is inserted immediately below amino acid 2332, corresponding to SEQ ID NO: 65.

In other embodiments, the FVIII protein contains four XTEN sequences, a first XTEN sequence is inserted into a first XTEN insertion site, a second XTEN sequence is inserted into a second XTEN insertion site, a third XTEN sequence is inserted into a third XTEN insertion site, and a fourth XTEN sequence is inserted into a fourth site. embedding XTEN. The first, second, third or fourth XTEN sequences may be the same, different, or a combination thereof. In some embodiments, the FVIII protein containing four XTEN sequences may additionally carry a mutation or substitution, for example, amino acid 1648 corresponding to SEQ ID NO: 65, for example, R1648A. Non-limiting examples of first, second, third and fourth XTEN embedding sites are listed in Table. 12.

Table 13

Typical embedding sites for four XTENs

Embedding 1 Embedding 2 Embedding 3 Embedding 4 Embed site Domain Embed site Domain Embed site Domain Embed site Domain 26 A1 403 A2 1656 a3 1720 AZ 26 A1 403 A2 1656 a3 1900 AZ 26 A1 403 A2 1720 AZ 1900 AZ 26 A1 1656 a3 1720 AZ 1900 AZ 403 A2 1656 a3 1720 AZ 1900 AZ 0040 A1 0403 A2 745 IN 2332 ST 0040 A1 0403 A2 745 IN 2332 st 0018 A1 0409 A2 745 V 2332 st 0040 A1 0409 A2 745 V 2332 st 0040 A1 0409 A2 745 V 2332 st

- 78 044115

0018 Al 0409 A2 745 IN 2332 ST 0040 Al 1720 A3 745 IN 2332 st 0026 Al 1720 A3 745 IN 2332 st 0018 Al 1720 A3 745 V 2332 st 0018 Al 1720 A3 745 V 2332 st 0018 Al 1720 A3 745 V 2332 st 0026 Al 1720 A3 745 V 2332 st 0018 Al 1720 A3 745 V 2332 st 0018 Al 1900 A3 745 V 2332 st 0018 Al 1900 A3 745 V 2332 st 0026 Al 1900 A3 745 V 2332 st 0040 Al 1900 A3 745 V 2332 st 0040 Al 1905 A3 745 V 2332 st 0018 Al 1905 A3 745 V 2332 st 0040 Al 1905 A3 745 V 2332 st 0026 Al 1905 A3 745 V 2332 st 0018 Al 1905 A3 745 V 2332 st 0018 Al 1905 A3 745 V 2332 st 0018 Al 1910 A3 745 V 2332 st 0018 Al 1910 A3 745 V 2332 st 0040 Al 1910 A3 745 V 2332 st 0026 Al 1910 A3 745 V 2332 st 0018 Al 1910 A3 745 V 2332 st 0026 Al 1910 A3 745 V 2332 st 0040 Al 1910 A3 745 V 2332 st 0018 Al 1910 A3 745 V 2332 st 0409 A2 1720 A3 745 V 2332 st 0403 A2 1720 A3 745 V 2332 st 0409 A2 1720 A3 745 V 2332 st 0403 A2 1720 A3 745 V 2332 st

- 79 044115

0403 A2 1720 AZ 745 IN 2332 ST 0403 A2 1900 AZ 745 IN 2332 st 0403 A2 1900 AZ 745 IN 2332 st 0409 A2 1900 AZ 745 V 2332 st 0403 A2 1900 AZ 745 V 2332 st 0403 A2 1900 AZ 745 V 2332 st 0409 A2 1900 AZ 745 V 2332 st 0409 A2 1905 AZ 745 V 2332 st 0403 A2 1905 AZ 745 V 2332 st 0403 A2 1905 AZ 745 V 2332 st 0403 A2 1905 AZ 745 V 2332 st 0409 A2 1905 AZ 745 V 2332 st 0403 A2 1905 AZ 745 V 2332 st 0409 A2 1910 AZ 745 V 2332 st 0403 A2 1910 AZ 745 V 2332 st 0403 A2 1910 AZ 745 V 2332 st 0403 A2 1910 AZ 745 V 2332 st 0403 A2 1910 AZ 745 V 2332 st 1720 AZ 1900 AZ 745 V 2332 st 1720 AZ 1905 AZ 745 V 2332 st 1720 AZ 1910 AZ 745 V 2332 st 1720 AZ 1910 AZ 745 V 2332 st 0403 A2 1656 a3 1720 AZ 2332 st 0403 A2 1656 a3 1900 AZ 2332 st 0403 A2 1720 AZ 1900 AZ 2332 st 1656 a3 1720 AZ 1900 AZ 2332 st 0018 A1 0403 A2 1656 a3 2332 st 0018 A1 0403 A2 1720 AZ 2332 st 0018 A1 0403 A2 1900 AZ 2332 st 0018 A1 1656 a3 1720 AZ 2332 st 0018 A1 1656 a3 1900 AZ 2332 st 0018 A1 1720 AZ 1900 AZ 2332 st 0018 A1 0403 A2 0745 IN 2332 st 0018 A1 0745 IN 1720 AZ 2332 st 0018 A1 0745 IN 1900 AZ 2332 st 0403 A2 0745 IN 1720 AZ 2332 st 0403 A2 0745 V 1900 AZ 2332 st 0745 IN 1720 AZ 1900 AZ 2332 st 0188 A1 1900 AZ 0745 IN 2332 st 0599 1900 AZ 0745 IN 2332 st 2068 1900 AZ 0745 V 2332 st 2171 1900 AZ 0745 V 2332 st 2227 1900 AZ 0745 V 2332 st 2277 1900 AZ 0745 V 2332 st

In other embodiments, the FVIII protein contains five sequences

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XTEN, the first XTEN sequence is inserted into the first XTEN insertion site, the second XTEN sequence is inserted into the second XTEN insertion site, the third XTEN sequence is inserted into the third XTEN insertion site, the fourth XTEN sequence is inserted into the fourth XTEN insertion site, the fifth XTEN sequence is inserted into the fifth insertion site XTEN. The first, second, third, fourth or fifth XTEN sequences may be the same, different, or combinations thereof. Non-limiting examples of first, second, third, fourth and fifth XTEN embedding sites are listed in Table. 14.

Table 14

Typical embedding sites for five XTENs

Embedding XTEN 1 Embedding XTEN 2 Embedding XTEN3 Embedding XTEN 4 Embedding XTEN 5 0403 1656 1720 1900 2332 0018 0403 1656 1720 2332 0018 0403 1656 1900 2332 0018 0403 1720 1900 2332 0018 1656 1720 1900 2332 0018 0403 0745 1720 2332 0018 0403 0745 1900 2332 0018 0745 1720 1900 2332 0403 0745 1720 1900 2332

In certain embodiments of the invention, the FVIII protein contains six XTEN sequences, a first XTEN sequence is inserted into a first XTEN insertion site, a second XTEN sequence is inserted into a second XTEN insertion site, a third XTEN sequence is inserted into a third XTEN insertion site, and a fourth XTEN sequence is inserted into a fourth insertion site. XTEN, a fifth XTEN sequence is inserted into a fifth XTEN insertion site, and a sixth XTEN sequence is inserted into 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 six XTEN embedding sites include, but are not limited to, the embedding sites listed in Table. 15.

Table 15

Typical XTEN embed sites for six XTENs

Built-in XTEN 1 Embedding XTEN 2 XTEN 3 installation Installation XTEN 4 Embedding XTEN 5 Embedding XTEN 5 0018 0403 1656 1720 1900 2332 0018 0403 0745 1720 1900 2332

In a specific example, a first XTEN sequence is inserted between amino acids corresponding to amino acids 26 and 27 of SEQ ID NO: 65, and a second XTEN sequence is inserted between amino acids corresponding to amino acids 1720 and 1721 of 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 sequence is inserted between amino acids corresponding to amino acids 1720 and 1721 of 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 a second XTEN sequence is inserted between amino acids corresponding to amino acids 1720 and 1721 of SEQ ID NO: 65. In other examples, the 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, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 65. In yet 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 yet other embodiments, the first XTEN is inserted between the amino acids 403 and 404, corresponding to SEQ ID NO: 65, a second XTEN is inserted between amino acids 1656 and 1657, corresponding to SEQ ID NO: 65, and a third XTEN is inserted between amino acids 1720 and 1721, corresponding to SEQ ID NO: 65. In some 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 1720 and 1721, corresponding

- 81 044115 of 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, 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, 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.

In particular embodiments, the XTEN sequence is inserted between amino acids 745-746 of full-length Factor VIII or the corresponding Factor VIII insertion site with the B domain removed.

In some embodiments, the chimeric protein of the invention comprises two polypeptide sequences, the first polypeptide sequence comprising an amino acid sequence that is at least about 80%, 90%, 95%, or 100% identical to a sequence selected from

FVIII-161 (SEQ ID NO: 69), FVIII-169 (SEQ Ш NO: 70), FVIII-170 (SEQ ID

NO: 71), FVni-173 (SEQ ID NO: 72); FVIII-195 (SEQ ID NO: 73); FVIII-196 (SEQ ID NO:

74), FVIII199 (SEQ ID NO: 75), FVIII-201 (SEQ ID NO: 76); FVIII-203 (SEQ ID NO: 77), FVin-204 (SEQ ID NO: 78), FVIII-205 (SEQ ID NO: 79), FVIII-266 (SEQ ID NO: 80), FVIII267 (SEQ ID NO: 81), FVIII-268 (SEQ ID NO: 82), FVIII-269 (SEQ ID NO: 83), FVIII-271 (SEQ ID NO: 84) or FVIII-272 (SEQ ID NO: 85), and a second polypeptide a sequence comprising an amino acid sequence that is at least about 80%, 90%, 95%, or 100% identical to a sequence selected from VF031 (SEQ ID NO: 86), VF034 (SEQ ID NO: 87), or VW-036.

II.D. IG constant region or part thereof.

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 region or part thereof may improve the pharmacokinetic or pharmacodynamic properties of the chimeric protein in combination with the XTEN sequence and the VWF protein. In certain embodiments, the first IG constant region or portion thereof prolongs the half-life of the molecule fused to the IG constant region or portion thereof.

The IG constant region consists of domains designated as CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype (i.e. IgG, IgM, IgA, IgD or IgE), the constant region may consist of three or four CH domains. Some isotype (eg IgG) constant regions also contain a hinge region. See Janeway et al., 2001, Immunobiology, Garland Publishing, New York, NY

The IG constant region or portion thereof for producing the chimeric protein of this invention can be obtained from several different sources. In some embodiments, the IG constant region or a portion thereof is derived from human IG. However, it is contemplated that the IG constant region, or a portion thereof, may be derived from the IG of other mammalian species, including, for example, rodent species (eg, mouse, rat, rabbit, guinea pig) or lower primate species (eg, chimpanzee, macaque). In addition, the Ig constant region or portion thereof may be derived from any class of Ig, including IgM, IgG, IgD, IgA, and IgE, and any isotype of Ig, including IgG, IgG2, IgG3, and IgG4. In one embodiment of the invention, human IgG1 is used.

A variety of IG constant region gene sequences (eg, human constant region gene sequences) are available in publicly available sources. The constant region domain sequences may be selected to have a particular effector function (or to lack an effector function) or to be modified to reduce immunogenicity. Many antibody and antibody-encoding gene sequences have been published, and suitable IG constant region sequences (eg, hinge, CH2 and/or CH3 sequences or portions thereof) can be derived from these sequences using methods known in the art. Genetic material obtained using any of the methods described above can then be modified or synthesized to produce the polypeptides of this invention. It should also be understood that the scope of the present invention includes alleles, variants and mutations of constant region DNA sequences.

IG constant region sequences or portions thereof can be cloned, for example, using polymerase chain reaction and primers that are selected to amplify the domain of interest. To clone the Ig constant region sequence or part thereof from an antibody, mRNA can be isolated from hybridoma, spleen or lymphatic cells by reverse trans

- 82 044115 transcriptions are transcribed into DNA and antibody genes are amplified using PCR. PCR amplification methods are described in detail in US patents No. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, PCR Protocols: A Guide to Methods and Applications Innis et al., ed., Academic Press, San Diego, California (1990); But et al., 1989. Gene 77:51; Horton et al., 1993. Methods Enzymol. 217:270). PCR can be initiated using consensus constant region primers or more specific primers based on published 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, libraries can be screened using consensus primers or large homologous probes such as mouse constant region probes. Numerous sets of primers suitable for amplification of antibody genes are known in the art (eg, 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 Newman et al. al., US Patent No. 5,658,570, filed January 25, 1995, which is incorporated herein by reference.

The IG constant region used herein may include all or part of the domains and the hinge region. In one embodiment of the invention, the IG constant region or a portion thereof comprises a CH2 domain, a CH3 domain, and a hinge region, i.e. Fc fragment or FcRn binding partner.

In this context, the term Fc fragment is defined as the part of the polypeptide that corresponds to the Fc fragment of native Ig, i.e. as formed through the dimeric association of the corresponding Fc domains of its two heavy chains. The native Fc fragment forms a homodimer with another Fc fragment. In contrast, the term genetically fused Fc fragment or single chain Fc fragment (scFc fragment) as used herein means a synthetic dimeric Fc fragment consisting of Fc domains genetically linked on a single polypeptide chain (ie, encoded in a single contiguous genetic sequence).

In one embodiment, the Fc fragment is the portion of a single heavy chain of an Ig starting in the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, if residue 114 is taken to be the first residue of the heavy chain constant region) and ending at the C terminus antibodies. Accordingly, a complete Fc domain contains at least a hinge domain, a CH2 domain, and a CH3 domain.

The Fc fragment of the Ig constant region, depending on the Ig isotype, may include the CH2, CH3 and CH4 domains, as well as the hinge region. Chimeric proteins containing the Fc fragment of IG provide 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 No. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which are incorporated herein by reference.

The IG constant region or part thereof may be a binding partner of FcRn. FcRn is active in adult epithelial tissues and is expressed in the intestinal lumen, respiratory tract, nasal surfaces, vaginal surfaces, colon and rectal surfaces (US Patent No. 6,485,726). The FcRn binding partner is the part of the IG that binds to FcRn.

The FcRn receptor has been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al., 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other classes of Ig such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports IgG transcellularly from the lumen to the serosa, and then releases IgG at the relatively high pH that characterizes interstitial fluids. It is expressed in mature epithelial tissue (US Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536 A1), including pulmonary and intestinal epithelium (Israel et al., 1997, Immunology 92:69) proximal renal tuba polar epithelium (Kobayashi et al., 2002, Am. J. Physiol. Renal Physiol. 282:F358), as well as nasal epithelium, vaginal surfaces, and bile duct surfaces.

FcRn binding partners useful in the present invention include molecules that can specifically bind to the FcRn receptor, including whole IgG, an Fc fragment of IgG, and other fragments that have a complete FcRn receptor binding region. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., 1994, Nature 372:379). The main area of contact between Fc and FcRn is located near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within one IgG heavy chain. FcRn binding partners include whole IgG, the Fc fragment of IgG, and other IgG fragments that have a complete FcRn binding region. The main contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 83044115 385-387, 428 and 433-436 of the CH3 domain. All references to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions of immunoglobulins are based on Kabat et al.,

1991, Sequences of Proteins of Immunological Interest, Department of Public Health

USA, Bethesda, Maryland.

The Fc fragment or FcRn binding partner bound to FcRn can be efficiently translocated across epithelial barriers by FcRn, thereby providing non-invasive methods for systemic administration of the desired therapeutic molecule. In addition, fusion proteins containing an Fc moiety or an FcRn binding partner are endocytosed by FcRn-expressing cells. But rather than being tagged for degradation, these fusion proteins are released back into the bloodstream, thereby increasing the in vivo half-life of these proteins. In certain embodiments, portions of the IG constant regions are an Fc moiety or FcRn binding partner, which typically associate through disulfide bonds and other nonspecific interactions with another Fc moiety or other FcRn binding partner to form dimers and higher order multimers.

Two FcRn receptors can bind to one Fc molecule.

Crystallographic evidence suggests that each FcRn molecule binds to one polypeptide in the Fc homodimer. In one embodiment, the binding of an FcRn binding partner, e.g., an IgG Fc fragment, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginally, in nasal aerosol or inhalation via the pulmonary route, or via the eye. In another embodiment of the invention, the chimeric protein can be administered in an invasive manner, for example, subcutaneously, intravenously.

An FcRn binding partner is a molecule or part thereof that can specifically bind to an FcRn receptor, followed by active trafficking of an Fc moiety through the FcRn receptor. Specifically bound means two molecules forming a complex that exhibits relative stability under physiological conditions. Specific binding is characterized by high affinity and low to moderate binding capacity, which is different from nonspecific binding, which generally has low affinity and moderate to high binding capacity. Generally, binding is considered specific when the affinity constant KA is greater than 106 M ^-1 or greater than 10 ⁸ M ^-1 . If necessary, nonspecific binding can be reduced without significantly affecting specific binding by varying the binding conditions. Appropriate binding conditions such as concentration of molecules, ionic strength of solution, temperatures, time required for binding, concentration of blocking agent (eg, whey albumin, milk casein), etc. can be optimized by one skilled in the art using conventional methods.

In some embodiments, the chimeric protein of the present invention contains one or more truncated Fc fragments that are still sufficient to impart Fc receptor (FcR) binding properties to the Fc fragment. For example, the portion of the Fc fragment that binds to FcRn (i.e., the FcRn binding portion) contains from about amino acids 282-438 of IgG1, EU numbering (primary contact sites are amino acids 248, 250-257, 272, 285, 288, 290,291, 308-311, 314 of the CH2 domain and amino acid residues 385-387, 428, 433-436 of the CH3 domain. Thus, the Fc fragment of the present invention may contain or include an FcRn binding moiety. The FcRn binding moiety may be derived from any heavy chain isotype, including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn binding moiety from a human IgG1 isotype antibody is used. In another embodiment, an FcRn binding moiety from a human IgG4 isotype antibody is used.

In another embodiment, the Fc fragment includes the amino acid sequence of an Fc domain or derived from an Fc domain. In some embodiments, the Fc fragment comprises at least one: hinge region (e.g., upper, middle, and/or lower hinge region), CH1 domain (about 216-230 amino acids of the antibody Fc fragment according to EU numbering) , CH2 domain (about 231-340 amino acids of the antibody Fc fragment according to EU numbering), CH3 domain (about 341-438 amino acids of the antibody Fc fragment according to EU numbering), CH4 domain or a variant, part, or fragment thereof. In other embodiments, the Fc fragment comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc fragment comprises, consists essentially of, or consists of a hinge domain (or a portion thereof) fused with a CH3 domain (or part thereof), a hinge domain (or part thereof) fused to a CH2 domain (or part thereof), a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof), a CH2 domain (or part thereof) part) fused to both the hinge domain (or part thereof) and the CH3 domain (or part thereof). In yet other embodiments, the Fc domain lacks at least a portion of a CH2 domain (eg, all or part of a CH2 domain). In a specific embodiment of the invention, the Fc fragment contains or consists of amino acids corresponding to EU numbers 221-447.

- 84 044115

Fc fragments, designated F, F1 or F2 herein, can be obtained from several different sources. In one embodiment of the invention, the Fc fragment polypeptide is derived from human IG. However, it is contemplated that the Fc fragment may be derived from the IG of other mammalian species, including, for example, rodent species (eg, mouse, rat, rabbit, guinea pig), or lower primate species (eg, chimpanzee, macaque). In addition, the Fc domain polypeptide or portions thereof may be derived from any class of Ig, including IgM, IgG, IgD, IgA, and IgE, and any isotype of Ig, including IgG1, IgG2, IgG3, and IgG4. In another embodiment, the human IgG1 isotype is used.

In certain embodiments, the Fc variant provides a change in at least one effector function conferred on the Fc fragment containing said wild-type Fc domain (e.g., improving or impairing the ability of the Fc fragment to bind to Fc receptors (e.g., FcyRI, FcyRII, or FcyRIII), or complement proteins (eg C1q) or to cause antibody-dependent cytotoxicity (ADCC), phagocytosis and complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant contains a engineered cysteine residue.

The Fc fragments of the present invention may use Fc variants accepted in the art with a known ability to cause changes (eg, improvement or impairment) in effector function and/or FcR or FcRn binding. In particular, the binding molecule of this invention may have, for example, a change (eg, substitution) of one or more amino acid positions described in international publications

PCT WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO0 2/44215A2, WO02/060919A2, WO03/074569A2, WO04 /016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO 05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1 , WO06/047350A2 and WO06/085967A2; US patent publications No. US2007/0231329, US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767, US2007/0243188, US20070248603, US20070286859, US20080 057056;

or 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 of the invention, a specific change (eg, a specific substitution of one or more amino acids described in the art) can be made at one or more described amino acid positions. In another embodiment, various changes to one or more of the described amino acid positions (eg, various substitutions of one or more amino acid positions described in the art) can be made.

The Fc fragment or FcRn binding partner of an IgG can be modified according to well-known procedures such as site-directed mutagenesis and the like to produce modified IgG or Fc fragments, or portions thereof, that will bind to FcRn. Such modifications include modifications at contact sites distant from FcRn, as well as modifications at contact sites that maintain or even enhance binding to FcRn. For example, the following single amino acid residues in the human IgG1 Fc (Fcy1) can be replaced without significant loss of Fc binding affinity to FcRn:

P238A, S239A, K246A,

K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T2 89A, K290A,

R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V3O3A, V305A,

T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A,

A327Q, P329A, A33OQ, P331A, EZZZA, K334A, T335A, S337A, K338A, K340A, Q342A,

R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A,

S375A, D376A, A378Q, E38OA, E382A, S383A, N384A, Q386A, E388A, N389A, N390A,

Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A,

V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where, for example, P238A represents a substitution of wild-type proline for alanine at position number 238.

As an example, a particular embodiment of the invention includes the N297A mutation, where

- 85 044115 laying a highly conserved N-glycosylation site. In addition to alanine, other amino acids may be replaced with wild-type amino acids at the positions indicated above. Mutations can be introduced into Fc one at a time, resulting in the formation of more than one hundred Fc fragments different from the native Fc. Additionally, combinations of two, three, or more of these individual mutations can be introduced together, resulting in the formation of hundreds more Fc fragments. Moreover, one of the Fc fragments of the construct of this invention may be mutated and the other Fc fragment of the construct is not mutated at all, or they may both be mutated but have different mutations.

Some of the above mutations may impart new functionality to the Fc fragment or FcRn binding partner. For example, one embodiment of the invention includes the N297A mutation, which removes a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby increasing the blood half-life of the Fc fragment, and render the Fc fragment incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising FcRn affinity (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 new functionality arising from the mutations described above, affinity for FcRn can be increased beyond wild-type levels in some cases. This increased affinity may correspond to an increased rate of association, a decreased rate of dissociation, or both an increased rate of association and a decreased rate of dissociation. 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).

In addition, at least three human Fc gamma receptors appear to recognize a binding site on IgG in the lower hinge region, which typically corresponds to amino acids 234-237. Thus, another example of new functionality and potential reduction in immunogenicity may arise from mutations in this region, such as by replacing amino acids 233-236 of human IgG1 ELLG with the corresponding sequence from IgG2 PVA (removing one amino acid). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions, will not bind to IgG1 when such mutations are introduced. Ward and Ghetie 1995 Therapeutic Immunology 2:77 and Armor et al. 1999 Eur. J. Immunol. 29:2613.

In one embodiment, the IG constant region or portion thereof, e.g., an Fc fragment, is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO: 89 or SEQ ID NO: 3 of US Pat. No. 5,739,277) and in some cases further including the sequence selected from HQLGTQ (SEQ ID NO: 90), HQNLSDGK (SEQ ID NO: 91), HQNTSDGK (SEQ ID NO: 92), or V1SSHLGQ (SEQ ID NO: 93) (or SEQ ID NO: 11, 1, 2 and 31, respectively from US patent No. 5,739,277).

In another embodiment, an immunoglobulin constant region or portion thereof comprises an amino acid sequence in a hinge region or portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or portion thereof. The disulfide bond of the immunoglobulin constant region or portion thereof connects the first polypeptide containing FVIII and the second polypeptide containing the VWF fragment such that endogenous VWF does not replace the VWF fragment and does not bind to FVIII. Thus, a disulfide bond between a first immunoglobulin constant region or portion thereof and a second immunoglobulin constant region or portion thereof prevents interaction between endogenous VWF and the FVIII protein. Inhibiting the interaction between VWF and the FVIII protein makes it possible to double the half-life limit of the chimeric protein. The hinge region or portion thereof may be further associated with one or more CH1, CH2, CH3 domains, fragments thereof, or any combination thereof. In a particular embodiment of the invention, the IG constant region or part thereof is a hinge region and a CH2 domain.

In some embodiments, the Ig constant region or portion thereof is hemi-glycosylated. For example, a chimeric protein containing two Fc fragments or an FcRn binding partner may comprise a first glycosylated Fc fragment (e.g., a CH2 glycosylated region) or FcRn binding partner and a second glycosylated Fc fragment (e.g., a CH2 glycosylated region) or binding partner FcRn. In one embodiment of the invention, the linker can be placed between glycosylated and aglycosylated Fc moieties. In another embodiment, the Fc fragment or FcRn binding partner is fully glycosylated, i.e. all Fc fragments are glycosylated. In other embodiments, the Fc fragment may be aglycosylated, i.e. none of the Fc fragments are glycosylated.

In some embodiments, the chimeric protein of the present invention contains an amino acid substitution in the Ig constant region or a portion thereof (e.g., an Fc variant) that alters the antigen-independent effector functions of the Ig constant region, particularly the half-life of the protein from the blood.

Such proteins exhibit increased or decreased ability to bind to FcRn compared to proteins that lack these substitutions and therefore have increased or

- 86 044115 reduced half-life in serum, respectively. Fc variants with improved affinity for FcRn are expected to have a longer half-life in serum, and such molecules have useful applications in mammalian treatments where a long half-life of the administered polypeptide is desired, for example, for the treatment of a chronic disease or disorder (see eg US Patents 7,348,004, 7,404,956 and 7,862,820). In contrast, Fc variants with reduced binding affinity to FcRn are expected to have shorter half-lives and such molecules may also be used, for example, for administration to a mammal where a reduced residence time in the bloodstream may be more advantageous, e.g. for diagnostic imaging in vivo or in situations where the initial polypeptide has toxic side effects if present in the bloodstream for an extended period of time. Also less likely are Fc variants with reduced binding affinity to FcRn to cross the placental barrier and thus also be useful in the treatment of diseases or disorders in pregnant women. Additionally, other applications in which reduced binding affinity to FcRn may be necessary include those applications in which localization to the brain, kidney and/or liver is required. In one exemplary embodiment of the invention, the chimeric protein herein exhibits a reduced ability to pass through the glomerular epithelium from the vascular system. In another embodiment, the chimeric protein of the invention exhibits a reduced ability to cross the blood-brain barrier (BBB) from the brain into the vasculature. In one embodiment, the protein with modified FcRn binding ability comprises at least one Fc fragment or FcRn binding partner (e.g., one or two Fc fragments or FcRn binding partner) having one or more amino acid substitutions within the binding loop FcRn constant region of IG. The FcRn binding loop consists of amino acid residues 280-299 (according to EU numbering) of the wild type, full-length Fc fragment. In other embodiments, an IG constant region or portion thereof in a chimeric protein of the invention having a modified FcRn binding affinity comprises at least one Fc fragment or FcRn binding partner having one or more amino acid substitutions within 15 k of the FcRn contact region. In this context, the term 15 to FcRn contact zone includes residues occupying the following positions in the full-length wild-type Fc fragment: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering). In other embodiments, an Ig constant region or portion thereof of the invention having a modified FcRn binding affinity comprises at least one Fc fragment or FcRn binding partner having one or more amino acid substitutions corresponding to any of the following EU positions: 256, 277 -281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (eg, N434A or N434K), and 438. Typical amino acid substitutions that modify FcRn binding activity are are described in PCT International Publication No. 05/047327, which is incorporated herein by reference in its entirety.

The Fc fragment or FcRn binding partner used in this invention may also contain an amino acid substitution known in the art that alters the level of glycosylation of the chimeric protein. For example, an Fc fragment or FcRn binding partner of a chimeric protein linked to a VWF fragment or an FVIII protein may contain an Fc fragment that has a mutation that results in reduced levels of glycosylation (eg, N- or O-linked glycosylation) or may contain a modified glycoform wild-type Fc fragment (eg, low fucose or fucose-free glycan).

In one embodiment, the full-length chimeric protein of the present invention may comprise a genetically fused Fc fragment (e.g., an scFc fragment) having two or more IG constant regions or portions thereof independently selected from the IG constant region or portion thereof described in this document. In one embodiment of the invention, the Fc fragments of the dimeric Fc fragments are the same. In another embodiment of the invention, at least two Fc fragments are different. For example, the Fc fragment or FcRn binding partner of the proteins of the present invention contain the same number of amino acid residues, or they may differ in the length of one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues) residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues or about 50 residues). In yet other embodiments, the Fc fragment or FcRn binding partner of the invention may differ at one or more positions in the amino acid sequence. For example, at least two of the Fc fragment or FcRn binding partner may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions or about 50 positions).

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II. E. Linkers.

The chimeric protein of this invention further contains 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 a fragment, for example a VWF protein, to be cleaved from the XTEN sequence, hence the chimeric protein, at the site of the coagulation cascade, thereby allowing activated FVIII (FVIIIa) to exhibit its FVIIIa activity. Another type of linker is a processable linker that contains an intracellular cleavage site and thus can be cleaved by an intracellular processing enzyme in a host cell, thereby facilitating expression of the polypeptide and formation of the chimeric protein.

One or more linkers may be present between any two proteins in a 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 portion thereof, and a second polypeptide comprising (III) an FVIII protein, (IV) a linker (e.g., a cleavable linker), (V) an XTEN sequence, and (VI) a second IG constant region or 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 portion thereof, and a second polypeptide comprising (III) a VWF protein, (IV) an XTEN sequence, (V) a linker ( for example, a cleavable linker) and (VI) a second IG constant region or part thereof. In other embodiments, the chimeric protein comprises a first polypeptide comprising (I) an FVIII protein and (II) a first IG constant region or portion thereof, and a second polypeptide comprising (III) an FVIII protein, (IV) a first linker (e.g., a cleavable linker) , (V) an XTEN sequence, (VI) a second linker (eg, a cleavable linker), and (VII) a second IG constant region or portion thereof. In certain embodiments, the first polypeptide further comprises a linker, for example, a cleavable linker between the FVIII protein and the first IG constant region.

In some embodiments, the chimeric protein comprises a single chain comprising (I) an FVIII protein, (II) a first IG constant region or portion thereof, (III) a linker (e.g., a processable linker), (IV) a VWF protein, (V) a sequence XTEN and (VI) the second constant region of the IG or part thereof. In other embodiments, the chimeric protein comprises a single chain comprising (I) an FVIII protein, (II) a first IG constant region or portion thereof, (III) a first linker (e.g., a processable linker), (IV) a VWF protein, (V) a second linker (eg, a cleavable linker), (VI) an XTEN sequence, and (VII) a second IG constant region or portion thereof. The processable linker can be processed after the chimeric protein is expressed in the host cell; thus, the chimeric protein produced in the host cell may be in a final form consisting of two or three polypeptide chains.

A linker useful in the present invention may comprise an organic molecule. In one embodiment of the invention, the linker contains a polymer, for example, polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment of the invention, 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 of the invention, the linker contains the sequence XTEN. Additional examples of XTEN sequences may be used herein and are disclosed in US Patent Publication No. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1 or 2011/0172146 A1, or international patent publications No. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, or WO 2011028344 A2. In another embodiment of the invention, the linker is a PAS sequence.

In one embodiment of the invention, the linker is a polymer, for example, polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment of the invention, 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.

Examples of linkers are well known in the art. In one embodiment of the invention, the linker comprises a G _n sequence. The linker may contain the sequence (GA) _n . The linker may contain the sequence (GGS) _n . In other embodiments, the linker contains (GGGS) _n (SEQ ID NO: 101). In yet other embodiments, the linker comprises the sequence (GGS) _n (GGGGS) _n (SEQ ID NO: 95). In these cases n can be an integer

- 88 044115 from 1 to 100. In other cases, n can be an integer from 1 to 20, for example, 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 Ш NO: 96),

GGSGGSGGSGGSGGG (SEQ ID NO: 97), GGSGGSGGGGSGGGGS (SEQ ID NO: 98), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 99), or GGGGSGGGGSGGGGS (SEQ ID NO: 100).

The linker does not eliminate or reduce VWF protein activity or factor VIII coagulation activity. In some cases, the linker increases the activity of the VWF protein or the coagulation activity of the factor VIII protein, for example, by further reducing the effect of steric mismatch and makes the VWF protein or part of the factor VIII more accessible to its target binding site.

In one embodiment of the invention, the linker useful for the chimeric protein is 1525 amino acids in length. In another embodiment of the invention, the linker useful for the chimeric protein is 15-20 amino acids in length. In some embodiments, the linker for the chimeric protein is 10-25 amino acids in length. In another embodiment, the linker for the chimeric protein is 15 amino acids in length. In yet other embodiments, the linker for the chimeric protein is (GGGGS) _n (SEQ ID NO: 94) wherein G is glycine, S is series, and N is an integer from 1 to 20.

II. F. Cleavage Sites

Cleavable linkers may include a moiety capable of being cleaved either chemically (eg, hydrolysis of an ester bond), enzymatically (eg, inclusion of a sequence for protease cleavage), or photolytically (eg, a chromophore such as 3-amino-3-(2- nitrophenyl) propionic acid (ANP)) in order to free one molecule from the other.

In one embodiment of the invention, the cleavable linker contains one or more cleavage sites at the N-terminus or the C-terminus, or both. In another embodiment, the cleavable linker is substantially composed of or consists of one or more cleavable sites. In other embodiments, the linker comprises heterologous linker amino acid sequences described herein, or polymers, and one or more cleavage sites.

In some embodiments, the cleavable linker contains one or more cleavage sites that can be cleaved in the host cell (ie, intracellular processing sites). Non-limiting examples of cleavage sites include: RRRR (SEQ ID NO: 102), RKRRKR (SEQ ID NO: SW) and RRRRS (SEQ ID NO: 104).

In some embodiments, the cleavable linker comprises a FVIII a1 region, a FVIII a2 region, a FVIII a3 region, a thrombin cleavage site that contains X-V-P-R (SEQ ID NO: 105), and a PAR1 exosite interaction motif in which X is an aliphatic amino acid or any combinations thereof, contains an a2 region containing an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the sequence Glu720 to Arg740 corresponding to full-length FVIII, in which region a2 is capable of being cleaved by thrombin. In a specific embodiment, the cleavable linker useful in this invention contains two a2 regions containing ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106).

In other embodiments, the cleavable linker of the invention comprises an a1 region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the sequence from Met337 to Arg372 , corresponding to full-length FVIII, in which the a1 region is capable of being cleaved by thrombin. In a specific embodiment of the invention, region a1 contains ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 107).

In some embodiments, the cleavable linker of the invention comprises an a3 region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, or 100% identical to the sequence from Glu1649 to Arg1689 , corresponding to full-length FVIII, in which the a3 region is capable of being cleaved by thrombin. In a specific embodiment of the invention, the cleavable linker useful in this invention contains two a3 regions containing

ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 108).

In other embodiments, the cleavable linker comprises a thrombin cleavage site containing X-V-P-R (SEQ ID NO: 105) and a PAR1 exosite interaction motif, wherein the PAR1 exosite interaction motif contains S-F-L-L-R-N (SEQ ID NO: 109). The PAR1 exosite interaction motif may further comprise an amino acid sequence selected from

- 89 044115

Ρ,

P-N, P-N-D, P-N-D-K (SEQ ID NO: 110), P-N-D-K-Y (SEQ ID NO: 111), P-N-D-K-Y-E (SEQ ID NO: 112), P-N-D-K-Y-E-P (SEQ ID NO: 113), P-N-D-K-Y-E-P-F (SEQ ID NO: 114 ) , P-ND-K-Y-E-P-F-W (SEQ ID NO: 115), P-N-D-K-Y-E-P-F-W-E (SEQ ID NO: 116), P-N-D-K-YE-P-F-W-E-D (SEQ ID NO: 117), P-N-D-K-Y-E-P-F-W-E-D-E (SEQ ID NO: 118 ), P-N-D-KY-E-P-F-W-E-D-E-E (SEQ ID NO: 119), P-N-D-K-Y-E-P-F-W-E-D-E-E-S (SEQ ID NO: 120) or any combination thereof. In some embodiments, the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine.

In other embodiments, the cleavable linker contains one or more cleavage sites that are cleaved by a protease after the chimeric protein containing the cleavable linker is administered to a subject. In one embodiment, the cleavage site is cleavable by a protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), elastase 2, MMP-12, MMP13, MMP- 17 and MMR-20. In another embodiment of the invention, the cleavage site is selected from the group consisting of an FXIa cleavage site (e.g., KLTR^AET (SEQ ID NO: 121)), an FXIa cleavage site (e.g., DFTR^VVG (SEQ ID NO: 122)), a cleavage site FXIIa (e.g., TMTR^IVGG (SEQ ID NO: 123)), kallikrein 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. LGPVkSGVP (SEQ ID NO: 129)), granzyme B cleavage site (e.g. VAGD^SLEE (SEQ ID NO: 130)), MMP cleavage site -12 (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)), a 3C protease cleavage site (PRESCISSION™) (e.g., LEVLFQ^GP (SEQ ID NO: 137)), and a sortase A cleavage site (e.g., LPKT^GSES) (SEQ ID NO: 138). In some embodiments, the FXIa cleavage site includes, but is not limited to, TQSFNDFTR (SEQ ID NO: 1) or SVSQTSKLTR (SEQ ID NO: 3).

Non-limiting examples of 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 a sequence comprising essentially consisting of or consisting of ALRPR (SEQ ID NO: 7) (for example, ALRPRVVGGA (SEQ ID NO: 145)). In exemplary embodiments of the invention, the cleavage site is the sequence

TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 146). in another embodiment of the invention, the cleavage site contains

DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ GO NO: 88) or a fragment thereof. In one particular embodiment of the invention, the cleavage site comprises IEPRSFS (SEQ ID NO: 194). in another embodiment, the cleavage site comprises EPRSFS (SEQ ID NO: 195), and the cleavage site is not the full-length a2 region of the FVIII protein. In yet another embodiment, the cleavage site comprises an IEPR (SEQ ID NO: 200). In another embodiment, the cleavage site comprises an IEPR (SEQ ID NO: 200), wherein the cleavage site is not a full-length a2 region of the FVIII protein or does not contain a full-length a2 region of FVIII. In other embodiments, the cleavage site comprises

- 90 044115

DKNTGDYYED SYEDISAYLLSKNNAIEPRSFS (SEQ GO NO

88), KNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 139), NTGDYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 140), TGDYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 141), GDYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 142), DYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 143), YYEDSYEDISAYLLS KNNAIEPRSFS (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 site comprises DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ GO NO: 88),

KNTGDYYEDSYED ISAYLLSKNNAIEPRSFS (SEQ ID NO: 139), NTGDYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 140), TGDYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 141), GDYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 142), DYYEDSYEDISAYLLS KNNAIEPRSFS (SEQ ID NO: 143), YYEDSYEDISAYLLS KNNAIEPRSFS (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), and the cleavage site is not the full-length a2 region of the FVIII protein. In certain embodiments of the invention, the cleavage site is amenable to cleavage in a thrombin hydrolysis test, as proposed herein or known in the art.

III. Polynucleotides, vectors and host cells.

Also provided according to this invention is a polynucleotide encoding a chimeric protein according to this invention. In one embodiment of the invention, the first polypeptide chain and the second polypeptide chain may be encoded by a single polynucleotide chain. In another embodiment, the first polypeptide chain and the second polypeptide chain are encoded by two different polynucleotides, for example, a first nucleotide sequence and a second nucleotide sequence. In another embodiment of the invention, the first nucleotide sequence and the second nucleotide sequence are two different polynucleotides (eg, different vectors).

The invention includes a polynucleotide encoding a single polypeptide chain (for example, FVIII(X2)-F1-L3-F2-L2-X1-L1-V), in which FVIII(X2), contains the FVIII protein, in which the sequence

- 91 044115 XTEN is inserted into one or more insertion sites, F1 contains the first IG constant region or part thereof, for example, the first Fc fragment, L1 contains the first linker, V contains the VWF protein, X1 contains the XTEN sequence having a length of less than 288 amino acids, L2 contains a second linker, L3 contains a third linker, and F2 contains a second IG constant region or part thereof, for example, a second Fc fragment. The invention also includes two polynucleotides, a first polynucleotide sequence encoding a first polypeptide that contains an FVIII protein fused to a first IG constant region or a portion thereof, and a second polynucleotide sequence encoding a second polypeptide that contains a VWF protein, an XTEN sequence having a length of less than 288 amino acids, and a second IG constant region or part thereof. In some embodiments, a chimeric protein comprising two polypeptide chains or three polypeptide chains may be encoded by a single polynucleotide chain and then processed into two or three (or more) polypeptide chains. In yet other embodiments, a chimeric protein comprising these polypeptide chains may be encoded using two or three polynucleotide chains.

In other embodiments, the set of polynucleotides further comprises an additional nucleotide strand (e.g., a second nucleotide strand when the chimeric polypeptide is encoded by a first polynucleotide strand or a third nucleotide strand when the chimeric protein is encoded by two polynucleotide strands) that encodes a convertase protein. The convertase protein may be selected from the group consisting of proprotein convertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertase subtilisin/kexin type 7 (PCSK7 or PC5), yeast Kex 2, proprotein convertase subtilisin/kexin type 3 (PACE or PCSK3), and two or more combinations of them. In some embodiments, the convertase protein is PACE, PC5, or PC7. In an exemplary embodiment of the invention, the convertase protein is PC5 or PC7. See international application No. PCT/US 2011/043568.

As used herein, an expression vector means any nucleic acid construct that contains elements necessary for transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, elements necessary for replication and translation upon introduction into an appropriate host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of this invention will include polynucleotides encoding the chimeric protein described herein. In one embodiment, one or more coding sequences for a first polypeptide comprising an FVIII protein and a first IG constant region, for a second polypeptide comprising an VWF protein, an XTEN sequence having less than 288 amino acids and a second IG constant region or a portion thereof, or both polypeptides are functionally linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that preserves the functionality of each component of the nucleic acid sequence. A coding sequence and a gene expression control sequence are said to be operably linked if they are covalently linked such that expression or transcription and/or translation of the coding sequence is influenced or controlled by the gene expression control sequence. Two DNA sequences are said to be functionally related if induction of a promoter at the 5' end of the gene expression sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence or (3) does not interfere with the corresponding RNA transcript being translated into protein. Thus, a gene expression sequence will be operably linked to a nucleic acid coding sequence if the gene expression sequence is capable of transcribing the sequence encoding the nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

As used herein, a gene expression control sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter enhancer combination, that promotes efficient transcription and translation of the encoding nucleic acid to which it is operably linked. The gene expression control sequence may, for example, be a mammalian promoter or a viral promoter, such as a constitutive or inducible promoter. Mammalian constitutive promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminases, pyruvate kinases, beta-actin promoter, and other constitutive promoters. Typical viral promoters that function constitutively in eukaryotic cells include, for example, those from cytomegalovirus (CMV), simian virus (eg, SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the herpes simplex virus thymidine kinase promoter. Other constitutive promoters are known to those skilled in the art.

- 92 044115 technology. Promoters useful as gene expression sequences of the present invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter induces transcriptional and translational activation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

In general, the gene expression control sequence, as required, should include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, and the like. In particular, such 5' untranscribed sequences include a promoter region, which includes a promoter sequence for transcriptional control of the operably linked coding nucleic acid. Optionally, in some cases, the gene expression sequence includes enhancer sequences or upstream activator sequences.

The viral vector includes, but is 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; adenovirus, adeno-associated virus; SV40 type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; cowpox virus; polio virus; and an RNA virus such as a retrovirus. Other vectors well known in the art can easily be used. Some viral vectors are based on noncytopathic eukaryotic viruses in which nonessential genes have been replaced with genes of interest. Noncytopathic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into the DNA of the host cell. Retroviruses have been approved for human gene therapy research. The most useful are replication-defective retroviruses (i.e., capable of directing the synthesis of necessary proteins, but not capable of synthesizing an infectious particle). Such genetically engineered retroviral expression vectors are useful for highly efficient gene transduction in vivo. Standard protocols for the production of replication-defective retroviruses (including the steps of introducing exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retroviruses through the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with viral particles) are presented in Kriegler , M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Clifton, NJ (1991).

In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication defective and capable of infecting a wide range of cell types and species. In addition, it has the advantages of heat stability and lipid dissolution resistance; high frequencies of transduction in cells of various lineages, including hematopoietic cells; and lack of inhibition of superinfection, thus allowing multiple series of transduction. It is reported that the adeno-associated virus can site-specifically integrate into human cellular DNA, thereby minimizing the possibility of insertional mutagenesis and variability in the expression characteristics of the integrated retroviral infection gene. In addition, wild-type adeno-associated virus infections were carried out in tissue culture for over 100 passages in the absence of selective pressure, implying that adeno-associated virus genomic integration is a relatively stable event. Adeno-associated virus may also function as an extrachromosomal factor.

Other vectors include plasmid vectors. Plasmid vectors are widely 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, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo due to their inability to integrate and replicate within the host genome. However, these plasmids have a promoter compatible with the host cell that can express a peptide from a gene functionally encoded in the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, PRC/CMV, various PCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/Myc-His, cat. no. V86320; and pBudCE4.1, part number V53220, all from Invitrogen (Carlsbad, CA). Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be specifically designed using standard molecular biology techniques to remove and/or add specific DNA fragments.

In one insect-based expression system that can be used to produce

- 93 044115 proteins of this invention, Autographa californica nuclear hyperhidrosis virus (AcNPV) is used as a vector for the expression of foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned into minor regions (eg, polyhedron gene) of the virus and placed under the control of the AcNPV promoter (eg, polyhedron promoter). Successful introduction of the coding sequence will result in inactivation of the polyhedron gene and the formation of an uninterspersed recombinant virus (i.e., the virus lacks the protein layer encoded by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells that express the inserted gene. (See, eg, Smith et al. (1983) J Virol 46:584; US Pat. No. 4,215,051). Additional examples of these expression systems can be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

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

A number of virus-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the coding sequence may be ligated to the adenovirus transcription/translation control complex, eg, a late promoter and a tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a minor region of the viral genome (eg, the E1 or E3 region) results in a recombinant virus that is viable and capable of expressing the peptide in infected hosts. (See, for example, Logan & Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, a 7.5K vaccinia virus promoter may be used. (See, for example, Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415; Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.

To improve production efficiency, polynucleotides can be designed to encode multiple protein units of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (eg, by treatment with an appropriate enzyme) in order to restore the polypeptide units. This may increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, translation of each polypeptide encoded by the mRNA is directed toward the interior of the transcript; for example, using the internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of one large polycistronic mRNA, which in turn directs the translation of several, separate polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and can significantly increase the yield of polypeptides driven by a single promoter.

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

Selectable markers can be introduced into a cell via a separate plasmid at the same time as the gene of interest, or they can be introduced via the same plasmid. While on the same plasmid the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, late placement produces a bicistronic message. Designs of this type are known in the art (eg, US Pat. No. 4,713,339).

Expression vectors can encode tags that allow easy purification of the recombinantly produced protein. Examples include, but are not limited to, vector pUR278 (Ruther et al. (1983) EMBO J 2:1791), in which coding sequences for an expressed protein can be ligated into the vector in frame with the lac z coding region so that a tagged protein is produced. fusion protein; pGEX vectors can be used to express the proteins of this invention using glutathione S-transferase (GST) as a tag. Typically, these proteins are soluble and can be easily purified from cells by adsorption onto glutathione-agarose microcarriers followed by elution in the presence of free glutathione. Vectors include cleavage sites (thrombin or factor Xa protease or PRESCISSION™ PROTEASE

- 94 044115 (Pharmacia, Peapack, NJ) for easy tag removal after cleaning.

The expression vector or vectors are then transfected or cotransfected into a suitable target cell that will express the polypeptides. Transfection methods 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 cell expression vector systems can be used to express the proteins described herein, including both prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (eg E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing the appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or filamentous fungal expression vectors containing the appropriate coding sequence; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing the appropriate coding sequence; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing the appropriate coding sequence; or animal cell systems, including mammalian cells (eg, HEK293, CHO, Cos, HeLa, HKB11 and BHK cells).

In one embodiment of the invention, the host cell is a eukaryotic cell. In this context, a eukaryotic cell refers to any animal or plant cell that has a well-defined nucleus. Eukaryotic animal cells include vertebrate cells, such as mammals, and invertebrate cells, such as insects. Eukaryotic plant cells may include, but are not limited to, yeast cells. A eukaryotic cell is different from a prokaryotic cell, such as a bacterium.

In some embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells may particularly include, but are not limited to, mammalian cell lines. In one embodiment of the invention, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK293 cell, which is a human embryonic kidney cell line. HEK293 cells, available as CRL-1533 from the American Type Culture Collection, Manassas, VA, and as 293-H cells, catalog no. 11631-017, or 293-F cells, catalog no. 11625-019 from Invitrogen (Carlsbat, California State). In some embodiments, the mammalian cell is a PER.C6® cell, which is a retinal-derived cell line. PER.C6® cells are available from Krusell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a cell from the Chinese hamster ovary (CHO) cell line. CHO cells are available from the American Type Culture Collection, Manassas, VA (eg, CHO-Ki; CCL-61). In yet other embodiments, the mammalian cell is a cell from a neonatal Syrian hamster kidney (SHK) cell line. BHK cells are available from the American Type Culture Collection, Manassas, VA (eg, CRL-1632). In some embodiments, the mammalian cell is an HKB11 cell, which is a hybrid cell line from the HEK293 cell line and a human B cell line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

In one embodiment, a plasmid containing an FVIII(X2)-Fc fusion coding sequence, a VWF-L1-X1-L2-Fc protein coding sequence, or both, and a selectable marker, e.g., zeocin resistance, is transfected into HEK293 cells to produce chimeric protein.

In another embodiment, a plasmid containing an FVIII-Fc fusion coding sequence, a VWF-L1-X-L2-Fc protein coding sequence, or both, and a selectable marker, such as zeocin resistance, is transfected into HEK293 cells to produce a chimeric protein.

In some embodiments, a first plasmid comprising an FVIII(X2)-Fc fusion coding sequence and a first selectable marker, such as zeocin resistance, and a second plasmid comprising the VWF-L1-X1-L2-Fc protein coding sequence and a second selectable marker , for example, a neomycin resistance gene and a third plasmid containing a convertase protein coding sequence and a third selectable marker, for example, a hygromycin resistance gene, were transfected into HEK293 cells to produce a chimeric protein. The first and second plasmids may be administered in equal amounts (eg, a 1:1 molar ratio), or they may be administered in unequal amounts.

In yet other embodiments, the first plasmid comprising an FVIII-Fc fusion

- 95 044115 a coding sequence and a first selectable marker, for example, resistance to zeocin, and a second plasmid containing the VWF-L1-X-L2-Fc protein, a coding sequence and a second selectable marker, for example, a neomycin resistance gene and a third plasmid containing the coding sequence convertase protein and a third selectable marker, such as the hygromycin resistance gene, were transfected into HEK293 cells to produce the chimeric protein. The first and second plasmids may be administered in equal amounts (eg, a 1:1 molar ratio), or they may be administered in unequal amounts.

In still other embodiments, a first plasmid comprising an FVIII(X2)-Fc fusion coding sequence and a first selectable marker, such as zeocin resistance, and a second plasmid comprising the VWF-L1-X1-L2-Fc protein fusion coding sequence and a second a selectable marker, eg, a neomycin resistance gene; and a third plasmid containing a convertase protein coding sequence and a third selectable marker, eg, a hygromycin resistance gene, were transfected into HEK293 cells to produce a chimeric protein. The first and second plasmids may be administered in equal amounts (eg, a 1:1 molar ratio), or they may be administered in unequal amounts.

In some embodiments, a first plasmid comprising a FVIII (with or without XTEN)-F1-L3-F2-L2-X-L1-V coding sequence and a first selectable marker, such as zeocin resistance, and a second plasmid comprising the coding sequence convertase protein and a second selectable marker, such as a hygromycin resistance gene, were transfected into HEK293 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.

In yet other embodiments, the transfected cells are stably transfected. These cells can be selected and maintained as a stable cell line using standard methods known to those skilled in the art.

Host cells containing the protein DNA constructs are grown in a suitable growth medium. As used herein, the term appropriate culture medium means a medium containing nutrients necessary for cell growth. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. In some cases, the environment may contain one or more selection factors. In some cases, the medium may contain bovine serum or fetal bovine serum (FBS). In one embodiment of the invention, the medium contains substantially no IgG. The culture medium is typically selected for cells containing the DNA construct, through, for example, selection for drug sensitivity or lack of an essential nutrient, which is supplemented through a selectable marker on the DNA construct or cotransfected with the DNA construct. Cultured mammalian cells are typically grown in commercially available serum-containing or serum-free media (eg, MEM, DMEM, DMEM/F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbath, Calif.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbath, Calif.). The choice of medium suitable for the particular cell line used is determined by the level of those of ordinary skill in the art.

In order to co-express two polypeptide chains of a chimeric protein, host cells are cultured under conditions that allow expression of both chains. As used herein, culture means maintaining living cells in vitro for at least a certain period of time. Maintenance may include, but does not necessarily include, increasing the population of living cells. For example, cells maintained in culture may be static in the population but still be viable and capable of producing the desired product, such as a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include proper selection of culture media, culture media additives, temperature, pH, oxygenation, and the like. For commercial purposes, cultivation may involve the use of any of various types of industrial scale systems, including shake flasks, spinner flasks, hollow fiber bioreactors, mechanically stirred bioreactors, airlift bioreactors, wave bioreactors, and others.

Cell culture conditions are also selected to allow the VWF fragment to combine with the FVIII protein. Conditions that allow expression of the VWF fragment and/or FVIII protein may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK293 cells are cultured in CD293 medium (Invitrogen, Carlsbath, Calif.) or OptiCHO medium (Invitrogen, Carlsbath, Calif.) California) with the addition of 4 mM glutamine.

In one aspect, the present invention relates to a method of expressing, making or producing a chimeric protein of the present invention, which includes a) transfecting a host cell,

- 96 044115 containing a polynucleotide encoding a chimeric protein and b) culturing the host cell in a culture medium, under conditions suitable for expression of the chimeric protein, wherein the chimeric protein is expressed.

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

In some aspects, the present invention relates to a chimeric protein produced by the methods described herein.

In vitro production allows for industrial scale production of large quantities of the desired modified polypeptides of the present invention. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, for example, in an airlift reactor or in a continuous stirred tank reactor, or immobilized or encapsulated cell culture, for example, in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, reverse phase chromatography and adsorption chromatography (Strategies for Protein Purification and Characterization:

IV. Pharmaceutical composition.

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

The pharmaceutical composition can be prepared for parenteral administration (ie, intravenously, subcutaneously or intramuscularly) by bolus injection. Injectable dosage forms may be presented in single dosage form, such as ampoules, or in multi-dose containers with an added preservative. The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous liquid vehicles, and contain auxiliary agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable carrier, for example, pyrogen-free water.

Dosage forms suitable for parenteral administration also include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds can be administered in the form of suitable oily injection suspensions. Suitable lipophilic solvents and carriers include fatty oils, for example sesame oil, or synthetic fatty acid esters, for example ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and dextran. In some cases, 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 include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments, the composition contains isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions contain pharmaceutically acceptable agents such as wetting agents or small amounts of excipients such as wetting or emulsifying agents, preservatives or buffers that increase the shelf life or potency of the active ingredients.

The compositions of this invention may be presented in various forms, including, for example, liquids (eg, injectable and insoluble solutions), dispersions, suspensions, semi-solid and solid dosage forms. The choice of the preferred form depends on the route of administration and therapeutic use.

The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentrations. Sterile injection solutions can be prepared by combining the active ingredient in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as necessary, followed by sterilizing filtration. In general, dispersions are prepared by introducing the active ingredient into a sterile diluent that contains the basic dispersion medium and the necessary other ingredients listed above. In the case of using sterile powders for the preparation of sterile injection solutions, it is preferable

- 97 044115 methods of preparation are vacuum drying and lyophilization, which results in the formation of a powdered substance of the active ingredient, as well as any necessary additional ingredient, from a solution previously sterilized by filtration. Proper fluidity of the solution can be maintained, for example, by coating with a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition a substance that delays absorption, for example, monostearate and gelatin salts.

The active ingredient may be formulated in a dosage form or device that has a controlled release. Examples of such dosage forms and devices include implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparing such dosage forms and devices are known in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Long-acting injectable dosage forms can be prepared by forming microencapsulated drug matrices in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio and the nature of the polymer used, the rate of drug release can be controlled. Other typical biodegradable polymers are polyorthoesters and polyanhydrides. Long-acting injectable dosage forms can also be prepared by incorporating the drug into liposomes or microemulsions.

Additional active compounds may be included in the compositions. In one embodiment, the chimeric protein of the invention is formulated with another coagulation factor or a variant, fragment, analog or derivative thereof. For example, coagulation factor includes, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF ) or activated forms of any of the previous factors. The blood clotting factor of the hemostatic agent may also include antifibrinolytic drugs, for example, aminocaproic acid, tranexamic acid.

Dosing regimens can be adjusted to provide the optimal desired response. So, for example, a single dose can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased, as indicated by the severity of the therapeutic situation. It is advantageous to formulate parenteral compositions in unit dosage form for ease of administration and dose uniformity. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA. 1980).

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

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

For oral administration, the pharmaceutical composition may take the form of tablets or capsules prepared by conventional methods. The composition may also be formulated as a liquid, such as a syrup or suspension. The liquid may contain suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or gum acacia), non-aqueous vehicles (for example, almond oil, fatty esters, ethyl alcohol or fractionated vegetable oils) and preservatives ( for example, methyl or propyl p-hydroxybenzoates or sorbic acid). The preparations may also include flavorings, colors and sweeteners. Alternatively, the composition may be presented as a dry product for reconstitution with water or other suitable carrier.

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

For administration by inhalation, the compounds used in accordance with the present invention are typically delivered as a nebulized aerosol with or without excipients or as an aerosol spray from an aerosol container or nebulizer, in some cases with a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane , carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dose unit can be determined by using a valve to deliver the metered quantity. For use in an inhaler or insufflator, capsules and cartridges may be formulated from, for example, gelatin containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition may also be prepared for rectal administration in the form of a suppository or retention enema, for example containing conventional suppository bases such as cocoa butter or other glycerides.

In one embodiment, the pharmaceutical composition comprises a chimeric protein, a polynucleotide encoding the chimeric protein, a vector containing the polynucleotide, or a host cell containing the vector, and a pharmaceutically acceptable carrier. The FVIII protein in the chimeric protein has a longer half-life compared to wild-type FVIII protein or the corresponding FVIII protein without the VWF fragment. In one embodiment of the invention, wherein 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 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 about 10 times, at least about 11 times, or at least about 12 times longer than that of wild-type FVIII protein. In another embodiment, 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.

In some embodiments, the composition is administered by a route selected from the group consisting of local administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. Parenteral administration may be intravenous or subcutaneous administration.

In other embodiments, the composition is used to treat a hemorrhagic disease or condition in a patient indicated for it. The hemorrhagic disease or condition is selected from the group consisting of bleeding due to a bleeding disorder, hemarthrosis, muscle hemorrhage, oral hemorrhage, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, head injury, gastrointestinal hemorrhage, intracranial hemorrhage, intra-abdominal hemorrhage , intrathoracic hemorrhage, bone fracture, bleeding of the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the sheath of the iliopsoas muscle and any combinations thereof. In other embodiments, the subject is planning to undergo surgery. In yet other embodiments, the treatment is prophylactic or on demand.

V. Gene therapy.

The chimeric protein of the present invention can be produced in vivo in a mammal, such as a human patient, using gene therapy approaches, and may also be therapeutically useful for treating a hemorrhagic disease or disorder selected from the group consisting of bleeding caused by a bleeding disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, head injury, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, bleeding of the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space and bleeding in the sheath of the iliopsoas muscle. In one embodiment of the invention, the bleeding disease or disorder is hemophilia. In another embodiment of the invention, the hemorrhagic disease or disorder is hemophilia A. This involves the introduction of a suitable chimeric protein encoding nucleic acid operably linked to suitable regulatory sequences. In a certain embodiment of the invention, these sequences are included in the viral vector. Suitable viral vectors for such gene therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, and adeno-associated virus (AAV) vectors. . The viral vector may be a replication-defective viral vector. In other embodiments, the adenoviral vector has a deletion in its

- 99 044115

E1 gene or E3 gene. When an adenoviral vector is used, the mammal may not be exposed to the nucleic acid encoding the selectable marker gene. In other embodiments, the sequences are included in a non-viral vector known to those skilled in the art.

VI. Methods of using chimeric protein.

The present invention relates to a method of using the chimeric protein described herein to prevent or inhibit the binding of endogenous VWF to the FVIII protein. The present invention relates to a method of using a chimeric protein having an FVIII protein associated with XTEN and an IG constant region or part thereof.

One aspect of the present invention relates to preventing or inhibiting the interaction of FVIII with endogenous VWF by blocking or shielding the VWF binding site on FVIII from endogenous VWF and at the same time prolonging the half-life of the chimeric protein using the XTEN sequence in combination with the IG constant region or part thereof, which may also increase the half-life. In one embodiment, the invention relates to a method for constructing an FVIII protein having a longer half-life than wild-type FVIII. A chimeric protein that is suitable for use in the method includes any one or more chimeric proteins described herein.

Another embodiment of the present invention includes a method of administering to a subject having an indication for this purpose a chimeric protein comprising an FVIII protein having a longer half-life than that of wild type FVIII, the method comprising administering the chimeric protein described herein to the subject .

In one embodiment, the invention relates to a method of using an XTEN sequence and an IG constant region or portion thereof to prolong the half-life of a chimeric protein comprising an FVIII protein and a VWF protein, thereby preventing or inhibiting the interaction of endogenous VWF with the FVIII protein. The FVIII protein is associated with an XTEN sequence (eg, FVIII(X)) and is then associated or coupled to a VWF protein fused to XTEN and the IG constant region or part thereof, is shielded or protected from the VWF clearance pathway, and therefore has reduced clearance compared to FVIII protein not related to VWF protein. Thus, shielded FVIII protein has the maximum half-life extension compared to FVIII protein unbound or coupled to the XTEN sequence and VWF protein. In some embodiments, the FVIII protein coupled to or protected by the VWF protein and linked to the XTEN sequence is not cleared through the VWF clearance receptor. In some embodiments, FVIII protein coupled to or protected by the VWF protein and linked to the XTEN sequence is cleared from the system more slowly than FVIII protein not coupled to or protected by the VWF protein and linked to the XTEN sequence.

In one aspect, a chimeric protein comprising an FVIII protein linked to an XTEN sequence, or a FVIII protein bound or fused to a VWF protein linked to XTEN, has reduced clearance from the bloodstream because the VWF protein does not contain a binding site for the VWF clearance receptor. The VWF protein prevents or inhibits the clearance of FVIII bound or coupled to the VWF protein from the system through the VWF clearance pathway. VWF proteins useful for the present invention may also provide at least one or more VWF-like protective properties that are provided by endogenous VWF. In some embodiments, the VWF protein or XTEN sequence may also conceal one or more FVIII clearance receptor binding sites, thereby preventing clearance of FVIII through its own clearance pathway.

In some embodiments, preventing or inhibiting the binding of FVIII protein to endogenous VWF via the VWF protein or XTEN sequence may occur in vitro or in vivo.

Also provided is a method of prolonging the half-life of a chimeric protein, comprising administering the chimeric protein described herein to a subject having an indication therefor. The half-life of inactivated FVIII bound or conjugated to full-length VWF is approximately 12-14 hours in plasma. In VWD type 3, in which there is virtually no VWF in the bloodstream, the half-life of FVIII is only about six hours, resulting in mild to moderate hemophilia A symptoms in these patients due to decreased concentrations of FVIII. The half-life of a chimeric protein of the present invention bound or linked to a VWF fragment or an XTEN sequence can become at least about 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2 ,0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, or 4.0 times times longer than the half-life of inactivated FVIII bound or coupled to full-length VWF.

In one embodiment, a chimeric protein comprising a first polypeptide comprising the FVIII protein and a first IG constant region or a portion thereof, and a second polypeptide containing the VWF protein, XTEN, containing less than 288 amino acids, and the IG constant region or a portion thereof

- 100 044115 part, has a half-life of at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times , 6.0 times, 7 times, 8 times, 9 times, or 10 times longer than the corresponding chimeric protein containing the same first polypeptide and a second polypeptide without the wild-type XTEN or FVIII sequence. In another embodiment, a chimeric protein comprising a first polypeptide comprising FVIII protein and a first IG constant region or a portion thereof, and a second polypeptide comprising the VWF protein, XTEN, containing less than 288 amino acids and an IG constant region or portion thereof, has a half-life about 2-5 times, about 3-10 times, about 5-15 times, about 10-20 times, about 15-25 times, about 20-30 times, about 25-35 times, about 30-40 times, about 35-45 times longer than the corresponding chimeric protein containing the same first polypeptide and a second polypeptide without the wild-type XTEN or FVIII sequence. In a particular embodiment, the half-life of the chimeric protein of the present invention becomes 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 in FVIII and VWF double knockout mice.

In certain embodiments of the invention, the chimeric protein exhibits a half-life of about 40 hours in mice.

In some embodiments, the half-life of the chimeric protein is longer than the half-life of FVIII coupled to endogenous VWF. In other embodiments, the half-life of the chimeric protein is at least about 1.5 times, 2 times, 2.5 times, 3.5 times, 3.6 times, 3.7 times, 3.8-fold, 3.9-fold, 4.0-fold, 4.5-fold, or 5.0-fold greater than the half-life of wild-type FVIII or endogenous VWF-bound FVIII protein.

In some embodiments, the result of the invention is that the half-life of the chimeric protein is prolonged compared to FVIII protein without VWF protein or wild-type FVIII. The half-life of the chimeric protein of the 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 at least about 11 times or at least about 12 times longer than the half-life of the chimeric protein without wild-type VWF or FVIII protein. 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 FVIII wild type. In another embodiment, the half-life of FVIII is extended by about 2 times to about 10 times, about 2 times to about 9 times, about 2 times to about 8 times, about 2 times to about 7 times, about 2 times to about 7 times. about 6 times, from about 2 times to about 5 times, from about 2 times to about 4 times, from about 2 times to about 3 times, from about 2.5 times to about 10 times, from about 2.5 times to about 9 times, from about 2.5 times to about 8 times, from about 2.5 times to about 7 times, from about 2.5 times to about 6 times, from about 2.5 times to about 5 times, from about 2 ,5 times to about 4 times, from about 2.5 times to about 3 times, from about 3 times to about 10 times, from about 3 times to about 9 times, from about 3 times to about 8 times, from about 3 times up to 7 times, from about 3 times to about 6 times, from about 3 times to about 5 times, from about 3 times to about 4 times, from about 4 times to about 6 times, from about 5 times to about 7 times or from about 6-fold to about 8-fold compared to wild-type FVIII or FVIII protein without VWF protein. In other embodiments, the half-life of the chimeric protein of the 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 yet other embodiments, the half-life of the chimeric protein of the invention is from about 15 hours to about two weeks, from about 16 hours to about one week, from about 17 hours to about one week, from about 18 hours to about one week, from about 19 hours to about one week, from about 20 hours to about one week, from about 21 hours to about one week, from about 22 hours to about one week, from about 23 hours to about one week, from about 24 hours to about one week, from about 36 hours to about one week, from about 48 hours to about one week, from about 60 hours to about one week, from about 24 hours to about six days, from about 24 hours to about five days, from about 24 hours to about four days, from about 24 hours to about three days, or from about 24 hours to about two days.

In some embodiments, the average half-life of the chimeric protein of the invention per subject is 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 h, about 36 h, about 40 h, about 44 h, about 48 h (2 days), about 54 h, about 60 h, about 72 h (3 days), about 84 h, about 96 h (4 days),

- 101 044115 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.

In addition, the invention provides a method of treating or preventing a hemorrhagic disease or disorder, comprising administering an effective amount of a chimeric protein. In one embodiment of the invention, the hemorrhagic disease or disorder is selected from the group consisting of bleeding caused by a bleeding disorder, hemarthrosis, muscular bleeding, oral bleeding, hemorrhage, muscular hemorrhage, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding and iliopsoas sheath bleeding. In a specific embodiment of the invention, the hemorrhagic disease or disorder is hemophilia A.

A chimeric protein containing an XTEN sequence and an IG constant region or portion thereof in combination with a VWF protein described herein that prevents or inhibits the interaction of the FVIII protein with endogenous VWF produced by the present invention has many uses as will be recognized by one skilled in the art. of the art, including, but not limited to, methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent. In one embodiment, the invention provides a method of treating a subject having a hemostatic disorder, comprising administering an effective amount of a chimeric protein.

The factor FVIII protein portion of the chimeric protein treats or prevents a hemostatic disorder by acting as a cofactor to factor IX on the negatively charged phospholipid surface, thereby forming the Xase complex. Binding of activated blood coagulation factors on the phospholipid surface localizes this process to sites of vascular damage. At the phospholipid surface, factor VIIa increases the maximum rate of activation of factor X by factor IXa by about 200,000-fold, resulting in a second major pulse of thrombin generation.

The chimeric protein of the invention can be used to treat any hemostatic disorder. Hemostatic disorders that can be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, as well as deficiencies or structural abnormalities related to factor VIII. In one embodiment of the invention, the hemostatic disorder is hemophilia A.

The chimeric protein of the invention can be used prophylactically to treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute episode of bleeding in a subject with a hemostatic disorder. In another embodiment, the hemostatic disorder may be the result of a defective coagulation factor, such as von Willebrand factor. In one embodiment of the invention, the hemostatic disorder is an inherited disorder. In another embodiment of the invention, the hemostatic disorder is an acquired disorder. An acquired disorder may result from an underlying secondary disease or condition. A non-disease condition may include, by way of example and not limitation, cancer, an autoimmune disease, or pregnancy. An acquired disorder may result from old age or from medications to treat an underlying secondary disorder (eg, cancer chemotherapy).

The invention also relates to methods of treating a subject who does not have a congenital hemostatic disorder, but has a secondary disease or condition leading to the acquisition of a hemostatic disorder, for example, due to the development of anti-FVIII antibodies or surgery. Thus, the invention relates to a method of treating a subject in need of a general hemostatic agent, comprising administering a therapeutically effective amount of a chimeric protein produced by the present methods.

The present invention also provides methods for reducing the immunogenicity of FVIII or inducing less immunogenicity against FVIII, comprising administering an effective amount of chimeric proteins described herein, or polynucleotides encoding the same.

In one embodiment of the invention, the subject in need of a general hemostatic agent is undergoing surgery or is about to undergo surgery. The chimeric protein of the invention may be administered before, during or after surgery as a prophylactic course of treatment. The chimeric protein of the invention can be administered before, during or after surgery to control an episode of acute bleeding.

The chimeric protein of the invention can be used to treat a subject having an acute bleeding episode and not having a hemostatic disorder. An episode of acute bleeding may occur as a result of severe trauma, such as surgery, a car accident, a wound, a gunshot laceration, or any other traumatic event that results in uncontrolled bleeding. Non-limiting examples of bleeding episodes include:

- 102 044115 bleeding disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, oral hemorrhage, trauma, head injury, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the sheath of the iliopsoas muscle and any combination thereof.

In prophylactic use, one or more compositions containing a chimeric protein of the present invention, or a mixture thereof, is administered to a patient who is not already in a disease state to enhance the patient's resistance or reduce symptoms associated with a disease or disorder. This amount is defined as a prophylactically effective dose. For therapeutic purposes, relatively high doses are sometimes required (eg, about 1 to 400 mg/kg polypeptide per dose, with doses of 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxic drug-modified polypeptides) via relatively short periods of time until disease progression decreases or stops, and until the patient shows partial or complete relief of disease symptoms. The patient can then be placed on a prophylactic regimen.

In some embodiments, the chimeric protein or composition of the invention is used for treatment as needed, which includes treatment of an acute episode of bleeding, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, muscle hemorrhage, intraoral bleeding, trauma, head trauma (cranial brain injury), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, bleeding in the central nervous system, hemorrhage in the retropharyngeal space, hemorrhage in the retroperitoneal space, or bleeding in the iliopsoas muscle sheath. The subject may require surgical prophylaxis, perioperative management, or care for surgery. Such operations include, for example, minor operations, major surgery, tooth extraction, tonsillectomy, inguinal hernia repair, synovectomy, total knee replacement, craniotomy, osteosynthesis, traumatology, intracranial surgery, intra-abdominal surgery, intrathoracic surgery or joint replacement surgery. .

In one embodiment, the chimeric protein of the present invention is administered intravenously, subcutaneously, intramuscularly, or through any mucosal surface, for example, orally, sublingually, buccally, nasally, rectally, vaginally, or inhalation. The chimeric protein containing the VWF and FVIII protein fragment of the present invention can be incorporated into or associated with a biopolymer solid support that allows slow release of the chimeric protein to the site of bleeding or insertion into a bandage/ligation. The dose of the chimeric protein will vary depending on the subject and the specific route of administration. Doses can vary from 0.1 to 100,000 mcg/kg body weight. In one embodiment of the invention, the dosage range is 0.1-1000 μg/kg. In another embodiment of the invention, the dosage range is 0.1500 μg/kg. Protein can be administered continuously or at regular intervals. In vitro tests can be used to determine optimal dose ranges and/or schedules for administration. In vitro tests that measure clotting factor activity are known in the art, for example the STA-CLOT VIIa-rTF clotting test or the ROTEM clotting test. In addition, effective doses can be extrapolated from dose-response curves obtained in animal models, such as the hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are hereby included for purposes of illustration only and are not intended to limit the present invention. All patents, publications and articles referenced herein are expressly and specifically incorporated herein by reference.

Examples

The following materials and methods were used in all examples unless otherwise noted.

Materials and methods.

In general, unless otherwise indicated, the practice of the present invention uses conventional methods of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (particularly, for example, antibody technology), and standard electrophoresis techniques. See, for example,

- 103 044115

Sambrook, Fritsch and

Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody

Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996);

Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty,

Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow cocoaem.., CS.H.L. Press,

Pub. (1999); and Current Protocols in Molecular Biology, ed. Ausubel co coaem.., John

Wiley & Sons (1992).

Example 1. FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers.

The present invention relates to the production of a chimeric FVIII molecule linked to the D'D3 domain of von Willebrand factor (VWF) protein via the Fc domain of IgG. The docked D'D3 domain prevents FVIII from interacting with endogenous VWF multimers. This molecule serves as a platform to incorporate other half-life extension technologies to improve the pharmacokinetics of the chimeric protein. XTEN sequences were included in the FVIII B domain and between D'D3 and the Fc region to increase the half-life of the FVIII/VWF heterodimer.

The thrombin cleavage site between D'D3 and Fc mediates the release of the D'D3 domain upon activation of the FVIII molecule by thrombin.

Example 2. Plasmid construction of heterodimers FVIII-XTEN-Fc/D'D3-Fc Cloning of the triple mutation FV050-IHH into FV031.

The IHH triple mutation in Fc prevents interaction with FcRn, so the Fc-containing molecule is not recycled through the FcRn pathway. The three mutations in Fc are I253A, H310A, H435A.

FB050 was created by replacing the Fc region of plasmid FB031 with an Fc fragment containing a triple IHH mutation between the RsRII and Not 1 restriction sites.

Cloning FV057- Cloning FV-Fc with 144 AE XTEN +35 a.k. thrombin-cleavable linker.

Oligonucleotides.

ESC 155-Oligonucleotide for 144 AE XTEN in FV034-rev

CCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCC

ACCGGAACCTCCACCGCCGCTCGAGGCACCTTCTTCAGTGCTGGTGGGCGAGCCCGC

TGGTGACCCTTCCTC

ESC 155-Oligonucleotide for 144 AE XTEN-GS linker in FV034-rev

GGGGAAGAGGAAGACTGACGGTCCGCCCAGGAGTTCTGGAGCTGGGCACGG

TGGGCATGTGTGAGTTTTGTCGCCTCCGCTGCCCCGGGGGACCAGGGATCCCCCGCC

ACCGGATCCCCCGCCACCGGATCCCCCGCCACCGGATCCCCCGCC

ESC 157-Oligonucleotide for 144 AE XTEN in FV031-Fwd

GTGAAGCCTGCCAGGAGCCGATATCGGGCGCGCAACATCAGAGAGCGCCA

CCCCTGAAAGTGGTCCCGGGAGCGAGCCAGC

To receive 144 AE-XTEN + 35 a.k. The GS linker with the thrombin cleavage site was PCR performed twice.

The first PCR reaction was carried out using 144-AE XTEN coding DNA as a template and the primer pair ESC157/ESC155. The PCR product obtained as a result of this reaction is approximately 550 bp in length. was used as a template for the second PCR reaction, and was amplified using the primer pair ESC 157/156. This reaction yielded a product of ~700 bp. This PCR product is 700 bp long. and plasmid FV034 were then treated with EcoRV-HF and RsRII. Plasmid backbone from split.

VWF034 was then used to ligate 700 bp. PCR product.

Cloning of the triple mutation FV058-IHH into FV034.

The IHH triple mutation in Fc prevents interaction with FcRn, so the Fc-containing molecule is not recycled through the FcRn pathway. The three mutations in Fc are I253 A, H310A, H435 A.

FV058 was created by replacing the Fc region of plasmid FV034 with an Fc fragment containing a triple IHH mutation between the RsRII and Not 1 restriction sites.

Cloning of FVIII-263-FVIII 205 with triple IHH mutation.

The IHH triple mutation in Fc prevents interaction with FcRn, so the Fc-containing molecule is not recycled through the FcRn pathway. The three mutations in Fc are I253A, H310A, H435A.

FVIII-263 was created by replacing the Fc region of the FVIII 205 plasmid with an Fc fragment containing

- 104 044115 triple mutation IHH between the RsRII and Not 1 restriction sites.

Cloning of FVIII-282-FVIII-Fc with 144 AE XTEN in domain B.

ESC 158-Oligonucleotide for 144 AE XTEN in the B-fwd domain

AAGAAGCTTCTCTCAAAACGGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTG

GTCCCGGGAGCGAGCCAGCCACATCTGGGTCGGAAACGCCAGGC

ESC 159-Oligonucleotide for 144 AE XTEN in the B-rev domain

GGTATCATCATAATCGATTTCCTCTTGATCTGACTGAAGAGTAGTACGAGTTATTTC

AGCTTGATGGCGTTTCAAGACTGGTGGGCTCGAGGCACCTTCTTCAGTGCTGGTGGG

CGAGCCCGCTGGTGACCCTTCCTCAGTGGACGTAGG

The first PNR reaction was carried out using 144-AE XTEN coding DNA as a template and the primer pair ESC158/ESC159. From this reaction a PCR product of approximately 550 bp is obtained, and the FVIII 169 plasmid is then processed with ASCii and Cla1. The plasmid backbone from the digested FVIII 169 is then used for 550 bp ligation. PCR product to obtain FVIII 282.

Cloning of FVIII-283-FVIII169 with triple IHH mutation.

The IHH triple mutation in Fc prevents interaction with FcRn, so the Fc-containing molecule is not recycled through the FcRn pathway. The three mutations in Fc are I253A, H310A, H435A.

FVIII-283 was created by replacing the Fc region of plasmid FVIII 169 with an Fc fragment containing a triple IHH mutation between the RsRII and Not 1 restriction sites.

Example 3. Production of FVIII-XTEN-Fc/D'D3-XTEN-Fc in HEK293 cells.

Fig. 2. Schematic representation illustrating the expression of the FVIII-XTENFc/D'D3-XTEN-Fc construct. Using Polyethylenimine (PEI), three plasmids were contransfected into HEK293 cells. The first plasmid expresses FVIII-XTEN-Fc, the second plasmid expresses D1D2D'D3-XTEN-Fc, and the third plasmid expresses PACE/furin, which is required to enzymatically remove the propeptide, i.e., the D1D2 domain from D1D2D'D3-XTEN-Fc. The products of this three-plasmid expression system include the FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimer, the D'D3-XTEN-Fc homodimer, and traces of the hemizygous-appearing FVIII-XTEN-Fc species.

Example 4. Purification of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers.

In order to purify the FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers for the first 10-fold concentration of the conditioned medium, a tangential flow filtration (TFF) step is used. The products in the filtrate are then further purified using affinity chromatography followed by a desalting column. The purity of the molecule is acceptable by size exclusion HPLC and is further confirmed by Western blotting. The specific activity of the molecule is comparable to the B domain of deleted FVIII, as measured by the FVIII activity assay (Example 5) and OD280 measurement.

Example 5. Specific activity of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers.

The activity of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers is measured using the FVIII chromogenic assay and the FVIII activated partial thromboplastin time (aPTT) assay. The specific chromogenic activity and specific APTT activity of SQ BDD-FVIII, rFVIШ69/FV034 and rFVIШ169/FV057 are presented in Table. 16. Compared with SQ BDD-FVIII, we observed comparable specific chromogenic activities and a 60% reduction in specific aPTT activity for rFVIIII69/FV034 and rFVIIII169/FV057.

Table 16

Specific activity of heterodimeric variants

FVIII SQ BDD-FVIII rFVIII169/OB034 rFVIII160/OB057 Specific chromogenic activity (IU/pmol) 0.9 - 2.0 1.1-1.2 0.8-1.6 Specific activity APTT (IU/pmol) 0.75-1.7 0.4 0.3-0.6

Chromogenic analysis of FVIII.

FVIII activity was measured using the COATEST SP FVIII kit from DiaPharma (products

- 105 044115 #: K824086), all incubations were performed on a 37°C plate heater with shaking.

The WHO Eighth International Standard for Blood Clotting Factor VIII:C, Concentrate, code name 07/350 is used as the assay standard, the range of the standard was from 100 mIU/ml to 0.78 mIU/ml. Normal human plasma pool assay control and test samples (diluted in 1X Coatest buffer) are added to 2HB 96-well Immulon plates in duplicate (25 µl/well). Freshly prepared IXa/FX/Phospholipid mixture (50 μl), 25 μl of 25 mM CaCl ₂ , and 50 μl of FXa substrate were sequentially added to each well with a 5-minute incubation between each addition. After incubation with the substrate, 25 µl of 20% acetic acid was added to complete the color reaction, and the OD ₄₀₅ absorbance was measured using a SpectraMax plus (Molecular Devices). Data were analyzed using SoftMax Pro software (version 5.2). The lower limit of quantification (LOQ) is 7.8 mIU/ml.

APTT FVIII analysis.

APTT FVIII analysis was performed on a Sysmex CA-1500 coagulation analyzer as follows: First, 50 μl of manually diluted samples, standards and Controls in APTT buffer (50 mM Tris, 100 mM NaCl, 1% HSA, pH 7.4) was added by the instrument to the reaction cuvette , followed by the addition of 50 μl of FVIII-deficient plasma (George King Bio-Medical, product #: 0800). After incubation at 37°C for 1 min, 50 µl of APTT reagent (activated cephaloplastin reagent Actin® FSL - Dade Behring, reference # B4219-2) was added to the reaction mixture, and incubated at 37°C for 4 min . Subsequently, 50 µl of 20 mM CaCl ₂ (Dade Behring, reference #ORFO37) was added and the reaction cuvette was immediately transferred to one of the four positions of the spectrophotometer channel to measure the amount of refracted light in the mixture, which, using the instrument's software algorithm, was converted into the occurrence of clotting . The recorded clotting time was the length of time from the addition of CaCl2 to the onset of clot formation. The assay standard is generated by diluting the WHO Eighth International Standard FVIII in aPTT buffer ranging from 100 mIU/mL to 0.78 mIU/mL. The calibration curve was plotted as clotting time (in seconds) on the Y-axis versus the logarithm (base 10) of FVIII activity (mIU/mL) on the X-axis in MS Excel, with the activity of individual samples calculated using the formula for linear regression of this calibration curve. Based on the assay, the lower limit of quantitation (LOQ) was 7.8 mIU/mL.

Example 6: Additive effect of XTEN insertions on prolonging heterodimer half-life.

XTEN inserts were incorporated into heterodimers to prolong the half-life. Insertion of a single amino acid (aa) 288 of AE-XTEN in the B domain of FVIII results in a heterodimer half-life of 16.7 hours in hemophilia A mice, as demonstrated by rFVΠΠ69/FV031 in FIG. 3. To further improve the half-life of the heterodimer, a second 144 aa long XTEN insert. or 288 a.k. was included in FVIII169/OB031 both in the FVIII A1 domain and immediately downstream of the D'D3 fragment, respectively, the heterodimer variants were named FVIII205/OB031 and FVII169/OB034.

The half-lives of rFVIIII69/FV031, rFVIIII205/FV031 and rFVIIII69/FV034 were assessed in FVIII-deficient (HemA) mice by single intravenous administration of the test molecules at a dose of 200 IU/kg. Plasma samples were collected at specific time points as shown in FIG. 3, FVIII activity of the samples was determined using the FVIII chromogenic assay; PK parameters were calculated using the WinNonlin-Phoenix program and listed in Table. 17.

As shown in FIG. 3 and table. 17, addition of a second XTEN insert in both the A1 domain of FVIII and downstream of D'D3 further improves the half-life of the heterodimer by 29.45 or 31.10, respectively. In addition, both XTEN inserts demonstrated a greater than 2-fold improvement in clearance and AUC.

Table 17

PK parameter of HemA mouse heterodimers

FVIII XTEN inserts T 1/2 (h) MRT (h) C1 (ml/h/kg) Vss (ml/kg) AUC_D (kg*h/ml) Installation 1 Embed 2 rFVIII169/OB031 B*-AE288 16.65 18.44 3.57 85.72 0.28 rFVIII205/OB031 B*-AE288 A1-AE144 29.45 36.02 1.76 63.56 0.57 rFVIII169/OB034 B*-AE288 D'D3- AE288 31.10 34.57 1.73 59.77 0.58

Example 7. 144 a.k. AE-XTEN provides a better half-life than 288 aa. AE-XTEN when inserted between the D'D3 and Fc domains.

Another heterodimer, FVIIII69/FV057, was constructed in an attempt to determine the optimal

- 106 044115 length of the XTEN insertion in the D'D3-XTEN-Fc chain, in which the length of the XTEN insertion was reduced from 288 aa. up to 144 a.k. As shown in FIG. 4, compared to rFVΠΠ169/FV034, the half-life of gFVΠΠ69/FV057 was increased from 31 hours to 42 hours. Improved clearance and AUC were also observed for gFVΠΠ69/FV057, data are shown in table. 18. Thus, when inserted between D'D3 and the Fc domain of the FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers, 144 aa. insert AE-XTEN is more optimal than AE-288 a.k. XTEN.

Table 18

PK parameters of gFVSh169/FV034 and gFVSh169/FV057 in HemA mice

FVIII T 1/2 (h) MRT (h) C1 (ml/h/kg) Vss (ml/kg) AUC_D (kg*h/ml) GRUSH169/FV034 31.10 34.57 1.73 59.77 0.58 rFVIIIl 69/FV057 42.23 53.24 0.97 51.44 1.03

Example 8: The Fc domain prolongs the half-life of the heterodimer.

Fc domains extend the half-life of their fusion proteins through the FcRn-mediated recycling pathway. To confirm the requirement of the Fc domain for prolonging the half-life of the heterodimer, the wild-type Fc domains were replaced by a triple mutant (I253A/H310A/H435A; IHH) in gFVIII205/FV031 to form gFVIII263/FV050, complete elimination of FcRn binding was confirmed by Surface assay. Plasmonic Resonance (Biacor) for gFVSh263/FV050. The half-life of FVIII263/FV050 was assessed in HemA mice compared with gFVIII205/FV031. As shown in FIG. 5 and in table. 19, an improved clearance rate, as well as a reduced half-life and AUC, were observed for gFVIII263/FV050. This result demonstrated that in addition to mediating covalent binding of FVIII and D'D3, Fc domains are also required to improve the half-life of the heterodimer.

Table 19

PK parameters of gFVSh205/FV031 and gFVSh263/FV040 in HemA mice

FVIII Mutation in the Fc domain t 1/2 (h) MRT (h) C1 (ml/h/kg) Vss (ml/kg) AUC_D (kg*h/ml) GRUSH205/FV031 Absence 29.45 36.02 1.76 63.56 0.57 GRUSH263/FV050 IHH 22.96 26.15 2.36 61.69 0.42

Example 9. Instantaneous efficacy of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers in the HemA mouse tail bleeding model.

The immediate efficacy of the leading heterodimer candidates was assessed using the HemA mouse tail bleeding model.

Male HemA mice, 8-12 weeks of age, were randomized into 4 treatment groups and received one intravenous injection of SQ BDD-FVIII, gFVΠΠ69/FV034, gFVIII169/FV057, or vehicle solution, respectively. In order to simulate episodic FVIII treatment (to recreate 50-100% of normal plasma FVIII levels), the selected FVIII treatment dose is 75 IU/kg, as measured by FVIII aPTT activity. At this dose level, all FVIII variants tested would reproduce ~70% of normal murine plasma FVIII activity within 5 min of dosing.

The amount of blood loss from each individual animal in the study is plotted in FIG. 6. A significant reduction in blood loss was observed for all FVIII treatment groups compared to vehicle-treated animals. Within the three FVIII treatment groups, no statistically significant differences were found in the reduction of blood loss, suggesting that heterodimer molecules could potentially be as effective as SQ BDD-FVIII for treatment when needed.

The amount of blood loss from each individual animal in the study is plotted in FIG. 6. A significant reduction in blood loss was observed for all FVIII treatment groups compared to vehicle-treated animals. Within the three FVIII treatment groups, no statistically significant differences were found in the reduction of blood loss, suggesting that heterodimer molecules could potentially be as effective as SQ BDD-FVIII for treatment when needed.

In addition, HemA mice were treated with a lower dose (37.5 IU/kg) of rBDD-FVIII or FVIII169/FV034, the results are illustrated in Fig. 6B. As at 75 IU/kg, gFVΠΠ69/FV034 provided the same protection in HemA mice after tail injury as BDD-FVIII, showing that the molecule is still effective in treating severe bleeding episodes at ~35% of normal circulating murine FVIII levels in HemA mice.

- 107 044115

The tail injury procedure was carried out as follows.

Briefly, mice were anesthetized with a 50 mg/kg ketamine/0.5 mg/kg dexmedetomidine cocktail until the onset of tail injury and placed on a 37°C electric heating pad to help maintain body temperature. The tails of the mice were then immersed in 37°C saline for 10 min to dilate the lateral vein. Once the vein was dilated, FVIII variants or vehicle solution was injected via the tail vein, and 5 min after dosing, the distal 5 mm of the tail was cut away using a #11 straight edge scalpel. The spilled blood was collected in 13 ml of 37°C saline solution for 30 min, and the volume of blood loss was determined by changing the weight of the blood collection tube: volume of blood loss = (final weight of the blood collection tube - initial weight +0.10) ml. Statistical analysis was performed using t-test (Mann-Whitney test) and one of the analysis of variance methods (Kruskal-Wallis test, post hoc: Dunn's multiple comparison test).

Example 10. Preventive efficacy of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers in the HemA mouse cut tail vein bleeding model.

The preventive efficacy of FVIII169/FV057 was tested in the HemA mouse tail vein transection (TVT) model. The TVT model induces bleeding by damaging the lateral vein of the mouse's tail, an injury that mimics spontaneous bleeding episodes in patients with hemophilia (a bleeding disorder).

Male HemA mice, 8–10 weeks old, were randomized into 4 treatment groups and treated with either FVIII169/FV057 72 h before tail vein injury or SQ BDD-FVIII 24 h or 48 h before injury. Animals that were treated with vehicle were used as negative controls. On the graph, in Fig. Figure 7 shows cases of rebleeding or euthanasia due to excessive blood loss within 24 hours after injury.

As illustrated in FIG. 7, in contrast to mice treated with SQ BDD-FVIII 48 h before TVT, which showed only limited protection after injury, mice treated with rFVIII169/FV057 72 h before tail injury had similar protection against rebleeding and survival compared with mice receiving SQ BDD-FVIII treatment 24 h before TVT, which means that rFVIII169/FV057 can provide at least 3-fold or greater (eg, 4-fold) long-term protection in HemA mice in the TVT model. Thus, FVIII169/FV057 may significantly reduce the treatment frequency of current FVIII prophylaxis.

Similarly, HemA mice were treated with FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimers: rFVIII169/FV034 and rFVIII169/FV057 24 h or 96 h before tail vein injury. Treatment data for rebleeding and survival were compared with data for BDD-FVIII treatment 24 or 48 hours before injury and vehicle. While rebleeding in rBDD-FVIII-treated mice within 24 hours before tail vein injury was similar to that in vehicle-treated mice, data on rebleeding in mice treated with heterodimers 24 hours before injury are significantly better. than in the vehicle treatment group. In addition, rebleeding data from heterodimer-treated mice 96 h before injury were comparable to mice treated with rBDD-FVIII 24 h before injury. Regarding the survival rate 24 h after TVT injury, in contrast to the requirement of less than 50% survival rate of mice treated with rBDD-FVIII, more than 90% of mice survived after TVT injury with treatment with FVIII-XTEN-Fc/D'D3- heterodimers XTEN-Fc when FVIII molecules were administered 24 h before injury. In addition, survival in mice treated with FVIII-XTEN-Fc/D'D3XTEN-Fc heterodimers 96 hours before tail vein injury was better (in the case of rFVIII169/FV034) or comparable (in the case of rFVIII169/FV057) compared with mice , treated with rBDD-FVIII 24 hours before injury. Both rebleeding and survival data indicated a 4-fold prolongation efficacy of FVIII-XTEN-Fc/D'D3-XTEN-Fc heterodimer treatment compared with rBDD-FVIII treatment.

HemA mouse cut tail vein model.

The procedure for cutting the caudal vein of the tail was carried out as follows. Mice were anesthetized with a cocktail containing 50 mg/kg ketamine, 0.125 mg/kg dexmedetomidine, and 0.1 mg/kg buprenex. With an adequate depth of anesthesia, the lateral tail vein of the mice was cut with a number 11 straight edge surgical scalpel in the area where the tail diameter is approximately 2.7 mm. To ensure clear observation of the wound, spilled blood was washed off with warm saline. Treated mice were then housed once in a clean cage with white paper bedding for the next 24 hours. Up to 12 hours after tail injury, mice experienced repeated tail bleeding and physical activity. Dying mice were immediately euthanized and final observation was made 24 h after tail injury. To simulate the bleeding situation in patients with hemophilia and ensure complete recovery of the animal from anesthesia, 1 mg/kg atipamezole solution was provided to reverse the effect of dexmedetomidine at the beginning of the tail vein dissection procedure. For nighttime analgesia, an additional dose of 0.1 mg/kg buprenex was administered at the end of the 12-hour observation period. Survival curve of time to rebleeding and

- 108 044115 time to euthanasia was generated for data analysis, and the log-rank (Cox-Mantel) test was used for statistical evaluation.

Example 11. Preparation of FVIII169/FV059 and other constructs Cloning pSYN FVIII 310.

A synthetic DNA fragment flanked by a BamH1 site at the N-terminus and a Cla1 site at the C-terminus was commercially produced. This synthetic DNA was used to replace the BamHl region with Cla1 in the pSYN FVIII 169 construct (SEQ ID NO: 155). Both synthetic DNA and pSYN FVIII 169 DNA were double-digested using BamH1 and Cla1, and the digested synthetic DNA was incorporated into digested pSYN FVIII 169 to create pSYN FVIII 310 (SEQ ID NO:168; Table 20).

Cloning of pSYN FVIII 312.

A synthetic DNA fragment flanked by a BamH1 site at the N-terminus and an Afe 1 site at the C-terminus was commercially produced. This synthetic DNA was used to replace BamH1 with the Afe1 region in the pSYN FVIII 169 construct (SEQ ID NO: 155). Both synthetic DNA and pSYN FVIII 169 DNA were double-digested using BamH1 and Afe1, and the digested synthetic DNA was incorporated into digested pSYN FVIII 169 to create pSYN FVIII 312 (SEQ ID NO: 169; Table 20). pSYN FVIII 312A (SEQ ID NO: 2; Table 20) was created from pSYN FVIII312 to remove the AscI site encoding the amino acid residue gap at the junction of FVIII and XTEN.

Table 20

Synthetic FVIII constructs

Construct Protein sequence pSYN FVIII 169 PRSFSQNGAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPASSPP VLKRHQAEITR (SEQ ID NO: 167) (Underlined = XTEN residues; not underlined = FVIII residues) pSYN FVIII 310 PRSFGAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPASSEITR (SEQ ID NO: 168) (Underlined = XTEN residues; not underlined = FVIII residues) pSYN FVIII 312 PRSFSONGAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPASSEI

- 109 044115

TR (SEQ ID NO: 169) (Underlined = XTEN residues; not underlined = FVIII residues) pSYN FVIII 312A PRSFSONGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPASSEITR (SEQ ID NO: 2) (similar sequence to pSYNFVIII 312, only the residues corresponding to the Asci site, i.e. the break, have been removed) (Underlined = XTEN residues; not underlined = FVIII residues)

Cloning of pSYN VF059 and VF073.

Various synthetic DNA fragments were generated encoding different linker regions between DD3-XTEN and Fc. These synthetic DNA fragments are flanked by an ASC1 site at the N-terminus and a Not 1 site at the C-terminus. These synthetic DNAs were used to replace the Asc1 region with Not1 in the pSYN FB057 construct (SEQ ID NO: 152). The FV059 pSYN construct (Table 21) contains a linker region (SEQ ID NO: 13) that includes the entire FVIII acidic region 2(a2). This site is reported to be cleaved by thrombin and upon FVIII activation to release DD3XTEN. The FV073 pSYN construct (Table 21) contains only the thrombin cleavage site of the FVIII acidic region 2 (a2) (ie, IEPRSFS) (SEQ ID NO: 23). Both synthetic DNA and pSYN FB057 DNA were double-digested using Asc1 and Not1. The digested synthetic DNA was inserted into the digested pSYN VWF057 to create pSYN VWF059 and pSYN VWF073. The FB59A pSYN construct (Table 21) was generated from FB059 pSYN by removing the EcoRV restriction site. The heterodimeric proteins FVIIII69/FV057 and FVIIII69/FV059 were created by coexpression of FVIII169 and FVIII169 and VF057 or VF059 in HEK293 cells.

- 110 044115

Table 21

Synthetic VWF constructs - cleavable linker regions

Construct Protein sequence pSYN FV057 TSTEEGASSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSLVPR GSGGDKTH (SEQ ID NO: 12) The italicized and underlined sequence shows the GS linker and the LVPR thrombin cleavage site (also shaded). pSYN FV059 TSTEEGASISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSDKTH (SEQ ID NO: 13) The italicized and underlined sequence shows 32 aa. from acidic region 2 of FVIII (a2). The shaded sequence shows the thrombin cleavage site used in pSYN FB059A. pSYN FV059A TSTEEGASSDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSDKTH (SEQ ID NO: 22) The italicized and underlined sequence shows 32 aa. from acidic region 2 of FVIII (a2). This sequence is similar to that of VF059, except that the residues corresponding to the EcoRV site (ie, IS) are deleted. pSYN FV073 TSTEEGASSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSIEPRSFSGSG GDKTH (SEQ ID NO: 23) The italicized and underlined sequence shows the GS linker with a truncated thrombin cleavage site from FVIII acidic region 2 (shaded 7 amino acids-IEPRSFS).

Example 12 Thrombin cleavage of FVIII heterodimer to assay release of D'D3 from Fc.

Two heterodimeric FVIII proteins were tested in thrombin cleavage experiments and their levels of thrombin cleavage were also tested. The two heterodimer constructs used in this experiment were the FVIII169/FV057 heterodimer and the FVIIII69/FV059 heterodimer plus FVIIIFc. The heterodimers FVIIII69/FV057 and FVIIH69/OB059 are described above. Three cleavage reactions were carried out: i) FVIIIFc ii) FVIII169^B057 (Fig. 11), and iii) FVIII 169/FB059 (Fig. 12). Test samples were treated with human α-thrombin at a molar ratio of approximately 22:1 FVIII:tpom6uh. Each reaction was incubated in a water bath at 37°C. At each specified time point (t=5, 15, 30, 45, 60 min), a 22.5 µL sample was removed, stopped with 22.5 µL of 2xSDS dye under non-reducing conditions, and heated for 3 min. The digested protein was then run on an SDS-PAGE gel. Western blotting was performed using anti-FVIII heavy chain antibody (GMA012) and anti-VWF-D3 antibody (Ab 96340) using the LICOR system.

As shown in FIG. 11, exposure of FVIII169/FV057 to thrombin results in a gradual decrease in the detectable level of D'D3-XTEN-Fc, correlating with an increase in the level of D'D3-144 XTEN cleaved product. After 15 minutes, uncleaved FVШ169/FV057 remains. On the other hand, FIG. 12 shows that FVIII 169/VWF059 is rapidly cleaved by thrombin, as evidenced by the virtually undetectable uncleaved FVIII 169/VWF059 after 5 minutes. Accordingly, FVIII 169/VWF059, compared to FVIII 169/VFW057, shows better release of D'D3 from Fc upon thrombin activation.

To investigate thrombin cleavage, parallel experiments were performed using mass spectroscopy (MS). Using MS, FVIII 169/VF059, compared to FV057, again showed better release of D'D3 from Fc.

Example 13: In vivo determination of FVIII169/FV059 in HemA mice.

To further evaluate the pharmacokinetic profile and in vivo efficacy of FVIII169/FV059,

- 111 044115 HemA mice were treated with FVIII 169/FV059 by intravenous administration at a dose of 150 IU/kg. Plasma samples were collected by vena cava blood draw at 5 min, 24, 48, 72, 96, and 120 h postinjection. FVIII activity in plasma samples was measured using the FVIII chromogenic assay, and PK parameters were calculated using Phoenix software. As illustrated in table. 22, compared to FVIII169/®B057, FVIII 169/®B059 exhibited a similar PK profile, indicating that the thrombin a2 cleavage linker does not negatively impact the PK profile of the heterodimer.

Table 22

PK profile of FVIII 169/FV057 and FVIII 169/FV059 in HemA mice

Heterodimer Tl/2 (h) AUC/D (h*kg*mIU/ml/mIU) Cl (ml/h/kg) MRT (h) Vss (ml/kg) ΡνΐΙΙ169/ΦΒ057 38.53 0.80 1.26 44.92 56.38 ΡνΐΙΙ169/ΦΒ059 40.51 0.74 1.35 49.22 66.26

The immediate efficacy of FVIII 169/FV059 was assessed in the HemA mouse tail model (described in Example 9), compared to wild type BDD-FVIII. HemA mice were treated with 75 IU/kg of either FVIII 169/FV059 or BDD-FVIII, and the amount of blood loss from each experimental mouse was plotted in FIG. 13. Compared with BDD-FVIII, FVIII169/®B059 provided the same degree of protection in HemA mice (p=0.9883), indicating that ΡνΐΙΙΙΙ169/ΦΒ059 is fully functional in vivo.

Plasmid construction of FVIII-XTEN-Fc/D'D3-Fc heterodimers.

Nucleotide sequence of FB031 (SEQ ID NO: 147).

ATGAT TCCTG CCAGA TTTGC CGGGG TGCTG CTTGC TCTGG CCCTC ATTTT

GCCAG GGACC CTTTG TGC AG AAGGA ACTCG CGGCA GGTC A TCC AC GGCCC

101 GATGC AGCCT TTTCG GA AGT GACTT CGTC A AC ACC TTTGA TGGG A GC ATG

151 TACAG CTTTG CGGGA TACTG CAGTT ACCTC CTGGC AGGGG GCTGC CAGAA

201 ACGCT CCTTC TCGAT T ATTG GGGAC TTCC A GA ATG GCA AG AGAGT GAGCC

251 TCTCC GTGTA TCTTG GGGAA TTTTT TGACA TCCAT TTGTT TGTCA ATGGT

301 ACCGT GACAC AGGGG GACCA AAGAG TCTCC ATGCC CTATG CCTCC AAAGG

351 GCTGT ATCTA GAAAC TGAGG CTGGG TACTA CAAGC TGTCC GGTGA GGCCT

401 ATGGC TTTGT GGCCA GGATC GATGG CAGCG GCA AC TTTCA AGTCC TGCTG

451 TCAGA CAGAT ACTTC AACAA GACCT GCGGG CTGTG TGGCA ACTTT AACAT

501 CTTTG CTGAA GATGA CTTTA TGACC CAAGA AGGGA CCTTG ACCTC GGACC

551 CTTAT GACTT TGCCA ACTCA TGGGC TCTGA GCAGT GGAGA ACAGT GGTGT

601 GAACG GGCAT CTCCT CCCAG CAGCT CATGC A AC AT CTCCT CTGGG GA A AT

651 GCAGA AGGGC CTGTG GGAGC AGTGC CAGCT TCTGA AGAGC ACCTC GGTGT

701 TTGCC CGCTG CCACC CTCTG GTGGA CCCCG AGCCT TTTGT GGCCC TGTGT

751 GAGAA GACTT TGTGT GAGTG TGCTG GGGGG CTGGA GTGCG CCTGC CCTGC

801 CCTCC TGGAG TACGC CCGGA CCTGT GCCCA GGAGG GAATG GTGCT GTACG

851 GCTGG ACCGA CCACA GCGCG TGC AG CCCAG TGTGC CCTGC TGGTA TGGAG

901 TAT AG GCAGT GTGTG TCCCC TTGCG CC AGG ACCTG CCAGA GCCTG C AC AT

951 CAATG AAATG TGTCA GGAGC GATGC GTGGA TGGCT GCAGC TGCCC TGAGG

1001 GACAG CTCCT GGATG AAGGC CTCTG CGTGG AGAGC ACCGA GTGTC CCTGC

1051 GTGCA TTCCG GAAAG CGCTA CCCTC CCGGC ACCTC CCTCT CTCGA GACTG

1101 C A AC A CCTGC ATTTG CCGA A AC AGC C AGTG GATCT GCAGC A ATGA AGA AT

1151 GTCCA GGGGA GTGCC TTGTC ACTGG TCAAT CCCAC TTCAA GAGCT TTGAC

- 112 044115

1201 A AC AG AT ACT TC ACC TTC AG TGGGA TCTGC C AGT A CCTGC TGGCC CGGGA

1251 TTGCC AGGAC CACTC CTTCT CCATT GTCAT TGAGA CTGTC CAGTG TGCTG 1301 ATGAC CGCGA CGCTG TGTGC ACCCG CTCCG TCACC GTCCG GCTGC CTGGC 1351 CTGC A C A AC A GCCTT GTGA A ACTGA AGC AT GGGGC AGGAG TTGCC ATGGA 1401 TGGCC AGGAC ATCCA GCT CC CCCTC CTGAA AGGTG ACCTC CGCAT CCAGC 1451 ATASA GTGAC GGCCT CCGTG CGCCT CAGCT ACGGG GAGGA CCTGC AGATG 1501 GACTG GGATG GCCGC GGGAG GCTGC TGGTG AAGCT GTCCC CCGTC TATGC

1551 CGGGA AGACC TGCGG CCTGT GTGGG A ATT A C A ATG GCA AC C AGGG CGACG 1601 ACTTC CTTAC CCCCT CTGGG CTGGC GGAGC CCCGG GTGGA GGACT TCGGG 1651 AACGC CTGGA AGCTG CACGG GGACT GCCAG GACCT GCAGA AGCAG CACAG 1701 CGATC CCTGC GCCCT CAACC CGCGC ATGAC CAGGT TCTCC GAGGA GGCGT 1751 GCGCG GTCCT GACGT CCCCC ACATT CGAGG CCTGC CATCG TGCCG TCAGC 1801 CCGCT GCCCT ACCTG CGGAA CTGCC GCTAC GACGT GTGCT CCTGC TCGGA 1851 CGGCC GCGAG TGCCT GTGCG GCGCC CTGGC CAGCT ATGCC GCGGC CTGCG 1901 CGGGG AGAGG CGTGC GCGTC GCGTG GCGCG AGCCA GGCCG CTGTG AGCTG 1951 AACTG CCCGA AAGGC CAGGT GT ACC TGCAG TGCGG GACCC CCTGC AACCT 2001 GACCT GCCGC TCTCT CTCTT ACCCG GATGA GGAAT GCA AT GAGGC CTGCC 2051 TGGAG GGCTG CTTCT GCCCC CCAGG GCTCT ACATG GATGA GAGGG GGGAC 2101 TGCGT GCCCA AGGCC CAGTG CCCCT GTTAC TATGA CGGTG AGATC TTCCA 2151 GCCAG A AGAC ATCTT CTC AG ASSAT CAC AC C ATGT GCTAC TGTGA GGATG 2201 GCTTC ATGCA CTGTA CCATG AGTGG AGTCC CCGGA AGCTT GCTGC CTGAC 2251 GCTGT CCTCA GCAGT CCCCT GTCTC ATCGC AGCAA AAGGA GCCTA TCCTG 2301 TCGGC CCCCC ATGGT CAAGC TGGTG TGTCC CGCTG ACAAC CTGCG GGCTG 2 351 A AGGG CTCGA GTGT A CCA A A ACGTG CC AGA ASTAT GACCT GGAGT GC ATG 2401 AGCAT GGGCT GTGTC TCTGG CTGCC TCTGC CCCCC GGGCA TGGTC CGGCA 2451 TGAGA ACAGA TGTGT GGCCC TGGAA AGGTG TCCCT GCTTC CATCA GGGCA 2501 AGGAG TATGC CCCTG GAGAA ACAGT GAAGA TTGGC TGCAA CACTT GTGTC 2551 TGTCG GGACC GGAAG TGGAA CTGCA CAGAC CATGT GTGTG ATGCC ACGTG 2601 CTCCA CGATC GGCAT GGCCC ACT AC CTCAC CTTCG ACGGG CTCAA AT ACC 2651 TGTTC CCCGG GGAGT GCCAG TACGT TCTGG TGCAG GATTA CTGCG GCAGT 2701 AACCC TGGGA CCTTT CGGAT CCTAG TGGGG AATAA GGGAT GCAGC CACCC 2751 CTCAG TGAAA TGCAA GAAAC GGGTC ACCAT CCTGG TGG AG GGAGG AGAGA 2801 TTGAG CTGTT TGACG GGGAG GTGAA TGTGA AGAGG CCCAT GAAGG ATGAG 2851 ACTCA CTTTG AGGTG GTGGA GTCTG GCCGG TACAT CATTC TGCTG CTGGG 2901 CAAAG CCCTC TCCGT GGTCT GGGAC CGCCA CCTGA GCATC TCCGT GGTCC 2951 TGAAG CAGAC AT ACC AGGAG AAAGT GTGTG GCCTG TGTGG GAATT TTGAT 300 1 GGCAT CCAGA ACAAT GACCT SASSA GCAGC AACCT CCAAG TGGAG GAAGA 3051 CCCTG TGGAC TTTGG GAACT CCTGG AAAGT GAGCT CGCAG TGTGC TGACA

- 113 044115

3101 CC AGA A A AGT GCCTC TGGAC TS ATS CCCTG CC ACC TGCC A TA AC A AC ATS

3151 ATGAA GCAGA CGATG GTGGA TTCCT CCTGT AGAAT CCTTA CCAGT GACGT

3201 CTTCC AGGAC TGCAA CAAGC TGGTG GACCC CGAGC CAT AT CTGGA TGTCT 3251 GCATT TACGA CACCT GCTCC TGTGA GTCCA TTGGG GACTG CGCCG CATTC 3301 TGCGA SASSA TTGCT GCCTA TGCCC ACGTG TGTGC CCAGC ATGGC AAGGT 3351 GGTGA CCTGG A GGAC GGCCA CATTG TGCCC CCAGA GCTGC GAGGA GAGGA 3401 ATCTC CGGGA GAACG GGTAT GAGGC TGAGT GGCGC TATAA CAGCT GTGCA 3451 CCTGC CTGTC AAGTC ACGTG TCAGC ACCCT GAGCC ACTGG CCTGC CCTGT 3501 GCAGT GTGTG GAGGG CTGCC ATGCC CACTG CCCTC CAGGG AAAAT CCTGG 3551 ATGAG CTTTT GCAGA CCTGC GTTGA CCCTG AAGAC TGTCC AGTG T GTGAG 3601 GTGGC TGGCC GGCGT TTTGC CTCAG GAAAG AAAGT CACCT TGAAT CCCAG 3651 TGACC CTGAG CACTG CCAGA TTTGC CACTG TGATG TTGTC AACCT CACCT 3701 GTGAA GCCTG CCAGG AGCCG ATATC TGGCG GTGGA GGTTC CGGTG GCGGG 3751 GGATC CGGCG GTGGA GGTTC CGGCG GTGGA GGTTC CGGTG GCGGG GGATC 3801 CGGTG GCGGG GGATC CCTGG TCCCC CGGGG CAGCG GCGGT GGAGG TTCCG 3851 GTGGC GGGGG ATCCG AC A A A ACTC A C AC AT GCCC A CCGTG CCCAG CTCC A 3901 GAACT CCTGG GCGGA CCGTC AGTCT TCCTC TTCCC CCCAA AACCC AAGGA 3951 CACCC TCATG ATCTC CCGGA CCCCT GAGGT CACAT GCGTG GTGGT GGACG 4001 TGAGC CACGA AGACC CTGAG GTCAA GTTCA ACTGG TACGT G GACG GCGTG 4051 GAGGT GCATA ATGCC AAGAC AAAGC CGCGG GAGGA GCAGT ACAAC AGCAC 4101 GT ACC GTGTG GTC AG CGTCC TC ACC GTCCT GC ACC AGGAC TGGCT GA ATG 4151 GCA AG GAGT A C A AGT GCA AG GTCTC C A AC A A AGCC CTCCC AGCCC CC ATC 4201 GAGA A A ACC A TCTCC AAAGC C A A AG GGC AG CCCCG A.G. A AC C AC AG GTGT A 4251 CACCC TGCCC CCATC CCGCG ATGAG CTGAC CAAGA ACC AG GTC AG CCTGA 4301 CCTGC CTGGT CAAAG GCTTC TATCC CAGCG ACATC GCCGT GGAGT GGGAG 4351 AGC A A TGGGC AGCCG GAGA A C A ACT AC A AG ACC AC GCCTC CCGTG TTGGA 4401 CTCCG ACGGC TCCTT CTTCC TCT AC AGC A A GCTCA CCGTG GAC A A GAGCA 4451 GGTGG CAGCA GGGGA ACGTC TTCTC ATGCT CCGTG ATGCA TGAGG CTCTG 4501 CACAA CCACT ACACG CAGAA GAGCC TCTCC CTGTC TCCGG GTAAA TGA VWF031 protein sequence (SEQ ID NO: 86).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

- 114 044115

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTIGMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGGGGSGGG

1251 GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP

1301 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV

1351 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 1401 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE

1451 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL

1501 HNHYTQKSLSLSPGK*

Nucleotide sequence of FB034 (SEQ ID NO: 148).

ATGAT TCCTG CCAGA TTTGC CGGGG TGCTG CTTGC TCTGG CCCTC ATTTT

GCCAG GGACC CTTTG TGC AG AAGGA ACTCG CGGCA GGTC A TCC AC GGCCC 101 GATGC AGCCT TTTCG GAAGT GACTT CGTCA AC ACC TTTGA TGGGA GCATG 151 TACAG CTTTG CGGGA TACTG CAGTT ACCTC CTGGC AGGGG GCTGC CAGAA 201 ACGCT CCTTC TCGAT TATTG GGGAC TTCCA GAATG GCAAG AGAGT GAGCC 251 TCTCC GTGTA TCTTG GGGAA TTTTT TGACA TCCAT TTGTT TGTCA ATGGT

301 ACCGT GACAC AGGGG GACCA AAGAG TCTCC ATGCC CTATG CCTCC AAAGG 351 GCTGT ATCTA GAAAC TGAGG CTGGG TACTA CAAGC TGTCC GGTGA GGCCT 401 ATGGC TTTGT GGCCA GGATC GATGG CAGCG GCAAC TTTCA AGTCC TGCTG 451 TCAGA CAGAT ACTTC AAC AA GACCT GCGGG CTGTG TGGCA ACTTT AACAT 501 CTTTG CTGAA GATGA CTTTA TGACC CAAGA AGGGA CCTTG ACCTC GGACC 551 CTTAT GACTT TGCCA ACTCA TGGGC TCTGA GCAGT GGAGA ACAGT GGTGT

- 115 044115

601 GA ACG GGC AT CTCCT CCC AG C AGCT C ATGC A AC AT CTCCT CTGGG GA A AT

651 GCAGA AGGGC CTGTG GGAGC AGTGC CAGCT TCTGA AGAGC ACCTC GGTGT

701 TTGCC CGCTG CCACC CTCTG GTGGA CCCCG AGCCT TTTGT GGCCC TGTGT

751 GAGAA GACTT TGTGT GAGTG TGCTG GGGGG CTGGA GTGCG CCTGC CCTGC 801 CCTCC TGGAG TACGC CCGGA CCTGT GCCCA GGAGG GAATG GTGCT GTACG

851 GCTGG ACCGA CCACA GCGCG TGCAG CCCAG TGTGC CCTGC TGGTA TGGAG

901 TAT AG GC AGT GTGTG TCCCC TTGCG CC AGG ACCTG CC AGA GCCTG C AC AT

951 CAATG AAATG TGTCA GGAGC GATGC GTGGA TGGCT GCAGC TGCCC TGAGG

1001 GACAG CTCCT GGATG AAGGC CTCTG CGTGG AGAGC ACCGA GTGTC CCTGC 1051 GTGCA TTCCG GAAAG CGCTA CCCTC CCGGC ACCTC CCTCT CTCGA GACTG 1101 C A AC A CCTGC ATTTG CCGA A AC AGC C AGTG GATCT GCAGC A ATGA AGA AT 1151 GTCCA GGGGA GTGCC TTG TC ACTGG TCAAT CCCAC TTCAA GAGCT TTGAC 1201 A AC AG AT ACT TC ACC TTC AG TGGGA TCTGC C AGT A CCTGC TGGCC CGGGA 1251 TTGCC AGGAC CACTC CTTCT CCATT GTCAT TGAGA CTGTC CAGTG TGCTG 1301 ATGAC CGCGA CGCTG TGTGC ACCCG CTCCG TCACC GTCCG GCTGC CTGGC 1351 CTGC A C A AC A GCCTT GTGA A ACTGA AGC AT GGGGC AGGAG TTGCC ATGGA 1401 TGGCC AGGAC ATCCA GCTCC CCCTC CTGAA AGGTG ACCTC CGCAT CCAGC 1451 ATASA GTGAC GGCCT CCGTG CGCCT CAGCT ACGGG GAGGA CCTGC AGATG 1501 GACTG GGATG GCCGC GGGAG GCTGC TGGTG AAGCT GTCCC CCGTC TATGC 15 51 CGGGA AGACC TGCGG CCTGT GTGGG A ATT A CAATG GCA AC C AGGG CGACG 1601 ACTTC CTTAC CCCCT CTGGG CTGGC GGAGC CCCGG GTGGA GGACT TCGGG 1651 AACGC CTGGA AGCTG CACGG GGACT GCCAG GACCT GCAGA AGCAG CACAG 1701 CGATC CCTGC GCCCT CAACC CGCGC ATGAC CAGGT TCTCC GAGGA GGCGT 1751 GCGCG GTCCT GACGT CCCCC ACATT CGAGG CCTGC CATCG TGCCG TCAGC 1801 CCGCT GCCCT ACCTG CGGAA CTGCC GCTAC GACGT GTGCT CCTGC TCGGA 1851 CGGCC GCGAG TGCCT GTGCG GCGCC CTGGC CAGCT ATGCC GCGGC CTGCG 1901 CGGGG AGAGG CGTGC GCGTC GCGTG GCGCG AGCCA GGCCG CTGTG AGCTG 1951 AACTG CCCGA AAGGC CAGGT GT ACC TGCAG TGC GG GACCC CCTGC AACCT 2001 GACCT GCCGC TCTCT CTCTT ACCCG GATGA GGAAT GCA AT GAGGC CTGCC 2051 TGGAG GGCTG CTTCT GCCCC CCAGG GCTCT ACATG GATGA GAGGG GGGAC 2101 TGCGT GCCCA AGGCC CAGTG CCCCT GTTAC TATGA CGGTG AGATC TTCCA 2151 GCCAG A AGAC ATCTT CTC AG ACCAT CAC AC C ATGT GCTAC TGTGA GGATG 2201 GCTTC ATGCA CTGTA CCATG AGTGG AGTCC CCGGA AGCTT GCTGC CTGAC 2251 GCTGT CCTCA GCAGT CCCCT GTCTC ATCGC AGCAA AAGGA GCCTA TCCTG 2301 TCGGC CCCCC ATGGT CAAGC TGGTG TGTCC CGCTG ACAAC CTGCG GGCTG 2351 A AGGG CTCGA GTGT A CCA A A ACGTG CC AGA ASTAT GACCT GGAGT GC ATG 2401 AGCAT GGGCT GTGTC TCTGG CTGCC TCTGC CCCCC GGGCA TGGTC CGGCA 2451 TGAGA ACAGA TGTGT GGCCC TGGAA AGGTG TCCCT GCTTC CATCA GGGCA

- 116 044115

2501 AGGAG TATGC CCCTG GAGAA ACAGT GAAGA TTGGC TGCAA CACTT GTGTC

2551 TGTCG GGACC GGAAG TGGAA CTGCA CAGAC CATGT GTGTG ATGCC ACGTG

2601 CTCCA CGATC GGCAT GGCCC ACT AC CTCAC CTTCG ACGGG CTCAA AT ACC

2651 TGTTC CCCGG GGAGT GCCAG TACGT TCTGG TGCAG GATTA CTGCG GCAGT 2701 AACCC TGGGA CCTTT CGGAT CCTAG TGGGG AATAA GGGAT GCAGC CACCC 2751 CTCAG TGAAA TGCAA GAAAC GGGTC ACCAT CCTGG TGGAG GGAGG AGAGA 2801 TTGAG CTGTT TGACG GGGAG GTGAA TGTGA AGAGG CCCAT GAAGG ATGAG 2851 ACTCA CTTTG AGGTG GTGGA GTCTG GCCGG TACAT CATTC TGCTG CTGGG 2901 CAAAG CCCTC TCCGT GGTCT GGGAC CGCCA CCTGA GCATC TCCGT GGTCC 2951 TGAAG CAGAC AT ACC AGGAG AAAGT GTGTG GCCTG TGTGG GAATT TTGAT 3001 GGCAT CCAGA ACAAT GACCT SASSA GCAGC AACCT CCAAG TGGAG GAAGA 3051 CCCTG TGGAC TTTGG GAACT CCTGG AAAGT GAGCT CGCAG TGTGC TGACA 3101 CCAGA AAAGT GCCTC TGGAC TS PBX CCCTG CC ACC TGCC A TA AC A AC PBX 3151 ATGAA GCAGA CGATG GTGGA TTCCT CCTGT AGAAT CCTTA CCAGT GACGT 3201 CTTCC AGGAC TGCAA CAAGC TGGTG GACCC CGAGC CAT AT CTGGA TGTCT 3251 GCATT TACGA C ACCT GCTCC TGTGA GTCCA TTGGG GACTG CGCCG CATTC 3301 TGCGA SASSA TTGCT GCCTA TGCCC ACGTG TGTGC CCAGC ATGGC AAGGT

3351 GGTGA CCTGG AGGAC GGCCA CATTG TGCCC CCAGA GCTGC GAGGA GAGGA 3401 ATCTC CGGGA GAACG GGTAT GAGGC TGAGT GGCGC TATAA CAGCT GTGCA 3451 CCTGC CTGTC AAGTC ACGTG TCAGC ACCCT GAGCC ACTGG CCTGC CCTGT 3501 GCAGT GTGTG GAGGG CTGCC ATGCC CACTG CCCTC CAGGG AAAAT CCTGG 3551 ATGAG CTTTT GCAGA CCTGC GTTGA CCCTG AAGAC TGTCC AGTGT GTGAG 3601 GTGGC TGGCC GGCGT TTTGC CTCAG GAAAG AAAGT CACCT TGAAT CCCAG 3651 TGACC CTGAG CACTG CCAGA TTTGC CACTG TGATG TTGTC AACCT CACCT 3701 GTGAA GCCTG CCAGG AGCCG ATATC GGGTA CCTCA GAGTC TGCTA C CCCC

3751 GAGTC AGGGC CAGGA TCAGA GCCAG CCACC TCCGG GTCTG AGACA CCCGG 3801 GACTT CCGAG AGTGC CACCC CTGAG TCCGG ACCCG GGTCC GAGCC CGCCA 3851 CTTCC GGCTC CGAAA CTCCC GGCAC AAGCG AGAGC GCTAC CCCAG AGTCA 3901 GGACC AGGAA CATCT AC AGA GCCCT CTGAA GGCTC CGCTC CAGGG TCCCC 3951 AGCCG GCAGT CCCAC TAGCA CCGAG GAGGG AACCT CTGAA AGCGC CACAC 4001 CCGAA TCAGG GCCAG GGTCT GAGCC TGCTA CCAGC GGCAG CGAGA SASSA 4051 GGCAC CTCTG AGTCC GCC AC ACC AG AGTCC GGACC CGGAT CTCCC GCTGG

4101 GAGCC CCACC TCC AC TGAGG AGGGA TCTCC TGCTG GCTCT CCA AC ATCT A 4151 CTGAG GAAGG T ACCT C A ACC GAGCC ATCCG AGGGA TCAGC TCCCG GC ACC 4201 TCAGA GTCGG C A ACC CCGGA GTCTG GACCC GGA AC TTCCG AAAGT GCC AC 4251 ACC AG AGTCC GGTCC CGGGA CTTCA GAATC AGCAA CACCC GAGTC CGGCC 4301 CTGGG TCTGA ACCCG CCACA AGTGG TAGTG AGACA CCAGG ATCAG AACCT 4351 GCTAC CTCAG GGTCA GAGAC ACCCG GATCT CCGGC AGGCT SASSA ACCTC

- 117 044115

4401 C ACTG AGGAG GGC AC C AGC A C AGA A CCA AG CGAGG GCTCC GC ACC CGGA A

4451 CAAGC ACTGA ACCCA GTGAG GGTTC AGCAC CCGGC TCTGA GCCGG CCACA

4501 AGTGG CAGTG AGACA CCCGG CACTT CAGAG AGTGC CACCC CCGAG AGTGG 4551 CCCAG GCACT AGTAC CGAGC CCTCT GAAGG CAGTG CGCCA GATTC TGGCG

4601 GTGGA GGTTC CGGTG GCGGG GGATC CGGTG GCGGG GGATC CGGTG GCGGG

4651 GGATC CGGTG GCGGG GGATC CCTGG TCCCC CGGGG CAGCG GAGGC GACAA

4701 AACTC ACACA TGCCC ACCGT GCCCA GCTCC AGAAC TCCTG GGCGG ACCGT

4751 CAGTC TTCCT CTTCC CCCCA AAACC CAAGG ACACC CTCAT GATCT CCCGG

4801 ACCCC TGAGG TCACA TGCGT GGTGG TGGAC GTGAG CCACG AAGAC CCTGA

4851 GGTC A AGTTC AACTG GT ACG TGGAC GGCGT GGAGG TGC AT AATGC C AAGA

4901 CAAAG CCGCG GGAGG AGCAG TACAA CAGCA CGTAC CGTGT GGTCA GCGTC

4951 CTCAC CGTCC TGC AC CAGGA CTGGC TGAAT GGCAA GGAGT ACAAG TGCAA

5001 GGTCT CCAAC AAAGC CCTCC CAGCC CCCAT CGAGA AAACC ATCTC CAAAG

5051 CCAAA GGGCA GCCCC GAGAA CCACA GGTGT ACACC CTGCC CCCAT CCCGG

5101 GATGA GCTGA CCA AG A ACC A GGTCA GCCTG ACCTG CCTGG TC A A A GGCTT

5151 ST ATS CC AGC GAC AT CGCCG TGGAG TGGGA GAGCA ATGGG CAGCC GGAGA

5201 ACAAC TACAA GACCA CGCCT CCCGT GTTGG ACTCC GACGG CTCCT TCTTC

5251 CTCTA CAGCA AGCTC ACCGT GGACA AGAGC AGGTG GCAGC AGGGG AACGT

5301 CTTCT CATGC TCCGT GATGC ATGAG GCTCT GCACA ACCAC TACAC GCAGA 5351 AGAGC CTCTC CCTGT CTCCG GGTAA ATGA VWF034 protein sequence (SEQ ID NO: 87).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQ D HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

- 118 044115

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTIGMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGTSESATP 1251 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES

1301 GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP 1351 GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT 1401 SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP 1451 ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTS TEPSE GSAPGSEPAT

1501 SGSETPGTSE SATPESGPGT STEPSEGSAP DIGGGGGSGG GGSLVPRGSG 1551 GDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

1601 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

1651 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

1701 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYS TO LTVDKSRWQQ

1751 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

Nucleotide sequence of FB050 (IHH triple mutant) (SEQ ID NO: 149).

ATGAT TCCTG CCAGA TTTGC CGGGG TGCTG CTTGC TCTGG CCCTC ATTTT

GCCAG GGACC CTTTG TGC AG AAGGA ACTCG CGGCA GGTC A TCC AC GGCCC 101 GATGC AGCCT TTTCG GAAGT GACTT CGTC A AC ACC TTTGA TGGGA GCATG 151 TACAG CTTTG CGGGA TACTG CAGTT ACCTC CTGGC AGGGG GCTGC CAGAA 201 ACGCT CCTTC TCGAT T ATTG GGGAC TTCC A GA ATG GCA AG AGAGT GAGCC 251 TCTCC GTGTA TCTTG GGGAA TTTTT TGACA TCCAT TTGTT TGTCA ATGGT

301 ACCGT GACAC AGGGG GACCA AAGAG TCTCC ATGCC CTATG CCTCC AAAGG 351 GCTGT ATCTA GAAAC TGAGG CTGGG TACTA CAAGC TGTCC GGTGA GGCCT 401 ATGGC TTTGT GGCCA GGATC GATGG CAGCG GCA AC TTTCA AGTCC TGCTG 451 TCAGA CAGAT ACTTC AAC AA GACCT GCGGG CTGTG TGGCA ACTTT AACAT 501 CTTTG CTGAA GATGA CTTTA TGACC CAAGA AGGGA CCTTG ACCTC GGACC 551 CTTAT GACTT TGCCA ACTCA TGGGC TCTGA GCAGT GGAGA ACAGT GGTGT 601 GA ACG GGCAT CTCCT CCC AG C AGCT C ATGC A AC AT CTCCT CTGGG GA A AT

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651 GCAGA AGGGC CTGTG GGAGC AGTGC CAGCT TCTGA AGAGC ACCTC GGTGT

701 TTGCC CGCTG CCACC CTCTG GTGGA CCCCG AGCCT TTTGT GGCCC TGTGT 751 GAGAA GACTT TGTGT GAGTG TGCTG GGGGG CTGGA GTGCG CCTGC CCTGC 801 CCTCC TGGAG TACGC CCGGA CCTGT GCCCA GGAGG GAATG GTGCT GTACG

851 GCTGG ACCGA CCACA GCGCG TGCAG CCCAG TGTGC CCTGC TGGTA TGGAG

901 TAT AG GC AGT GTGTG TCCCC TTGCG CC AGG ACCTG CC AGA GCCTG C AC AT

951 CAATG AAATG TGTCA GGAGC GATGC GTGGA TGGCT GCAGC TGCCC TGAGG

1001 GACAG CTCCT GGATG AAGGC CTCTG CGTGG AGAGC ACCGA GTGTC CCTGC 1051 GTGCA TTCCG GAAAG CGCTA CCCTC CCGGC ACCTC CCTCT CTCGA GACTG 1101 C A AC A CCTGC ATTTG CCGA A AC AGC C AGTG GATCT GCAGC A ATGA AGA AT 1151 GTCCA GGGGA GTGCC TTG TC ACTGG TCAAT CCCAC TTCAA GAGCT TTGAC 1201 A AC AG AT ACT TC ACC TTC AG TGGGA TCTGC C AGT A CCTGC TGGCC CGGGA 1251 TTGCC AGGAC CACTC CTTCT CCATT GTCAT TGAGA CTGTC CAGTG TGCTG 1301 ATGAC CGCGA CGCTG TGTGC ACCCG CTCCG TCACC GTCCG GCTGC CTGGC 1351 CTGC A C A AC A GCCTT GTGA A ACTGA AGC AT GGGGC AGGAG TTGCC ATGGA 1401 TGGCC AGGAC ATCCA GCTCC CCCTC CTGAA AGGTG ACCTC CGCAT CCAGC 1451 ATASA GTGAC GGCCT CCGTG CGCCT CAGCT ACGGG GAGGA CCTGC AGATG 1501 GACTG GGATG GCCGC GGGAG GCTGC TGGTG AAGCT GTCCC CCGTC TATGC 15 51 CGGGA AGACC TGCGG CCTGT GTGGG A ATT A CAATG GCA AC C AGGG CGACG 1601 ACTTC CTTAC CCCCT CTGGG CTGGC GGAGC CCCGG GTGGA GGACT TCGGG 1651 AACGC CTGGA AGCTG CACGG GGACT GCCAG GACCT GCAGA AGCAG CACAG 1701 CGATC CCTGC GCCCT CAACC CGCGC ATGAC CAGGT TCTCC GAGGA GGCGT 1751 GCGCG GTCCT GACGT CCCCC ACATT CGAGG CCTGC CATCG TGCCG TCAGC 1801 CCGCT GCCCT ACCTG CGGAA CTGCC GCTAC GACGT GTGCT CCTGC TCGGA 1851 CGGCC GCGAG TGCCT GTGCG GCGCC CTGGC CAGCT ATGCC GCGGC CTGCG 1901 CGGGG AGAGG CGTGC GCGTC GCGTG GCGCG AGCCA GGCCG CTGTG AGCTG 1951 AACTG CCCGA AAGGC CAGGT GT ACC TGCAG TGC GG GACCC CCTGC AACCT 2001 GACCT GCCGC TCTCT CTCTT ACCCG GATGA GGAAT GCA AT GAGGC CTGCC 2051 TGGAG GGCTG CTTCT GCCCC CCAGG GCTCT ACATG GATGA GAGGG GGGAC 2101 TGCGT GCCCA AGGCC CAGTG CCCCT GTTAC TATGA CGGTG AGATC TTCCA 2151 GCCAG A AGAC ATCTT CTC AG ACCAT CAC AC C ATGT GCTAC TGTGA GGATG 2201 GCTTC ATGCA CTGTA CCATG AGTGG AGTCC CCGGA AGCTT GCTGC CTGAC 2251 GCTGT CCTCA GCAGT CCCCT GTCTC ATCGC AGCAA AAGGA GCCTA TCCTG 2301 TCGGC CCCCC ATGGT CAAGC TGGTG TGTCC CGCTG ACAAC CTGCG GGCTG 2351 A AGGG CTCGA GTGT A CCA A A ACGTG CC AGA ASTAT GACCT GGAGT GC ATG 2401 AGCAT GGGCT GTGTC TCTGG CTGCC TCTGC CCCCC GGGCA TGGTC CGGCA 2451 TGAGA ACAGA TGTGT GGCCC TGGAA AGGTG TCCCT GCTTC CATCA GGGCA 2501 AGGAG TATGC CCCTG GAGAA ACAGT GAAGA TTGGC TGCAA CACTT GTGTC

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2551 TGTCG GGACC GGAAG TGGAA CTGCA CAGAC CATGT GTGTG ATGCC ACGTG 2601 CTCCA CGATC GGC AT GGCCC ACT AC CTCAC CTTCG ACGGG CTCAA AT ACC 2651 TGTTC CCCGG GGAGT GCCAG TACGT TCTGG TGCAG GATTA CTGCG GCAGT 2701 AACCC TGGGA CCT TT CGGAT CCTAG TGGGG AATAA GGGAT GCAGC CACCC 2751 CTCAG TGAAA TGCAA GAAAC GGGTC ASSAT CCTGG TGGAG GGAGG AGAGA 2801 TTGAG CTGTT TGACG GGGAG GTGAA TGTGA AGAGG CCCAT GAAGG ATGAG 2851 ACTCA CTTTG AGGTG GTGGA GTCTG GCCGG TACAT CATTC TGCTG CTGGG 2901 CAAAG CCCTC TCCGT GGTCT GGGAC CGCCA CCTGA GCATC TCCGT GGTCC 2951 TGAAG CAGAC AT ACC AGGAG AAAGT GTGTG GCCTG TGTGG GAATT TTGAT 3001 GGCAT CCAGA ACAAT GACCT SASSA GCAGC AACCT CCAAG TGGAG GAAGA 3051 CCCTG TGGAC TTTGG GAACT CCTGG AAAGT GAGCT CGCAG TGTGC TGACA 3101 CCAGA AAAGT GCCTC TGGAC TS ATS CCCTG CC ACC TGCC A TA AC A AC ATS 3 151 ATGAA GCAGA CGATG GTGGA TTCCT CCTGT AGAAT CCTTA CCAGT GACGT 3201 CTTCC AGGAC TGCAA CAAGC TGGTG GACCC CGAGC CAT AT CTGGA TGTCT 3251 GCATT TACGA CACCT GCTCC TGTGA GTCCA TTGGG GACTG CGCCG CATTC 3301 TGCGA SASSA TTGCT GCCTA TGCCC ACGTG TGTGC CCAGC ATGGC AAGGT 33 51 GGTGA CCTGG AGGAC GGCCA CATTG TGCCC CCAGA GCTGC GAGGA GAGGA 3401 ATCTC CGGGA GAACG GGTAT GAGGC TGAGT GGCGC TATAA CAGCT GTGCA 3451 CCTGC CTGTC AAGTC ACGTG TCAGC ACCCT GAGCC ACTGG CCTGC CCTGT 3501 GCAGT GTGTG GAGGG CTGCC ATGCC CACTG CCCTC CAGGG AAAAT CCTGG 3551 ATGAG CTTTT GCAGA CCTGC GTTGA CCCTG AAGAC TGTCC AGTGT GTGAG 3601 GTGGC TGGCC GGCGT TTTGC CTCAG GAAAG AAAGT CACCT TGAAT CCCAG 3651 TGACC CTGAG CACTG CCAGA TTTGC CACTG TGATG TTGTC AACCT CACCT 3701 GTGAA GCCTG CCAGG AGCCG ATATC TGGCG GTGGA GGTTC CGGTG GCGGG 3751 GGATC CGGCG GTGGA GGTTC CGGCG GTGGA GGTTC CGGTG GCGGG GGATC 3801 CGGTG GCGGG GGATC CCTGG TCCCC CGGGG CAGCG GCGGT GGAGG TTCCG 3851 GTGGC GGGGG ATCCG AC A A A ACTCA C AC AT GCCC A CCGTG CCCAG CTCCA 3901 GAACT CCTGG GCGGA CCGTC AGTCT TCCTC TTCCC CCCAA AACCC AAGGA 3951 CACCC TCATG GCCTC CCGGA CCCCT GAGGT CACAT GCGTG GTGGT GGACG 4001 TGAGC CACGA AGACC CTGAG GTC A A GTTCA ACTGG TACGT GGACG GCGTG 4051 GAGGT GCATA ATGCC AAGAC AAAGC CGCGG GAGGA GCAGT ACAAC AGCAC 4101 GT ACC GTGTG GTC AG CGTCC TC ACC GTCCT GGCCC AGGAC TGGCT GA ATG 4151 GCA AG GAGT A C A AGT GCA AG GTCTC C A AC A A AGCC CTCCC AGCCC CC ATC 4201 GAGA A A ACC A TCTCC AAAGC CAAAG GGC AG CCCCG AG A AC C AC AG GTGT A 4251 CACCC TGCCC CCATC CCGCG ATGAG CTGAC CAAGA ACC AG GTC AG CCTGA 4301 CCTGC CTGGT CAAAG GCTTC TATCC CAGCG ACATC GCCGT GGAGT GGGAG

4351 AGC A A TGGGC AGCCG GAGA A C A ACT AC A AG ACC AC GCCTC CCGTG TTGGA

4401 CTCCG ACGGC TCCTT CTTCC TCT AC AGC A A GCTCA CCGTG GAC A A GAGCA 4451 GGTGG CAGCA GGGGA ACGTC TTCTC ATGCT CCGTG ATGCA TGAGG CTCTG

4501 CACAA CGCCT ACACG CAGAA GAGCC TCTCC CTGTC TCCGG GTAAA TGA

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Protein sequence of FB050 (IHH triple mutant) (SEQ ID NO: 150).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVY AGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGGGGSGGG

1251 GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP 1301 ELLGGPSVFL FPPKPKDTLM ASRTPEVTCV VVDVSHEDPE VKFNWYVDGV 1351 EVHNAKTKPR EEQYNSTYRV VSVLTVLAQD WLNGKEYKCK VSNKALPAPI

1401 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE

1451 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL

1501 HNAYTQKSLSLSPGK*

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Nucleotide sequence of FB057 (SEQ ID NO: 151).

ATGAT TCCTG CCAGA TTTGC CGGGG TGCTG CTTGC TCTGG CCCTC ATTTT

GCCAG GGACC CTTTG TGC AG AAGGA ACTCG CGGCA GGTC A TCC AC GGCCC

101 GATGC AGCCT TTTCG GA AGT GACTT CGTC A AC ACC TTTGA TGGG A GC ATG

151 TACAG CTTTG CGGGA TACTG CAGTT ACCTC CTGGC AGGGG GCTGC CAGAA

201 ACGCT CCTTC TCGAT T ATTG GGGAC TTCC A GA ATG GCA AG AGAGT GAGCC

251 TCTCC GTGTA TCTTG GGGAA TTTTT TGACA TCCAT TTGTT TGTCA ATGGT

301 ACCGT GACAC AGGGG GACCA AAGAG TCTCC ATGCC CTATG CCTCC AAAGG

351 GCTGT ATCTA GAAAC TGAGG CTGGG TACTA CAAGC TGTCC GGTGA GGCCT

401 ATGGC TTTGT GGCCA GGATC GATGG CAGCG GCA AC TTTCA AGTCC TGCTG

451 TCAGA CAGAT ACTTC AACAA GACCT GCGGG CTGTG TGGCA ACTTT AACAT

501 CTTTG CTGAA GATGA CTTTA TGACC CAAGA AGGGA CCTTG ACCTC GGACC 551 CTTAT GACTT TGCCA ACTCA TGGGC TCTGA GCAGT GGAGA ACAGT GGTGT

601 GAACG GGCAT CTCCT CCCAG CAGCT CATGC A AC AT CTCCT CTGGG GA A AT

651 GCAGA AGGGC CTGTG GGAGC AGTGC CAGCT TCTGA AGAGC ACCTC GGTGT

701 TTGCC CGCTG CCACC CTCTG GTGGA CCCCG AGCCT TTTGT GGCCC TGTGT

751 GAGAA GACTT TGTGT GAGTG TGCTG GGGGG CTGGA GTGCG CCTGC CCTGC

801 CCTCC TGGAG TACGC CCGGA CCTGT GCCCA GGAGG GAATG GTGCT GTACG

851 GCTGG ACCGA CCACA GCGCG TGCAG CCCAG TGTGC CCTGC TGGTA TGGAG

901 TAT AG GCAGT GTGTG TCCCC TTGCG CC AGG ACCTG CCAGA GCCTG C AC AT

951 CAATG AAATG TGTCA GGAGC GATGC GTGGA TGGCT GCAGC TGCCC TGAGG

1001 GACAG CTCCT GGATG AAGGC CTCTG CGTGG AGAGC ACCGA GTGTC CCTGC

1051 GTGCA TTCCG GAAAG CGCTA CCCTC CCGGC ACCTC CCTCT CTCGA GACTG

1101 C A AC A CCTGC ATTTG CCGA A AC AGC C AGTG GATCT GCAGC A ATGA AGA AT

1151 GTCCA GGGGA GTGCC TTGTC ACTGG TCAAT CCCAC TTCAA GAGCT TTGAC

1201 A AC AG AT ACT TC ACC TTC AG TGGGA TCTGC C AGT A CCTGC TGGCC CGGGA

1251 TTGCC AGGAC CACTC CTTCT CCATT GTCAT TGAGA CTGTC CAGTG TGCTG

1301 ATGAC CGCGA CGCTG TGTGC ACCCG CTCCG TCACC GTCCG GCTGC CTGGC

1351 CTGC A C A AC A GCCTT GTGA A ACTGA AGC AT GGGGC AGGAG TTGCC ATGGA

1401 TGGCC AGGAC ATCCA GCTCC CCCTC CTGAA AGGTG ACCTC CGCAT CCAGC

1451 ATASA GTGAC GGCCT CCGTG CGCCT CAGCT ACGGG GAGGA CCTGC AGATG

1501 GACTG GGATG GCCGC GGGAG GCTGC TGGTG AAGCT GTCCC CCGTC TATGC

1551 CGGGA AGACC TGCGG CCTGT GTGGG A ATT A CAATG GCA AC C AGGG CGACG

1601 ACTTC CTTAC CCCCT CTGGG CTGGC GGAGC CCCGG GTGGA GGACT TCGGG 1651 AACGC CTGGA AGCTG CACGG GGACT GCCAG GACCT GCAGA AGCAG CACAG

1701 CGATC CCTGC GCCCT CAACC CGCGC ATGAC CAGGT TCTCC GAGGA GGCGT 1751 GCGCG GTCCT GACGT CCCCC ACATT CGAGG CCTGC CATCG TGCCG TCAGC

1801 CCGCT GCCCT ACCTG CGGAA CTGCC GCTAC GACGT GTGCT CCTGC TCGGA

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1851 CGGCC GCGAG TGCCT GTGCG GCGCC CTGGC CAGCT ATGCC GCGGC CTGCG 1901 CGGGG AGAGG CGTGC GCGTC GCGTG GCGCG AGCCA GGCCG CTGTG AGCTG 1951 AACTG CCCGA AAGGC CAGGT GT ACC TGCAG TGCGG GACCC CCTGC AACCT 2001 GACCT GCCGC TCTCT CTCTT ACCCG GATGA GGAAT GCA AT GAGGC CTGCC 2051 TGGAG GGCTG CTTCT GCCCC CCAGG GCTCT ACATG GATGA GAGGG GGGAC 2101 TGCGT GCCCA AGGCC CAGTG CCCCT GTTAC TATGA CGGTG AGATC TTCCA 2151 GCC AG A AGAC ATCTT CTC AG ASSAT CAC AC C ATGT GCT AC TGTGA GGATG 2201 GCTTC ATGCA CTGTA CCATG AGTGG AGTCC CCGGA AGCTT GCTGC CTGAC 2251 GCTGT CCTCA GCAGT CCCCT GTCTC ATCGC AGCAA AAGGA GCCTA TCCTG 2301 TCGGC CCCCC ATGGT CAAGC TGGTG TGTCC CGCTG ACAAC CTGCG GGCTG 2351 A AGGG CTCGA GTGT A CCA A A ACGTG CC AGA ASTAT GACCT GGAGT GC ATG 2401 AGCAT GGGCT GTGTC TCTGG CTGCC TCTGC CCC CC GGGCA TGGTC CGGCA 2451 TGAGA ACAGA TGTGT GGCCC TGGAA AGGTG TCCCT GCTTC CATCA GGGCA 2501 AGGAG TATGC CCCTG GAGAA ACAGT GAAGA TTGGC TGCAA CACTT GTGTC 2551 TGTCG GGACC GGAAG TGGAA CTGCA CAGAC CATGT GTGTG ATGCC ACGTG 2601 CTCCA CGATC GGCAT GGCCC ACT AC CTCAC CTTCG ACGGG CTCAA AT ACC 2651 TGTTC CCCGG GGAGT GCCAG TACGT TCTGG TGCAG GATTA CTGCG GCAGT 2701 AACCC TGGGA CCTTT CGGAT CCTAG TGGGG AATAA GGGAT GCAGC CACCC 2751 CTCAG TGAAA TGCAA GAAAC GGGTC ASSAT CCTGG TGGAG GGAGG AGAGA 2801 TTGAG CTGTT TGACG GGGAG GTGAA TGTGA AGAGG CCCAT GAAGG ATGAG 2851 ACTCA CTT TG AGGTG GTGGA GTCTG GCCGG TACAT CATTC TGCTG CTGGG 2901 CAAAG CCCTC TCCGT GGTCT GGGAC CGCCA CCTGA GCATC TCCGT GGTCC 2951 TGAAG CAGAC AT ACC AGGAG AAAGT GTGTG GCCTG TGTGG GAATT TTGAT 3001 GGCAT CCAGA ACAAT GACCT SASSA GCAGC AACCT CCAAG TGGAG GAAGA 3051 CCCTG TGGAC TTTGG GAACT CCTGG AAAGT GAGCT CGCAG TGTGC TGACA 3101 CCAGA AAAGT GCCTC TGGAC TS ATS CCCTG CC ACC TGCC A TA AC A AC ATC 3151 ATGAA GCAGA CGATG GTGGA TTCCT CCTGT AGAAT CCTTA CCAGT GACGT 3201 CTTCC AGGAC TGCAA CAAGC TGGTG GACCC CGAGC CAT AT CTGGA TGTCT 3251 GCATT TACGA CACCT GCTCC TGTGA GTCCA TTGGG GACTG CGCCG CATTC 3 301 TGCGA SASSA TTGCT GCCTA TGCCC ACGTG TGTGC CCAGC ATGGC AAGGT 3351 GGTGA CCTGG AGGAC GGCCA CATTG TGCCC CCAGA GCTGC GAGGA GAGGA 3401 ATCTC CGGGA GAACG GGTAT GAGGC TGAGT GGCGC TATAA CAGCT GTGCA 3451 CCTGC CTGTC AAGTC ACGTG TCAGC ACCCT GAGCC ACTGG CCTGC CCTGT 3501 GCAGT GTGTG GAGGG CTGCC ATGCC CACTG CCCTC CAGGG AAAAT CCTGG 3551 ATGAG CTTTT GCAGA CCTGC GTTGA CCCTG AAGAC TGTCC AGTGT GTGAG 3601 GTGGC TGGCC GGCGT TTTGC CTCAG GAAAG AAAGT CACCT TGAAT CCCAG 3651 TGACC CTGAG CACTG CCAGA TTTGC CACTG TGATG TTGTC AACCT CACCT 3701 GTGAA GCCTG CCAGG AGCCG ATATC GGGCG C GCCA AC automatic telephone exchange AGAGA GCGCC

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3751 ACCCC TGAAA GTGGT CCCGG GAGCG AGCCA GCCAC ATCTG GGTCG GAAAC

3801 GCCAG GCACA AGTGA GTCTG CAACT CCCGA GTCCG GACCT GGCTC CGAGC

3851 CTGCC ACT AG CGGCT CCGAG ACTCC GGGA A CTTCC GAGAG CGCT A SASSA

3901 GAAAG CGGAC CCGGA ACCAG TACCG AACCT AGCGA GGGCT CTGCT CCGGG

3951 CAGCC CAGCC GGCTC TCCTA CATCC ACGGA GGAGG GCACT TCCGA ATCCG

4001 CCACC CCGGA GTCAG GGCCA GGATC TGAAC CCGCT ACCTC AGGCA GTGAG

4051 ACGCC AGGAA CGAGC GAGTC CGCTA CACCG GAGAG TGGGC CAGGG AGCCC

4101 TGCTG GATCT CCTAC GTCCA CTGAG GAAGG GTCAC CAGCG GGCTC GCCCA

4151 CC AGC ACTG A AGA AG GTGCC TCGAG CGGCG GTGGA GGTTC CGGTG GCGGG

4201 GGATC CGGTG GCGGG GGATC CGGTG GCGGG GGATC CGGTG GCGGG GGATC 4251 CCTGG TCCCC CGGGG CAGCG GAGGC GACAA AACTC AC ACA TGCCC ACCGT

4301 GCCCA GCTCC AGAAC TCCTG GGCGG ACCGT CAGTC TTCCT CTTCC CCCCA

4351 A A ACC C A AGG AC ACC CTC AT GATCT CCCGG ACCCC TGAGG TC AC A TGCGT

4401 GGTGG TGGAC GTGAG CCACG AAGAC CCTGA GGTCA AGTTC AACTG GTACG

4451 TGGAC GGCGT GGAGG TGCAT AATGC CAAGA CAAAG CCGCG GGAGG AGCAG

4501 TACAA CAGCA CGTAC CGTGT GGTCA GCGTC CTCAC CGTCC TGCAC CAGGA

4551 CTGGC TGA AT GGC A A GGAGT AC A AG TGC A A GGTCT CCA AC A A AGC CCTCC

4601 CAGCC CCCAT CGAGA AAACC ATCTC CAAAG CCAAA GGGCA GCCCC GAGAA

4651 CCACA GGTGT ACACC CTGCC CCCAT CCCGG GATGA GCTGA CCAAG AACCA

4701 GGTCA GCCTG ACCTG CCTGG TCAAA GGCTT ST ATS CCAGC GAC AT CGCCG

4751 TGGAG TGGGA GAGCA ATGGG CAGCC GGAGA ACAAC TACAA GACCA CGCCT

4801 CCCGT GTTGG ACTCC GACGG CTCCT TCTTC CTCTA CAGCA AGCTC ACCGT

4851 GGACA AGAGC AGGTG GCAGC AGGGG AACGT CTTCT CATGC TCCGT GATGC

4901 ATGAG GCTCT GCACA ACC AC TAC AC GCAGA AGAGC CTCTC CCTGT CTCCG

4951 GGTAAAATGA

Protein sequence of VF057 (SEQ ID NO: 152).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARIDGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

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501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTIGMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGAPTSESA

1251 TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE TPGTSESATP 1301 ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP GSEPATSGSE 1351 TPGTSESATP ESGPGSPAGS PSTEEGSPA GSPTSTEEGA SSGGGGSGGG

1401 GSGGGGSGGG GSGGGGSLVP RGSGGDKTHT CPPCPAPELL GGPSVFLFPP

1451 KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 1501 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE 1551 PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYK TTP

1601 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP

1651 GK*

Nucleotide sequence of VF058 (VF034 with IHH mutation) (SEQ ID NO: 153).

ATGAT TCCTG CCAGA TTTGC CGGGG TGCTG CTTGC TCTGG CCCTC ATTTT

GCCAG GGACC CTTTG TGC AG AAGGA ACTCG CGGCA GGTC A TCC AC GGCCC 101 GATGC AGCCT TTTCG GA AGT GACTT CGTC A AC ACC TTTGA TGGG A GC ATG

151 TACAG CTTTG CGGGA TACTG CAGTT ACCTC CTGGC AGGGG GCTGC CAGAA

201 ACGCT CCTTC TCGAT T ATTG GGGAC TTCC A GA ATG GCA AG AGAGT GAGCC

251 TCTCC GTGTA TCTTG GGGAA TTTTT TGACA TCCAT TTGTT TGTCA ATGGT

301 ACCGT GACAC AGGGG GACCA AAGAG TCTCC ATGCC CTATG CCTCC AAAGG

351 GCTGT ATCTA GAAAC TGAGG CTGGG TACTA CAAGC TGTCC GGTGA GGCCT

401 ATGGC TTTGT GGCCA GGATC GATGG CAGCG GCA AC TTTCA AGTCC TGCTG

451 TCAGA CAGAT ACTTC AACAA GACCT GCGGG CTGTG TGGCA ACTTT AACAT

501 CTTTG CTGAA GATGA CTTTA TGACC CAAGA AGGGA CCTTG ACCTC GGACC

- 126 044115

551 CTTAT GACTT TGCCA ACTCA TGGGC TCTGA GCAGT GGAGA ACAGT GGTGT

601 GA ACG GGC AT CTCCT CCCAG C AGCT C ATGC A AC AT CTCCT CTGGG GA A AT

651 GCAGA AGGGC CTGTG GGAGC AGTGC CAGCT TCTGA AGAGC ACCTC GGTGT

701 TTGCC CGCTG CCACC CTCTG GTGGA CCCCG AGCCT TTTGT GGCCC TGTGT

751 GAGAA GACTT TGTGT GAGTG TGCTG GGGGG CTGGA GTGCG CCTGC CCTGC 801 CCTCC TGGAG TACGC CCGGA CCTGT GCCCA GGAGG GAATG GTGCT GTACG

851 GCTGG ACCGA CCACA GCGCG TGCAG CCCAG TGTGC CCTGC TGGTA TGGAG

901 TAT AG GCAGT GTGTG TCCCC TTGCG CC AGG ACCTG CCAGA GCCTG C AC AT

951 CAATG AAATG TGTCA GGAGC GATGC GTGGA TGGCT GCAGC TGCCC TGAGG

1001 GACAG CTCCT GGATG AAGGC CTCTG CGTGG AGAGC ACCGA GTGTC CCTGC 1051 GTGCA TTCCG GAAAG CGCTA CCCTC CCGGC ACCTC CCTCT CTCGA GACTG 1101 C A AC A CCTGC ATTTG CCGA A AC AGC C AGTG GATCT GCAGC A ATGA AGA AT 1151 GTCCA GGGGA GTGCC TTG TC ACTGG TCAAT CCCAC TTCAA GAGCT TTGAC 1201 A AC AG AT ACT TC ACC TTC AG TGGGA TCTGC C AGT A CCTGC TGGCC CGGGA 1251 TTGCC AGGAC CACTC CTTCT CCATT GTCAT TGAGA CTGTC CAGTG TGCTG 1301 ATGAC CGCGA CGCTG TGTGC ACCCG CTCCG TCACC GTCCG GCTGC CTGGC 1351 CTGC A C A AC A GCCTT GTGA A ACTGA AGC AT GGGGC AGGAG TTGCC ATGGA 1401 TGGCC AGGAC ATCCA GCTCC CCCTC CTGAA AGGTG ACCTC CGCAT CCAGC 1451 ATASA GTGAC GGCCT CCGTG CGCCT CAGCT ACGGG GAGGA CCTGC AGATG 1501 GACTG GGATG GCCGC GGGAG GCTGC TGGTG AAGCT GTCCC CCGTC TATGC 15 51 CGGGA AGACC TGCGG CCTGT GTGGG A ATT A CAATG GCA AC C AGGG CGACG 1601 ACTTC CTTAC CCCCT CTGGG CTGGC GGAGC CCCGG GTGGA GGACT TCGGG 1651 AACGC CTGGA AGCTG CACGG GGACT GCCAG GACCT GCAGA AGCAG CACAG 1701 CGATC CCTGC GCCCT CAACC CGCGC ATGAC CAGGT TCTCC GAGGA GGCGT 1751 GCGCG GTCCT GACGT CCCCC ACATT CGAGG CCTGC CATCG TGCCG TCAGC 1801 CCGCT GCCCT ACCTG CGGAA CTGCC GCTAC GACGT GTGCT CCTGC TCGGA 1851 CGGCC GCGAG TGCCT GTGCG GCGCC CTGGC CAGCT ATGCC GCGGC CTGCG 1901 CGGGG AGAGG CGTGC GCGTC GCGTG GCGCG AGCCA GGCCG CTGTG AGCTG 1951 AACTG CCCGA AAGGC CAGGT GT ACC TGCAG TGC GG GACCC CCTGC AACCT 2001 GACCT GCCGC TCTCT CTCTT ACCCG GATGA GGAAT GCA AT GAGGC CTGCC 2051 TGGAG GGCTG CTTCT GCCCC CCAGG GCTCT ACATG GATGA GAGGG GGGAC 2101 TGCGT GCCCA AGGCC CAGTG CCCCT GTTAC TATGA CGGTG AG ATC TTCCA 2151 GCCAG AAGAC ATCTT CTC AG ACCAT CAC AC C ATGT GCTAC TGTGA GGATG 2201 GCTTC ATGCA CTGTA CCATG AGTGG AGTCC CCGGA AGCTT GCTGC CTGAC 2251 GCTGT CCTCA GCAGT CCCCT GTCTC ATCGC AGCAA AAGGA GCCTA TCCTG 2301 TCGGC CCCCC ATGGT CAAGC TGGTG TGTCC CGCTG ACAAC CTGCG GGCTG 2351 A AGGG CTCGA GTGT A CCA A A ACGTG CCAGA ASTAT GACCT GGAGT GC ATG 2401 AGCAT GGGCT GTGTC TCTGG CTGCC TCTGC CCCCC GGGCA TGGTC CGGCA

- 127 044115

2451 TGAGA ACAGA TGTGT GGCCC TGGAA AGGTG TCCCT GCTTC CATCA GGGCA

2501 AGGAG TATGC CCCTG GAGAA ACAGT GAAGA TTGGC TGCAA CACTT GTGTC

2551 TGTCG GGACC GGAAG TGGAA CTGCA CAGAC CATGT GTGTG ATGCC ACGTG

2601 CTCCA CGATC GGC AT GGCCC ACT AC CTCAC CTTCG ACGGG CTCAA AT ACC 2651 TGTTC CCCGG GGAGT GCCAG TACGT TCTGG TGCAG GATTA CTGCG GCAGT 2701 AACCC TGGGA CCTTT CGGAT CCTAG TGGGG AATAA GGGAT GCAGC CACCC 2751 CTCAG TGAAA TGCAA GAAAC GGGTC ACCAT CCTGG TGGAG GGAGG AGAGA 2801 TTGAG CTGTT TGACG GGGAG GTGAA TGTGA AGAGG CCCAT GAAGG ATGAG 2851 ACTCA CTTTG AGGTG GTGGA GTCTG GCCGG TACAT CATTC TGCTG CTGGG 2901 CAAAG CCCTC TCCGT GGTCT GGGAC CGCCA CCTGA GCATC TCCGT GGTCC 2951 TGAAG CAGAC AT ACC AGGAG AAAGT GTGTG G CCTG TGTGG GAATT TTGAT 3001 GGCAT CCAGA ACAAT GACCT SASSA GCAGC AACCT CCAAG TGGAG GAAGA 3051 CCCTG TGGAC TTTGG GAACT CCTGG AAAGT GAGCT CGCAG TGTGC TGACA 3101 CCAGA AAAGT GCCTC TGGAC TS ATS CCCTG CC ACC TGCC A TA AC A AC ATS 3151 ATGAA GCAGA CGATG GTGGA TTCCT CCTGT AGAAT CCTTA CCAGT GACGT 3 201 CTTCC AGGAC TGCAA CAAGC TGGTG GACCC CGAGC CAT AT CTGGA TGTCT 3251 GCATT TACGA CACCT GCTCC TGTGA GTCCA TTGGG GACTG CGCCG CATTC 3301 TGCGA SASSA TTGCT GCCTA TGCCC ACGTG TGTGC CCAGC ATGGC AAGGT

3351 GGTGA CCTGG AGGAC GGCCA CATTG TGCCC CCAGA GCTGC GAGGA GAGGA 3401 ATCTC CGGGA GAACG GGTAT GAGGC TGAGT GGCGC TATAA CAGCT GTGCA 3451 CCTGC CTGTC AAGTC ACGTG TCAGC ACCCT GAGCC ACTGG CCTGC CCTGT 3501 GCAGT GTGTG GAGGG CTGCC ATGCC CACTG CCCTC CAGGG AAAAT CCTGG 3551 ATGAG CTTTT GCAGA CCTGC GTTGA CCCTG AAGAC TGTCC AGTGT GTGAG 3601 GTGGC TGGCC GGCGT TTTGC CTCAG GAAAG AAAGT CACCT TGAAT CCCAG 3651 TGACC CTGAG CACTG CCAGA TTTGC CACTG TGATG TTGTC AACCT CACCT 3701 GTGAA GCCTG CCAGG AGCCG ATATC GGGTA CCTCA GAGTC TGCTA C CCCC 3751 GAGTC AGGGC CAGGA TCAGA GCCAG CCACC TCCGG GTCTG AGACA CCCGG 3801 GACTT CCGAG AGTGC CACCC CTGAG TCCGG ACCCG GGTCC GAGCC CGCCA 3851 CTTCC GGCTC CGAAA CTCCC GGCAC AAGCG AGAGC GCTAC CCCAG AGTCA 3901 GGACC AGGAA CATCT ACAGA GCCCT CTGAA GGCTC CGCTC CAGGG TCCCC

3951 AGCCG GCAGT CCCAC TAGCA CCGAG GAGGG AACCT CTGAA AGCGC CACAC 4001 CCGAA TCAGG GCCAG GGTCT GAGCC TGCTA CCAGC GGCAG CGAGA SASSA 4051 GGCAC CTCTG AGTCC GCC AC ACC AG AGTCC GGACC CGGAT CTCCC GCTGG 4101 GAGCC CCACC TCC AC TGAGG AGGGA TCTCC TGCTG GCTCT CCA AC ATCT A 4151 CTGAG GAAGG T ACCT C A ACC GAGCC ATCCG AGGGA TCAGC TCCCG GC ACC 4201 TCAGA GTCGG C A ACC CCGGA GTCTG GACCC GGA AC TTCCG AAAGT GCC AC 4251 ACC AG AGTCC GGTCC CGGGA CTTCA GAATC AGCAA CACCC GAGTC CGGCC 4301 CTGGG TCTGA ACCCG CCACA AGTG G TAGTG AGACA CCAGG ATCAG AACCT

- 128 044115

4351 GCTAC STS AG GGTC A GAGAC ACCCG GATCT CCGGC AGGCT SASSA ACCTC

4401 C ACTG AGGAG GGC AC C AGC A C AGA A CCA AG CGAGG GCTCC GC ACC CGGA A

4451 CAAGC ACTGA ACCCA GTGAG GGTTC AGCAC CCGGC TCTGA GCCGG CCACA

4501 AGTGG CAGTG AGACA CCCGG CACTT CAGAG AGTGC CACCC CCGAG AGTGG

4551 CCCAG GCACT AGTAC CGAGC CCTCT GAAGG CAGTG CGCCA GATTC TGGCG

4601 GTGGA GGTTC CGGTG GCGGG GGATC CGGTG GCGGG GGATC CGGTG GCGGG

4651 GGATC CGGTG GCGGG GGATC CCTGG TCCCC CGGGG CAGCG GAGGC GACAA

4701 AACTC ACACA TGCCC ACCGT GCCCA GCTCC AGAAC TCCTG GGCGG ACCGT

4751 CAGTC TTCCT CTTCC CCCCA AAACC CAAGG ACACC CTCAT GGCCT CCCGG

4801 ACCCC TGAGG TCACA TGCGT GGTGG TGGAC GTGAG CCACG AAGAC CCTGA

4851 GGTC A AGTTC AACTG GT ACG TGGAC GGCGT GGAGG TGC AT AATGC C AAGA

4901 CAAAG CCGCG GGAGG AGCAG TACAA CAGCA CGTAC CGTGT GGTCA GCGTC

4951 CTCAC CGTCC TGGCC CAGGA CTGGC TGAAT GGCAA GGAGT ACAAG TGCAA

5001 GGTCT CCAAC AAAGC CCTCC CAGCC CCCAT CGAGA AAACC ATCTC CAAAG

5051 CCAAA GGGCA GCCCC GAGAA CCACA GGTGT ACACC CTGCC CCCAT CCCGC

5101 GATGA GCTGA CCA AG A ACC A GGTCA GCCTG ACCTG CCTGG TC A A A GGCTT

5151 ST ATS CC AGC GAC AT CGCCG TGGAG TGGGA GAGCA ATGGG CAGCC GGAGA

5201 ACAAC TACAA GACCA CGCCT CCCGT GTTGG ACTCC GACGG CTCCT TCTTC

5251 CTCTA CAGCA AGCTC ACCGT GGACA AGAGC AGGTG GCAGC AGGGG AACGT

5301 CTTCT CATGC TCCGT GATGC ATGAG GCTCT GCACA ACGCC TACAC GCAGA

5351 AGAGC CTCTC CCTGT CTCCG GGTAA ATGA

Protein sequence of VWF058 (VWF034 with IHH mutation) (SEQ ID NO: 154).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 5 51 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

- 129 044115

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGTSESATP 1251 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES

1301 GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP 1351 GTSESATPES GGPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT

1401 SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP 1451 ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTSTEPSE GSAPGSEPAT

1501 SGSETPGTSE SATPESGPGT STEPSEGSAP DSGGGGSGGG GSGGGGSGGG 1551 GSGGGGSLVP RGSGGDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMASR

1601 TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV

1651 LTVLAQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 1701 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF

1751 LYSKLTVDKS RWQQGNVFSC SVMHEALHNA YTQKSLSLSP GK* Nucleotide sequence of FVIII 169 (SEQ ID NO: 155).

ATGCA AATAG AGCTC TCCAC CTGCT TCTTT CTGTG CCTTT TGCGA TTCTG

CTTTA GTGCC ACCAG AAGAT ACTAC CTGGG TGCAG TGGAA CTGTC ATGGG

101 ASTAT ATGCA AAGTG ATCTC GGTGA GCTGC CTGTG GACGC AAGAT TTCCT

151 CCTAG AGTGC CAAAA TCTTT TCCAT TCAAC ACCTC AGTCG TGTAC AAAAA

201 GACTC TGTTT GT AGA ATTCA CGGAT CACCT TTTCA AC ATC GCT A A GCC A A

251 GGCCA CCCTG GATGG GTCTG CTAGG TCCTA CCATS CAGGC TGAGG TTTAT

301 GAT AC AGTGG TCATT AC ACT TAAGA AC ATG GCTTC CCATC CTGTC AGTCT

351 TCATG CTGTT GGTGT ATCCT ACTGG AAAGC TTCTG AGGGA GCTGA ATATG

401 ATGAT CAGAC CAGTC A A AGG GAGA A AGA AG ATGAT AAAGT CTTCC CTGGT

451 GGAAG CCATA CAT AT GTCTG GCAGG TCCTG AAAGA GA ATG GTCCA ATGGC

501 CTCTG ACCC A CTGTG CCTTA CCTAC TC AT A TCTTT CTCAT GTGGA CCTGG

- 130 044115

551 TAAAA GACTT GAATT CAGGC CTCAT TGGAG CCCTA CTAGT ATGTA GAGAA

601 GGGAG TCTGG CCA AG GAAAA GACAC AGACC TTGCA CAAAT TTATA CT ACT

651 TTTTG CTGTA TTTGA TGAAG GGAAA AGTTG GCACT CAGAA AC AAA GAACT

701 CCTTG ATGC A GGAT A GGGAT GCTGC ATCTG CTCGG GCCTG GCCT A A A ATG

751 CACAC AGTCA ATGGT TATGT AAACA GGTCT CTGCC AGGTC TGATT GGATG

801 CCACA GGAAA TCAGT CTATT GGCAT GTGAT TGGAA TGGGC ACCAC TCCTG

851 AAGTG CACTC AATAT TCCTC GAAGG TCACA CATTT CTTGT GAGGA ASSAT

901 CGCC A GGCT A GCTTG GA AAT CTCGC C A AT A ACTTT CCTT A CTGCT C A A AC

951 ACTCT TGATG GACCT TGGAC AGTTT CT ACT GTTTT GTCAT ATCTC TTCCC

1001 ACCAA CATGA TGGCA TGGAA GCTTA TGTCA AAGTA GACAG CTGTC CAGAG 1051 GAACC CCAAC TACGA ATGAA AAATA ATGAA GAAGC GGAAG ASTAT GATGA 1101 TGATC TT ACT GATTC TGA A A TGGAT GTGGT C AGGT TTGAT GATGA C A ACT 1151 CTCCT TCCTT TAT CC AAATT CGCTC AGTTG CCAAG AAGCA TCCTA AAACT 1201 TGGGT AC ATT AC ATT GCTGC TGAAG AGGAG GACTG GGACT ATGCT CCCTT 1251 AGTCC TCGCC CCCGA TGACA GAAGT TATAA AAGTC AATAT TTGAA CAATG 1301 GCCCT CAGCG GATTG GTAGG AAGTA CAAAA AAGTC CGATT TATGG CATAC 1351 AC AGA TGA A A CCTTT A AGAC TCGTG A AGCT ATTC A GC ATG A ATC A GGA AT 1401 CTTGG GACCT TT ACT TTH GGGA AGAC AGAC ACTG TTGAT TTAT AT 1451 TTAAG A AGCA AGCA AGA CC AACA TCTC ACTC ACTC TCACT 1501 GATGT CCGTC CTTTG TC ATTC AAGGA GATTA CCAAA AGGTG TAAAA CATTTTTTTTTH 1551 GAAGG ATTTT CCA AT TCTGC C AGGA GA A AT ATTC A AATAT A A ATG GAC AG 1601 TGACT GT AGA AGATG GGCCA ACT AA ATCAG ATCCT CGGTG CCTGA CCCGC 1651 TATTA CTCTA GTTTC GTTAA TATGG AGAGA GATCT AGCTT CAGGA CTCAT 1701 TGGCC CTCTC CTCAT CTGCT AC AAA GAATC TGT AG ATCAA AGAGG AAACC 1751 AGATA ATGTC AGACA AGAGG AATGT CATCC TGTTT TCTGT ATTTG ATGAG 1801 AACCG AAGCT GGTAC CTCAC AGAGA AT ATA CAACG CTTTC TCCCC AATCC 1851 AGCTG GAGTG C AGCT TGAGG ATCC A GAGTT CCA AG CCTCC AAC AT C ATGC 1901 ACAGC ATCAA TGGCT ATGTT TTTGA T AGTT TGC AG TTGTC AGTTTT GTTTG 1951 CATGA GGTGG CATAC TGGTA CATTC TAAGC ATTGG AGCAC AGACT GACTT 2001 CCTTT CTGTC TTCTT CTCTG GAT AT ACCTT CAAAC AC AAA ATGGT CT ATG 2051 AAGAC AC ACT CACCC TATTC CCATT CTCAG GAGAA ACTGT CTTCA TG TCG 2101 ATGGA AAACC C AGGT CT ATG GATTC TGGGG TGCC A C A ACT C AGAC TTTCG 2151 GA AC A GAGGC ATGAC CGCCT T ACTG A AGGT TTCT A GTTGT GAC A A GA AC A 2201 CTGGT GATTA TT ACG AGGAC AGTT A TGAAG AT ATT TC AGC AT ACT TGCTG 2251 AGTAA AAACA ATGCC ATTGA ACCAA GAA G.C. TTCTC TCAAA ACGGC GCGCC 2301 AGGTA CCTCA GAGTC TGCTA CCCCC GAGTC AGGGC CAGGA TCAGA GCCAG 2351 CCACC TCCGG GTCTG AGACA CCCGG GACTT CCGAG AGTGC CACCC CTGAG 2401 TCCGG ACCCG GGTCC GAGCC CGCCA CTTCC GGCTC CGAAA CTCCC GGCAC

- 131 044115

2451 AAGCG AGAGC GCT AC CCCAG AGTCA GGACC AGGAA CATCT AC AGA GCCCT 2501 CTGAA GGCTC CGCTC CAGGG TCCCC AGCCG GCAGT CCCAC TAGCA CCGAG 2551 GAGGG AACCT CTGAA AGCGC CACAC CCGAA TCAGG GCCAG GGTCT GAGCC 2601 TGCTA CCAGC GGC AG CGAGA SASSA GGCAC CTCTG AGTCC GCCAC ACC AG 2651 AGTCC GGACC CGGAT CTCCC GCTGG GAGCC CCACC TCCAC TGAGG AGGGA 2701 TCTCC TGCTG GCTCT CCAAC ATCTA CTGAG GAAGG TACCT CAACC GAGCC

2751 ATCCG AGGGA TCAGC TCCCG GCACC TCAGA GTCGG CAACC CCGGA GTCTG 2801 GACCC GGAAC TTCCG AAAGT GCCAC ACCAG AGTCC GGTCC CGGGA CTTCA 2851 GAATC AGCAA CACCC GAGTC CGGCC CTGGG TCTGA ACCCG CCACA AGTGG 2901 TAGTG AGACA CCAGG A TCAG AACCT GCTAC CTCAG GGTCA GAGAC ACCCG 2951 GATCT CCGGC AGGCT SASSA ACCTC CACTG AGGAG GGCAC CAGCA CAGAA 3001 CCAAG CGAGG GCTCC GCACC CGGAA CAAGC ACTGA ACCCA GTGAG GGTTC 3051 AGCAC CCGGC TCTGA GCCGG CCACA AGTGG CAGTG AGACA CCCGG CACTT 3101 CAGAG AGTGC CACCC CCGAG AGTGG CCCAG GCACT AGTAC CGAGC C CTCT 3151 GAAGG CAGTG CGCCA GCCTC GAGCC SASSA GTCTT GAAAC GCCAT CAAGC 3201 TGAAA TAACT CGTAC TACTC TTCAG TCAGA TCAAG AGGAA ATCGA TTATG

3251 ATGAT ACCAT ATCAG TTGAA ATGAA GAAGG AAGAT TTTGA CATTT ATGAT 3301 GAGGA TGAAA ATCAG AGCCC CCGCA GCTTT CAAAA GAAAA CACGA CACTA 3351 TTTTA TTGCT GCAGT GGAGA GGCTC TGGGA TTATG GGATG AGTAG CTCCC 3401 CACAT GTTCT AAGAA ACAGG G CTCA GAGTG GCAGT GTCCC TCAGT TCAAG 3451 AAAGT TGTTT TCCAG GAATT TACTG ATGGC TCCTT TACTC AGCCC TTATA

3501 CCGTG GAGAA BECAME TGAAC ATTTG GGACT CCTGG GGCCA TATAT AAGAG 3551 CAGAA GTTGA AGATA ATATC ATGGT AACTT TCAGA AATCA GGCCT CTCGT 3601 CCCTA TTCCT TCTAT TCTAG CCTTA TTTCT TATGA GGAAG ATCAG AGGCA 3651 AGGAG CAGAA CCTAG AAA AA ACTTT GTCAA GCCTA ATGAA ACCAA AACTT 3701 ACTTT TGGAA AGTGC AAC AT CAT AT GGCAC CCACT AAAGA TGAGT TTGAC 3751 TGCAA AGCCT GGGCT TATTT CTCTG ATGTT GACCT GGAAA AAGAT GTGCA 3801 CTCAG GCCTG ATTGG ACCCC TTCTG GTCTG CCACA CTAAC ACACT GAACC 3851 CTGCT CATGG GAGAC AAGTG ACAGT ACAGG AATTT GCTCT GTTTT TCACC 3901 ATCTT TGATG AGACC AAAAG CTGGT ACTTC ACTGA ΑΛΑΤΑ TGGAA AGAAA 3951 CTGCA GGGCT CCCTG CAATA TCCAG ATGGA AGATC CCACT TTTAA AGAGA

4001 ATT AT CGCTT CCATG CAATC A ATGG STASA TAATG GAT AC ACT AC CTGGC 4051 TTAGT AATGG CTCAG GATCA AAGGA TTCGA TGGTA TCTGC TCAGC ATGGG 4101 CAGCA ATGAA A AC AT CC ATT CT ATT CATTT CAGTG GAC AT GTGTT CACTG 4151 TACGA AAAAA AGAGG AGT AT AAAAT GGCAC TGT AC A ATCT CT ATS C AGGT 4201 GTTTT TGAGA CAGTG GAAAT GTTAC CATCC A A AGC TGGAA TTTGG CGGGT 4251 GGAAT GCCTT ATTGG CGAGC ATCTA CATGC TGGGA TGAGC ACACT TTTTC 4301 TGGTG TACAG CAATA AGTGT CAGAC TCCCC TGGGA ATGGC TTCTG GACAC

- 132 044115

4351 ATT AG AGATT TTC AG ATT AC AGCTT C AGGA C A AT A TGGAC AGTGG GCCCC

4401 A A AGC TGGCC AGACT TCATT ATTCC GGATC A ATC A ATGCC TGGAG SASSA

4451 AGGAG CCCTT TTCTT GGATC AAGGT GGATC TGTTG GCACC AATGA TTATT 4501 CACGG CATCA AGACC CAGGG TGCCC GTCAG AAGTT CTCCA GCCTC TACAT

4551 CTCTC AGTTT ATS AT C ATGT AT AGT CTTGA TGGGA AGA AG TGGC A GACTT

4601 ATCGA GGAAA TTCCA CTGGA ACCTT AATGG TCTTC TTTGG CAATG TGGAT

4651 TCATC TGGGA TAAAA CACAA TATTT TTAAC CCTCC AATTA TTGCT CGATA

4701 CATCC GTTTG CACCC AACTC ATT AT AGC AT TCGCA GCACT CTTCG CATGG

4751 AGTTG ATGGG CTGTG ATTTA AATAG TTGCA GCATG CCATT GGGAA TGGAG

4801 AGTAA AGCAA TATCA GATGC ACAGA TTACT GCTTC ATCCT ACTTT ACCAA

4851 TATGT TTGCC ACCTG GTCTC CTTCA AAAGC TCGAC TTCAC CTCCA AGGGA

4901 GGAGT AATGC CTGGA GACCT CAGGT GAATA ATCCA AAAGA GTGGC TGCAA

4951 GTGGA CTTCC AGAAG ACAAT GAAAG TCACA GGAGT AACTA CTCAG GGAGT

5001 AAAAT CTCTG CTTAC CAGCA TGTAT GTGAA GGAGT TCCTC ATCTC CAGCA

5051 GTCAA GATGG CCATC AGTGG ACTCT CTTTT TTCAG AATGG CAAAG TAAAG

5101 GTTTT TC AGG GA A AT C A AGA CTCCT TCACA CCTGT GGTGA ACTCT CT AGA

5151 CCCAC CGTTA CTGAC TCGCT ACCTT CGAAT TCACC CCCAG AGTTG GGTGC

5201 ACC AG ATTGC CCTGA GGATG GAGGT TCTGG GCTGC GAGGC AC AGG ACCTC

5251 TACGA CAAAA CTCAC ACATG CCCAC CGTGC CCAGC TCCAG AACTC CTGGG 5301 CGGAC CGTCA GTCTT CCTCT TCCCC CCAAA ACCCA AGGAC ACCCT CATGA 5351 TCTCC CGGAC CCCTG AGGTC ACATG CGTGG TGGTG GACGT GAGCC ACGAA

5401 GACCC TGAGG TCAAG TTCAA CTGGT ACGTG GACGG CGTGG AGGTG CATAA

5451 TGCCA AGACA AAGCC GCGGG AGGAG CAGTA CAACA GCACG TACCG TGTGG

5501 TCAGC GTCCT CACCG TCCTG SASSA GGACT GGCTG AATGG CAAGG AGTAC

5551 AAGTG CAAGG TCTCC AACAA AGCCC TCCCA GCCCC CATCG AGAAA ASSAT

5601 CTCCA AAGCC AAAGG GCAGC CCCGA GAACC ACAGG TGTAC ACCCT GCCCC 5651 CATCC CGGGA TGAGC TGACC AAGAA CCAGG TCAGC CTGAC CTGCC TGGTC 5701 AAAGG CTTCT ATCCC AGCGA CATCG CCGTG GAGTG GGAGA GCA AT GGGCA 5751 GCCGG AGAAC AACTA CAAGA CCACG CCTCC CGTGT TGGAC TCCGA CGGCT

5801 STTTS TTCCT STASA GCAAG CTCAC CGTGG ACAAG AGCAG GTGGC AGCAG

5851 GGGAA CGTCT TCTC A TGCTC CGTGA TGC AT GAGGC TCTGC AC A AC C ACT A

5901 CACGC AGAAG AGCCT CTCCC TGTCT CCGGG TAAAT GA

FVIII169 protein sequence (SEQ ID NO: 70).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNIAKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

- 133 044115

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKN NE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE 801 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 851 EGTSESATPE SGPGSEPATS GSETPG TSES ATPESGPGSP AGSPTSTEEG

901 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 951 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1001 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1051 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1101 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1151 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1201 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1251 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1301 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1351 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1401 VFETVEMLPS KAGIWRVECLIGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1451 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1501 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1551 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1601 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1651 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1701 VF QGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1751 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

1801 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

1851 KCKVSNKALP APIEKTIS KA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

1901 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

1951 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

- 134 044115

Nucleotide sequence of FVIII 263 (IHH triple mutant) (SEQ ID NO: 156).

ATGCA AATAG AGCTC TCCAC CTGCT TCTTT CTGTG CCTTT TGCGA TTCTG

CTTTA GTGCC ACCAG AAGAT ACTAC CTGGG TGC AG TGGAA CTGTC ATGGG

101 ASTAT ATGCA AGGCG CGCC A AC ATS AGAGA GCGCC ACCCC TGA A A GTGGT

151 CCCGG GAGCG AGCC A GCCAC ATCTG GGTCG GAAAC GCCAG GC AC A AGTGA

201 GTCTG CAACT CCCGA GTCCG GACCT GGCTC CGAGC CTGCC ACT AG CGGCT

251 CCGAG ACTCC GGGAA CTTCC GAGAG CGCTA SASSA GAAAG CGGAC CCGGA

301 ACCAG TACCG AACCT AGCGA GGGCT CTGCT CCGGG CAGCC CAGCC GGCTC

351 TCCTA CATCC ACGGA GGAGG GCACT TCCGA ATCCG CCACC CCGGA GTCAG

401 GGCCA GGATC TGAAC CCGCT ACCTC AGGCA GTGAG ACGCC AGGAA CGAGC

451 GAGTC CGCTA CACCG GAGAG TGGGC CAGGG AGCCC TGCTG GATCT CCTAC

501 GTCCA CTGAG GAAGG GTCAC CAGCG GGCTC GCCCA CCAGC ACTGA AGAAG

551 GTGCC TCGAG CAGTG ATCTC GGTGA GCTGC CTGTG GACGC AAGAT TTCCT 601 CCTAG AGTGC CAAAA TCTTT TCCAT TCAAC ACCTC AGTCG TGTAC AAAAA

651 GACTC TGTTT GT AGA ATTCA CGGAT CACCT TTTCA ACATC GCTAA GCCAA

701 GGCCA CCCTG GATGG GTCTG CTAGG TCCTA CCATS CAGGC TGAGG TTTAT

751 GAT AC AGTGG TCATT ACACT TAAGA ACATG GCTTC CCATC CTGTC AGTCT

801 TCATG CTGTT GGTGT ATCCT ACTGG AAAGC TTCTG AGGGA GCTGA ATATG

851 ATGAT CAGAC CAGTC AAAGG GAGAA AGAAG ATGAT AAAGT CTTCC CTGGT

901 GGA AG CC AT A CAT AT GTCTG GC AGG TCCTG A A AGA GA ATG GTCC A ATGGC

951 CTCTG ACCCA CTGTG CCTTA CCTAC TCATA TCTTT CTCAT GTGGA CCTGG

1001 TAAAA GACTT GA ATT CAGGC CTCAT TGGAG CCCTA CT AGT ATGTA GAGAA

1051 GGGAG TCTGG CC AAG GAAAA GAC AC AGACC TTGC A C AAAT TT ATA CT ACT

1101 TTTTG CTGT A TTTGA TGA AG GGA A A AGTTG GCACT C AGA A AC A A A GA ACT

1151 CCTTG ATGCA GGATA GGGAT GCTGC ATCTG CTCGG GCCTG GCCTA AAATG

1201 CACAC AGTCA ATGGT TATGT A A AC A GGTCT CTGCC AGGTC TGATT GGATG

1251 CCACA GGAAA TCAGT CTATT GGCAT GTGAT TGGAA TGGGC ACCAC TCCTG

1301 AAGTG CACTC AATAT TCCTC GAAGG TCACA CATTT CTTGT GAGGA ASSAT

1351 CGCC A GGCT A GCTTG GA A AT CTCGC C A AT A ACTTT CCTTA CTGCT C A A AC

1401 ACTCT TGATG GACCT TGGAC AGTTT CT ACT GTTTT GTCAT ATCTC TTCCC

1451 ACCAA CATGA TGGCA TGGAA GCTTA TGTCA AAGTA GACAG CTGTC CAGAG

1501 GAACC CCAAC TACGA ATGAA AAATA ATGAA GAAGC GGAAG ASTAT GATGA 1551 TGATC TT ACT GATTC TGA A A TGGAT GTGGT C AGGT TTGAT GATGA CAACT

1601 CTCCT TCCTT TATCC AAATT CGCTC AGTTG CCAAG AAGCA TCCTA AAACT

1651 TGGGT ACATT ACATT GCTGC TGAAG AGGAG GACTG GGACT ATGCT CCCTT

1701 AGTCC TCGCC CCCGA TGACA GAAGT TATAA AAGTC AATAT TTGAA CAATG

1751 GCCCT CAGCG GATTG GTAGG AAGTA CAAAA AAGTC CGATT TATGG CATAC

- 135 044115

1801 ACAGA TGAAA CCTTT AAGAC TCGTG AAGCT ATTCA GCATG AATCA GGAAT

1851 CTTGG GACCT TT ACT TT ATG GGGA A GTTGG AGAC A C ACTG TTGAT TAT AT

1901 TTAAG AATCA AGCA A GCAGA CC AT A T AACA TCT AC CCTC A CGGAA TCACT

1951 GATGT CCGTC CTTTG TATTC AAGGA GATTA CCAAA AGGTG TAAAA CATTT

2001 GA AGG ATTTT CCA AT TCTGC CAGGA GA A AT ATTCA A AT AT A A ATG GAC AG

2051 TGACT GT AGA AGATG GGCCA ACTAA ATCAG ATCCT CGGTG CCTGA CCCGC

2101 TATTA CTCT A GTTTC GTT A A T ATGG AGAGA GATCT AGCTT CAGGA CTC AT

2151 TGGCC CTCTC CTC AT CTGCT AC A A A GA ATC TGT AG ATC A A AGAGG A A ACC

2201 AGATA ATGTC AGACA AGAGG AATGT CATCC TGTTT TCTGT ATTTG ATGAG

2251 AACCG AAGCT GGT AC CTCAC AGAGA AT ATA CAACG CTTTC TCCCC AATCC

2301 AGCTG GAGTG CAGCT TGAGG ATCCA GAGTT CCAAG CCTCC AACAT CATGC

2351 AC AGC ATC A A TGGCT ATGTT TTTGA T AGTT TGCAG TTGTC AGTTT GTTTG

2401 CATGA GGTGG CAT AC TGGTA CATTC TA AGC ATTGG AGC AC AGACT GACTT

2451 CCTTT CTGTC TTCTT CTCTG GAT AT ACCTT CAAAC AC AAA ATGGT CT ATG

2501 AAGAC ACACT CACCC TATTC CCATT CTCAG GAGAA ACTGT CTTCA TGTCG

2551 ATGGA A AACC CAGGT CT ATG GATTC TGGGG TGCC A C A ACT C AGAC TTTCG

2601 GAACA GAGGC ATGAC CGCCT TACTG AAGGT TTCTA GTTGT GACAA GAACA

2651 CTGGT GATTA TTACG AGGAC AGTTA TGAAG AT ATT TCAGC AT ACT TGCTG

2701 AGTAA AAACA ATGCC ATTGA ACCAA GAAGC TTCTC TCAAA ACGGC GCGCC

2751 AGGTA CCTCA GAGTC TGCTA CCCCC GAGTC AGGGC CAGGA TCAGA GCCAG

2801 CCACC TCCGG GTCTG AGACA CCCGG GACTT CCGAG AGTGC CACCC CTGAG

2851 TCCGG ACCCG GGTCC GAGCC CGCCA CTTCC GGCTC CGAAA CTCCC GGCAC

2901 AAGCG AGAGC GCTAC CCCAG AGTCA GGACC AGGAA CATCT ACAGA GCCCT

2951 CTGAA GGCTC CGCTC CAGGG TCCCC AGCCG GCAGT CCCAC TAGCA CCGAG

3001 GAGGG AACCT CTGAA AGCGC CACAC CCGAA TCAGG GCCAG GGTCT GAGCC

3051 TGCTA CCAGC GGC AG CGAGA SASSA GGC AC CTCTG AGTCC GCCAC ACC AG

3101 AGTCC GGACC CGGAT CTCCC GCTGG GAGCC CCACC TCCAC TGAGG AGGGA

3151 TCTCC TGCTG GCTCT CCAAC ATCTA CTGAG GAAGG TACCT CAACC GAGCC

3201 ATCCG AGGGA TCAGC TCCCG GCACC TCAGA GTCGG CAACC CCGGA GTCTG

3251 GACCC GGAAC TTCCG AAAGT GCCAC ACC AG AGTCC GGTCC CGGGA CTTCA

3301 GAATC AGCAA CACCC GAGTC CGGCC CTGGG TCTGA ACCCG CCACA AGTGG

3351 TAGTG AGACA CCAGG ATCAG AACCT GCTAC CTCAG GGTCA GAGAC ACCCG

3401 GATCT CCGGC AGGCT SASSA ACCTC CACTG AGGAG GGCAC CAGCA CAGAA 3451 CCAAG CGAGG GCTCC GCACC CGGAA CAAGC ACTGA ACCCA GTGAG GGTTC 3501 AGCAC CCGGC TCTGA GCCGG CCACA AGTGG CAGTG AGACA CCCGG CACTT

3551 CAGAG AGTGC CACCC CCGAG AGTGG CCCAG GCACT AGTAC CGAGC CCTCT

3601 GAAGG CAGTG CGCCA GCCTC GAGCC SASSA GTCTT GAAAC GCCAT CAAGC

3651 TGAAA TAACT CGTAC TACTC TTCAG TCAGA TCAAG AGGAA ATCGA TTATG

- 136 044115

3701 ATGAT ACCAT ATS AG TTGAA ATGAA GAAGG AAGAT TTTGA CATTT ATGAT 3751 GAGGA TGAAA ATS AG AGCCC CCGCA GCTTT CAAAA GAAAA CACGA CACTA 3801 TTTTA TTGCT GCAGT GGAGA GGCTC TGGGA TTATG GGATG AGTAG CTCCC 3851 C AC AT GTTCT A AGA A AC AGG GCTC A GAGTG GCAGT GTCCC TC AGT TCA AG 3901 AAAGT TGTTT TCCAG GAATT T ACTG ATGGC TCCTT T ACTC AGCCC TT AT A 3951 CCGTG GAGAA CT AAA TGAAC ATTTG GGACT CCTGG GGCCA TAT AT AAGAG 4001 CAGAA GTTGA AGATA ATATC ATGGT AACTT TCAGA AATCA GGCCT CTCGT 40 51 CCCTA TTCCT TCT AT TCTAG CCTTA TTTCT TATGA GGAAG ATS AG AGGCA

4101 AGGAG CAGAA CCT AG A A A A A ACTTT GTC A A GCCT A ATGAA ACC A A AACTT 4151 ACTTT TGGA A AGTGC A AC AT CAT AT GGC AC CC ACT A A AGA TGAGT TTGAC 4201 TGC A A AGCCT GGGCT TATTT CTCTG ATGTT GACCT GGA A A AAGAT GTGCA 4251 C TCAG GCCTG ATTGG ACCCC TTCTG GTCTG CCACA CTAAC ACACT GAACC 4301 CTGCT CATGG GAGAC AAGTG ACAGT ACAGG AATTT GCTCT GTTTT TCACC 4351 ATCTT TGATG AGACC A A AAG CTGGT ACTTC ACTGA A A AT A TGGA A AGA A A 4401 CTGCA GGGCT CCCTG CAATA TCCAG ATGGA AGATC CCACT T TT A A AGAGA 4451 ATT AT CGCTT CCATG CAATC AATGG STASA TAATG GAT AC ACTAC CTGGC 4501 TTAGT AATGG CTCAG GATCA AAGGA TTCGA TGGTA TCTGC TCAGC ATGGG 4551 C AGC A ATGAA A AC AT CC ATT CT ATT CATTT C AGTG GAC AT GTGTT C ACTG

4601 TACGA AAAAA AGAGG AGT AT AAAAT GGC AC TGT AC AATCT ST ATS CAGGT 4651 GTTTT TGAGA CAGTG GAAAT GTTAC CATCC AAAGC TGGAA TTTGG CGGGT 4701 GGAAT GCCTT ATTGG CGAGC ATCTA CATGC TGGGA TGAGC ACACT TTTTC 4751 TGGTG TACAG CAATA AGTGT CAGAC TCCCC TGGGA ATGGC TTCTG GACAC 4801 ATTAG AGATT TTCAG ATTAC AGCTT CAGGA CAATA TGGAC AGTGG GCCCC 4851 AAAGC TGGCC AGACT TCATT ATTCC GGATC AATCA ATGCC TGGAG SASSA 4901 AGGAG CCCTT TTCTT GGATC AAGGT GGATC TGTTG GCACC AATGA TTATT 4951 CACGG CATCA AGACC CAGGG TGCCC GTCAG AAGTT CTCCA GCCTC TACAT 5001 CTCTC AGTTT ATCAT CATGT ATAGT CTTGA TGGGA AGAAG TGGCA GACTT 5051 ATCGA GGAAA TTCCA CTGGA ACCTT AATGG TCTTC TTTGG CAATG TGGAT 5101 TC ATC TGGGA TA A A A C AC A A TATTT TTA AC CCTCC A ATT A TTGCT CGAT A 5151 CATCC GTTTG CACCC AACTC ATT AT AGC AT TCGC A GC ACT CTTCG CATGG 5201 AGTTG ATGGG CTGTG ATTTA AATAG TTGCA GCATG CCATT GGGAA TGGAG 5251 AGTAA AGCAA TATCA GATGC AC AGA TTACT GCTTC ATCCT ACTTT ACCAA 5301 TATGT TTGCC ACCTG GTCTC CTTCA AAAGC TCGAC TTCAC CTCCA AGGGA 5351 GGAGT AATGC CTGGA GACCT CAGGT GAATA ATCCA AAAGA GTGGC TGCAA 5401 GTGGA CTTCC AGAAG ACAAT GAAAG TCACA GGAGT AACTA CTCAG GGAGT 5451 AAAAT CTCTG CTTAC CAGCA TGTAT GTGAA GGAGT TCCTC ATCTC CAGCA 5501 GTCAA GATGG CCATC AGTGG ACTCT CTTTT TTCAG AATGG CAAAG TAAAG 5551 GTTTT TCAGG GAAAT CAAGA CTCCT TCACA CCTGT GGTGA ACTCT CTAGA

- 137 044115

5601 CCCAC CGTTA CTGAC TCGCT ACCTT CGAAT TCACC CCCAG AGTTG GGTGC

5651 ACC AG ATTGC CCTGA GGATG GAGGT TCTGG GCTGC GAGGC AC AGG ACCTC

5701 TACGA CAAAA CTCAC ACATG CCCAC CGTGC CCAGC TCCAG AACTC CTGGG

5751 CGGAC CGTCA GTCTT CCTCT TCCCC CCAAA ACCCA AGGAC ACCCT CATGG

5801 CCTCC CGGAC CCCTG AGGTC ACATG CGTGG TGGTG GACGT GAGCC ACGAA

5851 GACCC TGAGG TC AAG TTC A A CTGGT ACGTG GACGG CGTGG AGGTG C AT AA

5901 TGCCA AGACA AAGCC GCGGG AGGAG CAGTA CAACA GCACG TACCG TGTGG 5951 TCAGC GTCCT CACCG TCCTG GCCCA GGACT GGCTG AATGG CAAGG AGTAC

6001 AAGTG CAAGG TCTCC A AC A A AGCCC TCCCA GCCCC CATCG AGA A A ACCAT

6051 CTCCA AAGCC AAAGG GCAGC CCCGA GAACC ACAGG TGTAC ACCCT GCCCC

6101 C ATCC CGCGA TGAGC TGACC A AGA A CC AGG TCAGC CTGAC CTGCC TGGTC

6151 AAAGG CTTCT ATCCC AGCGA CATCG CCGTG GAGTG GGAGA GCA AT GGGCA

6201 GCCGG AGA AC A ACT A CAAGA CCACG CCTCC CGTGT TGGAC TCCGA CGGCT

6251 CCTTS TTCCT STASA GCAAG CTCAC CGTGG ACAAG AGCAG GTGGC AGCAG

6301 GGGAA CGTCT TCTCA TGCTC CGTGA TGCAT GAGGC TCTGC ACAAC GCCTA

6351 CACGC AGAAG AGCCT CTCCC TGTCT CCGGG TAAAT GA

FVIII 263 protein sequence (IHH triple mutant) (SEQ ID NO: 157).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS

151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTTLTLF PFSGETVFMS 851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE

- 138 044115

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE

1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMASRTPEV TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL AQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

2101 GNVFSCSVMH EALHNAYTQK SLSLSPGK*

Nucleotide sequence of FVIII 282 (SEQ ID NO: 158).

ATGCA AATAG AGCTC TCCAC CTGCT TCTTT CTGTG CCTTT TGCGA TTCTG

CTTTA GTGCC ACCAG AAGAT ACTAC CTGGG TGCAG TGGAA CTGTC ATGGG

101 ASTAT ATGCA AAGTG ATCTC GGTGA GCTGC CTGTG GACGC AAGAT TTCCT

151 CCTAG AGTGC CAAAA TCTTT TCCAT TCAAC ACCTC AGTCG TGTAC AAAAA 201 GACTC TGTTT GT AGA ATTCA CGGAT CACCT TTTCA ACATC GCT A A GCC A A 251 GGCCA CCCTG GATGG GTCTG CTAGG TCCTA CCATC CAGGC TGAGG TTTAT 301 GATAC AGTGG TCATT ACACT TAAGA ACATG GCTTC CCATC CTGTC AGTCT

51 TCATG CTGTT GGTGT ATCCT ACTGG AAAGC TTCTG AGGGA GCTGA ATATG

401 ATGAT CAGAC CAGTC A A AGG GAGA A AGA AG ATGAT AAAGT CTTCC CTGGT

451 GGAAG CCATA CAT AT GTCTG GCAGG TCCTG AAAGA GA ATG GTCCA ATGGC

501 CTCTG ACCCA CTGTG CCTTA CCTAC TCATA TCTTT CTCAT GTGGA CCTGG

- 139 044115

551 TAAAA GACTT GAATT CAGGC CTCAT TGGAG CCCTA CTAGT ATGTA GAGAA

601 GGGAG TCTGG CCA AG GAAAA GAC AC AGACC TTGCA CAAAT TTATA CT ACT

651 TTTTG CTGTA TTTGA TGAAG GGAAA AGTTG GCACT CAGAA AC AAA GAACT

701 CCTTG ATGC A GGAT A GGGAT GCTGC ATCTG CTCGG GCCTG GCCT A A A ATG

751 CACAC AGTCA ATGGT TATGT AAACA GGTCT CTGCC AGGTC TGATT GGATG

801 CCACA GGAAA TCAGT CTATT GGCAT GTGAT TGGAA TGGGC ACCAC TCCTG

851 AAGTG CACTC AATAT TCCTC GAAGG TCACA CATTT CTTGT GAGGA ACCAT

901 CGCC A GGCT A GCTTG GA AAT CTCGC C A AT A ACTTT CCTT A CTGCT CAA AC

951 ACTCT TGATG GACCT TGGAC AGTTT CT ACT GTTTT GTC AT ATCTC TTCCC

1001 ACCAA CATGA TGGCA TGGAA GCTTA TGTCA AAGTA GACAG CTGTC CAGAG 1051 GAACC CCAAC TACGA ATGAA AAATA ATGAA GAAGC GGAAG ASTAT GATGA 1101 TGATC TT ACT GATTC TGA A A TGGAT GTGGT C AGGT TTGAT GATGA C A ACT 1151 CTCCT TCCTT TAT CC AAATT CGCTC AGTTG CCAAG AAGCA TCCTA AAACT 1201 TGGGT AC ATT AC ATT GCTGC TGAAG AGGAG GACTG GGACT ATGCT CCCTT 1251 AGTCC TCGCC CCCGA TGACA GAAGT TATAA AAGTC AATAT TTGAA CAATG 1301 GCCCT CAGCG GATTG GTAGG AAGTA CAAAA AAGTC CGATT TATGG CATAC 1351 AC AGA TGA A A CCTTT A AGAC TCGTG A AGCT ATTC A GC ATG A ATC A GGA AT 1401 CTTGG GACCT TT ACT TTH GGGA AGAC AGAC ACTG TTGAT TTAT AT 1451 TTAAG A AGCA AGCA AGA CC AACA TCTC ACTC ACTC TCACT 1501 GATGT CCGTC CTTTG TC ATTC AAGGA GATTA CCAAA AGGTG TAAAA CATTTTTTTTTH 1551 GAAGG ATTTT CCA AT TCTGC C AGGA GA A AT ATTC A A AT AT A A ATG GAC AG 1601 TGACT GT AGA AGATG GGCCA ACT AA ATCAG ATCCT CGGTG CCTGA CCCGC 1651 TATTA CTCTA GTTTC GTTAA TATGG AGAGA GATCT AGCTT CAGGA CTCAT 1701 TGGCC CTCTC CTCAT CTGCT AC AAA GAATC TGT AG ATCAA AGAGG AAACC 1751 AGATA ATGTC AGACA AGAGG AATGT CATCC TGTTT TCTGT ATTTG ATGAG 1801 AACCG AAGCT GGT AC CTCAC AGAGA AT ATA CAACG CTTTC TCCCC AATCC 1851 AGCTG GAGTG C AGCT TGAGG ATCC A GAGTT CCA AG CCTCC AAC AT C ATGC 1901 AC AGC ATCAA TGGCT ATGTT TTTGA T AGTT TGC AG TTGTC AGTTT GTTTG 1951 CATGA GGTGG CATAC TGGTA CATTC TAAGC ATTGG AGCAC AGACT GACTT 2001 CCTTT CTGTC TTCTT CTCTG GAT AT ACCTT CAAAC AC AAA ATGGT CT ATG 2051 AAGAC AC ACT CACCC TATTC CCATT CTC AG GAGAA ACTGT CTTCA TGTCG 2101 ATGGA AAACC C AGGT CT ATG GATTC TGGGG TGCC A C A ACT C AGAC TTTCG 2151 GA AC A GAGGC ATGAC CGCCT T ACTG A AGGT TTCT A GTTGT GAC A A GA AC A 2201 CTGGT GATTA TT ACG AGGAC AGTT A TGAAG AT ATT TC AGC AT ACT TGCTG 2251 AGTAA AAACA ATGCC ATTGA ACCAA GAAGC TTCTC TCAAA ACGGC GCGCC 2301 AACAT CAGAG AGCGC CACCC CTGAA AGTGG TCCCG GGAGC GAGCC AGCCA 2351 C ATCT GGGTC GGAAA CGCC A GGC AC AAGTG AGTCT GCA AC TCCCG AGTCC 2401 GGACC TGGCT CCGAG CCTGC CACTA GCGGC TCC GA GACTC CGGGA ACTTC

- 140 044115

2451 CGAGA GCGCT ACACC AGAAA GCGGA CCCGG AACCA GT ACC GAACC TAGCG

2501 AGGGC TCTGC TCCGG GCAGC CCAGC CGGCT CTCCT ACATC CACGG AGGAG

2551 GGCAC TTCCG AATCC GCCAC CCCGG AGTCA GGGCC AGGAT CTGAA CCCGC

2601 TACCT CAGGC AGTGA GACGC CAGGA ACGAG CGAGT CCGCT ACACC GGAGA 2651 GTGGG CCAGG GAGCC CTGCT GGATC TCCTA CGTCC ACTGA GGAAG GGTCA 2701 CCAGC GGGCT CGCCC ACC AG CACTG AAGAA GGTGC CTCGA GCCCA CCAGT

2751 CTTGA AACGC CATCA AGCTG AAATA ACTCG TACTA CTCTT CAGTC AGATC

2801 AAGAG GAAAT CGATT ATGAT GATAC CATAT CAGTT GAAAT GAAGA AGGAA

2851 GATTT TGACA TTTAT GATGA GGATG AAAAT CAGAG CCCCC GCAGC TTTCA

2901 AAAGA AAACA CGACA CT ATT TTATT GCTGC AGTGG AGAGG CTCTG GGATT

2951 ATGGG ATGAG TAGCT CCCCA CATGT TCTAA GAAAC AGGGC TCAGA GTGGC

3001 AGTGT CCCTC AGTTC AAGAA AGTTG TTTTC CAGGA ATTTA CTGAT GGCTC

3051 CTTTA CTCAG CCCTT AT ACC GTGGA GAACT AAATG AAC AT TTGGG ACTCC

3101 TGGGG CC AT A TAT A A GAGC A GA AGT TGA AG AT A AT ATC AT GGT A A CTTTC

3151 AGAAA TCAGG CCTCT CGTCC CTATT CCTTC TATTC TAGCC TTATT TCTTA

3201 TGAGG AAGAT CAGAG GCAAG GAGCA GAACC TAGAA AAAAC TTTGT CAAGC 3251 CTAAT GAAAC CAAAA CTTAC TTTTG GAAAG TGCAA CATCA TATGG CACCC 3301 ACTAA AGATG AGTTT GACTG CAAAG CCTGG GCTTA TTTCT CTGAT GTTGA 3351 CCTGG AAAAA GATGT GCACT CAGGC CTGAT TGGAC CCCTT CTGGT CTGCC

3401 AC ACT AAC AC ACTGA ACCCT GCTCA TGGGA GACAA GTGAC AGTAC AGGAA

3451 TTTGC TCTGT TTTTC ACCAT CTTTG ATGAG ACCAA AAGCT GGTAC TTCAC

3501 TGAAA ATATG GAAAG AAACT GCAGG GCTCC CTGCA ATATC CAGAT GGAAG

3551 ATCCC ACTTT TAAAG AGAAT TATCG CTTCC ATGCA ATCAA TGGCT ACATA

3601 ATGGA TACAC TACCT GGCTT AGTAA TGGCT CAGGA TCAAA GGATT CGATG

3651 GT ATC TGCTC AGC AT GGGCA GCAAT GAAAA CATCC ATTCT ATTCA TTTCA

3701 GTGGA CATGT GTTCA CTGTA CGAAA AAAAG AGGAG TATAA AATGG CACTG

3751 TACAA TCTCT ATCCA GGTGT TTTTG AGACA GTGGA AATGT TACCA TCCAA

3801 AGCTG GAATT TGGCG GGTGG AATGC CTTAT TGGCG AGCAT STASA TGCTG

3851 GGATG AGC AC ACTTT TTCTG GTGT A C AGC A AT A AG TGTC A GACTC CCCTG

3901 GGAAT GGCTT CTGGA CACAT TAGAG ATTTT CAGAT TACAG CTTCA GGACA

3951 ATATG GACAG TGGGC CCCAA AGCTG GCC AG ACTTC ATT AT TCCGG ATCAA

4001 TCAAT GCCTG GAGCA CCAAG GAGCC CTTTT CTTGG ATCAA GGTGG ATCTG

4051 TTGGC ACCAA TGATT ATTCA CGGCA TCAAG ACCCA GGGTG CCCGT CAGAA

4101 GTTCT CCAGC CTCTA СATCT CTCAG TTTAT CATCA TGTAT AGTCT TGATG

4151 GGAAG A AGTG GC AGA CTTAT CGAGG A A ATT CC ACT GGA AC CTT A A TGGTC

4201 TTCTT TGGC A ATGTG GATTC ATCTG GGAT A AAACA C A AT A TTTTT A ACCC

4251 TCCAA TTATT GCTCG ATACA TCCGT TTGCA CCCAA CTCAT TAT AG CATTC 4301 GCAGC ACTCT TCGCA TGGAG TTGAT GGGCT GTGAT TTAAA TAGTT GCAGC

- 141 044115

4351 ATGCC ATTGG GA ATG GAGAG TA A AG C A AT A TC AGA TGC AC AGATT ACTGC

4401 TTCAT CCTAC TTTAC CAATA TGTTT GCCAC CTGGT CTCCT TCAAA AGCTC

4451 GACTT CACCT CCAAG GGAGG AGTAA TGCCT GGAGA CCTCA GGTGA ATAAT 4501 CCAAA AGAGT GGCTG CAAGT GGACT TCCAG AAGAC AATGA AAGTC ACAGG

51 AGTAA CT ACT C AGGG AGTAA A ATCT CTGCT T ACC A GC ATG T ATGT GAAGG

4601 AGTTC CTCAT CTCCA GCAGT CAAGA TGGCC ATS AG TGGAC TCTCT TTTTT

4651 CAGAA TGGCA AAGTA AAGGT TTTTC AGGGA AATCA AGACT CCTTC ACACC

4701 TGTGG TGAAC TCTCT AGACC CACCG TTACT GACTC GCTAC CTTCG AATTC

4751 ACCCC CAGAG TTGGG TGCAC CAGAT TGCCC TGAGG ATGGA GGTTC TGGGC

4801 TGCGA GGCAC AGGAC CTCTA CGACA AAACT CACAC ATGCC CACCG TGCCC

4851 AGCTC CAGAA CTCCT GGGCG GACCG TCAGT CTTCC TCTTC CCCCC AAAAC

4901 CCAAG GACAC CCTCA TGATC TCCCG GACCC CTGAG GTCAC ATGCG TGGTG

4951 GTGGA CGTGA GCCAC GAAGA CCCTG AGGTC AAGTT CAACT GGTAC GTGGA

5001 CGGCG TGGAG GTGC A T AATG CCAAG ACAA A GCCGC GGGAG GAGCA GT AC A

5051 ACAGC ACGTA CCGTG TGGTC AGCGT CCTCA CCGTC CTGCA CCAGG ACTGG

5101 CTGAA TGGCA AGGAG TACA A GTGC A AGGTC TCC A A C A A AG CCCTC CC AGC 5151 CCCCA TCGAG AAAAC CATCT CCAAA GCCAA AGGGC AGCCC CGAGA ACC AC 5201 AGGTG TACAC CCTGC CCCCA TCCCG GGATG AGCTG ACCAA GAACC AGGTC 5251 AGCCT GACCT GCCTG GTC AA AGGCT TCTAT CCCAG CGACA TCGCC GTGGA

5301 GTGGG AGAGC AATGG GCAGC CGGAG AACAA STASA AGACC ACGCC TCCCG

5351 TGTTG GACTC CGACG GCTCC TTCTT CCTCT ACAGC AAGCT CACCG TGGAC 5401 AAGAG CAGGT GGCAG CAGGG GAACG TCTTC TCATG CTCCG TGATG CATGA

5451 GGCTC TGCAC AACCA STASA CGCAG AAGAG CCTCT CCCTG TCTCC GGGTA

5501 AATGA

FVIII 282 protein sequence (SEO ID NO: 159).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

451 TDETFKTREAIQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

- 142 044115

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

751 SKNNAIEPRS FSQNGAPTSE SATPESGPGS EPATSGSETP GTSESATPES

801 GPGSEPATSG SETPGTSESA TPESGPGTST EPSEGSAPGS PAGSPTSTEE

851 GTSESATPES GPGSEPATSG SETPGTSESA TPESGPGSPA GSPTSTEEGS

901 PAGSPTSTEE GASSPPVLKR HQAEITRTTL QSDQEEIDYD DTISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1151 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI

1201 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1251 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1301 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1351 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1401 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1451 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1501 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1551 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1601 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1651 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

1701 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1751 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

1801 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* Nucleotide sequence of FVIII 283 (FVIII169 with triple IHH mutation) (SEQ ID NO: 160).

ATGCA AATAG AGCTC TCCAC CTGCT TCTTT CTGTG CCTTT TGCGA TTCTG

CTTTA GTGCC ACCAG AAGAT ACTAC CTGGG TGCAG TGGAA CTGTC ATGGG

101 ASTAT ATGCA AAGTG ATCTC GGTGA GCTGC CTGTG GACGC AAGAT TTCCT

151 CCTAG AGTGC CAAAA TCTTT TCCAT TCAAC ACCTC AGTCG TGTAC AAAAA

201 GACTC TGTTT GT AGA ATTCA CGGAT CACCT TTTCA AC ATS GCTAA GCC A A

251 GGCCA CCCTG GATGG GTCTG CTAGG TCCTA CCATS CAGGC TGAGG TTTAT

301 GAT AC AGTGG TCATT AC ACT TAAGA ACATG GCTTC CCATC CTGTC AGTCT

- 143 044115

351 TCATG CTGTT GGTGT ATCCT ACTGG AAAGC TTCTG AGGGA GCTGA ATATG

401 ATGAT CAGAC CAGTC A A AGG GAGA A AGA AG ATGAT A A AGT CTTCC CTGGT

451 GGAAG CCATA CAT AT GTCTG GCAGG TCCTG AAAGA GA ATG GTCCA ATGGC

501 CTCTG ACCC A CTGTG CCTTA CCTAC TC AT A TCTTT CTCAT GTGGA CCTGG

551 TAAAA GACTT GAATT CAGGC CTCAT TGGAG CCCTA CTAGT ATGTA GAGAA

601 GGGAG TCTGG CCA AG GAAAA GAC AC AGACC TTGCA CAAAT TTATA CT ACT

651 TTTTG CTGTA TTTGA TGAAG GGAAA AGTTG GCACT CAGAA AC AAA GAACT

701 CCTTG ATGC A GGAT A GGGAT GCTGC ATCTG CTCGG GCCTG GCCT A A A ATG

751 CACAC AGTCA ATGGT TATGT AAACA GGTCT CTGCC AGGTC TGATT GGATG

801 CCACA GGAAA TCAGT CTATT GGCAT GTGAT TGGAA TGGGC ACCAC TCCTG

851 AAGTG CACTC AATAT TCCTC GAAGG TCACA CATTT CTTGT GAGGA ASSAT

901 CGCC A GGCT A GCTTG GA AAT CTCGC C A AT A ACTTT CCTTA CTGCT C A A AC

951 ACTCT TGATG GACCT TGGAC AGTTT CT ACT GTTTT GTC AT ATCTC TTCCC

1001 ACCAA CATGA TGGCA TGGAA GCTTA TGTCA AAGTA GACAG CTGTC CAGAG 1051 GAACC CCAAC TACGA ATGAA AAATA ATGAA GAAGC GGAAG ASTAT GATGA 1101 TGATC TT ACT GATTC TGA A A TGGAT GTGGT C AGGT TTGAT GATGA C A ACT 1151 CTCCT TCCTT TAT CC AAATT CGCTC AGTTG CCAAG AAGCA TCCTA AAACT 1201 TGGGT AC ATT AC ATT GCTGC TGAAG AGGAG GACTG GGACT ATGCT CCCTT 1251 AGTCC TCGCC CCCGA TGACA GAAGT TATAA AAGTC AATAT TTGAA CAATG 1301 GCCCT CAGCG GATTG GTAGG AAGTA CAAAA AAGTC CGATT TATGG CATAC 1351 AC AGA TGA A A CCTTT A AGAC TCGTG A AGCT ATTC A GC ATG A ATC A GGA AT 1401 CTTGG GACCT TT ACT TT ATG GGGA A GTTGG AGAC A C ACTG TTGAT TAT AT 1451 TTAAG A ATC A AGCA A GC AGA CCATA T AACA TCT AC CCTC A CGGAA TCACT 1501 GATGT CCGTC CTTTG TATTC AAGGA GATTA CCAAA AGGTG TAAAA CATTT 1551 G AAGG ATTTT CCA AT TCTGC C AGGA GA A AT ATTC A AATAT A A ATG GAC AG 1601 TGACT GT AGA AGATG GGCCA ACT AA ATCAG ATCCT CGGTG CCTGA CCCGC 1651 TATTA CTCTA GTTTC GTTAA TATGG AGAGA GATCT AGCTT CAGGA CTCAT 1701 TGGCC CTCTC CTCAT CTGCT AC AAA GAATC TGT AG ATCAA AGAGG AAACC 1751 AGATA ATGTC AGACA AGAGG AATGT CATCC TGTTT TCTGT ATTTG ATGAG 1801 AACCG AAGCT GGTAC CTCAC AGAGA AT ATA CAACG CTTTC TCCCC AATCC 1851 AGCTG GAGTG C AGCT TGAGG ATCC A GAGTT CCA AG CCTCC AAC AT C ATGC 1 901 AC AGC ATCAA TGGCT ATGTT TTTGA T AGTT TGC AG TTGTC AGTTTT GTTTG 1951 CATGA GGTGG CATAC TGGTA CATTC TAAGC ATTGG AGCAC AGACT GACTT 2001 CCTTT CTGTC TTCTT CTCTG GAT AT ACCTT CAAAC AC AAA ATGGT CT ATG 2051 AAGAC AC ACT CACCC TATTC CCATT CTC AG GAGAA ACTGT CTTCA TGTCG 2101 ATGGA AAACC C AGGT CT ATG GATTC TGGGG TGCC A C A ACT CAGAC TTTCG 2151 GA AC A GAGGC ATGAC CGCCT T ACTG A AGGT TTCT A GTTGT GAC A A GA AC A 2201 CTGGT GATTA TT ACG AGGAC AGTT A TGAAG AT ATT TC AGC AT ACT TGCTG

- 144 044115

2251 AGTAA AAACA ATGCC ATTGA ACCAA GAAGC TTCTC TCAAA ACGGC GCGCC

2301 AGGTA CCTCA GAGTC TGCTA CCCCC GAGTC AGGGC CAGGA TCAGA GCCAG

51 CCACC TCCGG GTCTG AGACA CCCGG GACTT CCGAG AGTGC CACCC CTGAG

2401 TCCGG ACCCG GGTCC GAGCC CGCCA CTTCC GGCTC CGAAA CTCCC GGCAC

2451 AAGCG AGAGC GCTAC CCCAG AGTCA GGACC AGGAA CATCT ACAGA GCCCT 2501 CTGAA GGCTC CGCTC CAGGG TCCCC AGCCG GCAGT CCCAC TAGCA CCGAG

2551 GAGGG AACCT CTGAA AGCGC CACAC CCGAA TCAGG GCCAG GGTCT GAGCC 2601 TGCTA CCAGC GGC AG CGAGA SASSA GGCAC CTCTG AGTCC GCCAC ACC AG 2651 AGTCC GGACC CGGAT CTCCC GCTGG GAGCC CCACC TCCAC TGAGG AGGGA

2701 TCTCC TGCTG GCTCT CCAAC ATCTA CTGAG GAAGG TACCT CAACC GAGCC

2751 ATCCG AGGGA TCAGC TCCCG GCACC TCAGA GTCGG CAACC CCGGA GTCTG

2801 GACCC GGAAC TTCCG AAAGT GCCAC ACCAG AGTCC GGTCC CGGGA CTTCA

2851 GAATC AGCAA CACCC GAGTC CGGCC CTGGG TCTGA ACCCG CCACA AGTGG

2901 TAGTG AGACA CCAGG ATCAG AACCT GCTAC CTCAG GGTCA GAGAC ACCCG 2951 GATCT CCGGC AGGCT SASSA ACCTC CACTG AGGAG GGCAC CAGCA CAGAA 3001 CCAAG CGAGG GCTCC GCACC CGGAA CAAGC ACTGA ACCCA GTGAG GGTTC

3051 AGCAC CCGGC TCTGA GCCGG CCACA AGTGG CAGTG AGACA CCCGG CACTT

3101 CAGAG AGTGC CACCC CCGAG AGTGG CCCAG GCACT AGTAC CGAGC CCTCT

3151 GAAGG CAGTG CGCCA GCCTC GAGCC SASSA GTCTT GAAAC GCCAT CAAGC

3201 TGAAA TAACT CGTAC TACTC TTCAG TCAGA TCAAG AGGAA ATCGA TTATG

3251 ATGAT ACCAT ATCAG TTGAA ATGAA GAAGG AAGAT TTTGA CATTT ATGAT

3301 GAGGA TGAAA ATCAG AGCCC CCGCA GCTTT CAAAA GAAAA CACGA CACTA

3351 TTTTA TTGCT GCAGT GGAGA GGCTC TGGGA TTATG GGATG AGTAG CTCCC

3401 CACAT GTTCT AAGAA ACAGG GCTCA GAGTG GCAGT GTCCC TCAGT TCAAG

3451 AAAGT TGTTT TCCAG GAATT TACTG ATGGC TCCTT TACTC AGCCC TTATA

3501 CCGTG GAGAA CTAAA TGAAC ATTTG GGACT CCTGG GGCCA TATAT AAGAG

3551 CAGAA GTTGA AGATA ATATC ATGGT AACTT TCAGA AATCA GGCCT CTCGT

3601 CCCTA TTCCT TCTAT TCTAG CCTTA TTTCT TATGA GGAAG ATCAG AGGCA

3651 AGGAG CAGAA CCTAG AAAAA ACTTT GTCAA GCCTA ATGAA ACCAA AACTT

3701 ACTTT TGGAA AGTGC AAC AT CAT AT GGCAC CCACT AAAGA TGAGT TTGAC

3751 TGCAA AGCCT GGGCT TATTT CTCTG ATGTT GACCT GGAAA AAGAT GTGCA

3801 CTCAG GCCTG ATTGG ACCCC TTCTG GTCTG CCACA CTAAC ACACT GAACC

3851 CTGCT CATGG GAGAC AAGTG ACAGT ACAGG AATTT GCTCT GTTTT TCACC

3901 ATCTT TGATG AGACC AAAAG CTGGT ACTTC ACTGA AAATA TGGAA AGAAA

3951 CTGCA GGGCT CCCTG CAATA TCCAG ATGGA AGATC CCACT TTTAA AGAGA 4001 ATT AT CGCTT CCATG CAATC AATGG STASA TAATG GAT AC ACTAC CTGGC 4051 TTAGT AATGG CTCAG GATCA AAGGA TTCGA TGGTA TCTGC TCAGC ATGGG 4101 CAGCA ATGAA A AC AT CC ATT CT ATT CATTT CAGTG GAC AT GTGTT CACTG

- 145 044115

4151 T ACGA A A A A A AGAGG AGT AT A A A AT GGC AC TGT AC A ATCT ST ATS CAGGT 4201 GTTTT TGAGA CAGTG GAAAT GTTAC CATCC AAAGC TGGAA TTTGG CGGGT 4251 GGAAT GCCTT ATTGG CGAGC ATCTA CATGC TGGGA TGAGC AC ACT TTTTC 4301 TGGTG TAC AG CAATA AGTGT CAGAC TCCCC TGGGA ATGGC TTCTG GACAC 4351 ATT AG AGATT TTC AG ATT AC AGCTT C AGGA CAATA TGGAC AGTGG GCCCC 4401 AAAGC TGGCC AGACT TCATT ATTCC GGATC A ATC A ATGCC TGGAG SASSA 4451 AGGAG CCCTT TTCTT GGATC AAGGT GGATC TGTTG GCACC AATGA TTATT 4501 CACGG CATCA AGACC CAGGG TGCCC GTCAG AAGTT CTCCA GCCTC TACAT 4551 CTCTC AGTTT ATC AT C ATGT AT AGT CTTGA TGGGA AGA AG TGGC A GACTT 4601 ATCGA GGAAA TTCCA CTGGA ACCTT AATGG TCTTC TTTGG CAATG TGGAT 4651 TCATC TGGGA TAAAA CACAA TATTT TTAAC CCTCC AATTA TTGCT CGATA 4701 CAT CC GTTTG CACCC AACTC ATT AT AGC AT TCGCA GCACT CTTCG CATGG 4751 AGTTG ATGGG CTGTG ATTTA AATAG TTGCA GCATG CCATT GGGAA TGGAG 4801 AGTAA AGCAA TATCA GATGC ACAGA TTACT GCTTC ATCCT ACTTT ACCAA 4851 TATGT TTGCC ACCTG GTCTC CTTCA AAAGC TCGAC TTCAC CTCCA AGGGA 4901 GG AGT AATGC CTGGA GACCT CAGGT GAATA ATCCA AAAGA GTGGC TGCAA 4951 GTGGA CTTCC AGAAG ACAAT GAAAG TCACA GGAGT AACTA CTCAG GGAGT 5001 AAAAT CTCTG CTTAC CAGCA TGTAT GTGAA GGAGT TCCTC ATCTC CAGCA 5051 GTCAA GATGG CCATC AGTGG ACTCT CTTTT TTCAG AATGG CAAAG TAAAG 5101 GTTTT TC AGG GAAAT C A AGA CTCCT T CACA CCTGT GGTGA ACTCT CT AGA 5151 CCCAC CGTTA CTGAC TCGCT ACCTT CGAAT TCACC CCCAG AGTTG GGTGC 5201 ACC AG ATTGC CCTGA GGATG GAGGT TCTGG GCTGC GAGGC AC AGG ACCTC 5251 TACGA CAAAA CTCAC ACATG CCCAC CGTGC CCAGC TCCAG AACTC CTGGG 5301 CGGAC CGTCA GTCTT CCTCT TCCCC CCAAA ACCCA AGGAC ACCCT CATGG 5 351 CCTCC CGGAC CCCTG AGGTC ACATG CGTGG TGGTG GACGT GAGCC ACGAA 5401 GACCC TGAGG TCAAG TTCAA CTGGT ACGTG GACGG CGTGG AGGTG CATAA 5451 TGCCA AGACA AAGCC GCGGG AGGAG CAGTA CAACA GCACG TACCG TGTGG 5501 TCAGC GTCCT CACCG TCCTG GCCCA GGACT GGCTG AATGG CAAGG AGTAC 5551 AAGTG CAAG G TCTCC AACAA AGCCC TCCCA GCCCC CATCG AGAAA ACCAT 5601 CTCCA AAGCC AAAGG GCAGC CCCGA GAACC ACAGG TGTAC ACCCT GCCCC 5651 CATCC CGGGA TGAGC TGACC AAGAA CCAGG TCAGC CTGAC CTGCC TGGTC 5701 AAAGG CTTCT ATCCC AGCGA CATCG CCGTG GAGTG GGAGA GCA AT GGGCA 5751 GCCGG AGAAC AACTA CAAGA CCACG CCTCC CGTGT TGGAC TCCGA C GGCT 5801 STTTS TTCCT STASA GCAAG CTCAC CGTGG ACAAG AGCAG GTGGC AGCAG 5851 GGGAA CGTCT TCTC A TGCTC CGTGA TGC AT GAGGC TCTGC AC A AC GCCT A 5901 CACGC AGAAG AGCCT CTCCC TGTCT CCGGG TAAAT GA

- 146 044115

FVIII 283 protein sequence (FVIII169 with triple IHH mutation) (SEQ ID NO: 161).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNIAKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYS YLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

451 TDETFKTREAIQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

751 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE

801 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE

851 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG

901 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS

951 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE

1001 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS

1051 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1101 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1151 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1201 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1251 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1301 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1351 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1401 VFETVEMLPS KAGIWRVECLIGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1451 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1501 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1551 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSSCSMPLGME

1601 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1651 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFLISSSQDGHQW TLFFQNGKVK

1701 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1751 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMASRTPEV TCVVVDVSHE

1801 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL AQDWLNGKEY

1851 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

1901 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

1951 GNVFSCSVMH EALHNAYTQK SLSLSPGK*

- 147 044115

Nucleotide sequence of pSYNFVIII 010-(FVIIIFc double strand) (SEQ ID NO: 162).

ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG

CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG

101 ACT AT ATGC A AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT

151 CCTAGAGTGC СAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAAA

201 GACTCTGTTT GT AGA ATTC A CGGATC ACCT TTTC A AC ATC GCT A AGCC A A

251 GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301 GAT AC AGTGG TC ATT AC ACT T AAGAACATG GCTTCCC ATC CTGTCAGTCT

351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG

401 ATGATC AGAC C AGTC A A AGG GAGA A AGA AG ATGAT A A AGT CTTCCCTGGT

451 GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC

501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG

551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601 GGGAGTCTGG CC A AGGA A A A GAC AC AGACC TTGC AC A A AT TT AT ACT ACT

651 TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT

701 CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG

751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG

801 CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG

851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

901 CGCC AGGCGT CCTTGGA A AT CTCGCC A AT A ACTTTCCTT A CTGCTC A A AC

951 ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC

1001 ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG

1051 GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA

1101 TGATCTT ACT GATTCTGA A A TGGATGTGGT C AGGTTTGAT GATGAC A ACT

1151 CTCCTTCCTT TATCC AAATT CGCTC AGTTG CCAAGAAGCA TCCT AAA ACT

1201 TGGGT AC ATT AC ATTGCTGC TGA AGAGGAG G ACTGGGACT ATGCTCCCTT

1251 AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG

1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC

1351 AC AGATGA AA CCTTT AAGAC TCGTGA AGCT ATTC AGC ATG A ATC AGGA AT

1401 CTTGGGACCT TT ACTTT ATG GGGA AGTTGG AGAC AC ACTG TTGATT AT AT

- 148 044115

1451 TTAAGAATCA AGCAAGCAGA CCATATAACA TСTACCCTCA CGGAATCACT

1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT 1551 GA AGGATTTT CC A ATTCTGC C AGGAGA A AT ATTC A A AT AT A A ATGGAC AG

1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC

1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT 1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC

1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG 1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC 1851 AGCTGG AGTG C AGCTTGAGG ATCC AGAGTT CC A AGCCTCC A AC ATC ATGC

1901 ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG

1951 CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT 2001 CCTTTCTGTC TTCTTCTCTG GAT AT ACCTT C A A AC AC A A A ATGGTCT ATG

2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101 ATGGA A A ACC C AGGTCT ATG GATTCTGGGG TGCC AC A ACT C AGACTTTCG

2151 GA AC AGAGGC ATGACCGCCT T ACTGA AGGT TTCT AGTTGT GAC A AG A AC A

2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG

2251 AGTAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACCCACCAGT

2301 CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT CAGTCAGATC 2351 A AGAGGA A AT TGACTATGAT GAT ACC AT AT C AGTTGA A AT GA AGA AGGA A

2401 GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA 2451 AAAGAAAACA CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT

2501 ATGGGATGAG TAGCTCCCCA CATGTTCTAA GAAACAGGGC TCAGAGTGGC

2551 AGTGTCCCTC AGTTC A AG AA AGTTGTTTTC C AGGA ATTT A CTGATGGCTC

2601 CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT TTGGGACTCC 2651 TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATTCAT GGTAACTTTC

2701 AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA

2751 TGAGGAAGAT CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC 2801 CTAATGAAAC CAAAACTTAC TTTTGGAAAG TGCAACATCA TATGGCACCC 2851 ACTAAAGATG AGTTTGACTG CAAAGCCTGG GCTTATTTCT CTGATGTTGA 2901 CCTGGAAAAA GATGTGCACT CAGGCCTGAT TG GACCCCTT CTGGTCTGCC 2951 ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA 3001 TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC

3051 TGAAAATATG GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG

3101 ATCCC ACTTT T A A AGAGA AT T ATCGCTTCC ATGC A ATC A A TGGCT AC AT A

3151 ATGGAT AC AC T ACCTGGCTT AGT AATGGCT CAGGATCAAA GGATTCGATG

3201 GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT ATTCATTTCA

3251 GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG 3301 TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA

- 149 044115

3351 AGCTGGAATT TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG 3401 GGATGAGCAC ACTTTTTCTG GTGTACAGCA ATAAGTGTCA GACTCCCCTG 3451 GGAATGGCTT CTGGACACAT TAGAGATTTT CAGATTACAG CTTCAGGACA 3501 ATATGGACAG TGGGCCCCAA AGCTGGCCAG AC TTCATTAT TCCGGATCAA 3551 TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG 3601 TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA 3651 GTTCTCCAGC CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG

3701 GGAAGAAGTG GCAGACTTAT CGAGGAAATT CCACTGGAAC CTTAATGGTC 3751 TTCTTTGGCA ATGTGGATTC ATCTGGGATA AAACACAATA TTTTTAACCC 3801 TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT TATAGCATTC 3851 GCAGC ACTCT TCGC ATGGAG TTGATGGGCT GTG ATTT A A A T AGTTGC AGC 3901 ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC

3951 TTCATCCTAC TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC

4001 GACTTC ACCT CC A AGGGAGG AGT A ATGCCT GGAGACCTC A GGTGA AT A AT 4051 CCAAAAGAGT GGCTGCAAGT GGACTTCCAG AAGACAATGA AAGTCACAGG

4101 AGT A ACT ACT C AGGG AGT A A A ATCTCTGCT T ACC AGC ATG T ATGTGA AGG 4151 AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT

4201 C AGA ATGGC A AAGT A A AGGT TTTTC AGGGA A ATC A AGACT CCTTC AC ACC 4251 TGTGGTGAAC TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC 4301 ACCCCCAGAG TTGGGTGCAC CAGATTGCCC TGAGGATGGA GGTTCTGGGC

51 TGCGAGGCAC AGGACCTCTA CGACAAAACT CACACATGCC CACCGTGCCC 4401 AGCTCC AGA A CTCCTGGGCG GACCGTC AGT CTTCCTCTTC CCCCC A A A AC 4451 CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG 4501 GTGGACGTGA GCCACGAAGA CCCTGA GGTC AAGTTCAACT GGTACGTGGA

4551 CGGCGTGG AG GTGC AT A ATG CCA AGAC A A A GCCGCGGG AG GAGC AGT AC A 4601 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 4651 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC 4701 CCCCATCGAG AAAACCATCT CC AAAGCC AA AGGGCAGCCC CGAGA ACC AC 4751 AGGTGTACAC CCTGCCCCCA TCCCGGGATG AGCTGACCAA GAACCAGGTC 4801 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA 4851 GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG 4901 TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 4951 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA 5001 GGCTCTGCAC AACCACTACA CGCAGAAG AG CCTCTCCCTG TCTCCGGGTA 5051 AATGA

- 150 044115

Protein sequence of pSYNFVIII 010-(FVIIIFc double strand) (SEQ ID NO: 163).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451 TDETF KTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE 801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 851 SVPQFKK VVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1001 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI

1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1101 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSK LTVD

1651 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 151 044115

FVIII195 protein sequence (double chain FVIIIFc with two 144 AE XTENs per amino acid

1656 and 1900) (SEO ID NO: 73).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG TASSSPGASP

801 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG

851 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA

901 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1151 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET

1201 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

1251 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS

1301 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI

1351 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1401 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1451 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1501 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1551 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1601 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1651 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1701 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1751 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1801 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 152 044115

Sequence of the mature protein pSYN-FVIII-173 (SEQ ID NO: 72).

ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL

FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA

201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL

351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG

451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD

501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP

551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG

601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS

651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR

701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGAPGT

751 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE

801 SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT

851 SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE

901 GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE

951 TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP

1001 GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP PVLKRHQREI

1051 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI

1101 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG

1151 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA

1201 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG

1251 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR

1301 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN

1351 ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC

1401 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL

1451 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMIIHGIKTQGAR QKFSSLYISQ

1501 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR

1551 LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF

1601 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS

1651 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP

1701 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP CPAPELLGGP

1751 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK

1801 TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK

1851 AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE

1901 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ

1951 KSLSLSPGK

- 153 044115

FVIII196 protein sequence (double chain FVIIIFc with three 144 AU XTENs per amino acid

26, 1656 and 1900) (SEO ID NO: 74).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVGAPGS

SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGAPGT SSTGSPGSST

101 PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST GTGPGTPGSG

151 TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG SPASSDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG TASSSPGASP

951 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG

1001 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA

1051 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE

1101 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

1151 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

1201 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

1251 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1301 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET

1351 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

1401 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS

1451 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI

1501 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1551 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1601 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1651 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1701 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1751 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1801 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1851 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1901 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1951 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

2001 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

2051 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

2101 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 154 044115

FVIII199 protein sequence (single chain FVIIIFc with three 144 AU XTENs at amino acids 1656 and 1900) (SEQ ID NO: 75).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

751 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG TASSSPGASP

801 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG

851 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA

901 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1151 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET

1201 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

1251 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS

1301 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI

1351 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1401 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1451 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1501 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1551 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1601 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1651 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1701 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1751 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1801 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 155 044115

FVIII 201 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 26, 1656 &1900) (SEO ID NO: 76).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVGAPGS

SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGAPGT SSTGSPGSST

101 PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST GTGPGTPGSG

151 TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG SPASSDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG TASSSPGASP

951 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG

1001 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA

1051 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE

1101 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

1151 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

1201 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

1251 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1301 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET

1351 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

1401 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS

1451 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI

1501 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1551 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1601 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1651 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1701 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1751 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1801 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1851 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1901 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1951 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

2001 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

2051 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

2101 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 156 044115

FVIII 203 protein sequence (single-chain FVIIIFc with two AE XTENs; one 288AE

XTEN in domain B and one 144 AE XTEN in amino acid 1900) (SEQ ID NO: 77).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVS SC DKNTGDYYED SYEDISAYLL

751 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE

801 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE

851 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG

901 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS

951 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE

1001 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS

1051 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1101 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1151 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1201 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1251 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1301 ^iIFDETKSWYF TENMERNCRG APTSESATPE SGPGSEPATS GSETPGTSES

1351 ATPESGPGSE PATSGSETPG TSESATPESG PGTSTEPSEG SAPGTSESAT

1401 PESGPGSPAG SPTSTEEGSP AGSPTSTEEG SPAGSPTSTE EGTSESATPE

1451 SGPGTSTEPS EGSAPGASSA PCNIQMEDPT FKENYRFHAINGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECLIGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

- 157 044115

FVIII 204 protein sequence (single-chain FVIIIFc with two AE XTENs; one 288AE

XTEN in domain B and one 144 AE of XTEN at amino acid 403) (SEQ ID NO: 78).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNIAKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPITFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDGAPTSTEP SEGSAPGSPA GSPTSTEEGT

451 STEPSEGSAP GTSTEPSEGS APGTSESATP ESGPGTSTEP SEGSAPGTSE

501 SATPESGPGS EPATSGSETP GTSTEPSEGS APGTSTEPSE GSAPGTSESA

551 TPESGPGTSE SATPESGPGA SSDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREAIQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE

1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

- 158 044115

FVIII 205 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE

XTEN in domain B and one 144 AE XTEN in amino acid 18) (SEQ ID NO: 79).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS

151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE

1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

- 159 044115

Protein sequence of pSYN FVIII 266 (FVIII Fc with 42 AE-XTEN at amino acid 18 and 288 AE

XTEN in domain B) SEO ID NO: 80).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST

EEGTSESATP ESGPGSEPAT SGSETPASS DLGELPVDAR FPPRVPKSFP

101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK

151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ

201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT 251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN

301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS

351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN

401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE 451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR

501 EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR 551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM

601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE 651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI 701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI

751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP

801 RSFSQNGAPG TSESATPESG PGSEPATSGS ETPGTSESAT PESGPGSEPA 851 TSGSETPGTS ESATPESGPG TSTEPSEGSA PGSPAGSPTS TEEGTSESAT

901 PESGPGSEPA TSGSETPGTS ESATPESGPG SPAGSPTSTE EGSPAGSPTS

951 TEEGTSTEPS EGSAPGTSES ATPESGPGTS ESATPESGPG TSESATPESG

1001 PGSEPATSGS ETPGSEPATS GSETPGSPAG SPTSTEEGTS TEPSEGSAPG 1051 TSTEPSEGSA PGSEPATSGS ETPGTSESAT PESGPGTSTE PSEGSAPASS

1101 PPVLKRHQAE ITRTTLQSDQ EEIDYDDTIS VEMKKEDFDI YDEDENQSPR

1151 SFQKKTRHYF IAAVERLWDY GMSSSPHVLR NRAQSGSVPQ FKKVVFQEFT

1201 DGSFTQPLYR GELNEHLGLL GPYIRAEVED NIMVTFRNQA SRPYSFYSSL

1251 ISYEEDQRQG AEPRKNFVKP NETKTYFWKV QHHMAPTKDE FDCKAWAYFS 1301 DVDLEKDVHS GLIGPLLVCH TNTLNPAHGR QVTVQEFALF FTIFDETKSW 1351 YFTENMERNC RAPCNIQMED PTFKENYRFH AINGYIMDTL PGLVMAQDQR

1401 IRWYLLSMGS NENIHSIHFS GHVFTVRKKE EYKMALYNLY PGVFETVEML

1451 PSKAGIWRVE CLIGEHLHAG MSTLFLVYSN KCQTPLGMAS GHIRDFQITA 1501 SGQYGQWAPK LARLHYSGSI NAWSTKEPFS WIKVDLLAPM IIHGIKTQGA 1551 RQKFSSLYIS QFIIMYSLDG KKWQTYRGNS TGTLMVFFGN VDSSGIKHNI 1601 F NPPIIARYI RLHPTHYSIR STLRMELMGC DLNSSCSMPLG MESKAISDAQ

1651 ITASSYFTNM FATWSPSKAR LHLQGRSNAW RPQVNNPKEW LQVDFQKTMK 1701 VTGVTTQGVK SLLTSMYVKE FLISSSQDGH QWTLFFQNGK VKVFQGNQDS 1751 FTPVVNSLDP PLLTRYLRIH PQSWVHQIAL RMEVLGCEAQ DLYDKTHTCP 1801 PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW 1851 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA 1901 LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI 1 951 AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV

2001 MHEALHNHYT QKSLSLSPGK *

- 160 044115

Protein sequence of pSYN FVIII 267 (FVIII Fc with 72 AE-XTEN at amino acid 18 and 288 AE

XTEN in domain B) SEQ ID NO: 81).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS VVYKKTLFVE

151 FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS HPVSLHAVGV

201 SYWKASEGAE YDDQTSQREK EDDKVFPPGGS HTYVWQVLKE NGPMASDPLC

251 LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH KFILLFAVFD

301 EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP GLIGCHRKSV

351 YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF LTAQTLLMDL

401 GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA EDYDDDLTDS

451 EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW DYAPLVLAPD

501 DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ HESGILGPLL

551 YGEVGDTLLIIFKNQASRPY NIYPHGITDV RPLYSRRLPK GVKHLKDFPI

601 LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL ASGLIGPLLI

651 CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR FLPNPAGVQL

701 EDPEFQASNIMHSINGYVFD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF

751 SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRGMT

801 ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS QNGAPGTSES

851 ATPESGPGSE PATSGSETPG TSESATPESG PGSEPATSGS ETPGTSESAT

901 PESGPGTSTE PSEGSAPGSP AGSPTSTEEG TSESATPESG PGSEPATSGS

951 ETPGTSESAT PESGPGSPAG SPTSTEEGSP AGSPTSTEEG TSTEPSEGSA

1001 PGTSESATPE SGPGTSESAT PESGPGTSES ATPESGPGSE PATSGSETPG

1051 SEPATSGSET PGSPAGSPTS TEEGTSTEPS EGSAPGTSTE PSEGSAPGSE

1101 PATSGSETPG TSESATPESG PGTSTEPSEG SAPASSPPVL KRHQAEITRT

1151 TLQSDQEEID YDDTISVEMK KEDFDIYDED ENQSPRSFQK KTRHYFIAAV

1201 ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV VFQEFTDGSF TQPLYRGELN

1251 EHLGLLGPYIRAEVEDNIMV TFRNQASRPY SFYSSLISYE EDQRQGAEPR

1301 KNFVKPNETK TYFWKVQHHM APTKDEFDCK AWAYFSDVDL EKDVHSGLIG

1351 PLLVCHTNTL NPAHGRQVTV QEFALFFTIF DETKSWYFTE NMERNCRAPC

1401 NIQMEDPTFK ENYRFHAING YIMDTLPGLV MAQDQRIRWY LLSMGSNENI

1451 HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF ETVEMLPSKA GIWRVECLIG

1501 EHLHAGMSTL FLVYSNKCQT PLGMASGHIR DFQITASGQY GQWAPKLARL

1551 HYSGSINAWS TKEPFSWIKV DLLAPMIIHGIKTQGARQKF SSLYISQFII

1601 MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS GIKHNIFNPP IIARYIRLHP

1651 THYSIRSTLR MELMGCDLNS CSMPLGMESK AISDAQITAS SYFTNMFATW

1701 SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD FQKTMKVTGV TTQGVKSLLT

1751 SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF QGNQDSFTPV VNSLDPPLLT

1801 RYLRIHPQSW VHQIALRMEV LGCEAQDLYD KTHTCPPCPA PELLGGPSVF

1851 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP

1901 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAPIEKTISKAKG

1951 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY

2001 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL

2051 SLSPGK*

- 161 044115

Protein sequence of pSYN FVIII 268 (FVIII Fc with 144 AE-XTEN at amino acid 18) SEQ

ID NO: 82).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS

151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QSDQEEIDYD DTISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1151 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI

1201 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1251 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1301 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1351 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1401 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1451 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1501 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1551 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1601 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1651 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

1701 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1751 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

1801 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 162 044115

Protein sequence pSYN FVIII 269 (FVIII Fc with 72 AE-XTEN at amino acid 18) SEQ ID

NO: 83).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS VVYKKTLFVE

151 FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS HPVSLHAVGV

201 SYWKASEGAE YDDQTSQREK EDDKVFPPGGS HTYVWQVLKE NGPMASDPLC

251 LTYSYLSHVD LVKDLNSGLIGALLVCREGS LAKEKTQTLH KFILLFAVFD

301 EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP GLIGCHRKSV

351 YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF LTAQTLLMDL

401 GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA EDYDDDLTDS

451 EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW DYAPLVLAPD

501 DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ HESGILGPLL

551 YGEVGDTLLIIFKNQASRPY NIYPHGITDV RPLYSRRLPK GVKHLKDFPI

601 LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL ASGLIGPLLI

651 CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR FLPNPAGVQL

701 EDPEFQASNI MHSINGYVFD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF

751 SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRGMT

801 ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS QNPPVLKRHQ

851 AEITRTTLQS DQEEIDYDDT ISVEMKKEDF DIYDEDENQS PRSFQKKTRH

901 YFIAAVERLW DYGMSSSPHV LRNRAQSGSV PQFKKVVFQE FTDGSFTQPL

951 YRGELNEHLG LLGPYIRAEV EDNIMVTFRN QASRPYSFYS SLISYEEDQR

1001 QGAEPRKNFV KPNETKTYFW KVQHHMAPTK DEFDCKAWAY FSDVDLEKDV

1051 HSGLIGPLLV CHTNTLNPAH GRQVTVQEFA LFTIFDETK SWYFTENMER

1101 NCRAPCNIQM EDPTFKENYR FHAINGYIMD TLPGLVMAQD QRIRWYLLSM

1151 GSNENIHSIH FSGHVFTVRK KEEYKMALYN LYPGVFETVE MLPSKAGIWR

1201 VECLIGEHLH AGMSTLFLVY SNKCQTPLGM ASGHIRDFQI TASGQYGQWA

1251 PKLARLHYSG SINAWSTKEP FSWIKVDLLA PMIIHGIKTQ GARQKFSSLY

1301 ISQFIIMYSL DGKKWQTYRG NSTGTLMVFF GNVDSSGIKH NIFNPPIIAR

1351 YIRLHPTHYS IRSTLRMELM GCDLNSCSMP LGMESKAISD AQITASSYFT

1401 NMFATWSPSK ARLHLQGRSN AWRPQVNNPK EWLQVDFQKT MKVTGVTTQG

1451 VKSLLTSMYV KEFLISSSQD GHQWTLFFQN GKVKVFQGNQ DSFTPVVNSL

1501 DPPLLTRYLR IHPQSWVHQI ALRMEVLGCE AQDLYDKTHT CPPCPAPELL

1551 GGPS VFLFPP KPKDTLMISR TPEVTC VVVD VSHEDPEVKF NWYVDGVEVH

1601 NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT

1651 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG

1701 QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH

1751 YTQKSLSLSP GK*

- 163 044115

Protein sequence of pSYN FVIII 271 (FVIII Fc with 42 AE-XTEN at amino acid 18) SEO ID

NO: 84).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST

EEGTSESATP ESGPGSEPAT SGSETPASS DLGELPVDAR FPPRVPKSFP

101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK

151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ

201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT

251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN

301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS

351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN

401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE

451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR

501 EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR

551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM

601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE

651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI

701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI

751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP

801 RSFSQNPPVL KRHQAEITRT TLQSDQEEID YDDTISVEMK KEDFDIYDED

851 ENQSPRSFQK KTRHYFIAAV ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV

901 VFQEFTDGSF TQPLYRGELN EHLGLLGPYI RAEVEDNIMV TFRNQASRPY

951 SFYSSLISYE EDQRQGAEPR KNFVKPNETK TYFWKVQHHM APTKDEFDCK

1001 AWAYFSDVDL EKDVHSGLIG PLLVCHTNTL NPAHGRQVTV QEFALFFTIF

1051 DETKSWYFTE NMERNCRAPC NIQMEDPTFK ENYRFHAING YIMDTLPGLV

1101 MAQDQRIRWY LLSMGSNENI HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF

1151 ETVEMLPSKA GIWRVECLIG EHLHAGMSTL FLVYSNKCQT PLGMASGHIR

1201 DFQITASGQY GQWAPKLARL HYSGSINAWS TKEPFSWIKV DLLAPMIIHG

1251 IKTQGARQKF SSLYISQFII MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS

1301 GIKHNIFNPP IIARYIRLHP THYSIRSTLR MELMGCDLNS CSMPLGMESK

1351 AISDAQITAS SYFTNMFATW SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD

1401 FQKTMKVTGV TTQGVKSLLT SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF

1451 QGNQDSFTPV VNSLDPPLLLT RYLRIHPQSW VHQIALRMEV LGCEAQDLYD

1501 KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP

1551 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC

1601 KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG

1651 FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN

1701 VFSCSVMHEA LHNHYTQKSL SLSPGK*

- 164 044115

Protein sequence of pSYN FVIII 272 (FVIII with 144 AE XTEN at amino acid 18 and 244 AE

XTEN in domain B without Fc) SEQ ID NO: 85).

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS

151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE

1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD

1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII

1651 HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL

1901Y*

Protein sequence of pSYN-FVIII-161 (SEQ ID NO: 69) (1-1457 position of the FVIII amino acid sequence; underlined region represents the Fc region; curved underline represents the cleavable linker between the first Fc and the VWF fragment; double-underlined area represents the VWF fragment; The area in bold indicates the cleavable linker between the VWF moiety and Fc).

- 165 044115

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNIAKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL

- 166 044115

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL

751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE

801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG

851 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP

951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1001 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI

1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1101 YNLYPGVFET VEMLPSKAGIWRVECLIGEH LH AGMSTLFL V YSNKCQTPL

1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

1651 KSRWOOGNVF SCSVMHEALH NHYTQKSLSL SPGKRRRRSG GGGSGGGGSG

1701 GGGSGGGGSG GGGSGGGGSR KRRKRSLSCR PPMVKLVCPA DNLRAEGLEC

1751 TKTCONYDLE CMSMGCVSGC LCPPGMVRHE NRCVALERCP CFHOGKEYAP

1801 GETVKIGCNT CVCRDRKWNC TDHVCDATCS TIGMAHYLTF DGLKYLFPGE

1851 COYVLVODYC GSNPGTFRIL VGNKGCSHPS VKCKKRVTIL VEGGEIELFD

1901 GEVNVKRPMK DETHFEVVES GRYIILLLGK ALSVVWDRHL SISVVLKOTY

1951 OEKVCGLCGN FDGIONNDLT SSNLOVEEDP VDFGNSWKVS SOCADTRKVP

2001 LDSPATCHN NIMKOTMVDS SCR1LTSDVF ODCNKLVDPE PYLDVCIYDT

2051 CSCESIGDCA AFCDTIAAYA HVCAOHGKVV TWRTATLCPO SCEERNLREN

2101 GYEAEWRYNS CAPACOVTCO HPEPLACPVO CVEGCHAHCP PGKILDELLO

2151 TCVDPEDCPV CEVAGRRFAS GKKVTLNPSD PEHCOICHCD VVNLTCEACO

2201 EEISGTSESA TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE

2251 TPGTSESATP ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP

2301 GSEPATSGSE TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGT

2351 STEPSEGSAP GTSESATPES GPGTSESATP ESGPGTSESA TPESGPGSEP

2401 ATSGSETPGS EPATSGSETP GSPAGSPTST EEGTSTEPSE GSAPGTSTEP

2451 SEGSAPGSEP ATSGSETPGT SESATPESGP GTSTEPSEGS APDSGGGGSG

2501 GGGSGGGGSG GGGSGGGGSL VPRGSGGDKT HTCPPCPAPE LLGGPSVFLF

2551 PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE

2601 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP 2651 REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 2701 TPPVLDSDGS FFLYSKLTVD KSRWOOGNVF SCSVMHEALH NHYTQKSL SL

SPGK

- 167 044115

Protein sequence of pSYN-FVIII-170 (SEO ID NO: 71).

SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG

MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC 101 DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG 151 CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII

201 LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ 251 VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL 301 TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCAAFCDT IAAYAHVCAQ 351 HGKVVTWRTA TLCPQSCEER NLRENGYEAE WRYNSCAPAC QVTCQHPEPL 401 ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT 451 LNPSDPEHCQ ICHCDVVNLT CEACQEPISG TSESATPESG PGSEPATSGS

501 ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG TSTEPSEGSA

551 PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

601 SPAGSPTSTE EGSPAGSPTS TEEGTSTEPS EGSAPGTSES ATPESGPGTS

651 ESATPESGPG TSESATPESG PGSEPATSGS ETPGSEPATS GSETPGSPAG

701 SPTSTEEGTS TEPSEGSAPG TSTEPSEGSA PGSEPATSGS ETPGTSESAT

751 PESGPGTSTE PSEGSAPDSG GGGSGGGGSG GGGSGGGGSG GGGSLVPRGS

801 GGASATRRYY LGAVELSWDY MQSDLGELPV DARFPPRVPK SFPFNTSVVY 851 KKTLFVEFTD HLFNIAKPRP PWMGLLGPTI QAEVYDTVVI TLKNMASHPV 901 SLHAVGVSYW KASEGAEYDD QTSQREKEDD KVFPGGSHTY VWQVLKENGP 951 MASDPLCLTY SYLSHVDLVK DLNSGLIGAL LVCREGSLAK EKTQTLHKFI 1001 LLFAVFDEGK SWHSETKNSL MQDRDAASAR AWPKMHTVNG YVNRSLPGLI 1051 GCHRKSVYWH VIGMGTTPEV HSIFLEGHTF LVRNHRQASL EISPITFLTA 1101 QTLLMDLGQF LLFCHISSHQ HDGMEAYV KV DSCPEEPQLR MKNNEEAEDY 1151 DDDLTDSEMD VVRFDDDNSP SFIQIRSVAK KHPKTWVHYI AAEEEDWDYA 1201 PLVLAPDDRS YKSQYLNNGP QRIGRKYKKV RFMAYTDETF KTREAIQHES 1251 GILGPLLYGE VGDTLLIIFK NQASRPYNIY PHGITDVRPL YSRRLPKGVK 1301 HLKDFPILPG EIFKYKWTVT VEDGPTKSDP RCLTRYYSSF VNMERDLASG

1351 LIGPLLICYK ESVDQRGNQI MSDKRNVILF SVFDENRSWY LTENIQRFLP

1401 NPAGVQLEDP EFQASNIMHS INGYVFDSLQ LSVCLHEVAY WYILSIGAQT 1451 DFLSVFFSGY TFKHKMVYED TLTLFPFSGE TVFMSMENPG LWILGCHNSD

- 168 044115

1501 FRNRGMTALL KVSSCDKNTG DYYEDSYEDI SAYLLSKNNA IEPRSFSQNP

1551 PVLKRHQREITRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS

1601 FQKKTRHYFIAAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD

1651 GSFTQPLYRG ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI

1701 SYEEDQRQGA EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD

1751 VDLEKDVHSG LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY

1801 FTENMERNCR APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI

1851 RWYLLSMGSN ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP

1901 SKAGIWRVEC LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS

1951 GQYGQWAPKL ARLHYSGSIN AWSTKEPFSW IKVDLLAPMIIHGIKTQGAR

2001 QKFSSLYISQ FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF

2051 NPPIIARYIR LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI

2101 TASSYFTNMF ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV

2151 TGVTTQGVKS LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF

2201 TPVVNSLDPP LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LY

Nucleotide sequence of pSYN FVIII 310 (encoding FVIII with complete deletion of domain B, except for 2 amino acid residues and 288 AE-XTEN inserted after 742 aa) (SEO ID NO: 170).

ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG

CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG

101 ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT

151 CCTAGAGTGC СAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAAA

201 GACTCTGTTT GT AGA ATTC A CGGATC ACCT TTTC A AC ATC GCT A AGCC A A

251 GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301 GAT AC AGTGG TC ATT AC ACT T AAGAACATG GCTTCCC ATC CTGTCAGTCT

351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG

401 ATGATC AGAC C AGTCA A AGG GAGA A AGA AG ATGAT A A AGT CTTCCCTGGT

451 GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC

501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG

551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601 GGGAGTCTGG CCA AGGAA A A GAC AC AGACC TTGC AC A A AT TT AT ACT ACT

651 TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT

701 CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG

751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG

801 CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG

851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

- 169 044115

901 CGCC AGGCGT CCTTGGA A AT CTCGCC A AT A ACTTTCCTT A CTGCTC A A AC

951 ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC 1001 ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG 1051 GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA 1101 TGATCTT ACT GATTCTGA A A TGGATGTGGT C AGGTTTGAT GATGAC A ACT 1151 CTCCTTCCTT TATCC AAATT CGCTC AGTTG CCAAGAAGCA TCCT AAA ACT 1201 TGGGT AC ATT AC ATTGCTGC TGA AGAGGAG G ACTGGGACT ATGCTCCCTT 1251 AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG 1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC 1351 AC AGATGA AA CCTTT AAGAC TCGTGA AGCT ATTC AGC ATG A ATC AGGA AT 1401 CTTGGGACCT TT ACTTT A TG GGGA AGTTGG AGAC AC ACTG TTGATT AT AT 1451 TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT 1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT 1551 GA AGGATTTT CC A ATTCTGC C AGGAGA A AT ATTC A A AT AT A A ATGGAC AG 1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC 1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT 1701 TGGCCCTC TC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC 1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG 1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC 1851 AGCTGG AGTG C AGCTTGAGG ATCC AGAGTT CC A AGCCTCC A AC ATC ATGC 1901 ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG 1951 CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT 2001 CCTTTCTGTC TTCTTCTCTG GAT AT ACCTT C A A AC AC A A A ATGGTCT ATG 2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101 ATGGA A A ACC C AGGTCT ATG GATTCTGGGG TGCC AC A ACT C AGACTTTCG 2151 GA AC AGAGGC ATGACCGCCT T ACTGA AGGT TTCT AGTTGT GAC A AG A AC A 2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG 2251 AGTAAAACA ATGCCATTGA ACCAAGAAGC TTCGGTACCT CAGAG TCTGC 2301 TACCCCCGAG TCAGGGCCAG GATCAGAGCC AGCCACCTCC GGGTCTGAGA 2351 CACCCGGGAC TTCCGAGAGT GCCACCCCTG AGTCCGGACC CGGGTCCGAG 2401 CCCGCCACTT CCGGCTCCGA AACTCCCGGC ACAAGCGAGA GCGCTACCCC 2451 AGAGTCAGGA CCAGGAACAT CTACAGAGCC CTCTGAAGGC TCCGCTCCAG 2501 GGTCCCCAGC CGGCAGTCCC ACTAGCACCG AGGAGGGAAC CTCTGAAAGC 2551 GCC AC ACCCG AATC AGGGCC AGGGTCTGAG CCTGCT ACC A GCGGC AGCGA 2601 GACACCAGGC ACCTCTGAGT CCGCCACACC AGAGTCCGGA CCCGGATCTC 2651 CCGCTGGGAG CCCCACCTCC ACTGAGGAGG GATCTCCTGC TGGCTCTCCA 2701 ACATCTACTG AGGAAGGTAC CTCAACCGAG CCATCCGAGG GATCAGCTCC 2751 CGGCACCTCA GAGTCGGCAA CCCCGGAGTC TGGACCCGGA ACTTCCGAAA

- 170 044115

2801 GTGCCACACC AGAGTCCGGT CCCGGGACTT CAGAATCAGC AACACCCGAG 2851 TCCGGCCCTG GGTCTGAACC CGCCACAAGT GGTAGTGAGA CACCAGGATC 2901 AGAACCTGCT ACCTC AGGGT C AG AG AC ACC CGGATCTCCG GC AGGCTCAC 2951 CAACCTCCAC TGAGGAGGGC ACC AGCACAG AACCAAGCGA GGGCTCCGCA 3001 CCCGGAACAA GCACTGAACC CAGTGAGGGT TCAGCACCCG GCTCTGAGCC 3051 GGCCACAAGT GGCAGTGAGA CACCCGGCAC TTCAGAGAGT GCCACCCCCG 3101 AGAGTGGCCC AGGCACTAGT ACCGAGCCCT CTGAAGGCAG TGCGCCAGCC 3151 TCGAGCGAAA TAACTCGTAC TACTCTTCAG TCAGATCAAG AGGAAATCGA 3201 TTATGATGAT ACC AT ATS AG TTGAAATGAA GAAGGAAGAT TTTGACATTT 3251 ATGATGAGGA TGAAAATCAG AGCCCCCGCA GCTTTCAAAA GAAAACACGA 3301 CACTATTTTA TTGCTGCAGT GGAGAGGCTC TGGGATTATG GGATGAGTAG 3 351 CTCCCCACAT GTTCTAAGAA ACAGGGCTCA GAGTGGCAGT GTCCCTCAGT 3401 TCAAGAAAGT TGTTTTCCAG GAATTTACTG ATGGCTCCTT TACTCAGCCC 3451 TTATACCGTG GAGAACTAAA TGAACATTTG GGACTCCTGG GGCCATATAT 3501 AAGAGCAGAA GTTGAAGATA ATATCATGGT AACTTTCAGA AATCAGGCCT 3551 CTCGTCCCTA TTCCTTCTAT TCTAGCCTTA TTTCTTATGA GGAAGATCAG

3601 AGGCAAGGAG CAGAACCTAG AAAAAACTTT GTCAAGCCTA ATGAAACCAA 3651 AACTTACTTT TGGAAAGTGC AACATCATAT GGCACCCACT AAAGATGAGT 3701 TTGACTGCAA AGCCTGGGCT TATTTCTCTG ATGTTGACCT GGAAAAAGAT 3751 GTGCACTCAG GCCTGATTGG ACCCCTTCTG GTCTGCCACA CTAACACACT 3801 GAACCCTGCT CATGGGAGAC AAGTGACAGT ACAGGAATTT GCTCTGTTTT 3851 TC ACC ATCTT TGATGAGACC A A AAGCTGGT ACTTC ACTGA A A AT ATGGA A 3901 AGAAACTGCA GGGCTCCCTG CAATATCCAG ATGGAAGATC CCACTTTTAA 3951 AGAGAATTAT CGCTTCCATG CAATCAATGG CTACATAATG GAT AC ACT AC 4001 CTGGCTT AGT A ATGGCTC AG GATC A A AGGA TTCGATGGT A TCTGCTC AGC 4051 ATGGGCAGCA ATGAAAACAT CCATTCTATT CATTTCAGTG GACATGTGTT 4101 C ACTGT ACGA AA AA AAGAGG AGT AT A A A AT GGC ACTGT AC A ATCTCT ATC 4151 C AGGTGTTTT TGAGAC AGTG GA A ATGTT AC CATCC A A AGC TGGA ATTTGG 4201 CGGGTGGA AT GCCTT ATTGG CGAGC ATCT A C ATGCTGGGA TGAGC AC ACT 4251 TTTTCTGGTG TACAGCAATA AGTGTCAGAC TCCCCTGGGA ATGGCTTCTG 4301 GACACATTAG AGATTTTCAG ATTACAGCTT CAGGACAATA TGGACAGTGG 43 51 GCCCCAAAGC TGGCCAGACT TCATTATTCC GGATCAATCA ATGCCTGGAG 4401 SASSA AGG AG CCCTTTTCTT GGATC A AGGT GGATCTGTTG GC ACC A ATGA 4451 TTATTCACGG CATCAAGACC CAGGGTGCCC GTCAGAAGTT CTCCAGCCTC 4501 TACATCTCTC AGTTTATCAT CATGTATAGT CTTGATGGGA AGAAGTGGCA 4551 GACTTATCGA GGAAATTCCA CTGGAACCTT AATGGTCTTC TTTGGCAATG 4601 TGGATTC ATC TGGGATAAAA C A CA ATATTT TTAACCCTCC AATT ATTGCT 4651 CGATACATCC GTTTGCACCC AACTCATTAT AGCATTCGCA GCACTCTTCG

- 171 044115

4701 CATGGAGTTG ATGGGCTGTG ATTTAAATAG TTGCAGCATG CCATTGGGAA

4751 TGGAGAGTAA AGCAATATCA GATGCACAGA TTACTGCTTC ATCCTACTTT

4801 ACCAATATGT TTGCCACCTG GTCTCCTTCA AAAGCTCGAC TTCACCTCCA

4851 AGGGAGGAGT AATGCCTGGA GACCTCAGGT GAATAATCCA AAAGAGTGGC

4901 TGCAAGTGGA CTTCCAGAAG ACAATGAAAG TCACAGGAGT AACTACTCAG

4951 GGAGTAAAAT CTCTGCTTAC CAGCATGTAT GTGAAGGAGT TCCTCATCTC

5001 CAGCAGTCAA GATGGCCATS AGTGGACTCT CTTTTTTCAG AATGGCAAAG

5051 TAAAGGTTTT TCAGGGAAAT CAAGACTCCT TCACACCTGT GGTGAACTCT

5101 CTAGACCCAC CGTTACTGAC TCGCTACCTT CGAATTCACC CCCAGAGTTG

5151 GGTGCACCAG ATTGCCCTGA GGATGGAGGT TCTGGGCTGC GAGGCACAGG

5201 ACCTCTACGA СAAAACTCAC ACATGCCCAC CGTGCCCAGC TCCAGAACTC

5251 CTGGGCGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT

5301 CATGATCTCC CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC

5351 ACGAAGACCC TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG

5401 CATAATGCCA AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG

5451 TGTGGTCAGC GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG

5501 AGTACAAGTG CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA

5551 ACCATTCCA AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT

5601 GCCCCCATCC CGGGATGAGC TGACCAAGAA CCAGGTCAGC CTGACCTGCC

5651 TGGTCAAAGG CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT

5701 GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGT TGGACTCCGA

5751 CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC

5801 AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC

5851 C ACT AC ACGC AGA AGAGCCT CTCCCTGTCT CCGGGT A A AT GA

Protein sequence of pSYN FVIII 310 (FVIII with complete deletion of domain B, except for 2 amino acid residues and 288 AE-XTEN inserted after 742 aa) (SEQ ID NO: 171).

ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL

FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA

201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL

351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL

- 172 044115

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG

451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD

K MVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR 701 GMTALLKVSS CDKNTGDYYE DSYED1SAYL LSKNNA1EPR SFGTSESATP 751 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES

801 GPGTSTEPSE GSAPGPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP

851 GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT 901 SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP 951 ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTSTEPSE GSAPGSEPAT

1001 SGSETPGTSE SATPESGPGT STEPSEGSAP ASSEITRTTL QSDQEEIDYD

1051 DTISVEMKKE DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP 1101 HVLRNRAQSG SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA 1151 EVEDNIMVTF RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

1201 FWKVQHHMAP TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

1251 AHGRQVTVQE FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN

1301 YRFHAINGYI MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV 1351 RKKEEYKMAL YNLYPGVFET VEMLPSKAGIWRVECLIGEH LHAGMSTLFL 1401 VYSNKCQTPL GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

1451 EPFSWIKVDL LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY 1501 RGNSTGTLMV FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLME

1551 LMGCDLNSCS MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR 1601 SNAWRPQVNN PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS 1651 QDGHQWTLFF QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH 1701 QIALRMEVLG CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

1751 SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV 1801 SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

1851 SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 1901 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

- 173 044115

Nucleotide sequence of pSYN FVIII 312 (encoding FVIII with complete deletion of the domain

B, with the exception of 5 amino acid residues and 288 AE-XTEN, inserted after aa. 745-B5 version) (SEQ ID NO: 172).

ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG

CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG

101 ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT

151 CCT AGAGTGC C A AAATCTTT TCC ATTCA AC ACCTC AGTCG TGTAC A AAA A

201 GACTCTGTTT GT AGA ATTCA CGGATC ACCT TTTC A AC ATC GCT A AGCC A A

251 GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301 GAT AC AGTGG TC ATT AC ACT T AAG A AC ATG GCTTCCCATC CTGTCAGTCT

351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG

401 ATGATC AGAC C AGTC A A AGG GAGA A AGA AG ATGAT A A AGT CTTCCCTGGT

451 GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC

501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG

551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601 GGGAGTCTGG CCAAGGAAAA GAC AC AG ACC TTGCACAAAT TT AT ACT ACT

651 TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT

701 CCTTGATGC A GGAT AGGGAT GCTGC ATCTG CTCGGGCCTG GCCT A A A ATG

751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG

801 CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG

851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

901 CGCC AGGCGT CCTTGGA A AT CTCGCC A AT A ACTTTCCTT A CTGCTC A A AC

951 ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC

1001 ACC AAC ATG A TGGC ATGG A A GCTT ATGTC A A AGT AGAC AG CTGTCC AGAG

1051 GAACCCC AAC T ACGA ATG AA A A ATA ATGAA GAAGCGGAAG ACTATGATGA

1101 TG ATCTT ACT G ATTCTGA A A TGGATGTGGT C AGGTTTGAT GATGAC A ACT

1151 CTCCTTCCTT TATCC AA ATT CGCTC AGTTG CCA AGAAGC A TCCT AAA ACT

1201 TGGGT AC ATT AC ATTGCTGC TGA AGAGG AG G ACTGGGACT ATGCTCCCTT

1251 AGTCCTCGCC CCCGATGACA GAAGTT ATA A AAGTCAAT AT TTGAAC AATG

1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC

1351 ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAT

1401 CTTGGGACCT TT ACTTT ATG GGGA AGTTGG AGAC AC ACTG TTGATT AT AT

1451 TT A AGA ATCA AGCAAGC AGA CC AT AT A AC A TCT ACCCTC A CGGA ATC ACT

1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT

1551 GA AGGATTTT CC A ATTCTGC C AGGAG A A AT ATTC A A AT AT A A ATGGAC AG

1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC

1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT

1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC

1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG

1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC

1851 AGCTGG AGTG C AGCTTGAGG ATCC AG AGTT CC A AGCCTCC A AC ATC ATGC

1901 AC AGC ATC A A TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG

- 174 044115

1951 CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT 2001 CCTTTCTGTC TTCTTCTCTG GAT AT ACCTT C A A AC AC A A A ATGGTCT ATG 2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101 ATGGAAAACC CAGGTCTATG GATTCTGGGG TGC CACAACT CAGACTTTCG 2151 GA AC AGAGGC ATGACCGCCT T ACTGA AGGT TTCT AGTTGT GAC A AG A AC A 2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG 2251 AGTAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACGGTACCTC 2301 AGAGTCTGCT ACCCCCGAGT CAGGGCCAGG ATCAGAGCCA GCCACCTCCG 2351 GGTCTGAGAC ACCCGGGACT TCCGAGAGTG CCACCCCTGA GTCCGGACCC 2401 GGGTCCGAGC CCGCCACTTC CGG CTCCGAA ACTCCCGGCA CAAGCGAGAG 2451 CGCTACCCCA GAGTCAGGAC CAGGAACATC TACAGAGCCC TCTGAAGGCT 2501 CCGCTCCAGG GTCCCCAGCC GGCAGTCCCA CTAGCACCGA GGAGGGAACC 2551 TCTGA AAGCG CC AC ACCCGA ATC AGGGCC A GGGTCTGAGC CTGCT ACC AG 2601 CGGCAGCGAG ACACC AGGC A CCTCTGAGTC CGCCAC ACC A GAGTCCGGAC 2651 CCGGATCTCC CGCTGGGAGC CCCACCTCCA CTGAGGAGGG ATCTCCTGCT 2701 GGCTCTCCAA CATCTACTGA GGAAGGTACC TCAACCGAGC CATCCGAGG 2751 ATCAGCTCCC GGCACCTCAG AGTCGG CAAC CCCGGAGTCT GGACCCGGAA 2801 CTTCCGAAAG TGCCACACCA GAGTCCGGTC CCGGGACTTC AGAATCAGCA 2851 ACACCCGAGT CCGGCCCTGG GTCTGAACCC GCCACAAGTG GTAGTGAGAC 2901 ACCAGGATCA GAACCTGCTA CCTCAGGGTC AGAGACACCC GGATCTCCGG 2951 CAGGCTCACC AACCTCCACT GAGGAGGGCA CCAGCACAGA ACCAAGCGAG 3001 GGCTCCGCAC CCGGAACAAG CACTGAACCC AGTGAGGGTT CAGCACCCGG 3051 CTCTGAGCCG GCCACAAGTG GCAGTGAGAC ACCCGGCACT TCAGAGAGTG 3101 CCACCCCCGA GAGTGGCCCA GGCACTAGTA CCGAGCCCTC TGAAGGCAGT 3151 GCGCCA GCCT CGAGCGAAAT AACTCGTACT ACTCTTCAGT CAGATCAAGA 3201 GGAAATCGAT TATGATGATA CCATATCAGT TGAAATGAAG AAGGAAGATT 3251 TTGACATTTA TGATGAGGAT GAAAATCAGA GCCCCCGCAG CTTTCAAAAG 3301 AAAACACGAC ACTATTTTAT TGCTGCAGTG GAGAGGCTCT GGGATTATGG 3351 GATGAGTAGC TCCCCACATG TTCTAAGAAA CAGGGCTCAG AGTGGCAGTG 3401 TCCCTCAGTT CAAGAAAGTT GTTTTCCAGG AATTTACTGA TGGCTCCTTT 3451 ACTCAGCCCT TATACCGTGG AGAACTAAAT GAACATTTGG GACTCCTGGG 3501 GCCATATATA AGAGCAGAAG TTGAAGATAA TATCATGGTA ACTTTCAGAA 3551 ATCAGGCCTC TCGTCCCTAT TCCTTCTA TT CTAGCCTTAT TTCTTATGAG

3601 GAAGATCAGA GGCAAGGAGC AGAACCTAGA AAAAACTTTG TCAAGCCTAA 3651 TGAAACCAAA ACTTACTTTT GGAAAGTGCA AC ATC AT ATG GCACCCACTA 3701 AAGATGAGTT TGACTGCAAA GCCTGGGCTT ATTTCTCTGA TGTTGACCTG 3751 GAAAAAGATG TGCACTCAGG CCTGATTGGA CCCC TTCTGG TCTGCCACAC 3801 TAACACACTG AACCCTGCTC ATGGGAGACA AGTGACAGTA CAGGAATTTG

- 175 044115

3851 CTCTGTTTT CACCATCTTT GATGAGACCA AAAGCTGGTA CTTCACTGAA 3901 AATATGGAAA GAAACTGCAG GGCTCCCTGC AATATCCAGA TGGAAGATCC 3951 CACTTTTAAA GAGAATTATC GCTTCCATGC AATCAATGGC TACATAATGG 4001 AT AC ACT ACC TGGCTTAGT A ATGGCTC AGG A TC A AAGG AT TCGATGGT AT 4051 CTGCTCAGCA TGGGCAGCAA TGAAAACATC CATTCTATTC ATTTCAGTGG 4101 AC ATGTGTTC ACTGT ACGA A A A A A AGAGGA GT AT A A A ATG GC ACTGT AC A 4151 ATCTCT ATCC AGGTGTTTTTT GAGAC AGTGG A A ATGTT ACC ATCC A A AGCT 4201 GGAATTTGGC GGGTGGAATG CCTTATTGGC GAGCATCTAC ATGCTGGGAT 4251 GAGCACACTT TTTCTGGTGT ACAGCAATAA GTGTCAGACT CCCCTGGGAA 4301 TGGCTTCTGG ACACATTA GA GATTTTCAGA TTACAGCTTC AGGACAATAT 4351 GGAC AGTGGG CCCC A A AGCT GGCC AGACTT C ATT ATTCCG GATC A ATC A A 4401 TGCCTGGAGC ACC A AGG AGC CCTTTTCTTG GATC A AGGTG GATCTGTTGG 4451 CACCAATGAT TATTCACGGC ATCAAGACCC AGGGTGCCCG TCAGAAGTTC 4501 TCCAGCCTCT ACATCTCTCA GTTTATCATC ATGTATAGTC TTGATGGGAA 4551 GA AGTGGC AG ACTT ATCGAG GA A ATTCC AC TGGA ACCTT A ATGGTCTTCT 4601 TTGGCAATGT GGATTCATCT GGGATAAAAAC ACAATATTTT TAACCCTCCA 4651 ATTATTGCTC GATACATCCG TTTGCACCCA ACTCATTATA GCATTCGCAG 4701 CACTCTTCGC ATGGAGTTGA TGGGCTGTGA TTTAAATAGT TGCAGCATGC 4751 CATTGGGAAT GGAGAGTAAA GCAATATCAG ATGCACAGAT TACTGCTTCA 4801 TCCTACTTTA CCAATATGTT TGCCACCTGG TCTCCTTCAA AAGCTCGACT 4851 TCACCTCCAA GGGAGGAGTA ATGCCTGGAG ACCTCAGGTG AATAATCCAA 490 1 AAGAGTGGCT GCAAGTGGAC TTCCAGAAGA CAATGAAAGT CACAGGAGTA 4951 ACTACTCAGG GAGTAAAATC TCTGCTTACC AGCATGTATG TGAAGGAGTT 5001 CCTCATCTCC AGCAGTCAAG ATGGCCATCA GTGGACTCTC TTTTTTCAGA 5051 ATGGCAAAGT AAAGGTTTT CAGGGAAATC AAGACTCCTT CA CACCTGTG 5101 GTGAACTCTC TAGACCCACC GTTACTGACT CGCTACCTTC GAATTCACCC 5151 CCAGAGTTGG GTGCACCAGA TTGCCCTGAG GATGGAGGTT CTGGGCTGCG 5201 AGGCACAGGA CCTCTACGAC AAAACTCACA CATGCCCACC GTGCCCAGCT 5251 CCAGAACTCC TGGGCGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA 5301 GGACACCCTC ATGA TCTCCC GGACCCCTGA GGTCACATGC GTGGTGGTGG 5351 ACGTGAGCCA CGAAGACCCT GAGGTCAAGT TCAACTGGTA CGTGGACGGC 5401 GTGGAGGTGC ATAATGCCAA GACAAAGCCG CGGGAGGAGC AGTACAACAG 5451 CACGTACCGT GTGGTCAGCG TCCTCACCGT CCTGCACCAG GACTGGCTGA 5 501 ATGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC 5551 ATCGAGAAAA CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT 5601 GTACACCCTG CCCCCATCCC GGGATGAGCT GACCAAGAAC CAGGTCAGCC 5651 TGACCTGCCT GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG 5701 GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGTT 5751 GGACTCCGAC GGCTCCTTCT TCCTCTACAG CAAGCTCACC GTGGACAAGA

5801 GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT 5851 CTGC AC A ACC ACT AC ACGC A GA AGAGCCTC TCCCTGTCTC CGGGT A A ATG

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Protein sequence of pSYN FVIII 312 (FVIII with complete deletion of domain B, except for 5 amino acid residues and 288 AE-XTEN inserted after aa 745-B5 version) (SEO ID NO: 173).

ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL

FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA

201 VFDEGKS WHS ETKNSLMQDR D A AS AR AWPK MHT VNGY VNR SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL

351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG

451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD

501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP

551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG

601 VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS

651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR

701 GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGTSES

751 ATPESGPGSE PATSGSETPG TSESATPESG PGSEPATSGS ETPGTSESAT

801 PESGPGTSTE PSEGSAPGSP AGSPTSTEEG TSESATPESG PGSEPATSGS

851 ETPGTSESAT PESGPGSPAG SPTSTEEGSP AGSPTSTEEG TSTEPSEGSA

901 PGTSESATPE SGPGTSESAT PESGPGTSES ATPESGPGSE PATSGSETPG

951 SEPATSGSET PGSPAGSPTS TEEGTSTEPS EGSAPGTSTE PSEGSAPGSE

1001 PATSGSETPG TSESATPESG PGTSTEPSEG SAPASSEITR TTLQSDQEEI

1051 DYDDTISVEM KKEDFDIYDE DENQSPRSFQ KKTRHYFIAA VERLWDYGMS

1101 SSPHVLRNRA QSGSVPQFKK VVFQEFTDGS FTQPLYRGEL NEHLGLLGPY

1151 IRAEVEDNIM VTFRNQASRP YSFYSSLISY EEDQRQGAEP RKNFVKPNET

1201 KT YFWK VQHH MAPTKDEFDC TO AWA YFSD VD LEKD VHSGLI GPLL VCHTNT

1251 LNPAHGRQVT VQEFALFFTI FDETKSWYFT ENMERNCRAP CNIQMEDPTF

1301 KENYRFHAIN GYIMDTLPGL VMAQDQRIRW YLLSMGSNEN IHSIHFSGHV

1351 FTVRKKEEYK MALYNLYPGV FETVEMLPSK AGIWRVECLI GEHLHAGMST

1401 LFLVYSNKCQ TPLGMASGHHI RDFQITASGQ YGQWAPKLAR LHYSGSINAW

1451 STKEPFSWIK VDLLAPMIIH GIKTQGARQK FSSLYISQFI IMYSLDGKKW

1501 QTYRGNSTGT LMVFFGNVDS SGIKHNIFNP PIIARYIRLH PTHYSIRSTL

1551 RMELMGCDLN SCSMPLGMES KAISDAQITA SSYFTNMFAT WSPSKARLHL

1601 QGRSNAWRPQ VNNPKEWLQV DFQKTMKVTG VTTQGVKSLL TSMYVKEFLI

1651 SSSQDGHQWT LFFQNGKVKV FQGNQDSFTP VVNSLDPPLL TRYLRIHPQS

1701 WVHQIALRME VLGCEAQDLY DKTHTCPPCP APELLGGPSV FLFPPKPKDT

1751 LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

1801 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT

1851 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

1901 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

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Nucleotide sequence of pSYN VWF059 (encoding VWF D'D3-Fc with thrombin site acidic site 2 (a2) in the linker) (SEQ ID NO: 196).

ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT

GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC

101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG

151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA

201 ACGCTCCTTC TCGATT ATTG GGGACTTCC A GA ATGGC A AG AGAGTG AGCC

251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT

301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG

351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT

401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG

451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT

501 CTTTGCTGAA GATGACTTT A TGACCC AAGA AGGGACCTTG ACCTCGGACC

551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT

601 GA ACGGGC AT CTCCTCCC AG C AGCTC ATGC A AC ATCTCCT CTGGGGA A AT

651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT

701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT

751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC

801 CCTCCTGGAG T ACGCCCGGA CCTGTGCCC A GGAGGGAATG GTGCTGT ACG

851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG

901 T AT AGGC AGT GTGTGTCCCC TTGCGCC AGG ACCTGCC AGA GCCTGC AC AT

951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG

1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC

1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG

1101 C A AC ACCTGC ATTTGCCGA A AC AGCC AGTG GATCTGC AGC A ATGA AGA AT

- 178 044115

1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC

1201 A AC AGAT ACT TC ACCTTC AG TGGGATCTGC C AGT ACCTGC TGGCCCGGGA 1251 TTGCCAGGAC СACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGC AC A AC A GCCTTGTGA A ACTGA AGC AT GGGGC AGG AG TTGCC ATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGA AGACC TGCGGCCTGT GTGGGA ATT A C A ATGGC A AC C AGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 165 1 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTG TGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTC TT ACCCGGATGA GGA ATGC A AT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAG AAGAC ATCTTCTC AG ACC ATC AC AC C ATGTGCT AC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 A AGGGC TCGA GTGT ACC A A A ACGTGCC AGA ACT ATGACCT GGAGTGC ATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAA GCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATAC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA

- 179 044115

3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGA AAAGT GCCTCTGGAC TC ATCCCCTG CC ACCTGCC A TA AC A AC ATS 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC T GGTGGACCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACT GTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG ATATCGGGCG CGCCAACTC AGAGAGCGCC 3751 ACCCCTGAAA GTGGTCCCGG GAGCGAGCCA GCCACATCTG GGTCGGAAAC 3801 GCCAGGCACA AGTGAGTCTG CAACTCCCGA GTCCGGACCT GGCTCCGAGC 3851 CTGCC ACT AG CGGCTCCGAG ACTCCGGGA A CTTCCGAGAG CGCT AC ACC A 3901 GAAAGCGGAC CCGGA ACC AG T ACCGAACCT AGCGAGGGCT CTGCTCCGGG 3951 CAGCCCAGCC GGCTCTCCTA CATCCACGGA GGAGGGCACT TCCGAATCC G 4001 CCACCCCGGA GTCAGGGCCA GGATCTGAAC CCGCTACCTC AGGCAGTGAG 4051 ACGCCAGGAA CGAGCGAGTC CGCTACACCG GAGAGTGGGC CAGGGAGCCC 4101 TGCTGGATCT CCTACGTCCA CTGAGGAAGG GTCACCAGCG GGCTCGCCCA 4151 CCAGC ACTGA AGA AGGTGCC TCGAT ATCTG AC A AGA AC AC TGGTGATT AT 4201 T ACGAGGAC A GTT ATGA AGA T ATTTC AGC A T ACTTGCTGA GT A A A A AC A A 4251 TGCCATTGAA CCAAGAAGCT TCTCTGACAA AACTCACACA TGCCCACCGT 4301 GCCCAGCTCC AGAACTCCTG GGCGGACCGT CAGTCTTCCT CTTCCCCCCA 4351 A A ACCC A AGG AC ACCCTC AT GATCTCCCGG ACCCCTGAGG TC AC ATGCGT 4401 GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG 4451 TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG 4501 TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA 4551 C TGGCTG A AT GGC AAGG AGT AC A AGTGC A A GGTCTCC A AC A A AGCCCTCC 4601 CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 4651 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA 4701 GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG 4751 TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT 4801 CCCGTGTTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT 4851 GGACAAGAGC AGGTGGCAGC AGGGGAACG T CTTCTCATGC TCCGTGATGC 4901 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 4951 GGTAAATGA

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Protein sequence of pSYN VF059 (VWF D'D3-Fc with the a2 region of the FVIII thrombin site in the linker) - the underlined region in bold represents the a2 region (SEQ ID NO: 197).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGAPTSESA

1251 TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE TPGTSESATP

1301 ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP GSEPATSGSE

1351 TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGA SISDKNTGDY

1401 YEDSYEDISA YLLSKNNAIE PRSFSDKTHT CPPCPAPELL GGPSVFLFPP

1451 KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ

1501 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE

1551 PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

1601 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP

1651 GK*

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Nucleotide sequence of pSYN VWF062 (encoding VWF D'D3-Fc without thrombin site in the linker) (SEQ ID NO: 198).

ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT

GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC

101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG

151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA

201 ACGCTCCTTC TCGATT ATTG GGGACTTCC A GA ATGGC A AG AGAGTG AGCC

251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT

301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG

351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT

401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG

451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT

501 CTTTGCTGAA GATGACTTT A TGACCC AAGA AGGGACCTTG ACCTCGGACC

551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT

601 GA ACGGGC AT CTCCTCCC AG C AGCTC ATGC A AC ATCTCCT CTGGGGA A AT

651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT

701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT

751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC

801 CCTCCTGGAG T ACGCCCGGA CCTGTGCCC A GGAGGGAATG GTGCTGT ACG

851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG

901 T AT AGGC AGT GTGTGTCCCC TTGCGCC AGG ACCTGCC AGA GCCTGC AC AT

951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG

1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC

1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG

1101 C A AC ACCTGC ATTTGCCGA A AC AGCC AGTG GATCTGC AGC A ATGA AGA AT

1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC

1201 A AC AGAT ACT TC ACCTTC AG TGGGATCTGC C AGT ACCTGC TGGCCCGGGA

1251 TTGCCAGGAC СACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG

1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC

1351 CTGC AC A AC A GCCTTGTGA A ACTGA AGC AT GGGGC AGG AG TTGCC ATGGA

1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC

1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG

1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC

1551 CGGGA AGACC TGCGGCCTGT GTGGGA ATT A C A ATGGC A AC C AGGGCGACG

1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG

- 182 044115

1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCT AC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCC GC TCTCTCTCTT ACCCGGATGA GGA ATGC A AT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAG AAGAC ATCTTCTC AG ACC ATC AC AC C ATGTGCT AC TGTGAGGATG 2201 GCTTCATGCA CTGTAC CATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 A AGGGCTCGA GTGT ACC A A A ACGTGCC AGA ACT ATGACCT GGAGTGC ATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 255 1 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGG AT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATTC TCCGTG GTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CC AGA AA AGT GCCTCTGG AC TC ATCCCCTG CC ACCTGCC A TA AC A AC ATS 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGC TGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3 501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG

- 183 044115

3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG

3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG

3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT

3701 GTGAAGCCTG CCAGGAGCCG ATATCGGGCG CGCCAACATS AGAGAGCGCC

3751 ACCCCTGAAA GTGGTCCCGG GAGCGAGCCA GCCACATCTG GGTCGGAAAC

3801 GCCAGGCACA AGTGAGTCTG CAACTCCCGA GTCCGGACCT GGCTCCGAGC

3851 CTGCC ACT AG CGGCTCCGAG ACTCCGGGA A CTTCCGAGAG CGCT AC ACC A

3901 GAAAGCGGAC CCGGA ACC AG T ACCGAACCT AGCGAGGGCT CTGCTCCGGG

3951 CAGCCCAGCC GGCTCTCCTA CATCCACGGA GGAGGGCACT TCCGAATCCG

4001 CCACCCCGGA GTCAGGGCCA GGATCTGAAC CCGCTACCTC AGGCAGTGAG

4051 ACGCCAGGAA CGAGCGAGTC CGCTACACCG GAGAGTGGGC CAGGGAGCCC

4101 TGCTGGATCT CCTACGTCCA CTGAGGAAGG GTCACCAGCG GGCTCGCCCA

4151 CC AGC ACTGA AGA AGGTGCC TCGAGCGAC A A A ACTC AC AC ATGCCC ACCG

4201 TGCCCAGCTC CAGAACTCCT GGGCGGACCG TCAGTCTTCC TCTTCCCCCC

4251 AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCTGAG GTCACATGCG

4301 TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC

4351 GTGGACGGCG TGGAGGTGC A T A ATGCC A AG AC A A AGCCGC GGGAGGAGC A

4401 GT AC AAC AGC ACGT ACCGTG TGGTC AGCGT CCTC ACCGTC CTGC ACC AGG

4451 ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTC

4501 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA

4551 ACC AC AGGTG T AC ACCCTGC CCCC ATCCCG GGATGAGCTG ACC A AGA ACC

4601 AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC

4651 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC

4701 TCCCGTGTTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG

4751 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG

4801 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC

4851 GGGTAAAATGA

Protein sequence of pSYN VWF062 (VWF D'D3-Fc without thrombin site in linker) (SEQ ID NO: 199).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

- 184 044115

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPS VK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEPISGAPTSESA

1251 TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE TPGTSESATP

1301 ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP GSEPATSGSE

1351 TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGA SSDKTHTCPP

1401 CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY

1451 VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL

1501 PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA

1551 VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM

1601 HEALHNHYTQ KSLSLSPGK*

Nucleotide sequence of pSYN VWF073 (encoding VWF D1D2D'D3 - 144 AE XTEN shortened a2 thrombin site FVIII-Fc) (SEQ ID NO: 174).

ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT

151 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC

101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG

151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA

201 ACGCTCCTTC TCGATT ATTG GGGACTTCC A GA ATGGC A AG AGAGTG AGCC

251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT

- 185 044115

301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG

351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT

401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG

451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT

501 CTTTGCTGAA GATGACTTT A TGACCC AAGA AGGGACCTTG ACCTCGGACC

551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GA ACGGGC AT CTCCTCCC AG C AGCTC ATGC A AC ATCTCCT CTGGGGA A AT

651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT

751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC

801 CCTCCTGGAG T ACGCCCGGA CCTGTGCCC A GGAGGGAATG GTGCTGT ACG

851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 T AT AGGC AGT GTGTGTCCCC TTGCGCC AGG ACCTGCC AGA GCCTGC AC AT

951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101 C A AC ACCTGC ATTTGCCGA A AC AGCC AG TG GATCTGC AGC A ATGA AGA AT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 A AC AGAT ACT TC ACCTTC AG TGGGATCTGC C AGT ACCTGC TGGCCCGGGA 1251 TTGCCAGGAC СACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGC AC A AC A GCCTTGTGA A ACTGA AGC AT GGGGC AGG AG TTGCC ATGGA 1401 TGGCCA GGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGA AGACC TGCGGCCTGT GTGGGA ATT A C A ATGGC A AC C AGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCT CC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGA ATGC A AT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCC AG AAGAC ATCTTCTC AG ACC ATC AC AC C ATGTGCT AC TGTGAGGATG

- 186 044115

2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 23 51 AAGGGCTCGA GTGT ACC AAA ACG TGCCAGA ACTATGACCT GGAGTGC ATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC

2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGG GG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TC CGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT

3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA

3101 CCAGA AAAGT GCCTCTGGAC TC ATCCCCTG CC ACCTGCC A TA AC A AC ATS 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT

3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT

3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT

3701 GTGAAGCCTG CC AGGAGCCG GGCGCGCC AA WITH ATC AGAGAG CGCC ACCCCT 3751 GAAAGTGGTC CCGGGAGCGA GCCAGCCACA TCTGGGTCGG AAACGCCAGG

3801 CACAAGTGAG TCTGCAACTC CCGAGTCCGG ACCTGGCTCC GAGCCTGCCA 3851 CT AGCGGCTC CGAGACTCCG GGA ACTTCCG AGAGCGCT AC ACC AG AA AGC 3901 GGACCCGGAA CCAGTACCGA ACCTAGCGAG GGCTCTGCTC CGGGCAGCCC 3951 AGCCGGCTCT CCTACATCCA CGGA GGAGGG CACTTCCGAA TCCGCCACCC 4001 CGGAGTCAGG GCCAGGATCT GAACCCGCTA CCTCAGGCAG TGAGACGCCA 4051 GGAACGAGCG AGTCCGCTAC ACCGGAGAGT GGGCCAGGGA GCCCTGCTGG

- 187 044115

4101 ATCTCCTACG TCCACTGAGG AAGGGTCACC AGCGGGCTCG CCCACCAGCA

4151 CTG A AG AAGG TGCCTCGAGC GGCGGTGGAG GATCCGGTGG CGGGGGATCC

4201 GGTGGCGGGG GATCCGGTGG CGGGGGATCC GGTGGCGGGG GATCCGGTGG

4251 CGGGGGATCC ATTGAACCAA GAAGCTTCTC TGGCAGCGGA GGCGACAAAA

4301 CTCACACATG CCCACCGTGC CCAGCTCCAG AACTCCTGGG CGGACCGTCA

4351 GTCTTCCTCT TCCCCCC A A A ACCC A AGGAC ACCCTC ATGA TCTCCCGG AC

4401 CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG

4451 TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA

4501 AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT

4551 CACCGTCCTG CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG

4601 TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAGCC

4651 AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC CATCCCGGGA

4701 TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT

4751 ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC

4801 AACTACAAGA CCACGCCTCC CGTGTTGGAC TCCGACGGCT CCTTCTTCCT

4851 CTACAGC AAG CTC ACCGTGG AC AAGAGCAG GTGGC AGCAG GGGAACGTCT

4901 TCTCATGCTC CGTGATGC AT GAGGCTCTGC AC AACC ACT A C ACGC AGAAG

4951 AGCCTCTCCC TGTCTCCGGG TAAATGA

Protein sequence pSYN VWF073 (VWF D1D2D'D3- 144 AE XTEN- shortened a2 thrombin-Fc site) (SEO ID NO: 175).

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARIDGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

51 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

- 188 -

Claims (22)

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP GAPTSESATP 1251 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES 1301 GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP 1351 GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGASS GGGGSGGGGS 1401 GGGGSGGGGS GGGGSGGGGS IEPRSFSGSG GDKTHTCPPC PAPELLGGPS 1451 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT 1501 KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA 1551 KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN 1601 NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 1651 SLSLSPGK* The foregoing description of specific embodiments of the invention so fully discloses the general nature of the invention that others can, by applying knowledge in the art, easily modify and/or adapt for various applications such specific embodiments of the invention, without undue experimentation, without departing from the general concept of the present invention . Thus, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teaching and guidance provided herein. It should be understood that the phraseology and terminology used herein are for purposes of description and not limitation, so the terminology or phraseology of this specification should be interpreted by those skilled in the art in light of the instructions and guidance provided. Other embodiments of the present invention will be apparent to those skilled in the art in view of the present description and the practice of the present invention described herein. It is intended that the description and examples be considered as illustrative only, with the true scope and spirit of the present invention set forth in the following claims. All patents and publications cited in their entirety herein are incorporated by reference. CLAIM 1. A chimeric protein comprising (i) a first polypeptide chain that contains, from its N-terminus to its C-terminus, (a) a factor VIII (FVIII) protein comprising an N-terminal portion and a C-terminal portion; wherein the N-terminal portion of the FVIII protein contains an A1 domain, an A2 domain, and a portion of the B domain of full-length mature FVIII (SEQ ID NO: 65); wherein the N-terminal portion contains the amino acid sequence of residues 1-745 of SEQ ID NO: 65 fused to the first XTEN sequence inserted immediately downstream of amino acid 745 of SEQ ID NO: 65; and wherein the C-terminal portion comprises an A3 domain, a C1 domain, and a C2 domain such that the C-terminal portion comprises residues 1690-2332 of SEQ ID NO: 65; (b) a first Fc fragment, wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and (ii) a second polypeptide chain that contains, from its N-terminus to its C-terminus, (a) a von Willebrand factor (VWF) protein containing a D' domain and a D3 domain of VWF, wherein the VWF protein contains an alanine substitution at residues corresponding to residues 1099 and 1142 of SEQ ID NO: 21; (b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues; (c) a cleavable linker containing the a2 region of FVIII, which contains the amino acid post- - 189 044115 the sequence from Glu720 to Arg740 corresponding to SEQ ID NO: 65, wherein the a2 region is cleavable by thrombin; and (d) a second Fc fragment, the first polypeptide chain being connected to the second polypeptide chain through the first Fc fragment and the second Fc fragment. 2. The chimeric protein according to claim 1, characterized in that the C-terminal part of the FVIII protein contains an amino acid sequence that is at least 95% identical to residues 1641-2332 from SEQ ID NO: 65. 3. The chimeric protein according to claim 2, characterized in that the second XTEN sequence has a length of about 144 amino acid residues. 4. The chimeric protein according to claim 3, characterized in that the second XTEN sequence consists of an amino acid sequence having a length of from 144 amino acid residues to 287 amino acid residues. 5. Chimeric protein according to claim 3, characterized in that the first Fc fragment is connected to the second Fc fragment by a covalent bond. 6. Chimeric protein according to claim 5, characterized in that the first Fc fragment is connected to the second Fc fragment by a disulfide bond. 7. Chimeric protein according to claim 1, characterized in that the first Fc fragment and the second Fc fragment are the same. 8. The chimeric protein according to claim 7, characterized in that the first Fc fragment and the second Fc fragment are derived from human IgG1. 9. Chimeric protein according to claim 8, characterized in that the VWF protein consists of a D' domain and a D3 domain. 10. The chimeric protein according to claim 8, characterized in that the VWF protein additionally contains domain D1 and D2 from VWF. 11. The chimeric protein according to claim 10, characterized in that the VWF protein additionally contains a VWF signal peptide. 12. The chimeric protein according to claim 11, characterized in that the FVIII protein additionally contains an FVIII signal peptide. 13. Chimeric protein according to claim 7, characterized in that the first Fc fragment is connected to the second Fc fragment by two disulfide bonds. 14. The chimeric protein of claim 1, wherein the cleavable linker comprises a FVIII a2 region containing an amino acid sequence at least 90% identical to SEQ ID NO: 106. 15. A pharmaceutical composition containing the chimeric protein according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier. 16. A chimeric protein comprising (i) a first polypeptide chain that contains, from its N-terminus to its C-terminus, (a) a factor VIII (FVIII) protein containing an amino acid sequence that is at least 99% identical to SEQ ID NO: 67, with the first XTEN sequence inserted immediately downstream of the residue corresponding to residue 745 of SEQ ID NO: 67; and (b) a first Fc fragment; wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and (ii) a second polypeptide chain that contains, from its N-terminus to its C-terminus, (a) a von Willebrand factor (VWF) protein containing a D' domain and a D3 domain of VWF, wherein the VWF protein contains an alanine substitution at residues corresponding to residues 1099 and 1142 of SEQ ID NO: 21; (b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues; (c) a cleavable linker containing the a2 region of FVIII, which contains the amino acid sequence from Glu720 to Arg740 corresponding to SEQ ID NO: 65, and the a2 region is amenable to thrombin cleavage; and (d) a second Fc moiety, wherein the first Fc moiety is linked to the second Fc moiety by a disulfide bond. 17. The chimeric protein according to claim 16, characterized in that the second sequence XTEN has a length of about 144 amino acid residues. 18. The chimeric protein according to claim 16, characterized in that the second XTEN sequence consists of an amino acid sequence having a length of from 144 amino acid residues to 287 amino acid residues. 19. The chimeric protein according to claim 16, characterized in that the first Fc fragment and the second Fc fragment are the same. 20. The chimeric protein according to claim 19, characterized in that the first Fc fragment and the second Fc fragment are derived from human IgG1. 21. The chimeric protein according to claim 20, characterized in that the VWF protein consists of a D' domain and a D3 domain. 22. Chimeric protein according to claim 20, characterized in that the VWF protein additionally contains a D1 domain - 190 -