阅读说明：本技术 具有xten的凝血酶可裂解连接子和其用途 (Thrombin cleavable linker with XTEN and uses thereof ) 是由 埃克塔·赛斯·恰布拉 约翰·库曼 刘童瑶 于 2014-06-27 设计创作，主要内容包括：本发明涉及具有XTEN的凝血酶可裂解连接子和其用途。本发明提供包含经由VWF连接子与异源部分融合的VWF蛋白的嵌合分子。本发明提供可在凝血酶存在下裂解的有效的VWF连接子。所述嵌合分子还可包含包括FVIII蛋白和第二异源部分的多肽链,其中包含所述VWF蛋白的链与包含所述FVIII蛋白的链彼此缔合。本发明还包括核苷酸、载体、宿主细胞、使用所述嵌合蛋白的方法。(The present invention relates to thrombin cleavable linkers with XTEN and uses thereof. The present invention provides a chimeric molecule comprising a VWF protein fused to a heterologous moiety via a VWF linker. The present invention provides an effective VWF linker that can be cleaved in the presence of thrombin. The chimeric molecule can further comprise a polypeptide chain comprising a FVIII protein and a second heterologous moiety, wherein a chain comprising the VWF protein and a chain comprising the FVIII protein are associated with each other. The invention also includes nucleotides, vectors, host cells, methods of using the chimeric proteins.)

1. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII169(SEQ ID NO:88) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, 90%, 95%, or 100% identity to VWF059(SEQ ID NO: 82).

2. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO:86) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to VWF059(SEQ ID NO: 82).

3.A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO:86) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, 90%, 95%, or 100% identity to VWF062(SEQ ID NO: 84).

4. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO:86) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, 90%, 95%, or 100% identity to VWF057(SEQ ID NO: 80).

5. A polynucleotide or polynucleotides encoding the chimeric molecule of any one of claims 1 to 4.

6. A polynucleotide encoding a nucleotide sequence having at least about 80%, about 90%, about 95%, or 100% identity to VWF057(SEQ ID NO: 79).

7. The polynucleotide of claim 6, further comprising a second polynucleotide comprising a second nucleotide sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII169(SEQ ID NO: 87).

8. The polynucleotide of claim 6, further comprising a second polynucleotide comprising a second nucleotide sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO: 85).

9. The polynucleotide of claim 7 or 8, wherein the first polynucleotide is on the same vector as the second polynucleotide or on two different vectors.

10. A method of reducing the frequency or extent of bleeding events in a subject in need thereof, comprising administering an effective amount of the chimeric molecule of any one of claims 1 to 4 or the polynucleotide or polynucleotides of any one of claims 5 to 9.

Background

Hemophilia a is a bleeding disorder caused by a deficiency in the gene encoding coagulation factor viii (fviii) and has a prevalence of 1-2 parts per million in male newborns. Graw et al, nat. rev. genet.6 (6): 488-501(2005). Patients suffering from hemophilia a can be treated by infusion of purified or recombinantly produced FVIII. However, all commercial FVIII products are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to the patient. See Weiner m.a. and Cairo, m.s., Pediatric Hematology secretes, Lee, m.t.,12.Disorders of coargulation, Elsevier Health Sciences, 2001; lilibrrap, d.Thromb.Res.122 suppl 4: s2-8 (2008). In addition, various approaches have been attempted to extend FVIII half-life. For example, methods developed to extend the half-life of coagulation factors include pegylation, glycosylation, and binding to albumin. See Dumont et al, blood.119 (13): 3024-3030 (published online at 1/13/2012). However, regardless of the protein engineering used, long-acting FVIII products currently under development have an improved half-life, which is reported to be limited, in preclinical animalsOnly about a 1.5 to 2 fold improvement in the model was achieved. See above. Consistent results have been demonstrated in humans, e.g., in hemophilia A patientsIn contrast, rFVIIIFc is reported to increase half-life by up to about 1.7-fold. See above. Thus, while the improvement is slight, an increase in half-life may indicate other t_1/2The presence of a limiting factor.

Due to frequent dosing and the inconvenience caused by the dosing regimen, there is still a need to develop FVIII products that require less frequent administration, i.e. FVIII products having a half-life longer than the 1.5 to 2 fold half-life limit.

Disclosure of Invention

The present invention is directed to a chimeric molecule comprising a wenweibull Factor (Von Willebrand Factor, VWF) protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker linking the VWF protein and the heterologous moiety, wherein the VWF linker comprises a polypeptide selected from the group consisting of: (i) the a2 region from factor viii (fviii); (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof, and wherein the XTEN sequence is linked to the VWF protein, the heterologous moiety (H1), the VWF linker, or any combination thereof. In one embodiment, the XTEN sequence connects the VWF protein to the VWF linker or the VWF linker to the heterologous moiety. In another embodiment, the chimeric molecule further comprises a second polypeptide chain comprising a FVIII protein, wherein the first polypeptide chain and the second polypeptide chain are associated with each other. In other embodiments, the FVIII protein in the chimeric molecule further comprises an additional XTEN sequence. The additional XTEN sequence can be linked to the N-terminus or C-terminus of the FVIII protein or inserted between two FVIII amino acids adjacent to each other. In other embodiments, the second polypeptide chain further comprises a second heterologous moiety (H2).

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

For the chimeric molecules of the present disclosure, the XTEN sequence attached to a VWF protein, VWF linker, FVIII protein, or any other component in the chimeric molecule comprises about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 1044 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about, About 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. In some embodiments, the XTEN polypeptide is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG 144. In other embodiments, the XTEN polypeptide is selected from SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45, a first step of; SEQ ID NO: 44; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46. SEQ ID NO: 44 or SEQ ID NO: 42.

in other aspects, the additional XTEN sequence in the chimeric molecule comprises about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about, About 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. In some embodiments, the additional XTEN polypeptide is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG 144. In certain embodiments, the additional XTEN polypeptide is selected from SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45, a first step of; SEQ ID NO: 43; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46. SEQ ID NO: 44 or SEQ ID NO: 42.

in one embodiment, a VWF linker useful for linking a VWF protein in a chimeric molecule to a heterologous moiety comprises a2 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, or 100% identity to Glu720 to Arg740 corresponding to full-length FVIII, wherein the a2 region is capable of being cleaved by thrombin. In a particular embodiment, the a2 region comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4). In another embodiment, a VWF linker useful for linking a VWF protein to a heterologous moiety comprises a1 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, or 100% identity to Met 337-Arg 372 corresponding to full-length FVIII, wherein the a1 region is capable of being cleaved by thrombin. In some embodiments, the a1 region comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5).

In other embodiments, a VWF linker useful for linking a VWF protein to a heterologous moiety comprises a3 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, or 100% identity to Glu1649 to Arg1689 corresponding to full-length FVIII, wherein the a3 region is capable of being cleaved by thrombin. In a particular embodiment, the a3 region comprises ISEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQ (SEQ ID NO: 6).

In other embodiments, a VWF linker useful for linking a VWF protein to a heterologous moiety comprises a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and a PAR1 exosite interaction motif, and wherein the PAR1 exosite interaction motif comprises S-F-L-L-R-N (SEQ ID NO: 7). In one embodiment, the PAR1 exosite-interacting motif further comprises a sequence selected from: p, P-N, P-N-D, P-N-D-K (SEQ ID NO: 8), P-N-D-K-Y (SEQ ID NO: 9), P-N-D-K-Y-E (SEQ ID NO: 10), P-N-D-K-Y-E-P (SEQ ID NO: 11), P-N-D-K-Y-E-P-F (SEQ ID NO: 12), P-N-D-K-Y-E-P-F-W (SEQ ID NO: 13), P-N-D-K-Y-E-P-F-W-E (SEQ ID NO: 14), P-N-D-K-Y-E-P-F-W-E-D (SEQ ID NO: 20), P-N-D-K-Y-E-P-F-W-E-D-E (SEQ ID NO: 21), P-N-D-K-Y-E-P-F-W-E-D-E-E (SEQ ID NO: 22), P-N-D-K-Y-E-P-F-W-E-D-E-S (SEQ ID NO: 23), or any combination thereof. In other embodiments, wherein the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine. In a particular embodiment, the VWF linker comprises GGLVPRSFLLRNPNDKYEPFWEDEES (SEQ ID NO: 24).

In certain embodiments, if the VWF linker is replaced with a thrombin cleavage site in the chimeric molecule, the rate at which thrombin cleaves the VWF linker is faster than the rate at which thrombin would cleave the thrombin cleavage site. In other embodiments, if the VWF linker is replaced with a thrombin cleavage site in the chimeric molecule, the thrombin cleaves the VWF linker at a rate that is at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times the rate at which thrombin will cleave the thrombin cleavage site.

In some embodiments, the VWF linker further comprises one or more amino acids having a length of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. In one example, the one or more amino acids comprise a gly peptide. In another example, the one or more amino acids comprise GlyGly. In other examples, the one or more amino acids comprise a gly/ser peptide. In some examples, the Gly/ser peptide has (Gly)₄Ser) n or S (Gly)₄Ser) n, wherein n is a positive integer selected from 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80 or 100. In some examples, (Gly₄Ser) n linker is (Gly₄Ser)₃(SEQ ID NO: 89) or (Gly)₄Ser)₄(SEQ ID NO：90)。

The VWF protein useful for the chimeric molecule of the present invention may comprise the D 'domain and the D3 domain of VWF, wherein the D' domain and the D3 domain are capable of binding to a FVIII protein. In one embodiment, the D' domain of the VWF protein comprises a sequence identical to SEQ ID NO: 2, has an amino acid sequence of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In another embodiment, the D3 domain of the VWF protein comprises a sequence identical to SEQ ID NO: 2, has an amino acid sequence of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In other embodiments, the VWF protein is expressed in a sequence corresponding to SEQ ID NO: 2, residue 1099, residue 1142, or residues 1099 and 1142 comprises at least one amino acid substitution. In other embodiments, in the sequence of the VWF protein, amino acid substitutions corresponding to SEQ ID NO: 2, residue 1099, residue 1142, or residues 1099 and 1142. In other embodiments, the sequence of the VWF protein comprises SEQ ID NO: 2, amino acids 764 to 1240. In certain embodiments, the VWF protein further comprises the D1 domain, the D2 domain, or the D1 and D2 domains of VWF. In some embodiments, the VWF protein further comprises a VWF domain selected from the group consisting of the a1 domain, the a2 domain, the A3 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, or any combination thereof. In other embodiments, the VWF protein consists essentially of or consists of: (1) the D' and D3 domains of VWF or a fragment thereof; (2) the D1, D' and D3 domains of VWF or fragments thereof; (3) the D2, D' and D3 domains of VWF or fragments thereof; (4) the D1, D2, D' and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3 and A1 domains of VWF or a fragment thereof. In other embodiments, the VWF protein further comprises a signal peptide of VWF. In other embodiments, the VWF protein is pegylated, glycosylated, hydroxyethylated, or polysialylated. The term "pegylated" refers to having polyethylene glycol (PEG) on a protein; the term "glycosylation" refers to having glycosylation on a protein; the term "hydroxyethylation" refers to having hydroxyethyl starch (HES) on the protein; and the term "polysialylated" refers to having polysialic acid (PSA) on a protein. Examples of PEG, HES and PSA are shown elsewhere herein.

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

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

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

In certain embodiments, the FVIII protein is linked to the second heterologous moiety via a FVIII linker. In one embodiment, the second linker is a cleavable linker. In another embodiment, the FVIII linker is identical to the VWF linker. In other embodiments, the FVIII linker is not the same as the VWF linker.

In some aspects, the chimeric molecules of the invention comprise a formula selected from the group consisting of: (a) V-L1-X1-H1: H2-L2-X2-C; (b) V-X1-L1-H1: H2-L2-X2-C; (c) V-L1-X1-H1: H2-X2-L2-C; (d) V-X1-L1-H1: H2-X2-L2-C; (e) V-L1-X1-H1: H2-L2-C (X2); (f) V-X1-L1-H1: H2-L2-C (X2); (g) C-X2-L2-H2: H1-X1-L1-V; (h) C-X2-L2-H2: H1-L1-X1-V; (i) C-L2-X2-H2: H1-L1-X1-V; (j) C-L2-X2-H2: H1-L1-X1-V; (k) c (X2) -L2-H2: H1-X1-L1-V; or (L) C (X2) -L2-H2: H1-L1-X1-V; wherein V is a VWF protein; l1 is a VWF linker; l2 is an optional FVIII linker; h1 is a first heterologous moiety; h2 is a second heterologous moiety; x1 is an XTEN sequence; x2 is an optional XTEN sequence; c is a FVIII protein; c (X2) is a FVIII protein fused to an XTEN sequence, wherein the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (: is a covalent bond between H1 and H2).

In other aspects, the chimeric molecule comprises a formula selected from the group consisting of: (a) V-L1-X1-H1: H2-L2-X2-C; (b) V-X1-L1-H1: H2-L2-X2-C; (c) V-L1-X1-H1: H2-X2-L2-C; (d) V-X1-L1-H1: H2-X2-L2-C; (e) V-L1-X1-H1: H2-L2-C (X2); (f) V-X1-L1-H1: H2-L2-C (X2); (g) C-X2-L2-H2: H1-X1-L1-V; (h) C-X2-L2-H2: H1-L1-X1-V; (i) C-L2-X2-H2: H1-L1-X1-V; (j) C-L2-X2-H2: H1-L1-X1-V; (k) c (X2) -L2-H2: H1-X1-L1-V; or (L) C (X2) -L2-H2: H1-L1-X1-V; wherein V is a VWF protein; l1 is a VWF linker; l2 is an optional FVIII linker; h1 is a first heterologous moiety; h2 is a second heterologous moiety; x1 is an XTEN sequence; x2 is an optional XTEN sequence; c is a FVIII protein; c (X2) is a FVIII protein fused to an XTEN sequence, wherein the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (: is a covalent bond between H1 and H2).

In other aspects, the chimeric molecule comprises a formula selected from the group consisting of: (a) V-L1-X1-H1: H2-L2-X2-C; (b) V-X1-L1-H1: H2-L2-X2-C; (c) V-L1-X1-H1: H2-X2-L2-C; (d) V-X1-L1-H1: H2-X2-L2-C; (e) V-L1-X1-H1: H2-L2-C (X2); (f) V-X1-L1-H1: H2-L2-C (X2); (g) C-X2-L2-H2: H1-X1-L1-V; (h) C-X2-L2-H2: H1-L1-X1-V; (i) C-L2-X2-H2: H1-L1-X1-V; (j) C-L2-X2-H2: H1-L1-X1-V; (k) c (X2) -L2-H2: H1-X1-L1-V; or (L) C (X2) -L2-H2: H1-L1-X1-V; wherein V is a VWF protein; l1 is a VWF linker; l2 is an optional FVIII linker; h1 is a first heterologous moiety; h2 is a second heterologous moiety; x1 is an optional XTEN sequence; x2 is an XTEN sequence; c is a FVIII protein; c (X2) is a FVIII protein fused to an XTEN sequence, wherein the XTEN sequence is inserted in two strains of FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (: is a covalent bond between H1 and H2).

In the chimeric molecules of the invention, VWF proteins can inhibit or prevent the binding of endogenous VWF to the FVIII protein.

In certain aspects, the FVIII protein in the chimeric molecule may comprise a third heterologous moiety (H3). The third heterologous moiety (H3) can be an XTEN sequence. In other aspects, the FVIII protein comprises a fourth heterologous moiety (H4). The fourth heterologous moiety (H4) can be an XTEN sequence. In some aspects, the FVIII protein comprises a fifth heterologous moiety (H5). The fifth heterologous moiety can be an XTEN sequence. In other aspects, the FVIII protein comprises a sixth heterologous moiety (H6). The sixth heterologous moiety can be an XTEN sequence. In certain aspects, one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are capable of extending the half-life of the chimeric molecule. In other aspects, the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are linked to the C-terminus or N-terminus of FVIII or inserted between two amino acids of FVIII protein. In other aspects, one or more of the third heterologous moiety, the fourth heterologous moiety, the fifth heterologous moiety, and the sixth heterologous moiety comprises a sequence selected from the group consisting of about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, and about, A length of one or more of about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids. For example, the XTEN sequence of the third heterologous moiety, the fourth heterologous moiety, the fifth heterologous moiety or the sixth heterologous moiety can be selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288 or AG 144. More specifically, the XTEN sequence can be selected from SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45, a first step of; SEQ ID NO: 43; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46. SEQ ID NO: 44 or SEQ ID NO: 42.

in certain embodiments, the half-life of the chimeric molecule is extended at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to wild-type FVIII.

The disclosure also provides a polynucleotide or a set of polynucleotides encoding the chimeric molecule or its complement. The polynucleotide or the set of polynucleotides may further comprise a polynucleotide chain encoding PC5 or PC 7.

Also included is a vector or set of vectors comprising the polynucleotide or set of polynucleotides and one or more promoters operably linked to the polynucleotide or set of polynucleotides. In some embodiments, the vector or the set of vectors may further comprise one additional polynucleotide chain encoding PC5 or PC 7.

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

In some aspects, the invention includes a pharmaceutical composition comprising a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding the chimeric molecule, a vector or set of vectors comprising the polynucleotide or set of polynucleotides, or a host cell disclosed herein, and a pharmaceutically acceptable carrier. In one embodiment, the chimeric molecule in the composition has an extended half-life compared to a wild-type FVIII protein. In another embodiment, wherein the half-life of the chimeric molecule in the composition is extended at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold as compared to wild-type FVIII.

Also included is a method of reducing the frequency or extent of bleeding events in a subject in need thereof, comprising administering an effective amount of a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding the chimeric molecule, a vector or set of vectors disclosed herein, a host cell disclosed herein, or a composition disclosed herein. The invention also includes a method of preventing the occurrence of a bleeding event in a subject in need thereof comprising administering an effective amount of a chimeric molecule disclosed herein, a polynucleotide or set of polynucleotides encoding the chimeric molecule, a vector or set of vectors disclosed herein, a host cell disclosed herein, or a composition disclosed herein. In one embodiment, the bleeding event is from a bleeding coagulation disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, traumatic tinea capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, postpharyngeal interstitial hemorrhage, retroperitoneal interstitial hemorrhage, bleeding in the hip-lumbar sheath, or any combination thereof. In another embodiment, the chimeric molecule disclosed herein, the polynucleotide or set of polynucleotides encoding the chimeric molecule, the vector or set of vectors disclosed herein, the host cell disclosed herein, or the composition disclosed herein may be administered by a route selected from topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, oral administration, or any combination thereof.

The disclosure also includes a method of making a chimeric molecule comprising transfecting one or more host cells with a polynucleotide disclosed herein or a vector disclosed herein and expressing the chimeric molecule in the host cells. The method further comprises isolating the chimeric molecule. In some embodiments, FVIII activity of the chimeric molecule can be measured by the aPTT assay or ROTEM assay.

The present disclosure relates to the following embodiments.

1. A chimeric molecule comprising a wenweibull factor (VWF) protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein and the heterologous moiety, wherein the VWF linker comprises a polypeptide selected from the group consisting of:

i. the a2 region from factor viii (fviii);

a1 region from FVIII;

a3 region from FVIII;

a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or

Any combination thereof, and

wherein the XTEN sequence is linked to the VWF protein, the heterologous moiety (H1), the VWF linker, or any combination thereof.

2. The chimeric molecule of embodiment 1, wherein the XTEN sequence connects the VWF protein to the VWF linker or the VWF linker to the heterologous moiety.

3. The chimeric molecule of embodiment 1 or 2, further comprising a second polypeptide chain comprising a FVIII protein, wherein the first polypeptide chain and the second polypeptide chain are associated with each other.

4. The chimeric molecule of embodiment 3, wherein the FVIII protein further comprises an additional XTEN sequence.

5. The chimeric molecule of embodiment 4, wherein the additional XTEN sequence is linked to the N-terminus or C-terminus of the FVIII protein or is inserted between two FVIII amino acids adjacent to each other.

6. The chimeric molecule of any one of embodiments 3 to 5, wherein the second polypeptide chain further comprises a second heterologous portion (H2).

7. A chimeric molecule comprising a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting the VWF protein to the heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein the VWF linker in the first polypeptide chain comprises:

i. from the a2 region of FVIII;

a1 region from FVIII;

a3 region from FVIII;

a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or

v. any combination thereof,

and wherein the first polypeptide chain and the second polypeptide chain are associated with each other.

8. The chimeric molecule of embodiment 7, wherein the XTEN sequence is linked to the N-terminus or C-terminus of the FVIII protein or is inserted between two FVIII amino acids adjacent to each other.

9. The chimeric molecule of embodiment 7 or 8, further comprising an additional XTEN sequence linked to the VWF protein, the heterologous moiety, the VWF linker, or any combination thereof.

10. The chimeric molecule of any one of embodiments 7 to 9, further comprising a second heterologous moiety (H2).

11. The chimeric molecule of embodiment 10, wherein the second heterologous moiety is linked to the FVIII protein, the XTEN sequence, or both.

12. The chimeric molecule of any one of embodiments 1 to 11, wherein the XTEN sequence comprises about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 1236 amino acids, about, About 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids.

13. The chimeric molecule of any one of embodiments 1 to 12, wherein the XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG 144.

14. The chimeric molecule of any one of embodiments 1 to 13, wherein the XTEN sequence is selected from the group consisting of SEQ ID NOs: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45, a first step of; SEQ ID NO: 44; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46. SEQ ID NO: 44 or SEQ ID NO: 42.

15. the chimeric molecule of any one of embodiments 4 to 6 and 9 to 11, wherein the additional XTEN sequence comprises about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 756 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 1044 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, about 1140 amino acids, about 923 amino acids, about, About 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids.

16. The chimeric molecule of any one of embodiments 4 to 6,9 to 11, and 15, wherein the additional XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG 144.

17. The chimeric molecule of any one of embodiments 4 to 6,9 to 11, and 15 to 16, wherein the additional XTEN sequence is selected from SEQ ID NOs: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45, a first step of; SEQ ID NO: 43; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46. SEQ ID NO: 44 or SEQ ID NO: 42.

18. the chimeric molecule of any one of embodiments 1 to 17, wherein the VWF linker comprises the a2 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, or 100% identity to Glu720 to Arg740 corresponding to full-length FVIII, wherein the a2 region is capable of being cleaved by thrombin.

19. The chimeric molecule of embodiment 18, wherein the a2 region comprises SEQ ID NO: 4.

20. the chimeric molecule of any one of embodiments 1 to 17, wherein the VWF linker comprises the a1 region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% identity to Met337 to Arg372 corresponding to full-length FVIII, wherein the a1 region is capable of being cleaved by thrombin.

21. The chimeric molecule of embodiment 20, wherein the a1 region comprises SEQ ID NO: 5.

22. the chimeric molecule of any one of embodiments 1 to 17, wherein the VWF linker comprises the a3 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, or 100% identity to Glu1649 to Arg1689 corresponding to full-length FVIII, wherein the a3 region is capable of being cleaved by thrombin.

23. The chimeric molecule of embodiment 22, wherein the a3 region comprises SEQ ID NO: 6.

24. the chimeric molecule of any one of embodiments 1 to 17, wherein the VWF linker comprises the thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and the PAR1 exosite interacting motif and wherein the PAR1 exosite interacting motif comprises S-F-L-L-R-N (SEQ ID NO: 7).

25. The chimeric molecule of any one of embodiments 1 to 17 and 24, wherein the PAR1 exosite interaction motif further comprises a sequence selected from: p, P-N, P-N-D, P-N-D-K (SEQ ID NO: 8), P-N-D-K-Y (SEQ ID NO: 9), P-N-D-K-Y-E (SEQ ID NO: 10), P-N-D-K-Y-E-P (SEQ ID NO: 11), P-N-D-K-Y-E-P-F (SEQ ID NO: 12), P-N-D-K-Y-E-P-F-W (SEQ ID NO: 13), P-N-D-K-Y-E-P-F-W-E (SEQ ID NO: 14), P-N-D-K-Y-E-P-F-W-E-D (SEQ ID NO: 20), P-N-D-K-Y-E-P-F-W-E-D-E (SEQ ID NO: 21), P-N-D-K-Y-E-P-F-W-E-D-E-E (SEQ ID NO: 22), P-N-D-K-Y-E-P-F-W-E-D-E-S (SEQ ID NO: 23), or any combination thereof.

26. The chimeric molecule of any one of embodiments 1 to 17, 24, and 25, wherein the aliphatic amino acid is selected from glycine, alanine, valine, leucine, or isoleucine.

27. The chimeric molecule of any one of embodiments 1 to 26, wherein the VWF linker is cleaved by thrombin at a faster rate than the rate at which a thrombin cleavage site consisting of L-V-P-R (SEQ ID NO: 25) is cleaved by thrombin.

28. The chimeric molecule of any one of embodiments 1 to 27, wherein the VWF linker comprises SEQ ID NO: 24.

29. the chimeric molecule of embodiment 27, wherein the rate at which the VWF linker is cleaved by thrombin is at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold greater than the rate at which a thrombin cleavage site consisting of L-V-P-R (SEQ ID NO: 25) is cleaved by thrombin.

30. The chimeric molecule of any one of embodiments 1 to 29, wherein the VWF linker further comprises one or more amino acids, the VWF linker having a length of at least about 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids.

31. The chimeric molecule of embodiment 30, wherein the one or more amino acids comprise a gly peptide.

32. The chimeric molecule of embodiment 30 or 31, wherein the one or more amino acids comprise GlyGly.

33. The chimeric molecule of embodiment 30, wherein the one or more amino acids comprise a gly/ser peptide.

34. The chimeric molecule of embodiment 33, wherein the Gly/ser peptide has (Gly)₄Ser) n or S (Gly)₄Ser) n, wherein n is a positive integer selected from 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80 or 100.

35. The chimeric molecule of embodiment 34, wherein the (Gly)₄Ser) n linker is (Gly₄Ser)₃(SEQ ID NO: 89) or (Gly)₄Ser)₄(SEQ ID NO：90)。

36. The chimeric molecule of any one of embodiments 1 to 35, wherein the VWF protein comprises the D 'domain and the D3 domain of VWF, wherein the D' domain and the D3 domain are capable of binding to a FVIII protein.

37. The chimeric molecule of embodiment 36, wherein the D' domain of the VWF protein comprises a sequence identical to SEQ ID NO: 2, has an amino acid sequence of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

38. The chimeric molecule of embodiment 36 or 37, wherein the D3 domain of the VWF protein comprises a sequence identical to SEQ ID NO: 2, has an amino acid sequence of at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

39. The chimeric molecule of any one of embodiments 36 to 38, wherein the VWF protein is encoded within a polypeptide corresponding to SEQ ID NO: 2, residue 1099, residue 1142, or residues 1099 and 1142 comprises at least one amino acid substitution.

40. The chimeric molecule of any one of embodiments 1 to 39, wherein in the sequence of the VWF protein, an amino acid substitution corresponding to SEQ ID NO: 2, residue 1099, residue 1142, or residues 1099 and 1142.

41. The chimeric molecule of any one of embodiments 1 to 39, wherein the sequence of the VWF protein comprises SEQ ID NO: 2, amino acids 764 to 1240.

42. The chimeric molecule of any one of embodiments 36 to 41, wherein the VWF protein further comprises the D1 domain, D2 domain, or D1 and D2 domains of VWF.

43. The chimeric molecule of any one of embodiments 36 to 42, wherein the VWF protein further comprises a VWF domain selected from the group consisting of an A1 domain, an A2 domain, an A3 domain, a D4 domain, a B1 domain, a B2 domain, a B3 domain, a C1 domain, a C2 domain, a CK domain, one or more fragments thereof, or any combination thereof.

44. The chimeric molecule of any one of embodiments 36 to 43, wherein the VWF protein consists essentially of or consists of: (1) the D' and D3 domains of VWF or a fragment thereof; (2) the D1, D' and D3 domains of VWF or fragments thereof; (3) the D2, D' and D3 domains of VWF or fragments thereof; (4) the D1, D2, D' and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3 and A1 domains of VWF or a fragment thereof.

45. The chimeric molecule of any one of embodiments 1 to 44, further comprising a signal peptide of VWF.

46. The chimeric molecule of any one of embodiments 1 to 45, wherein the VWF protein is pegylated, glycosylated, hydroxyethylated, or polysialylated.

47. The chimeric molecule of any one of embodiments 1 to 46, wherein the heterologous moiety (H1) is capable of extending the half-life of the chimeric molecule.

48. The chimeric molecule of embodiment 47, wherein the heterologous moiety (H1) comprises an immunoglobulin constant region or a portion thereof, albumin, an albumin binding polypeptide, PAS, a C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding small molecule, or any combination thereof.

49. The chimeric molecule of embodiment 48, wherein the immunoglobulin constant region or portion thereof comprises an FcRn binding partner.

50. The chimeric molecule of embodiment 48, wherein the immunoglobulin constant region or portion thereof comprises an Fc region.

51. The chimeric molecule of any one of embodiments 1 to 47, wherein the heterologous moiety (H1) comprises a scavenger receptor or fragment thereof, wherein the scavenger receptor blocks binding of a FVIII protein to a FVIII scavenger receptor.

52. The chimeric molecule of embodiment 51, wherein the clearance receptor is low density lipoprotein receptor-related protein 1(LRP1) or a FVIII binding fragment thereof.

53. The chimeric molecule of any one of embodiments 6 and 10 to 52, wherein the second heterologous moiety comprises an immunoglobulin constant region or a portion thereof, albumin, an albumin binding polypeptide, PAS, a C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding small molecule, or any combination thereof.

54. The chimeric molecule of any one of embodiments 6 and 10 to 53, wherein the second heterologous moiety (H2) is capable of extending the half-life of the FVIII protein.

55. The chimeric molecule of embodiment 54, wherein the second heterologous moiety (H2) comprises a polypeptide, a non-polypeptide moiety, or both.

56. The chimeric molecule of embodiment 54 or 55, wherein the second heterologous moiety (H2) comprises an immunoglobulin constant region or a portion thereof.

57. The chimeric molecule of embodiment 56, wherein the second heterologous moiety comprises an FcRn binding partner.

58. The chimeric molecule of embodiment 56, wherein the second heterologous portion comprises a second Fc region.

59. The chimeric molecule of any one of embodiments 6 and 10 to 58, wherein the first heterologous moiety and the second heterologous moiety are associated with each other.

60. The chimeric molecule of embodiment 59, wherein the association between the first polypeptide chain and the second polypeptide is a covalent bond.

61. The chimeric molecule of embodiment 59, wherein the association between the first heterologous moiety and the second heterologous moiety is a disulfide bond.

62. The chimeric molecule of any one of embodiments 6 and 10 to 58, wherein the first heterologous moiety is an FcRn binding partner and the second heterologous moiety is an FcRn binding partner.

63. The chimeric molecule of any one of embodiments 6 and 10 to 58, wherein the first heterologous moiety is an Fc region and the second heterologous moiety is an Fc region.

64. The chimeric molecule of any one of embodiments 6 and 10 to 58, wherein the FVIII protein is linked to the second heterologous moiety through a FVIII linker.

65. The chimeric molecule of embodiment 64, wherein the FVIII linker is a cleavable linker.

66. The chimeric molecule of embodiment 64 or 65, wherein the FVIII linker and the VWF linker are the same.

67. The chimeric molecule of embodiment 64 or 65, wherein the FVIII linker is different from the VWF linker.

68. The chimeric molecule of any one of embodiments 6 and 10 to 67, comprising a formula selected from the group consisting of:

(a)V-L1-X1-H1：H2-L2-X2-C；

(b)V-X1-L1-H1：H2-L2-X2-C；

(c)V-L1-X1-H1：H2-X2-L2-C；

(d)V-X1-L1-H1：H2-X2-L2-C；

(e)V-L1-X1-H1：H2-L2-C(X2)；

(f)V-X1-L1-H1：H2-L2-C(X2)；

(g)C-X2-L2-H2：H1-X1-L1-V；

(h)C-X2-L2-H2：H1-L1-X1-V；

(i)C-L2-X2-H2：H1-L1-X1-V；

(j)C-L2-X2-H2：H1-L1-X1-V；

(k) c (X2) -L2-H2: H1-X1-L1-V; or

(l)C(X2)-L2-H2：H1-L1-X1-V；

Wherein V is the VWF protein;

l1 is the VWF linker;

l2 is an optional FVIII linker;

h1 is the first heterologous moiety;

h2 is the second heterologous moiety;

c is the FVIII protein;

c (X2) is the FVIII protein fused to the XTEN sequence, wherein the XTEN sequence is inserted between two FVIII amino acids that are adjacent to each other;

(-) is a peptide bond or one or more amino acids; and is

Is a covalent bond between said H1 and said H2.

69. The chimeric molecule of any one of embodiments 6 and 10 to 68, wherein the VWF protein inhibits or prevents endogenous VWF from binding to the FVIII protein.

70. The chimeric molecule of any one of embodiments 6 and 10 to 69, wherein the FVIII protein comprises a third heterologous moiety (H3).

71. The chimeric molecule of embodiment 70, wherein the third heterologous moiety (H3) is an XTEN sequence.

72. The chimeric molecule of embodiment 70 or 71, wherein the FVIII protein comprises a fourth heterologous moiety (H4).

73. The chimeric molecule of embodiment 72, wherein the fourth heterologous moiety (H4) is an XTEN sequence.

74. The chimeric molecule of embodiment 72 or 73, wherein the FVIII protein comprises a fifth heterologous moiety (H5).

75. The chimeric molecule of embodiment 74, wherein the fifth heterologous moiety is an XTEN sequence.

76. The chimeric molecule of embodiment 74 or 75, wherein the FVIII protein comprises the sixth heterologous moiety (H6).

77. The chimeric molecule of embodiment 76, wherein the sixth heterologous moiety is an XTEN sequence.

78. The chimeric molecule of embodiment 76 or 77, wherein one or more of the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) is capable of extending the half-life of the chimeric molecule.

79. The chimeric molecule of embodiment 76 or 77, wherein the third heterologous moiety (H3), the fourth heterologous moiety (H4), the fifth heterologous moiety (H5), and the sixth heterologous moiety (H6) are linked to the C-terminus or N-terminus of FVIII or inserted between two amino acids of the FVIII protein.

80. The chimeric molecule of any one of embodiments 71, 73, 75, and 77, wherein one or more of the XTEN sequences comprises a sequence selected from about 42 amino acids, about 72 amino acids, about 108 amino acids, about 144 amino acids, about 180 amino acids, about 216 amino acids, about 252 amino acids, about 288 amino acids, about 324 amino acids, about 360 amino acids, about 396 amino acids, about 432 amino acids, about 468 amino acids, about 504 amino acids, about 540 amino acids, about 576 amino acids, about 612 amino acids, about 624 amino acids, about 648 amino acids, about 684 amino acids, about 720 amino acids, about 792 amino acids, about 828 amino acids, about 836 amino acids, about 864 amino acids, about 875 amino acids, about 912 amino acids, about 923 amino acids, about 948 amino acids, about 1044 amino acids, and about, A length of one or more of about 1140 amino acids, about 1236 amino acids, about 1318 amino acids, about 1332 amino acids, about 1428 amino acids, about 1524 amino acids, about 1620 amino acids, about 1716 amino acids, about 1812 amino acids, about 1908 amino acids, or about 2004 amino acids.

81. The chimeric molecule of any one of embodiments 71, 73, 75, 77, and 80, wherein the XTEN sequence is selected from AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, or AG 144.

82. The chimeric molecule of embodiment 81, wherein the XTEN sequence is selected from the group consisting of SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 47; SEQ ID NO: 45, a first step of; SEQ ID NO: 44; SEQ ID NO: 41; SEQ ID NO: 48; SEQ ID NO: 46. SEQ ID NO: 44 or SEQ ID NO: 42.

83. the chimeric molecule of any one of embodiments 1 to 82, wherein the half-life of the chimeric molecule is extended at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold as compared to wild-type FVIII.

84. A polynucleotide or a set of polynucleotides encoding the chimeric molecule of any one of embodiments 1 to 83 or the complement thereof.

85. The polynucleotide or set of polynucleotides of embodiment 84, further comprising a polynucleotide chain encoding PC5 or PC 7.

86. A vector or set of vectors comprising a polynucleotide or set of polynucleotides according to embodiments 84 or 85 and one or more promoters operably linked to the polynucleotide or set of polynucleotides.

87. The vector or set of vectors of embodiment 86, further comprising an additional polynucleotide chain encoding PC5 or PC 7.

88. A host cell comprising a polynucleotide or a set of polynucleotides according to embodiment 86 or 87 or a vector or a set of vectors according to embodiment 86 or 87.

89. The host cell of embodiment 88, which is a mammalian cell.

90. The host cell of embodiment 89, wherein the mammalian cell is selected from a HEK293 cell, a CHO cell or a BHK cell.

91. A pharmaceutical composition comprising the chimeric molecule of any one of embodiments 1 to 83, the polynucleotide or set of polynucleotides of embodiment 84 or 85, the vector or set of vectors of embodiment 86 or 87, or the host cell of any one of embodiments 88 to 90, and a pharmaceutically acceptable carrier.

92. The composition of embodiment 91, wherein said chimeric molecule has an extended half-life as compared to a wild-type FVIII protein.

93. The composition of embodiment 91 or 92, wherein the half-life of the chimeric molecule is extended at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold as compared to wild-type FVIII.

94. A method of reducing the frequency or extent of bleeding events in a subject in need thereof, comprising administering an effective amount of a chimeric molecule of any one of embodiments 1 to 83, a polynucleotide of embodiment 84 or 85, a vector of embodiment 86 or 87, a host cell of any one of embodiments 88 to 90, or a composition of any one of embodiments 91 to 93.

95. A method of preventing the occurrence of a bleeding event in a subject in need thereof, comprising administering an effective amount of the chimeric molecule of any one of embodiments 1 to 83, the polynucleotide of embodiment 84 or 85, the vector of embodiment 86 or 87, the host cell of any one of embodiments 88 to 90, or the composition of any one of embodiments 91 to 93.

96. The method of embodiment 94 or 95, wherein the bleeding event is from a bleeding coagulation disorder, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage into a muscle, oral hemorrhage, trauma, traumatic tinea capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, postpharyngeal interstitial hemorrhage, retroperitoneal interstitial hemorrhage, hip-lumbar-sheath hemorrhage, or any combination thereof.

97. The method of any one of embodiments 95 to 96, wherein the chimeric molecule of any one of embodiments 1 to 83, the polynucleotide of embodiment 84 or 85, the vector of embodiment 86 or 87, the host cell of any one of embodiments 88 to 90, or the composition of any one of embodiments 91 to 93 is administered by a route selected from topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, oral administration, or any combination thereof.

98. A method of making a chimeric molecule comprising transfecting one or more host cells with a polynucleotide as in embodiment 84 or 85 or a vector as in embodiment 86 or 87 and expressing the chimeric molecule in the host cells.

99. The method of embodiment 98, further comprising isolating said chimeric molecule.

100. A method of increasing FVIII activity of a chimeric FVIII protein comprising a VWF protein, a heterologous moiety (H1) and a VWF linker connecting said VWF protein to said heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein said VWF linker in the first polypeptide chain comprises:

i. from the a2 region of FVIII;

a1 region from FVIII;

a3 region from FVIII;

a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or

v. any combination thereof.

101. The method of embodiment 100, wherein the FVIII activity is measured by the aPTT assay or ROTEM assay.

102. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII169(SEQ ID NO:88) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, 90%, 95%, or 100% identity to VWF059(SEQ ID NO: 82).

103. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO:86) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to VWF059(SEQ ID NO: 82).

104. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO:86) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, 90%, 95%, or 100% identity to VWF062(SEQ ID NO: 84).

105. A chimeric molecule comprising two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO:86) and a second polypeptide sequence comprising an amino acid sequence having at least about 80%, 90%, 95%, or 100% identity to VWF057(SEQ ID NO: 80).

106. A polynucleotide or polynucleotides encoding the chimeric molecule of any one of embodiments 102 to 105.

107. A polynucleotide encoding a nucleotide sequence having at least about 80%, about 90%, about 95%, or 100% identity to VWF057(SEQ ID NO: 79).

108. The polynucleotide of embodiment 107, further comprising a second polynucleotide comprising a second nucleotide sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII169(SEQ ID NO: 87).

109. The polynucleotide of embodiment 107, further comprising a second polynucleotide comprising a second nucleotide sequence having at least about 80%, about 90%, about 95%, or 100% identity to FVIII286(SEQ ID NO: 85).

110. The polynucleotide of embodiment 108 or 109, wherein said first polynucleotide is on the same vector as said second polynucleotide or on two different vectors.

111. A method of reducing the frequency or extent of bleeding events in a subject in need thereof, comprising administering an effective amount of a chimeric molecule of any one of embodiments 102 to 105 or a polynucleotide or polynucleotides of any one of embodiments 106 to 110.

Drawings

Figure 1 shows an illustrative diagram of a chimeric molecule comprising two polypeptide chains (FVIII-XTEN/VWF heterodimer), the first chain comprising a VWF protein fused to an Fc region via a thrombin-cleavable VWF linker (e.g., the D' domain and D3 domain of VWF) and the second chain comprising a FVIII protein fused to a second Fc region via a FVIII linker. The FVIII protein comprises one or more XTENs in multiple domains of FVIII.

Figure 2 shows various VWF constructs, each comprising a D' domain and a D3 domain fused to an Fc region via a thrombin-cleavable VWF linker, except for the control (i.e., VWF-052). VWF-031 contains a 48 amino acid linker that includes the thrombin cleavage site of L-V-P-R (SEQ ID NO: 25). VWF-034 comprises an XTEN sequence of 288 amino acids and a linker of 35 amino acids comprising the thrombin cleavage site of L-V-P-R (SEQ ID NO: 25). VWF-035 contains a linker of 73 amino acids that includes the thrombin cleavage site of L-V-P-R (SEQ ID NO: 25). VWF-036 comprises a linker of 98 amino acids that includes the thrombin cleavage site of L-V-P-R (SEQ ID NO: 25). VWF-039 comprises a VWF linker of 26 amino acids comprising the thrombin cleavage site of L-V-P-R (SEQ ID NO: 25) and the PAR1 exosite interaction motif. VWF-051 contains a 54 amino acid linker that includes the thrombin cleavage site of A-L-R-P-R-V (SEQ ID NO: 26). VWF-052 comprises a linker of 48 amino acids without any thrombin cleavage site (control). VWF-054 comprises a VWF linker of 40 amino acids comprising the a1 region from FVIII. VWF-055 comprises a VWF linker of 34 amino acids comprising the a2 region from FVIII. VWF-056 comprises a VWF linker of 46 amino acids comprising the a3 region from FVIII.

FIG. 3 panel A shows thrombin-mediated cleavage rates (in resonance units per second (RU/s)) as a function of capture density (in RU) for VWF-Fc fusion constructs, i.e., VWF-031, VWF-034, VWF-036, VWF-039, VWF-051, and VWF-052. FIG. 3 panel B shows thrombin-mediated cleavage rates (in resonance units per second (RU/s)) as a function of capture density (in RU) for VWF-Fc fusion constructs, i.e., VWF-031, VWF-034, VWF-036, VWF-051 and VWF-052. In these experiments, each VWF-Fc fusion construct was captured at different densities and subsequently exposed to a fixed concentration of human alpha-thrombin. The slope of each curve in panel a of fig. 3 and panel B of fig. 3 directly reflects the susceptibility to thrombin cleavage for each construct.

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

FIG. 5 shows the results of a linear regression analysis to determine the susceptibility of various VWF-Fc constructs VWF-031, VWF-034, VWF-036, VWF-039, VWF-051, VWF-052, VWF-054, VWF-055 and VWF-056 to thrombin-mediated cleavage. The value is given by s^-1Is expressed in units and reflects the slope of the curves shown in fig. 3 and 4. The relative susceptibility of the two different constructs is derived from the quotient of their respective slopes. Slope of_VWF-039Slope of_VWF-031Is 71, indicating that VWF-Fc fusion construct VWF-039 is 71-fold more susceptible to thrombin-mediated cleavage than VWF-031. Slope of_VWF-055Slope of_VWF-031Is 65, and the slope_VWF-051Slope of_VWF-031Is 1.8.

Fig. 6 shows the clotting times of various chimeric molecules in HemA patients as measured by the whole blood ROTEM assay. FVII155/VWF-031 comprises two polypeptide chains, the first chain comprising BDD FVIII fused to an Fc region and the second chain comprising the D' domain and D3 domain of VWF fused to the Fc region via a minimal thrombin cleavage site (i.e., L-V-P-R (SEQ ID NO: 25)). FVII155/VWF-039 comprises two polypeptide chains, the first chain comprising BDD FVIII fused to an Fc region and the second chain comprising the D' domain and D3 domain of VWF fused to an Fc region via a VWF linker comprising L-V-P-R (SEQ ID NO: 25) and a PAR1 exosite interacting motif. FVII155/VWF-055 comprises two polypeptide chains, the first chain comprising BDD FVIII fused to an Fc region and the second chain comprising the D' domain and the D3 domain of VWF fused to the Fc region via a VWF linker comprising the a2 region from FVIII.

Figure 7 shows a diagram of representative FVIII-VWF heterodimers as well as FVIII169, FVIII286, VWF057, VWF059, and VWF062 constructs. For example, the FVIII169 construct comprises a B domain deleted FVIII protein having a R164 1648A substitution fused to an Fc region with the XTEN sequence (e.g., AE288) inserted at amino acid 745 corresponding to mature full length FVIII (a1-a1-a2-a2-288 XTEN-A3-A3-C1-C2-Fc). The FVIII286 construct comprises a B domain deleted FVIII protein with a R1648 substitution fused to an Fc region, with an XTEN sequence (e.g., AE288) inserted at amino acid 745 corresponding to mature full length FVIII, with another a2 region between FVIII and Fc (a1-a1-a2-a2-288XTEN-A3-A3-C1-C2-a 2-Fc). VWF057 is a VWF-Fc fusion construct comprising the D 'D3 domain of the VWF protein (with two amino acid substitutions in the D' D3 domain, i.e., C336A and C379A) linked to the Fc region via a VWF linker comprising an LVPR thrombin site ("LVPR") and a GS linker ("GS"), wherein an XTEN sequence (i.e., 144XTEN) is inserted between the D 'D3 domain and the VWF linker (D' D3-144XTEN-GS + LVPR-Fc). VWF059 is a VWF-Fc fusion construct comprising the D ' D3 domain (with two amino acid substitutions in the D ' D3 domain, i.e., C336A and C379A) of the VWF protein linked to the Fc region via the acidic region 2(a2) region as VWF linker, wherein the XTEN sequence is inserted between the D ' D3 domain and the VWF linker. VWF062 is a VWF-Fc fusion construct comprising the D 'D3 domain of the VWF protein (with two amino acid substitutions in the D' D3 domain, i.e., C336A and C379A) linked to the Fc region, with the XTEN sequence inserted between the D 'D3 domain and the Fc region (D' D3-144 XTEN-Fc).

Figure 8 shows the acute efficacy of FVIII-XTEN-Fc/D' D3-linker-Fc heterodimers (i.e., FVIII169/VWF034, FVIII169/VWF059, and FVIII169/VWF057) compared to B-domain deleted FVIII ("SQ BDD FVIII" or "BDD-rFVIII") or vehicle control in the HemA mouse tail snip model. BDD-rFVIII is shown in circles, while FVIII169/VWF034 is shown in squares, FVIII169/VWF059 is shown in triangles, FVIII169/VWF057 is shown in hollow circles, and the vehicle is shown in inverted triangles. VWF034 is a VWF-Fc fusion construct comprising the D ' domain and the D3 domain of VWF fused to the Fc region via a VWF linker comprising the LVPR, wherein the XTEN sequence (i.e. 288XTEN) is inserted between the D ' D3 domain and the VWF linker (D ' D3-288 XTEN-LVPR-Fc). The constructs details of FVIII169, VWF059 and VWF057 are shown elsewhere herein. The median blood loss (uL) in mice after administration of 75IU/kg of construct in each treatment group is indicated by the horizontal line.

Detailed Description

The present invention is directed to chimeric molecules comprising an XTEN sequence and a thrombin-cleavable linker connecting a VWF protein or a FVIII protein to a heterologous moiety (e.g., a half-life extending moiety). The invention also provides a chimeric molecule comprising two polypeptide chains, a first chain comprising a VWF protein fused to a heterologous moiety, and a second chain comprising a FVIII protein and a second heterologous moiety, wherein the chimeric molecule comprises an XTEN sequence in the first or second polypeptide chain and wherein the VWF protein or the FVIII protein (or both) is fused to the heterologous moiety via a VWF linker or a FVIII linker (or both). The thrombin-cleavable linker (VWF linker or FVIII linker) is efficiently cleavable by thrombin at the site of injury where thrombin is readily available. Exemplary chimeric molecules are described in the specification and drawings. In some embodiments, the invention relates to chimeric molecules having a structure such as that shown in fig. 1-7. In other embodiments, the invention relates to polynucleotides encoding the chimeric molecule constructs disclosed herein.

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

I. Definition of

It should be noted that the terms "a" or "an" entity refer to one or more of the entity; for example, "nucleotide sequence" is understood to mean one or more nucleotide sequences. Thus, the terms "a", "an", "one or more" and "at least one" are used interchangeably herein.

The term "about" is used herein to mean about, approximately, left-right, or nearby. When the term "about" is used in conjunction with a range of values, it adjusts the range by extending the boundaries above and below the values recited. Generally, the term "about" is used herein to adjust numerical values above and below the stated value by a variation of ± 10% (higher or lower).

The term "polynucleotide" or "nucleotide" is intended to encompass a single nucleic acid as well as a plurality of nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger rna (mrna) or plasmid dna (pdna). In certain embodiments, the polynucleotide comprises a conventional phosphodiester bond or an unconventional bond (e.g., an amide bond, such as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide means a nucleic acid molecule, DNA or RNA that has been removed from its natural environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding a factor VIII polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides that are maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolating RNA molecules includes in vivo or in vitro RNA transcripts of the polynucleotides of the invention. Isolated polynucleotides or nucleic acids according to the invention also include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may include regulatory elements such as promoters, enhancers, ribosome binding sites or transcription termination signals.

As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons that can be translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not generally translated into an amino acid, it may be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of the coding region are generally determined at the 5 'end by a start codon, which codes for the amino terminus of the resulting polypeptide, and at the 3' end by a translation stop codon, which codes for the carboxy terminus of the resulting polypeptide. The two or more coding regions of the invention may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Conversely, a single vector may contain only a single coding region, or may contain two or more coding regions, e.g., a single vector may independently encode a first polypeptide chain and a second polypeptide chain of a chimeric molecule as described below. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, fused or unfused to the nucleic acid encoding the chimeric molecule of the invention. Heterologous coding regions include, without limitation, professional elements or motifs, such as secretion signal peptides or heterologous functional domains.

Certain proteins secreted by mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein once export of the growing protein chain through the rough endoplasmic reticulum has been initiated. One of ordinary skill in the art recognizes that a signal peptide is typically fused to the N-terminus of a polypeptide and cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as a FVIII signal peptide or a VWF signal peptide, or a functional derivative of such sequences that retains the ability to direct secretion of an operably associated polypeptide is used. Alternatively, a heterologous mammalian signal peptide may be used, for example, a human Tissue Plasminogen Activator (TPA) or a mouse β -glucuronidase signal peptide, or a functional derivative thereof.

The term "downstream" refers to a nucleotide sequence that is 3' to a reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence refers to a sequence after the transcription start point. For example, the translation initiation codon of a gene is located downstream of the transcription initiation site.

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

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

A polynucleotide encoding a gene product (e.g., a polypeptide) can include a promoter and/or other transcriptional or translational control elements operatively associated with one or more coding regions. In operable association, the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory regions in a manner such that expression of the gene product is under the influence or control of the regulatory region(s). For example, a coding region is "operably associated with" a promoter if induction of the promoter's function results in transcription of an mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the gene product or with the ability of the DNA template to be transcribed. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals may also be operably associated with a coding region to direct the expression of a gene product.

Various transcriptional control regions are known to those skilled in the art. It includes, but is not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, binding intron-a), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β -globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

Similarly, a variety of translational control elements are known to those of ordinary skill in the art. It includes, but is not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (especially internal ribosome entry sites or IRES, also known as CITE sequences).

The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product (e.g., RNA or polypeptide). Including, but not limited to, transcription of polynucleotides 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 produces a "gene product". As used herein, a gene product can be a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids with post-transcriptional modifications (e.g., polyadenylation or splicing), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage).

"vector" refers to any vehicle used to clone and/or transfer a nucleic acid into a host cell. A vector may be a replicon that can be linked to another nucleic acid segment to create a replication of the linked segment. "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions in vivo as an autonomously replicating unit, i.e., capable of replicating under its own control. The term "vector" includes viral and non-viral vectors used to introduce nucleic acids into cells in vitro, ex vivo or in vivo. A wide variety of vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. The polynucleotide may be inserted into a suitable vector by ligating appropriate polynucleotide fragments into the selected vector with complementary cohesive ends.

The vector may be engineered to encode a selectable marker or reporter that provides for the selection or identification of cells into which the vector has been incorporated. Expression of the selectable marker or reporter allows for identification and/or selection of host cells that incorporate and express additional coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: providing genes resistant to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, pyribenzolar herbicide, sulfonamide, and the like; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase genes, and the like. Examples of reporters known and used in the art include: luciferase (Luc), Green Fluorescent Protein (GFP), Chloramphenicol Acetyltransferase (CAT), -galactosidase (LacZ), -Glucuronidase (GUS), and the like. The selectable marker may also be considered a reporter.

The term "plasmid" refers to an extrachromosomal element that normally carries a gene that is not part of the central metabolism of the cell, and is usually in the form of a circular double-stranded DNA molecule. The elements may be linear, circular or supercoiled autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences derived from single-or double-stranded DNA or RNA of any origin, in which a plurality of nucleotide sequences have been joined or recombined into a unique configuration which enables introduction of a promoter fragment of a selected gene product and the DNA sequence into a cell together with appropriate 3' untranslated sequence.

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

"cloning vector" refers to a "replicon" which is a unit length of nucleic acid that is continuously replicating and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be ligated to bring about replication of the ligated segment. Certain cloning vectors are capable of replication in one cell type (e.g., bacterial) and expression in another cell type (e.g., eukaryotic). Cloning vectors typically comprise one or more sequences that can be used to select cells comprising the vector and/or one or more multiple cloning sites for insertion of a nucleic acid sequence of interest.

The term "expression vector" refers to a vehicle designed to achieve expression of an inserted nucleic acid sequence following insertion into a host cell. The inserted nucleic acid sequence is in operable association with a regulatory region as described above.

The vector is introduced into the host cell by methods well known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or DNA vector transporter.

As used herein, "culturing" refers to incubating cells under in vitro conditions that allow the cells to grow or divide or maintain the cells in a viable state. As used herein, "cultured cells" refers to cells propagated in vitro.

As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as a plurality of "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, rather than to a product of a particular length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from the designed nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

An "isolated" polypeptide or fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural environment. No specific degree of purification is required. For example, an isolated polypeptide may simply be removed from its natural or native environment. For the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated in that the native or recombinant polypeptide has been isolated, fractionated, or partially or substantially purified by any suitable technique.

Also included in the invention are fragments or variants of the polypeptides and any combination thereof. The term "fragment" or "variant" when referring to a polypeptide binding domain or binding molecule of the invention includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity of the FcRn binding domain or Fc variant, coagulation activity of the FVIII variant, or FVIII binding activity of the VWF protein). In addition to the specific antibody fragments discussed elsewhere herein, fragments of a polypeptide include proteolytic fragments, as well as deletion fragments, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of the polypeptide binding domains or binding molecules of the invention include fragments as described above, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutation-inducing techniques known in the art. The variant polypeptide may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

The term "VWF fragment" as used herein refers to any VWF fragment that interacts with FVIII and retains at least one or more properties normally provided to FVIII by full-length VWF, e.g., preventing premature activation of FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that can lead to premature clearance, preventing binding to FVIII-clearing receptors that can bind naked FVIII but not VWF-binding FVIII, and/or stabilizing FVIII heavy and light chain interactions. In a particular embodiment, a "VWF fragment" as used herein comprises the D' domain and the D3 domain of the VWF protein, but does not include the a1 domain, the a2 domain, the A3 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain and the CK domain of the VWF protein.

The term "half-life limiting factor" or "FVIII half-life limiting factor" as used herein indicates that the half-life of the FVIII protein is prevented compared to wild type FVIII (e.g.,or) Is a factor of 1.5 times or 2 times. For example, full-length or mature VWF can act as a FVIII half-life limiting factor by inducing clearance of FVIII and VWF complexes from the system using one or more VWF clearance pathways. In one example, endogenous VWF is a FVIII half-life limiting factor. In another example, the full length recombinant VWF molecule that is non-covalently bound to the FVIII protein is a FVIII half-life limiting factor.

The term "endogenous VWF" as used herein indicates the VWF molecules naturally occurring in the plasma. The endogenous VWF molecule may be multimeric, but may also be monomeric or dimeric. Endogenous VWF in plasma binds to FVIII and forms a non-covalent complex with FVIII.

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

As is known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, methods and Computer programs/software (such as, but not limited to, the BESTFIT program) known in the art (Wisconsin sequence analysis suite, Unix 8 th edition, Genetics Computer Group, University Research Park,575Science Drive, Madison, WI53711) can be used to determine whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to another polypeptide. BESTFIT uses Smith and Waterman, Advances in Applied Mathemitics 2: 482-489(1981) to find the optimal homology region between the two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, the parameters are of course set such that the percentage identity is calculated over the full length of the reference polypeptide sequence and allows a homology gap of at most 5% of the total number of amino acids in the reference sequence.

As used herein, an "amino acid corresponding to … …" or an "equivalent amino acid" in a VWF sequence or FVIII protein sequence is identified by alignment to maximize the identity or similarity between the first VWF or FVIII sequence and the second VWF or FVIII sequence. The numbering used to identify equivalent amino acids in the second VWF or FVIII sequence is based on the numbering used to identify the corresponding amino acids in the first VWF or FVIII sequence.

A "fusion" or "chimeric" molecule comprises a first amino acid sequence linked to a second amino acid sequence which is not naturally linked in nature. The amino acid sequences normally present in separate proteins may be assembled in a fusion polypeptide, or the amino acid sequences normally present in the same protein may be placed in a new arrangement in a fusion polypeptide, e.g., a fusion of a factor VIII domain of the invention with an immunoglobulin Fc domain. Fusion proteins are produced, for example, by chemical synthesis or by generating and translating polynucleotides that encode peptide regions in a desired relationship. The chimeric protein can further comprise a second amino acid sequence associated with the first amino acid sequence by covalent, non-peptide, or non-covalent bonds.

As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. The half-life can be expressed as the time required for half of the amount administered to the subject to clear from circulation and/or other tissues in the animal. When the clearance curve for a given polypeptide is understood as a function of time, the curve is typically biphasic with a fast alpha phase and a longer beta phase. The alpha phase generally represents the balance between intravascular and extravascular space of the administered polypeptide and is determined in part by the size of the polypeptide. The beta phase generally indicates catabolism of the polypeptide in the intravascular space. In some embodiments, the chimeric molecules of the invention are monophasic, and thus do not have an alpha phase, but only a single beta phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the beta phase. The typical beta-phase half-life of human antibodies in humans is 21 days.

The term "heterologous" as applied to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is derived from a different entity than the entity to which it is being compared. Thus, a heterologous polypeptide linked to a VWF protein refers to a polypeptide chain linked to a VWF protein other than the naturally occurring portion of the VWF protein. For example, heterologous polynucleotides or antigens can be derived from different species, individuals of different cell types, or different individuals of the same or different cell types.

The terms "linked", "fused" or "connected" as used herein refer to a first amino acid sequence or nucleotide sequence joined to a second amino acid sequence or nucleotide sequence (e.g., via a peptide bond or phosphodiester bond, respectively). The term "covalently linked" or "covalent linkage" refers to a covalent bond between two moieties that are linked together, e.g., a disulfide bond, a peptide bond, or one or more amino acids, e.g., a linker. The first amino acid or nucleotide sequence may be joined directly to the second amino acid or nucleotide sequence or the intervening sequence may join the first sequence to the second sequence. The terms "linked", "fused" or "linked" refer not only to the fusion of a first amino acid sequence to a second amino acid sequence at the C-terminus or N-terminus, but also include the insertion of the entire first amino acid sequence (or second amino acid sequence) into any two amino acids in the second amino acid sequence (or first amino acid sequence). In one embodiment, the first amino acid sequence may be joined to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be joined to the second nucleotide sequence by a phosphodiester bond or a linker. A linker can be a peptide or polypeptide (for polypeptide chains) or a nucleotide or nucleotide chain (for nucleotide chains) or any chemical moiety (for polypeptide and polynucleotide chains). Covalent attachment is sometimes indicated by (-) or hyphen.

As used herein, the term "associated with … …" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with … …" refers to covalent, non-peptide or non-covalent bonds. In some embodiments, this association is indicated by a colon (i.e., (:)). In another embodiment, it refers to a covalent bond other than a peptide bond. In other embodiments, the term "covalently associated" as used herein refers to an association between two moieties by a covalent bond (e.g., a disulfide bond, a peptide bond, or one or more amino acids (e.g., a linker)). For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metallic bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, unknown bonds, flexural bonds, dipole bonds, pi-reverse bonds, double bonds, triple bonds, quadruple bonds, quintuplet bonds, sextuplet bonds, conjugated, hyperconjugated, aromatic, haplottic, or anti-bonds. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cationic pi bonds or salt bonds), metallic bonds, hydrogen bonds (e.g., di-hydrogen bonds, dihydro complexes, low barrier hydrogen bonds, or symmetric hydrogen bonds), van der waals forces, london dispersion forces, mechanical bonds, halogen bonds, gold bonds (autophilicity), intercalation (intercalation), stacking, entropic forces, or chemical polarity.

As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. In one embodiment, the polypeptide has enzymatic cleavage sites that are cleaved by enzymes activated during the coagulation cascade, such that cleavage of these sites occurs at the site of clot formation. In another embodiment, the FVIII linker linking the FVIII protein to the second heterologous moiety may comprise a cleavage site. Exemplary such sites include, for example, those recognized by thrombin, factor XIa, or factor Xa. Exemplary FXIa cleavage sites include, for example, TQSFNDFTR (SEQ ID NO: 27) and SVSQTSKLTR (SEQ ID NO: 28). Exemplary thrombin cleavage sites include, for example, DFLAEGGGVR (SEQ ID NO: 29), TTKIKPR (SEQ ID NO: 30), LVPRG (SEQ ID NO: 31), and ALRPR (amino acids 1 to 5 of SEQ ID NO: 26). Other enzymatic cleavage sites are known in the art. The cleavage site cleavable by thrombin is herein referred to as "thrombin cleavage site".

As used herein, the term "processing site" or "intracellular processing site" refers to a class of enzymatic cleavage sites in a polypeptide that are targets for enzymes that function after translation of the polypeptide. In one embodiment, these enzymes function during transport from the Golgi lumen to the functional region of the transport Golgi. Intracellular processing enzymes cleave polypeptides prior to secretion of proteins from the cell. Examples of such processing sites include, for example, those targeted by the PACE/furin family of endopeptidases, where PACE is an abbreviation for Paired basic Amino acid Cleaving Enzyme (Paired basic Amino acid cleaning Enzyme). These enzymes localize to the golgi membrane and cleave proteins on the carboxy-terminal side of the sequence motif Arg- [ any residue ] - (Lys or Arg) -Arg. As used herein, the "furin" family of enzymes includes, for example, PCSK1 (also referred to as PC1/PC3), PCSK2 (also referred to as PC2), PCSK3 (also referred to as furin or PACE), PCSK4 (also referred to as PC4), PCSK5 (also referred to as PC5 or PC6), PCSK6 (also referred to as PACE4) or PCSK7 (also referred to as PC7/LPC, PC8 or SPC 7). Other processing sites are known in the art. The term "processable linker" as referred to herein refers to a linker comprising an intracellular processing site.

The term "furin" refers to an enzyme corresponding to EC No. 3.4.21.75. Furin is a subtilisin-like proprotein convertase, also known as Paired basic Amino acid lyase (PACE), Enzyme that cleans Amino acid. Furin lacks a portion of the inactive precursor protein to convert it to a biologically active protein. During its intracellular transport, the propeptide is cleaved from the mature VWF molecule by furin enzyme in the golgi apparatus.

In constructs comprising more than one processing or cleavage site, it will be appreciated that these sites may be the same or different.

Hemostatic disorder as used herein refers to a genetic or acquired condition characterized by a tendency to bleed spontaneously or as a result of trauma, due to an impaired ability to form or failure to form a fibrin clot. Examples of such disorders include hemophilia. The three major forms are hemophilia a (factor VIII deficiency), hemophilia B (factor IX deficiency or "christmas") and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand disease, factor XI deficiency (PTA deficiency), factor XII deficiency, fibrinogen, prothrombin, factor V, factor VII, factor X or factor XIII deficiency or structural abnormalities, Bernard-Soulier syndrome (which is GPIb deficient or deficient). GPIb is a receptor for VWF, which may be defective and result in a lack of primary clot formation (primary hemostasis) and increased tendency to bleed, as well as platelet insufficiency of Glanzman and Naegeli (Glanzmann's thromboplastia). In liver failure (both acute and chronic forms), the liver produces insufficient coagulation factors; this may increase the risk of bleeding.

The chimeric molecules of the invention may be used prophylactically. As used herein, the term "prophylactic treatment" refers to the administration of a molecule prior to a bleeding event. In one embodiment, a subject in need of a general hemostatic agent is undergoing surgery, or is about to undergo surgery. The chimeric proteins of the invention may be administered as a prophylactic agent, either before or after surgery. The chimeric proteins of the invention may be administered during or after surgery to control acute bleeding events. Surgery may include, but is not limited to, liver transplantation, liver resection, dental surgery, or stem cell transplantation.

The chimeric molecules of the invention are also useful in on-demand (also referred to as "episodic") therapy. The term "on-demand treatment" or "episodic treatment" refers to administration of the chimeric molecule in response to symptoms of a bleeding event or prior to an activity that can cause bleeding. In one aspect, on-demand (episodic) treatment can be administered to a subject who is beginning to appear (such as after surgery) or is expected to bleed (such as before surgery). In another aspect, the on-demand treatment may be administered prior to an activity that increases the risk of bleeding (such as contact sports).

As used herein, the term "acute bleeding" refers to a bleeding event without regard to the underlying cause. For example, the subject may have trauma, uremia, an inherited bleeding disorder (e.g., factor VII deficiency), a platelet disorder, or resistance due to the production of coagulation factor antibodies.

Treatment as used herein refers to, for example, a reduction in the severity of a disease or condition; the duration of the disease process is shortened; amelioration of one or more symptoms associated with the disease or condition; providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition, or preventing one or more symptoms associated with the disease or condition. In one embodiment, the term "treating" refers to maintaining FVIII trough levels in a subject at least about 1IU/dL, 2IU/dL, 3IU/dL, 4IU/dL, 5IU/dL, 6IU/dL, 7IU/dL, 8IU/dL, 9IU/dL, 10IU/dL, 11IU/dL, 12IU/dL, 13IU/dL, 14IU/dL, 15IU/dL, 16IU/dL, 17IU/dL, 18IU/dL, 19IU/dL, or 20IU/dL by administering a chimeric molecule of the invention. In another embodiment, treating refers to maintaining FVIII trough levels between about 1 to about 20IU/dL, about 2 to about 20IU/dL, about 3 to about 20IU/dL, about 4 to about 20IU/dL, about 5 to about 20IU/dL, about 6 to about 20IU/dL, about 7 to about 20IU/dL, about 8 to about 20IU/dL, about 9 to about 20IU/dL, or about 10 to about 20 IU/dL. Treatment of the disease or condition may also include maintaining FVIII activity in the subject to an extent equivalent to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% FVIII activity in a non-hemophilia subject. The minimum trough content required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each individual.

Chimeric molecules

The chimeric molecules of the invention are designed to improve the release of a VWF protein or FVIII protein from another moiety to which it is fused. The present invention provides thrombin-cleavable linkers that can be cleaved rapidly and efficiently at the site of injury. In one aspect of the invention, a chimeric molecule can comprise a VWF protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein and the heterologous moiety, wherein the VWF linker comprises a polypeptide selected from the group consisting of: (i) the a2 region from factor viii (fviii); (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof, and wherein the XTEN sequence is linked to the VWF protein, the heterologous moiety (H1), the VWF linker, or any combination thereof. In another aspect of the invention, a chimeric molecule can comprise a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting said VWF protein to said heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein said VWF linker in said first polypeptide chain comprises: (i) from the a2 region of FVIII; (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof, and wherein the first polypeptide chain and the second polypeptide chain are associated with each other.

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

Chimeric molecules with VWF, XTEN, VWF linker

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

In one embodiment, the chimeric molecule comprises a VWF protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein and the heterologous moiety, wherein the XTEN sequence is located between the VWF protein and the VWF linker and wherein the VWF linker comprises a polypeptide selected from the group consisting of: (i) the a2 region from factor viii (fviii); (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof. In another embodiment, the chimeric molecule comprises a VWF protein, a heterologous moiety (H1), an XTEN sequence, and a VWF linker connecting the VWF protein and the heterologous moiety, wherein the XTEN sequence is located between the VWF linker and the heterologous moiety and wherein the VWF linker comprises a polypeptide selected from the group consisting of: (i) from the a2 region of FVIII; (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof.

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

In certain embodiments, the chimeric molecule comprises a formula selected from the group consisting of: (i) V-L1-X1-H1: H2-L2-X2-C; (ii) V-X1-L1-H1: H2-L2-X2-C; (iii) V-L1-X1-H1: H2-X2-L2-C; (iv) V-X1-L1-H1: H2-X2-L2-C; (v) V-L1-X1-H1: H2-L2-C (X2); (vi) V-X1-L1-H1: H2-L2-C (X2); (vii) C-X2-L2-H2: H1-X1-L1-V; (viii) C-X2-L2-H2: H1-L1-X1-V; (ix) C-L2-X2-H2: H1-L1-X1-V; (x) C-L2-X2-H2: H1-L1-X1-V; (xi) C (X2) -L2-H2: H1-X1-L1-V; or (xii) C (X2) -L2-H2: H1-L1-X1-V; wherein V is a VWF protein; l1 is a VWF linker; l2 is an optional FVIII linker; h1 is a first heterologous moiety; h2 is a second heterologous moiety; x1 is an XTEN sequence; x2 is an optional XTEN sequence; c is a FVIII protein; c (X2) is a FVIII protein fused to an XTEN sequence, wherein the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (: is a covalent bond between H1 and H2).

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

Chimeric molecules with FVIII, XTEN, VWF protein, VWF linker

The invention also provides a chimeric molecule comprising a first polypeptide chain comprising a VWF protein, a heterologous moiety (H1), and a VWF linker connecting the VWF protein to the heterologous moiety (H1), and a second polypeptide chain comprising a FVIII protein and an XTEN sequence, wherein the VWF linker in the first polypeptide chain comprises: (i) from the a2 region of FVIII; (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof, and wherein the first polypeptide chain and the second polypeptide chain are associated with each other. In one embodiment, wherein the XTEN sequence is linked to the N-terminus or C-terminus of the FVIII protein or inserted between two FVIII amino acids adjacent to each other. In another embodiment, the chimeric molecule further comprises an additional XTEN sequence linked to the VWF protein, the heterologous moiety, the VWF linker, or any combination thereof. In other embodiments, the chimeric molecule further comprises a second heterologous moiety (H2). In other embodiments, the second heterologous portion of the chimeric molecule is linked to a FVIII protein, an XTEN sequence, or both. In other embodiments, the second heterologous moiety is linked to the FVIII protein or XTEN sequence via a FVIII linker. In other embodiments, the FVIII linker is the same as the VWF linker. In some embodiments, the FVIII linker is not the same as the VWF linker.

In certain embodiments, the chimeric molecule comprises a formula selected from the group consisting of: (i) V-L1-X1-H1: H2-L2-X2-C; (ii) V-X1-L1-H1: H2-L2-X2-C; (iii) V-L1-X1-H1: H2-X2-L2-C; (iv) V-X1-L1-H1: H2-X2-L2-C; (v) V-L1-X1-H1: H2-L2-C (X2); (vi) V-X1-L1-H1: H2-L2-C (X2); (vii) C-X2-L2-H2: H1-X1-L1-V; (viii) C-X2-L2-H2: H1-L1-X1-V; (ix) C-L2-X2-H2: H1-L1-X1-V; (x) C-L2-X2-H2: H1-L1-X1-V; (xi) C (X2) -L2-H2: H1-X1-L1-V; or (xii) C (X2) -L2-H2: H1-L1-X1-V; wherein V is a VWF protein; l1 is a VWF linker; l2 is an optional FVIII linker; h1 is a first heterologous moiety; h2 is a second heterologous moiety; x1 is an optional XTEN sequence; x2 is an XTEN sequence; c is a FVIII protein; c (X2) is a FVIII protein fused to an XTEN sequence, wherein the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (: is a covalent bond between H1 and H2). In one embodiment, the VWF linker and FVIII linker may be identical. In another embodiment, the VWF linker is different from the FVIII linker.

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

Chimeric molecules with FVIII, XTEN and FVIII linker

The chimeric molecules of the invention can comprise a FVIII protein, an XTEN sequence, and a heterologous moiety fused by a FVIII linker comprising (i) an a2 region from FVIII; (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof. In certain embodiments, the chimeric molecule comprises two polypeptide chains, a first chain comprising a FVIII protein fused to a first Fc region via a FVIII linker, and a second chain comprising a VWF protein fused to an Fc region (e.g., the D' domain and the D3 domain of VWF), wherein the FVIII linker in the first polypeptide chain comprises: (i) from the a2 region of FVIII; (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof, and wherein the first polypeptide chain and the second polypeptide chain are associated with each other and wherein the XTEN sequence is linked to the first polypeptide (e.g., N-terminus or C-terminus, linker or first Fc region of the FVIII protein or inside the FVIII protein), the second polypeptide (e.g., N-terminus or C-terminus or Fc region of the VWF protein or inside the FVIII protein), or both. In a particular embodiment, the linker in the first polypeptide chain comprises the a2 region from FVIII.

In certain embodiments, the chimeric molecule comprises a formula selected from the group consisting of: (i) V-L2-X2-H2: H1-L1-X1-C; (ii) V-X2-L2-H2: H1-L1-X1-C; (iii) V-L2-X2-H2: H1-X1-L1-C; (iv) V-X2-L2-H2: H1-X1-L1-C; (v) V-L2-X2-H2: H1-L1-C (X1); (vi) V-X2-L2-H2: H1-L1-C (X1); (vii) C-X1-L1-H1: H2-X2-L2-V; (viii) C-X1-L1-H1: H2-L2-X2-V; (ix) C-L1-X1-H1: H2-L2-X2-V; (x) C-L1-X1-H1: H2-L2-X2-V; (xi) C (X1) -L1-H1: H2-X2-L2-V; or (xii) C (X1) -L1-H1: H2-L2-X2-V, wherein V is a VWF protein; l1 is a FVIII linker; l2 is an optional VWF linker; h1 is a first heterologous moiety; h2 is a second heterologous moiety; x1 is an optional XTEN sequence; x2 is an optional XTEN sequence; c is a FVIII protein; c (X1) is a FVIII protein fused to an XTEN sequence, wherein the XTEN sequence is inserted between two FVIII amino acids adjacent to each other; (-) is a peptide bond or one or more amino acids; and (: is a covalent bond between H1 and H2, and wherein at least one XTEN sequence is present in the chimeric molecule. In one embodiment, the VWF linker is identical to the FVIII linker. In another embodiment, the VWF linker is different from the FVIII linker.

Components of chimeric molecules

II.C.1.VWF linker or FVIII linker

The VWF linker or FVIII linker useful in the chimeric molecules of the invention is a thrombin-cleavable linker fusing the VWF protein with a heterologous moiety or fusing the FVIII protein with a heterologous moiety. In one embodiment, the VWF linker or FVIII linker comprises the a1 region of FVIII. In another embodiment, the VWF linker or FVIII linker comprises the a2 region of FVIII. In other embodiments, the VWF linker or FVIII linker comprises the a3 region of FVIII. In other embodiments, the VWF linker or the FVIII linker comprises a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3), wherein X is an aliphatic amino acid, and a PAR1 exosite interaction motif.

In one embodiment, the VWF linker or FVIII linker comprises a1 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to Met337 to Arg372 corresponding to full length mature FVIII, wherein said a1 region is capable of being cleaved by thrombin. In another embodiment, the VWF linker or FVIII linker comprises a1 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to amino acids 337 to 374 corresponding to full length mature FVIII, wherein the a1 region is capable of being cleaved by thrombin. In other embodiments, the VWF linker or FVIII linker further comprises other amino acids, e.g., one, two, three, four, five, ten, or more. In a particular embodiment, the VWF linker or the FVIII linker comprises ISMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSV (SEQ ID NO: 5).

In some embodiments, the VWF linker or FVIII linker comprises a2 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to Glu720 to Arg740 corresponding to full length mature FVIII, wherein the a2 region is capable of being cleaved by thrombin. In other embodiments, the VWF linker or FVIII linker comprises the a2 region comprising an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to amino acids 712 to 743 corresponding to full length mature FVIII. In other embodiments, the VWF linker or FVIII linker further comprises other amino acids, e.g., one, two, three, four, five, ten, or more. In a particular embodiment, the VWF linker comprises ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 4).

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

In other embodiments, the VWF linker or the FVIII linker comprises a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) and an extra-PAR 1 site interaction motif and wherein the extra-PAR 1 site interaction motif comprises S-F-L-L-R-N (SEQ ID NO: 7). In some embodiments, the PAR1 exosite-interacting motif further comprises an amino acid sequence selected from the group consisting of: p, P-N, P-N-D, P-N-D-K (SEQ ID NO: 8), P-N-D-K-Y (SEQ ID NO: 9), P-N-D-K-Y-E (SEQ ID NO: 10), P-N-D-K-Y-E-P (SEQ ID NO: 11), P-N-D-K-Y-E-P-F (SEQ ID NO: 12), P-N-D-K-Y-E-P-F-W (SEQ ID NO: 13), P-N-D-K-Y-E-P-F-W-E (SEQ ID NO: 14), P-N-D-K-Y-E-P-F-W-E-D (SEQ ID NO: 20), P-N-D-K-Y-E-P-F-W-E-D-E (SEQ ID NO: 21), P-N-D-K-Y-E-P-F-W-E-D-E-E (SEQ ID NO: 22), P-N-D-K-Y-E-P-F-W-E-D-E-S (SEQ ID NO: 23), or any combination thereof. In other embodiments, the aliphatic amino acid comprising the thrombin cleavage site of X-V-P-R is selected from glycine, alanine, valine, leucine, or isoleucine. In a particular embodiment, the thrombin cleavage site comprises L-V-P-R. In some embodiments, if the VWF linker or FVIII linker, respectively, is replaced with a thrombin cleavage site (L-V-P-R) (i.e., the PAR1 exosite interaction motif is not present), the rate at which thrombin cleaves the VWF linker or FVIII linker is faster than the rate at which thrombin would cleave the thrombin cleavage site (e.g., L-V-P-R). In some embodiments, if the VWF linker or FVIII linker is replaced with a thrombin cleavage site (e.g., L-V-P-R), the rate at which thrombin cleaves the VWF linker or FVIII linker is at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times the rate at which thrombin will cleave the thrombin cleavage site (e.g., L-V-P-R).

In some embodiments, a VWF linker or FVIII linker comprising (i) the a1 region, (ii) the a2 region, (iii) the a3 region, or (iv) the thrombin cleavage site X-V-P-R and PAR1 exosite interaction motif further comprises one or more VWF linker or FVIII linker having at least about 2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 20Amino acids of 00 amino acids in length. In one embodiment, the one or more amino acids comprise a gly peptide. In another embodiment, the one or more amino acids comprise GlyGly. In other embodiments, the one or more amino acids comprise IleSer. In other embodiments, the one or more amino acids comprise a gly/ser peptide. In other embodiments, the one or more amino acids comprise a (Gly) amino acid having₄Ser) n or S (Gly)₄Ser) n, wherein n is a positive integer selected from 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80 or 100. In some embodiments, the one or more amino acids comprise (Gly)₄Ser)₃(SEQ ID NO: 89) or (Gly)₄Ser)₄(SEQ ID NO：90)。

II.C.2.VWF protein

VWF (also known as F8VWF) is a large, multimeric glycoprotein that is present in plasma and constitutively produced in the endothelium (in Weibel-Palade bodies), megakaryocytes (the alpha-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a protein with 2813 amino acids. Each monomer comprises a plurality of specific domains with specific functions, the D'/D3 domain (which binds to factor VIII), the a1 domain (which binds to platelet GPIb-receptor, heparin and/or possibly collagen), the A3 domain (which binds to collagen), the C1 domain (where the RGD domain binds to platelet integrin α IIb β 3 when it is activated), and the "cysteine knot" domain at the C-terminus of the protein (the VWF is shared with platelet-derived growth factor (PDGF), transforming growth factor- β (TGF β) and β -human chorionic gonadotropin (β HCG)).

The term "VWF protein" as used herein includes, but is not limited to, a full-length VWF protein comprising a D' domain and a D3 domain, or a functional VWF fragment, which is capable of inhibiting the binding of endogenous VWF to FVIII. In one embodiment, the VWF protein binds to FVIII. In another embodiment, the VWF protein blocks the VWF binding site on FVIII, thereby inhibiting interaction of FVIII with endogenous VWF. In other embodiments, the VWF protein is not cleared by the VWF clearance pathway. VWF proteins include derivatives, variants, mutants or analogs that retain these activities of VWF.

The 2813 monomeric amino acid sequence of human VWF is reported in Genbank as accession No. NP _000543.2 __. The nucleotide sequence encoding human VWF is reported in Genbank as accession number __ NM-000552.3. The nucleotide sequence of human VWF is designated SEQ ID NO: 1. SEQ ID NO: 2 is a polypeptide consisting of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. Each domain of VWF is listed in table 1.

TABLE 1 VWF sequences

A VWF protein as used herein may comprise the D' domain and the D3 domain of VWF, wherein the VWF protein binds to FVIII and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF protein comprising the D' domain and the D3 domain may further comprise a VWF domain selected from the group consisting of the a1 domain, the a2 domain, the A3 domain, the D1 domain, the D2 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, or any combination thereof. In one embodiment, the VWF protein comprises, consists essentially of, or consists of: (1) the D' and D3 domains of VWF or a fragment thereof; (2) the D1, D' and D3 domains of VWF or fragments thereof; (3) the D2, D' and D3 domains of VWF or fragments thereof; (4) the D1, D2, D' and D3 domains of VWF or fragments thereof; or (5) the D1, D2, D', D3 or a1 domain of VWF or a fragment thereof. The VWF proteins described herein do not comprise a VWF clearing receptor binding site. The VWF protein of the invention may comprise any other sequence linked or fused to the VWF protein. For example, the VWF protein described herein may further comprise a signal peptide.

In one embodiment, the VWF protein is bound to or associated with a FVIII protein. By binding to or associating with the FVIII protein, the VWF protein of the invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy and light chains of FVIII, and prevents FVIII from being cleared by scavenger receptors. In another embodiment, the VWF protein binds to or associates with the FVIII protein and blocks or prevents binding of the FVIII protein to the phospholipid and activated protein C. By preventing or inhibiting binding of FVIII protein to endogenous full length VWF, VWF proteins of the invention can reduce clearance of FVIII by endogenous VWF clearance receptors and thus prolong the half-life of FVIII protein. The half-life extension of the FVIII protein is thus due to the association of the FVIII protein with VWF proteins lacking a VWF scavenger receptor binding site and thereby shielding and/or protecting the FVIII protein from endogenous VWF comprising a VWF scavenger receptor binding site. FVIII proteins bound to or protected by VWF proteins may also allow recycling of FVIII proteins. By eliminating the VWF clearance pathway receptor binding site in the full-length VWF molecule, the FVIII/VWF heterodimers of the invention are shielded away from the VWF clearance pathway, thereby further extending the FVIII half-life.

In one embodiment, the VWF protein of the invention comprises the D 'domain and the D3 domain of VWF, wherein the D' domain is identical to the sequence of SEQ ID NO: 2, has at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity, wherein the VWF protein prevents endogenous VWF from binding to FVIII. In another embodiment, the VWF protein comprises the D' domain and the D3 domain of VWF, wherein the D3 domain is identical to SEQ ID NO: 2, wherein the VWF protein prevents endogenous VWF from binding to FVIII, is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, a VWF protein described herein comprises, consists essentially of, or consists of: the D' domain and D3 domain of VWF, which is identical to SEQ ID NO: 2, wherein the VWF protein prevents endogenous VWF from binding to FVIII, is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In other embodiments, the VWF protein comprises, consists essentially of, or consists of: a D1, D2, D' and D3 domain that hybridizes to SEQ ID NO: 2, wherein the VWF protein prevents endogenous VWF from binding to FVIII, is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In other embodiments, the VWF protein further comprises a signal peptide operably linked thereto.

In some embodiments, a VWF protein of the invention consists essentially of or consists of: (1) a D 'D3 domain, a D1D' D3 domain, a D2D 'D3 domain, or a D1D2D' D3 domain; and (2) up to about 10 amino acids (e.g., any sequence from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up to about 15 amino acids (e.g., any sequence from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about 20 amino acids (e.g., any sequence from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), an additional VWF sequence of up to about 25 amino acids (e.g., any sequence from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1265 of SEQ ID NO: 2), or up to about 30 amino acids (e.g., any sequence from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a particular embodiment, the VWF protein comprising or essentially consisting of the D' domain and the D3 domain is neither SEQ ID NO: 2, nor is 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 to the D' D3 domain.

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

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

In other embodiments, the VWF protein of the invention comprises, consists essentially of, or consists of: an amino acid sequence corresponding to the D' domain, the D3 domain, and the a1 domain, wherein the amino acid sequence is identical to SEQ ID NO: 2, wherein the VWF protein prevents endogenous VWF from binding to FVIII, has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In a particular embodiment, the VWF protein is not SEQ ID NO: 2, amino acids 764 to 1274.

In some embodiments, the VWF protein of the invention comprises a D' domain and a D3 domain, but does not comprise at least one VWF domain selected from the group consisting of: (1) a domain, (2) A domain, (3) A domain, (4) D domain, (5) B domain, (6) B domain, (7) B domain, (8) C domain, (9) C domain, (10) CK domain, (11) CK domain and C domain, (12) CK domain, C domain and C domain, (13) CK domain, C domain, B domain, (14) CK domain, C domain, B domain, (15) CK domain, C domain, B domain and B domain, (16) CK domain, C domain, B domain and D domain, (17) CK domain, C domain, B domain and D domain, A B1 domain, a D4 domain, and A3 domain, (18) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, and an a2 domain, (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, an a2 domain, and an a1 domain, or (20) any combination thereof.

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

In certain embodiments, the VWF proteins of the invention form multimers, e.g., dimers, trimers, tetramers, pentamers, hexamers, heptamers, or higher order multimers. In other embodiments, the VWF protein is a monomer with only one VWF protein. In some embodiments, the VWF protein of the invention may have one or more amino acid substitutions, deletions, additions or modifications. In one embodiment, the VWF protein may comprise amino acid substitutions, deletions, additions or modifications such that the VWF protein is unable to form disulfide bonds or to form dimers or multimers. In another embodiment, the amino acid substitution is within the D' domain and the D3 domain. In a particular embodiment, the VWF protein of the invention has a sequence corresponding to SEQ ID NO: 2, residue 1099, residue 1142, or residues 1099 and 1142 comprises at least one amino acid substitution. The at least one amino acid substitution may be any amino acid not naturally present in the wild-type VWF. For example, an amino acid substitution can be any amino acid other than cysteine, e.g., isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, or histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the formation of multimers of the VWF protein.

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

In certain embodiments, a VWF protein as used herein may be further modified to improve its interaction with FVIII, e.g., to improve binding affinity to FVIII. As a non-limiting example, a VWF protein is comprised in a polypeptide corresponding to SEQ ID NO: 2 and a serine residue at a residue corresponding to amino acid 764 of SEQ ID NO: 2, or a lysine residue at a residue of amino acid 773 of 2. Residues 764 and/or 773 may promote the binding affinity of the VWF protein to FVIII. In other embodiments, VWF proteins useful in the present invention may have other modifications, for example, the protein may be pegylated, glycosylated, hydroxyethylated, or polysialylated.

II.C.3. heterologous moieties

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

II.C.3.a immunoglobulin constant region or part thereof

The immunoglobulin constant region consists of domains called CH (constant weight) 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 (e.g., IgG) constant regions also comprise a hinge region. See Janeway et al, 2001, immunology, Garland Publishing, n.y., n.y.

The immunoglobulin constant regions, or portions thereof, used to produce the chimeric proteins of the invention may be obtained from a variety of different sources. In some embodiments, the immunoglobulin constant region or portion thereof is derived from a human immunoglobulin. However, it will be appreciated that the immunoglobulin constant region or a portion thereof may be derived from an immunoglobulin of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, the immunoglobulin constant region or portion thereof may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, as well as any immunoglobulin isotype, including IgGl, IgG2, IgG3, and IgG 4. In one embodiment, human isotype IgG1 is used.

A variety of immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) can be obtained in publicly accessible stock. Constant region domain sequences may be selected that have a particular effector function (or lack a particular effector function) or have particular modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been disclosed and suitable Ig constant region sequences (e.g., hinge, CH2, and/or CH3 sequences, or portions thereof) can be obtained from these sequences using art-recognized techniques. The genetic material obtained using any of the foregoing methods may then be altered or synthesized to obtain the polypeptides of the invention. It is further understood that alleles, variants and mutations of the constant region DNA sequence are encompassed within the scope of the present invention.

The sequence of the immunoglobulin constant region or a portion thereof can be cloned, for example, using the polymerase chain reaction and primers selected to amplify the domain of interest. To clone the sequence of an immunoglobulin constant region or a portion thereof from an antibody, mRNA can be isolated from a hybridoma, spleen, or lymphocyte, reverse-transcribed into DNA, and the antibody gene amplified by PCR. PCR amplification methods are described in detail in U.S. Pat. nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188; and for example "PCR Protocols: a Guide to Methods and Applications, eds. Innis et al, Academic Press, San Diego, Calif. (1990); ho et al, 1989.Gene 77: 51; horton et al, 1993.Methods enzymol.217: 270) are provided.

The immunoglobulin constant region used herein may include all domains and hinge regions or portions thereof. In one embodiment, the immunoglobulin constant region or portion thereof comprises a CH2 domain, a CH3 domain, and a hinge region, i.e., an Fc region or FcRn binding partner.

The term "Fc region" as used herein is defined as the portion of a polypeptide that corresponds to the Fc region of a native immunoglobulin, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. The native Fc region forms a homodimer with the other Fc region.

In one embodiment, an "Fc region" refers to the portion of a single immunoglobulin heavy chain that begins in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, the first residue of the heavy chain constant region taken as 114) and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

Depending on the immunoglobulin isotype, the Fc region of an immunoglobulin constant region may include CH2, CH3, and CH4 domains, as well as a hinge region. Chimeric proteins comprising an Fc region of an immunoglobulin confer several desirable properties on 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 neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which are incorporated herein by reference in their entirety.

The immunoglobulin constant region or portion thereof may be an FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the intestinal lumen, pulmonary trachea, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. patent No. 6,485,726). The FcRn binding partner is a part of an immunoglobulin that binds to FcRn.

The FcRn receptor has been isolated from several mammalian species, including humans. The sequences of human, monkey, rat and mouse FcRn are known (Story et al, 1994, J.Exp.Med.180: 2377). The FcRn receptor binds IgG at relatively low pH (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE), actively transports IgG intracellularly in the lumen to the serosal direction, and then releases IgG at the relatively higher pH found in interstitial fluid. It is expressed in adult epithelial tissues (U.S. Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1), including lung and intestinal epithelium (Israel et al, 1997, Immunology 92: 69), renal proximal tubule epithelium (Kobayashi et al, 2002, am. J. physiol. Renal physiol.282: F358), as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

FcRn binding partners useful in the present invention encompass molecules that can be specifically bound by the FcRn receptor, including intact IgG, Fc fragments of IgG, and other fragments including the intact binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al, 1994, Nature 372: 379). The main contact area of Fc to FcRn is near the intersection of CH2 and CH3 domains. The Fc-FcRn contacts are all within a single Ig heavy chain. FcRn binding partners include intact IgG, Fc fragments of IgG, and other fragments of IgG that include the intact binding region of FcRn. The primary contact sites include amino acid residues 248, 250, 257, 272, 285, 288, 290, 291, 308, 311 and 314 of the CH2 domain and amino acid residues 385, 387, 428 and 433, 436 of the CH3 domain. Reference to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions is based on Kabat et al, 1991, Sequences of Proteins of Immunological Interest, U.S. department of Public Health, Bethesda, Md.

The Fc region or FcRn binding partner bound to FcRn can be efficiently shuttled across epithelial barriers by FcRn, thus providing a non-invasive method for systemic administration of the desired therapeutic molecule. In addition, fusion proteins comprising an Fc region or an FcRn binding partner are endocytosed by cells expressing FcRn. But not indicative of degradation, these fusion proteins are recycled out to re-enter the circulation, thus increasing the in vivo half-life of these proteins. In certain embodiments, the portion of the immunoglobulin constant region is an Fc region or FcRn binding partner that associates, typically via disulfide bonds and other non-specific interactions, with another Fc region or another FcRn binding partner to form dimers and higher order multimers.

The FcRn binding partner region is a molecule or portion thereof that can be specifically bound by the FcRn receptor and subsequently transported by the FcRn receptor activity of the Fc region. Specific binding means that the two molecules form a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, which is distinguished from non-specific binding, which typically has low affinity and moderate to high capacity. Generally, when the affinity constant KA is higher than 10⁶M^-1Or higher than 10⁸M^-1When it comes toIs specific. If desired, non-specific binding can be reduced by altering the binding conditions without substantially affecting specific binding. One skilled in the art can optimize appropriate binding conditions such as concentration of molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of blocking agents (e.g., serum albumin, milk casein), and the like using routine techniques.

Numerous mutants, fragments, variants and derivatives are described, for example, in PCT publication nos. WO 2011/069164 a2, WO 2012/006623 a2, WO 2012/006635 a2 or WO 2012/006633 a2, all of which are incorporated herein by reference in their entirety.

II.C.3.b. albumin or fragments or variants thereof

In certain embodiments, the heterologous moiety that is linked to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is albumin or a functional fragment thereof. In some embodiments, the albumin fused to the VWF protein is covalently associated with the albumin fused to the FVIII protein.

Human serum albumin (HSA or HA) is a 609 amino acid protein in its full length form, which is responsible for a significant proportion of the serum osmolarity and also acts as a carrier for endogenous and exogenous ligands. The term "albumin" as used herein includes full length albumin or functional fragments, variants, derivatives or analogues thereof. Examples of albumin, or fragments or variants thereof, are disclosed in U.S. patent publication nos. 2008/0194481 a1, 2008/0004206 a1, 2008/0161243 a1, 2008/0261877 a1, or 2008/0153751 a1, or PCT application publication nos. 2008/033413 a2, 2009/058322 a1, or 2007/021494 a2, which are incorporated herein by reference in their entirety.

II.C.3.c. Albumin binding moieties

In certain embodiments, the heterologous moiety that is linked to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is an albumin binding moiety comprising an albumin binding peptide, a bacterial albumin binding domain, an albumin binding antibody fragment, or any combination thereof. For example, the albumin binding protein may be a bacterial albumin binding protein, an anti-albumin binding proteinBody or antibody fragments, including domain antibodies (see U.S. Pat. No. 6,696,245). The albumin binding protein may for example be a bacterial albumin binding domain such as one of streptococcal protein G (Konig, t. and Skerra, a. (1998) j.immunological. methods 218, 73-83). Other examples of albumin binding peptides that can be used as binding partners are e.g. those with Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄Cys consensus sequence, where Xaa₁Asp, Asn, Ser, Thr or Trp; xaa₂Is Asn, Gln, His, Ile, Leu or Lys; xaa₃Is Ala, Asp, Phe, Trp or Tyr; and Xaa₄Is Asp, Gly, Leu, Phe, Ser or Thr, as described in U.S. patent application 2003/0069395 or Dennis et al (Dennis et al, (2002) J.biol.chem.277, 35035-35043).

PAS sequence II.C.3.d.

In other embodiments, the heterologous moiety linked to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a PAS sequence. In one embodiment, the chimeric molecule comprises a VWF protein described herein fused to a PAS sequence via a VWF linker. In another embodiment, the chimeric molecule of the invention comprises a first chain comprising a VWF protein fused to a PAS sequence via a VWF linker and a second chain comprising a FVIII protein and another optional PAS sequence, wherein the PAS sequence shields or protects the VWF binding site on the FVIII protein, thereby inhibiting or preventing interaction of the FVIII protein with endogenous VWF. Two PAS sequences may be covalently associated with each other.

As used herein, a PAS sequence refers to an amino acid sequence comprising predominantly alanine and serine residues or comprising predominantly alanine, serine, and proline residues that form a random coil configuration under physiological conditions. Thus, a PAS sequence is an architectural block, amino acid polymer, or cassette comprising, consisting essentially of, or consisting of alanine, serine, and proline, which can be used as part of a heterologous moiety in a chimeric protein. However, those skilled in the art recognize that amino acid polymers can also form random coil configurations when residues other than alanine, serine, and proline are added as minor components in the PAS sequence. The term "minor component" as used herein means that amino acids other than alanine, serine, and proline may be added to the PAS sequence to some extent, for example, up to about 12% (i.e., about 12 out of 100 amino acids), up to about 10% (i.e., about 10 out of 100 amino acids), up to about 9% (i.e., about 9 out of 100 amino acids), up to about 8% (i.e., about 8 out of 100 amino acids), about 6% (i.e., about 6 out of 100 amino acids), about 5% (i.e., about 5 out of 100 amino acids), about 4% (i.e., about 4 out of 100 amino acids), about 3% (i.e., about 3 out of 100 amino acids), about 2% (i.e., about 2 out of 100 amino acids), about 1% (i.e., about 1 out of 100 amino acids) of the PAS sequence. The amino acid other than alanine, serine and proline may be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val.

Under physiological conditions, the PAS sequence stretches to form a random coil configuration and may thereby mediate an increased in vivo and/or in vitro stability of the VWF factor or the thrombogenically active protein. Since the random coil domain does not adopt a stable structure or act by itself, the biological activity mediated by the VWF protein or FVIII protein fused thereto is substantially retained. In other embodiments, the PAS sequence forming the random coil domain is biologically inert, particularly with respect to proteolysis in plasma, immunogenicity, isoelectric/electrostatic behavior, binding to cell surface receptors, or internalization, but is still biodegradable, which provides significant advantages over synthetic polymers such as PEG.

Non-limiting examples of PAS sequences that form a random coil configuration include amino acid sequences selected from the group consisting of: ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 32), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 33), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 34), APSSPSPSAPSSPSPASPS (SEQ ID NO: 35), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 36), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 37) and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 38) or any combination thereof. Other examples of PAS sequences are known, for example, from U.S. patent publication No. 2010/0292130 a1 and PCT application publication No. WO 2008/155134 a 1.

II.C.3.e.HAP sequence

In certain embodiments, the heterologous moiety attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a glycine-rich amino acid Homopolymer (HAP). The HAP sequence may comprise a repeat of glycine having a length of at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids. In one embodiment, the HAP sequence is capable of extending the half-life of the moiety fused or linked to the HAP sequence. Non-limiting examples of HAP sequences include, but are not limited to (Gly)_n、(Gly₄Ser)_nOr S (Gly)₄Ser)_nWherein n is 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See, e.g., Schlapschy M et al, Protein eng. design Selection, 20: 273-284(2007).

II.C.3.f. transferrin or a fragment thereof

In certain embodiments, the heterologous moiety attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is transferrin or a fragment thereof. Any transferrin can be used to prepare the chimeric molecules of the invention. For example, wild-type human tf (tf) is a 679 amino acid protein (not causing glycosylation) of about 75KDa with two major domains, N (about 330 amino acids) and C (about 340 amino acids), which appears to result from gene replication. See GenBank accession nos. NM001063, XM002793, M12530, XM039845, XM 039847, and S95936(www.ncbi.nlm.nih.gov /), all of which are incorporated herein by reference in their entirety. Transferrin comprises two domains, the N domain and the C domain. The N domain comprises two subdomains, the N1 domain and the N2 domain, and the C domain comprises two subdomains, the C1 domain and the C2 domain.

In one embodiment, the transferrin moiety of the chimeric molecule comprises a transferrin splice variant. In one example, the transferrin splice variant can be a splice variant of human transferrin, e.g., Genbank accession No. AAA 61140. In another embodiment, the transferrin moiety of the chimeric molecule comprises one or more domains of a transferrin sequence, e.g., an N domain, a C domain, an N1 domain, an N2 domain, a C1 domain, a C2 domain, or any combination thereof.

II.C.3.g polymers, e.g. polyethylene glycol (PEG)

In other embodiments, the heterologous moiety attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a soluble polymer known in the art, including, but not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, or polyvinyl alcohol. Heterologous moieties such as soluble polymers can be attached to any location within the chimeric molecule.

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

The present invention also provides chemically modified derivatives of the chimeric molecules of the invention, which may provide other advantages, such as increased polypeptide solubility, stability and circulation time, or reduced immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moiety used for modification may be selected from water soluble polymers including, but not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, or polyvinyl alcohol. The chimeric molecule may be modified at any position within the molecule or at the N-terminus or C-terminus, or at a predetermined position within the molecule and may include one, two, three, or more linked chemical moieties.

The polymer may have any molecular weight and may be branched or unbranched. For polyethylene glycol, in one embodiment, the molecular weight is between about 1kDa and about 100kDa for ease of handling and manufacture. Other dimensions may be used depending on the desired profile (e.g., duration of desired sustained release, effect on biological activity (if present), ease of handling, degree or absence of antigenicity, and other known effects of polyethylene glycol on proteins or the like). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

In some embodiments, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. patent nos. 5,643,575; morpurgo et al, appl.biochem.Biotechnol.56: 59-72 (1996); vorobjev et al, Nucleotides 18: 2745-2750 (1999); and Caliceti et al, bioconjugate. chem.10: 638-.

The number of polyethylene glycol moieties attached to each chimeric molecule (i.e., the degree of substitution) can also vary. For example, a pegylated protein of the invention can be linked to, on average, 1,2, 3, 4,5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution is within a range such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al, crit. 249-304 (1992).

In other embodiments, FVIII protein used in the invention is bound to one or more polymers. The polymer may be water soluble and covalently or non-covalently linked to factor VIII or bound to other moieties of factor VIII. Non-limiting examples of polymers may be poly (alkylene oxide), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, or poly (acryloylmorpholine). Other types of polymer-bound FVIII are disclosed in U.S. patent No. 7,199,223.

Hydroxyethyl starch (HES)

In certain embodiments, the heterologous moiety attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a polymer, for example, hydroxyethyl starch (HES) or a derivative thereof.

Hydroxyethyl starch (HES) is a derivative of naturally occurring amylopectin and is degraded in vivo by alpha-amylase. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95% by weight. HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in clinics for haemodilution therapy (Sommermeyer et al, Krankenhaus pharmazie,8(8),271-278 (1987); and Weidler et al, Arzneim. -Forschung/Drug Res.,41,494-498 (1991)).

Amylopectin contains glucose moieties in which alpha-1, 4-glucosidic bonds are present in the backbone and alpha-1, 6-glucosidic bonds are present at the branching sites. The physico-chemical properties of this molecule are mainly determined by the type of glycosidic bond. Due to the notched α -1, 4-glycosidic bond, a helical structure with about six glucose monomers per turn is produced. The physico-chemical as well as biochemical properties of the polymers can be modified by substitution. Introduction of the hydroxyethyl group can be achieved via basic hydroxyethylation. By adjusting the reaction conditions, it is possible to take advantage of the different reactivity of the individual hydroxyl groups in the unsubstituted glucose monomers with respect to hydroxyethylation. The person skilled in the art is thus able to influence the substitution pattern to a limited extent.

HES is primarily characterized by molecular weight distribution and degree of substitution. The degree of substitution (indicated as DS) relates to molar substitution and is known to the person skilled in the art. See Sommermeyer et al, Krankenhauspharmazie,8(8),271-278(1987), especially page 273, cited above.

In one embodiment, the hydroxyethyl starch has an average molecular weight (weight average) of 1 to 300kD, 2 to 200kD, 3 to 100kD or 4 to 70 kD. Hydroxyethyl starch may also exhibit a molar substitution degree of from 0.1 to 3, preferably from 0.1 to 2, more preferably from 0.1 to 0.9, preferably from 0.1 to 0.8, and the molar substitution degree with respect to the C2 of the hydroxyethyl group: the ratio between C6 substitutions was in the range of 2 to 20. Non-limiting examples of HES having an average molecular weight of about 130kD are HES having a degree of substitution of 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, preferably 0.4 to 0.7 such as 0.4, 0.5, 0.6 or 0.7. In a particular embodiment, HES having an average molecular weight of about 130kD is from Fresenius Are artificial colloids, e.g. for volume replacement as used in therapeutic indications for the treatment and prevention of hypovolemia.Is characterized by an average molecular weight of 130,000+/-20,000D, a molar substitution of 0.4 and an average molecular weight of about 9: c2 of 1: c6 ratio. In other embodiments, the average molecular weight of the hydroxyethyl starch ranges, for example, from 4 to 70kD or from 10 to 70kD or from 12 to 70kD or from 18 to 70kD or from 50 to 70kD or from 4 to 50kD or from 10 to 50kD or from 12 to 50kD or from 18 to 50kD or from 4 to 18kD or from 10 to 18kD or from 12 to 18kD or from 4 to 12kD or from 10 to 12kD or from 4 to 10 kD. In other embodiments, the hydroxyethyl starch used has an average molecular weight in the range above 4kD and below 70kD, such as about 10kD, or in the range of 9 to 10kD or 10 to 11kD or 9 to 11kD, or about 12kD, or in the range of 11 to 12kD or 12 to 13kD or 1l to 13kD, or about 18kD, or in the range of 17 to 18kD or 18 to 19kD or 17 to 19kD, or about 30kD, or in the range of 29 to 30, or 30 to 31kD, or about 50kD, or in the range of 49 to 50kD or 50 to 51kD, or 49 to 51 kD.

In certain embodiments, the heterologous moiety may be a peptide having a different average molecular weight and/or a different degree of substitution and/or a different C2: a mixture of hydroxyethyl starch with a C6 substitution ratio. Thus, polymers having different average molecular weights and different degrees of substitution and different C2: c6 substitution ratios, or C2 with different average molecular weights and different degrees of substitution and the same or about the same: c6 substitution ratios, or C2 with different average molecular weights and the same or approximately the same degree of substitution and different: c6 substitution ratios, or C2 having the same or about the same average molecular weight and different degrees of substitution: c6 substitution ratio, or C2 with different average molecular weights and the same or about the same degree of substitution and the same or about the same: c6 substitution ratios, or C2 having the same or about the same average molecular weight and different degrees of substitution and the same or about the same: c6 substitution ratios, or C2 having the same or about the same average molecular weight and the same or about the same degree of substitution and being different: a C6 substitution ratio, or a ratio having about the same average molecular weight and about the same degree of substitution and about the same C2: a mixture of hydroxyethyl starch with a C6 substitution ratio.

Polysialic acid (PSA)

In certain embodiments, the non-polypeptide heterologous moiety attached to the VWF protein via a VWF linker or to the FVIII protein via a FVIII linker is a polymer, e.g., polysialic acid (PSA) or a derivative thereof. Polysialic acid (PSA) is a naturally occurring unbranched sialic acid polymer produced by certain bacterial strains and in certain cells in mammals. Roth J. et al, (1993) Polysialic Acid: from Microbes to Man, Roth j., Rutishauser u., Troy f.a. (eds.) (Verlag, Basel, Switzerland), page 335-. They can be produced by limited acid hydrolysis or by neuraminidase digestion, or by fractionation of the natural bacterial source form of the polymer, in various degrees of polymerization from n-about 80 or more sialic acid residues down to n-2. The composition of the different polysialic acids was also varied so that a homopolymer form was present, i.e.comprising E.coli strain K1 and meningitis group BAlpha-2, 8-linked polysialic acid of the capsular polysaccharide of cocci, which is also present on the embryonic form of neuronal cell adhesion molecule (N-CAM). There are also heterogeneous polymer forms such as alternating alpha-2, 8 alpha-2, 9 polysialic acid of the E.coli strain K92 and N.meningitidis polysaccharide of group C. Sialic acids can also be found in alternating copolymers with monomers other than sialic acid, such as n.meningitidis group W135 or group Y. Polysialic acid has important biological functions including escape of the immune and complement systems by pathogenic bacteria and regulation of glial adhesion of immature neurons during fetal development, where the polymer has anti-adhesion functions (Cho and Troy, P.N.A.S., USA,91(1994)11427-11431), but the known receptor for polysialic acid is absent in mammals. The alpha-2, 8-linked polysialic acid of E.coli strain K1 is also known as 'colominic acid' and is used (in various lengths) to exemplify the invention. Various methods of linking or binding polysialic acid to A polypeptide have been described (see, e.g., U.S. Pat. No. 5,846,951, WO-A-0187922, and US 2007/0191597A 1, which are incorporated herein by reference in their entirety.

Sequence II.C.4. XTEN.

As used herein, "XTEN sequence" refers to an extended length polypeptide having a non-naturally occurring substantially non-repeating sequence consisting essentially of small hydrophilic amino acids, wherein the sequence has a low degree of secondary or tertiary structure or does not have secondary or tertiary structure under physiological conditions. As a chimeric protein partner, XTEN can serve as a carrier that confers certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when linked to a VWF protein or FVIII protein of the invention to produce a chimeric protein. These desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and dissolution properties. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as single chain antibodies or Fc fragments of light or heavy chains.

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

The XTEN sequences of the invention can comprise one or more sequence motifs having 9 to 14 amino acid residues or amino acid sequences having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence motif, wherein the motif comprises, consists essentially of or consists of: 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (a), serine (S), threonine (T), glutamic acid (E) and proline (P). See US 2010-0239554 a 1.

In some embodiments, XTEN comprises non-overlapping sequence motifs, wherein about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of a non-overlapping sequence selected from a single motif family selected from table 2A, thereby generating a family sequence. As used herein, "family" means that XTEN has motifs selected only from a single motif class from table 2A; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and any other amino acid in XTEN that is not from a family motif is selected to achieve the desired property, such as allowing incorporation of restriction sites by encoding nucleotides, incorporation of cleavage sequences, or achieving a better linkage to FVIII or VWF. In some embodiments of the XTEN family, the XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, wherein the resulting XTEN exhibits the above-described ranges of homology. In other embodiments, XTEN comprises multiple units of motif sequences from two or more of the motif families of table 2A. These sequences may be selected to achieve desired physical/chemical properties, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of reproducibility conferred by the amino acid composition of the motif, as described more fully below. In the above embodiments in this paragraph, the motifs incorporated into XTEN can be selected and assembled using the methods described herein to achieve XTEN having from about 36 to about 3000 amino acid residues.

Table 2a. XTEN sequence motifs and motif families with 12 amino acids

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

Individual motif sequences indicating that "family sequences" are generated when used together in various permutations

XTEN can have different lengths for insertion or attachment to FVIII or VWF or any other component of the chimeric molecule. In one embodiment, the length of one or more XTEN sequences is selected based on the property or function to be achieved in the fusion protein. XTEN can be a shorter or medium length sequence or a longer sequence that can act as a vector, depending on the desired properties or functions. In certain embodiments, XTEN comprises a short segment having from about 6 to about 99 amino acid residues, a medium length segment having from about 100 to about 399 amino acid residues, and a longer length segment having from about 400 to about 1000 and up to about 3000 amino acid residues. Thus, an XTEN inserted or attached to FVIII or VWF can have a length of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues. In other embodiments, the XTEN sequence is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The precise length of the XTEN inserted or attached to FVIII or VWF can be varied without adversely affecting the activity of FVIII or VWF. In one embodiment, one or more XTENs used herein have a length of 36 amino acids, 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids and can be selected from one or more of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD.

In some embodiments, the XTEN sequence used in the invention has at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from the group consisting of seq id no: AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE612, AG 624, AE648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD 828, 864, AE864, AF864, AG 912, AM923, AM AE8, BC864, BD 133948, AG1044, AG1140, AE1236, AE 1902, AG1908, AG1620, AG 598, AE18, AG1044, AG1140, AE 152864, AE864, AE12, AG8, AG1044, AG8, AG 0041140, AE 1758, AE 152864, AE 41013, AG 0048, AG 00413, AG 0048, AG 0043, AG 0047, aa 190864, AG 0048, AE 0048, AG 0047, AE 152864, AG 0048, AG 00413, AG 0043, AG 0048, AG 0043, AG 0047, AG8, AG 0047, AG 0048, aa 864, AG8, aa 864, AG 0043, AG8, AG 00413, aa 864, AG8, AG 598, AG 8. See US 2010-0239554 a 1.

In one embodiment, the XTEN sequence has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of AE42, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144, and any combination thereof. In another embodiment, the XTEN sequence is selected from the group consisting of AE42, AE864, AE576, AE288, AE144, AG864, AG576, AG288, AG144, and any combination thereof. In a particular embodiment, the XTEN sequence is AE 288. The amino acid sequences of certain XTEN sequences of the invention are shown in table 2B.

XTEN sequences of Table 2B.

In those embodiments in which less than 100% of the amino acids in the XTEN component used consist of 4,5 or 6 types of amino acids selected from glycine (G), alanine (a), serine (S), threonine (T), glutamic acid (E) and proline (P), or less than 100% of the sequence consists of the sequence motif from table 2A or the XTEN sequence of table 2B, the other amino acid residues of the XTEN are selected from any other 14 natural L-amino acids, but are preferably selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% hydrophilic amino acids. XTEN amino acids other than glycine (G), alanine (a), serine (S), threonine (T), glutamic acid (E), and proline (P) are interspersed throughout the XTEN sequence, located within or between sequence motifs, or concentrated in one or more short stretches of the XTEN sequence, e.g., to create linkers between the XTEN and other components (e.g., VWF proteins). In these cases where the XTEN component comprises amino acids other than glycine (G), alanine (a), serine (S), threonine (T), glutamic acid (E), and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids are hydrophobic residues such that the resulting sequence generally lacks secondary structure, e.g., does not have greater than 2% alpha helix or 2% beta sheet, as determined by the methods disclosed herein. Hydrophobic residues that are less favored in the XTEN configuration include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. In addition, the XTEN sequence can be designed to comprise less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or to be free 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, XTENs that comprise other amino acids besides glycine (G), alanine (a), serine (S), threonine (T), glutamic acid (E), and proline (P) have a sequence with less than 5% of residues contributing to the alpha helix and beta sheet as measured by the Chou-Fasman algorithm and have at least 90% or at least about 95% or more random coil formation as measured by the GOR algorithm.

In other embodiments, the XTEN sequence used in the invention affects a physical or chemical property, e.g., a pharmacokinetic property, of the chimeric protein of the invention. XTEN sequences used in the invention can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced water solubility, a high degree of protease resistance, low immunogenicity, low binding to mammalian receptors or increased hydrodynamic (or Stoke) radius. In a particular embodiment, the XTEN sequence linked to the FVIII protein in the present invention improves pharmacokinetic properties, such as longer terminal half-life or increased area under the curve (AUC), such that the chimeric protein described herein remains in vivo for an increased time compared to wild type FVIII. In other embodiments, the XTEN sequence used in the invention improves pharmacokinetic properties, such as longer terminal half-life or increased area under the curve (AUC), such that the time that the FVIII protein remains in vivo is increased compared to wild type FVIII.

A variety of methods and assays can be used to determine the physical/chemical properties of proteins comprising XTEN sequences. Such methods include, but are not limited to, analytical centrifugation, EPR, ion exchange HPLC, size exclusion HPLC, reverse phase HPLC, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, ion exchange HPLC, size exclusion HPLC, IR, NMR, raman spectroscopy, refractive index determination, and uv/vis spectroscopy. Other methods are disclosed in Amau et al, Prot Expr and Purif 48,1-13 (2006).

Other examples of XTEN sequences can be used according to the invention 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 publication No. WO 2010091122 a1, WO 2010144502 a2, WO 2010144508 a1, WO 2011028228 a1, WO 2011028229 a1, WO 2011028344 a2 or WO2013123457 a1, or international application No. PCT/US 2013/049989.

FVIII protein II.C.5.

Unless otherwise stated, noAs used herein, "FVIII protein" refers to a functional FVIII polypeptide that plays its normal role in coagulation. The term FVIII protein includes functional fragments, variants, analogues or derivatives thereof which retain the function of full length wild type factor VIII in the coagulation pathway. "FVIII protein" may be used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII function include, but are not limited to, the ability to activate coagulation, the ability to act as a cofactor for factor IX, or at Ca²⁺And the ability to form a tenase complex with factor IX in the presence of phospholipids and then convert factor X to the activated form Xa. The FVIII protein may be human, porcine, canine, rat or murine FVIII protein. In addition, comparisons between FVIII from humans and other species have identified conserved residues that may be required for function (Cameron et al, Thromb. Haemost.79: 317-22 (1998); US 6,251,632).

Various tests can be used to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, Prothrombin Time (PT) test (also used to determine INR), fibrinogen test (usually by the Clauss method), platelet count, platelet function test (usually by PFA-100), TCT, bleeding time, mixing test (if the patient's plasma is mixed with normal plasma if the abnormality is corrected), coagulation factor assay, antiphospholipid antibody, D-dimer, gene test (e.g. factor V Leiden, prothrombin mutation G20210A), diluted Russell viper venom time (dRVVT), miscellaneous platelet function test, thrombelastogram (TEG or Sonoclot), Thrombelastogram (TM)For example) Or euglobulin dissolution time (ELT).

The aPTT test is a potency indicator that measures the efficacy of both the "intrinsic" (also known as contact activation pathway) and common coagulation pathways. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors (e.g., FVIII or FIX). It is used in conjunction with measuring the Prothrombin Time (PT) of the external pathway.

The ROTEM analysis provides information about the complete kinetics of hemostasis: clotting time, clot formation, clot stability and lysis. Different parameters in the determination of thromboelastosis depend on the activity of the plasma coagulation system, platelet function, fibrinolysis or many factors that influence these interactions. This assay may provide complete insight into secondary hemostasis.

FVIII polypeptides and polynucleotide sequences are known, as are a number of functional fragments, mutants and modified forms. An example of a human FVIII sequence (full length) is shown as SEQ ID NO: 16 or 18.

TABLE 3 full-length FVIII (FVIII signal peptide underlined; FVIII heavy chain double underlined; B domain italicized; and FVIII light chain plain text)

Signal peptide: (SEQ ID NO: 15)

Mature factor VIII (SEQ ID NO: 16).)

TABLE 4 nucleotide sequence (SEQ ID NO: 17) encoding full-length FVIII

Underlined nucleic acids encode signal peptides.

FVIII polypeptides include full length FVIII, full length FVIII minus Met at the N-terminus, mature FVIII (minus signal sequence), mature FVIII with another Met at the N-terminus, and/or FVIII with a complete or partial deletion of the B domain. In certain embodiments, the FVIII variant comprises a B domain deletion, with partial or complete deletions.

The human FVIII gene was 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 U.S. Pat. No. 4,757,006). FVIII amino acid sequences were deduced from cdnas as shown in U.S. patent No. 4,965,199. In addition, partially or fully B-domain deleted FVIII is shown in U.S. patent nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B domain is replaced with a human factor V B domain as set forth in U.S. patent No. 5,004,803. The cDNA sequence and amino acid sequence encoding human factor VIII are shown in SEQ ID NO: 1 and 2.

Porcine FVIII sequences are disclosed in Toole, j.j. et al, proc.natl.acad.sci.usa83: 5939-. In addition, the complete porcine cDNA sequence obtained from PCR amplification of FVIII sequences from porcine spleen cDNA libraries has been reported in Healey, j.f. et al, Blood 88: 4209 and 4214 (1996). Substituted hybrid human/porcine FVIII with all domains, all subunits and specific amino acid sequences is disclosed in U.S. patent No. 5,364,771 to lorar and Runge and WO 93/20093. Recently, the a1 and a2 domains of porcine FVIII as well as the nucleotide and corresponding amino acid sequences of chimeric FVIII with the corresponding human domains replaced by porcine a1 and/or a2 domains have been reported in WO 94/11503. U.S. patent No. 5,859,204 to Lollar, j.s. also discloses porcine cDNA and deduced amino acid sequences. U.S. patent No. 6,458,563 discloses B-domain deleted porcine FVIII.

U.S. patent No. 5,859,204 to Lollar, j.s. reports functional mutants of FVIII with reduced antigenicity and reduced immunoreactivity. FVIII mutants with reduced immunoreactivity are also reported by Lollar, j.s. U.S. patent No. 6,376,463. U.S. application publication No. 2005/0100990 to Saenko et al reports functional mutations in the A2 domain of FVIII.

In one embodiment, the FVIII protein (or FVIII portion of the chimeric protein) may be identical to SEQ ID NO: 18 or amino acids 1 to 1438 of SEQ ID NO: 16 (without a signal sequence) has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the FVIII amino acid sequence of amino acids 1 to 2332, wherein the FVIII has coagulation activity, e.g., activation of factor IX as a cofactor to convert factor X to activated factor X. FVIII (or FVIII portion of the chimeric protein) may be compared to SEQ ID NO: 18 or amino acids 1 to 1438 of SEQ ID NO: 16 (without signal sequence) are identical for amino acids 1 to 2332. FVIII proteins may also comprise a signal sequence.

As used herein, the "B domain" of FVIII is identical to the B domain known in the art to be defined by internal amino acid sequence identity and proteolytic cleavage sites, e.g., residues Ser741-Arg1648 of full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: a1, residues Ala1-Arg 372; a2, residues Ser373-Arg 740; a3, residues Ser1690-Asn 2019; c1, residues Lys2020-Asn 2172; c2, residue Ser2173-Tyr 2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr 2332. The remaining sequence (residues Glu1649-Arg1689) is commonly referred to as the a3 acidic region. The border positions of all domains (including the B-domain of porcine, mouse and canine FVIII) are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B domain deleted factor VIII" or "BDD FVIII"). Examples of BDD FVIII are(recombinant BDD FVIII) having a sequence identical to the factor VIII portion of the sequence in table 5. (BDD FVIII heavy chain double underlined; B domain italicized; and BDD FVIII light chain common).

TABLE 5 BDD FVIII (SEQ ID NO: 18)

TABLE 6 BDD FVIII encoding nucleotide sequence (SEQ ID NO: 19)

Underlined nucleic acids encode signal peptides.

"B-domain deleted FVIII" may have complete or partial deletions as disclosed in U.S. patent nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112 and 6,458,563. In some embodiments, the B-domain deleted FVIII sequences of the invention comprise any one of the deletions disclosed in column 4, line 4 through column 5, line 28 and examples 1-5 of U.S. patent No. 6,316,226 (also in US 6,346,513). In another embodiment, the B domain deleted factor VIII is S743/Q1638B domain deleted factor VIII (SQ BDD FVIII) (e.g., factor VIII with a deletion of amino acids 744 to 1637, e.g., factor VIII with amino acids 1-743 of SEQ ID NO: 16 and amino acids 1638-2332, i.e., SEQ ID NO: 18). In some embodiments, the B-domain deleted FVIII of the present invention has the deletions disclosed in column 2, lines 26-51 and examples 5-8 of U.S. patent No. 5,789,203 (as well as US 6,060,447, US 5,595,886 and US 6,228,620). In some embodiments, the B domain deleted factor VIII has column 1, line 25 to column 2, line 40 of U.S. patent No. 5,972,885; column 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; column 2, lines 17-46 of U.S. patent No. 5,543,502; column 4, line 22 to column 5, line 36 of U.S. patent No. 5,171,844; column 2, lines 55-68 of U.S. patent No. 5,112,950, fig. 2 and example 1; column 2, line 2 to column 19, line 21 and table 2 of U.S. patent No. 4,868,112; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of U.S. patent No. 7,041,635; or the deletions described in column 4, lines 25-53 of U.S. patent No. 6,458,563.

In some embodiments, the B-domain deleted FVIII has a deletion of most of the B-domain, but still comprises the amino terminal sequence of the B-domain necessary for proteolytic processing of the main translation product into two polypeptide chains in vivo, as disclosed in WO 91/09122. In some embodiments, the B-domain deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., an almost complete deletion of the B-domain. Hoeben r.c. et al, j.biol.chem.265 (13): 7318-7323(1990). The B domain deleted factor VIII may also comprise a deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien p. et al, Protein eng.2 (4): 301-6(1988). Other B domain deletions that are part of the invention include: amino acids 982 to 1562 or 760 to 1639(Toole et al, Proc. Natl. Acad. Sci. U.S.A. (1986)83, 5939-: 8343-8347)), 741 to 1646(Kaufman (PCT published application 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 other embodiments, BDD FVIII includes FVIII polypeptides comprising a B domain fragment retaining one or more N-linked glycosylation sites (e.g., residues 757, 784, 828, 900, 963, or optionally 943, which correspond to the amino acid sequence of a full-length FVIII sequence). Examples of B domain fragments include 226 amino acids or 163 amino acids of the B domain, e.g., Miao, h.z, etc., Blood 103 (a): 3412 + 3419 (2004); kasuda, A et al, J.Thromb.Haemost.6: 1352 and 1359 (2008); and Pipe, s.w., et al, j.thromb.haemost.9: 2235-. In some embodiments, the FVIII with the partial B domain is FVIII 198. FVIII198 is a part of the B domain containing the single chain FVIIIFc molecule-226N 6. 226 represents the N-terminal 226 amino acids of the FVIII B domain, and N6 represents the six N-glycosylation sites in the B domain. In other embodiments, BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve expression of BDD FVIII protein. See Miao, h.z, et al, Blood 103 (a): 3412-3419(2004). In other embodiments, BDD FVIII includes a FVIII polypeptide comprising a portion of the B domain, but does not comprise one or more furin cleavage sites (e.g., Arg1313 and Arg 1648). See Pipe, s.w. et al, j.thromb.haemost.9: 2235-2242(2011). Each of the foregoing deletions may be made in any FVIII sequence.

FVIII proteins useful in the invention may include FVIII having one or more additional heterologous sequences or chemical or physical modifications therein which do not affect FVIII coagulation activity. These heterologous sequences or chemical or physical modifications may be fused to the C-terminus or N-terminus of the FVIII protein or inserted between one or more of two amino acid residues in the FVIII protein. These insertions in the FVIII protein do not affect the FVIII coagulation activity or FVIII function. In one embodiment, the insertion may improve the pharmacokinetic properties (e.g., half-life) of the FVIII protein. In another embodiment, the insertion may be at more than two, three, four, five or six sites.

In one embodiment, FVIII is cleaved immediately after arginine at amino acid 1648 (in full-length factor VIII or SEQ ID NO: 16), amino acid 754 (in S743/Q1638B domain deleted factor VIII or SEQ ID NO: 16), or the corresponding arginine residue (in other variants), thereby producing heavy and light chains. In another embodiment, FVIII comprises a heavy chain and a light chain which are connected or associated by a metal ion mediated non-covalent bond.

In other embodiments, FVIII is single chain FVIII that has not been cleaved immediately after arginine at amino acid 1648 (in full-length factor FVIII or SEQ ID NO: 16), amino acid 754 (in S743/Q1638B domain deleted factor FVIII or SEQ ID NO: 18), or the corresponding arginine residue (in other variants). Single chain FVIII may comprise 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 of full length mature factor VIII polypeptide (SEQ ID NO: 16) or residue 754, residue 751, or both of SQ BDD factor VIII (SEQ ID NO: 18). The amino acid substitution can be any amino acid other than arginine, for example, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine.

FVIII can be further cleaved by thrombin and then activated to FVIIIa, acting as a cofactor for activated factor IX (FIXa). And activated FIX forms a Xase complex with activated FVIII and converts factor X to activated factor X (fxa). For activation, FVIII is cleaved by thrombin after three arginine residues at amino acids 372, 740 and 1689 (corresponding to amino acids 372, 740 and 795 in the B domain deleted FVIII sequence), the cleavage yielding FVIIIa with 50kDa a1, 43kDa a2 and 73kDa A3-C1-C2 chains. In one embodiment, the FVIII protein used in the invention is non-active FVIII. In another embodiment, the FVIII protein is activated FVIII.

A protein having a FVIII polypeptide attached to or associated with a VWF protein may comprise an amino acid sequence as set forth in SEQ ID NO: 16 or 18, wherein the sequence has FVIII clotting activity, e.g., activation of factor IX as a cofactor to convert factor X to activated factor X (fxa).

In some embodiments, the FVIII protein further comprises one or more heterologous moieties fused to the C-terminus or N-terminus of the FVIII protein or inserted between two adjacent amino acids in the FVIII protein. In other embodiments, the heterologous moiety comprises an amino acid sequence having at least about 50 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, at least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 550 amino acids, at least about 600 amino acids, at least about 650 amino acids, at least about 700 amino acids, at least about 750 amino acids, at least about 800 amino acids, at least about 850 amino acids, at least about 900 amino acids, at least about 950 amino acids, or at least about 1000 amino acids. In some embodiments, the half-life of the chimeric molecule is extended at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, or at least about 12-fold compared to wild-type FVIII.

Other exemplary FVIII variants are also disclosed in U.S. publication No. US2013/0017997 published on day 17/1/2013, international publication No. WO 2013/122617 published on day 22/8/2013, or international publication No. WO 2014/011819 published on day 16/1/2014, or international publication No. WO2013123457 a1, or international application No. PCT/US 2013/049989.

Polynucleotides, vectors, host cells and methods of preparation

Also provided in the invention are polynucleotides encoding the chimeric molecules described herein. The present invention relates to a single polynucleotide encoding a single polypeptide chain when the VWF protein is linked to a heterologous moiety via a VWF linker and to the FVIII protein and the XTEN sequence in the chimeric protein as a single polypeptide chain. When the chimeric protein comprises a first polypeptide chain comprising a VWF protein, an XTEN sequence, and a first heterologous moiety (e.g., a first Fc region) connected via a VWF linker and a second polypeptide chain comprising a FVIII protein and a second heterologous moiety (e.g., a second Fc region), the polynucleotide can comprise a first nucleotide region and a second nucleotide region. In one embodiment, the first nucleotide region and the second nucleotide region are on the same polynucleotide. In another embodiment, the first nucleotide region and the second nucleotide region are on two different polynucleotides (e.g., different vectors). In certain embodiments, the invention is directed to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, wherein the first nucleotide strand encodes a VWF protein, an XTEN sequence, a VWF linker, and a heterologous portion of a chimeric protein and the second nucleotide strand encodes a FVIII protein and a second heterologous portion. In some embodiments, the invention is directed to a set of polynucleotides comprising a first nucleotide strand and a second nucleotide strand, wherein the first nucleotide strand encodes a VWF protein and a heterologous portion of a chimeric protein and the second nucleotide strand encodes a FVIII protein fused to a second heterologous portion via a FVIII linker, wherein at least one XTEN sequence is fused to the chimeric protein. In other embodiments, the invention is directed to a set of polynucleotides comprising a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes a VWF protein, a VWF linker, and a heterologous portion of a chimeric protein and the second nucleotide chain encodes a FVIII protein, a FVIII linker, and a second heterologous portion, wherein at least one XTEN sequence is fused to the chimeric protein.

In other embodiments, the set of polynucleotides further comprises another nucleotide strand encoding a protein converting enzyme (e.g., a second nucleotide strand when the chimeric polypeptide is encoded by a single polynucleotide strand, or a third nucleotide strand when the chimeric protein is encoded by two polynucleotide strands). The protein convertase may be selected from proprotein convertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertase subtilisin/kexin type 7 (PCSK7 or PC5), yeast Kex 2, proprotein convertase subtilisin/kexin type 3 (PACE or PCSK3), or a combination of two or more thereof. In some embodiments, the protease-converting enzyme is PACE, PC5, or PC 7. In a particular embodiment, the protein converting enzyme is PC5 or PC 7. See international application No. PCT/US2011/043568, which is incorporated herein by reference. In another embodiment, the protease converting enzyme is PACE/furin.

In certain embodiments, the invention includes a set of polynucleotides comprising a first nucleotide sequence encoding a VWF protein comprising the D' domain and the D3 domain of VWF, a second nucleotide sequence encoding a FVIII protein and a second heterologous moiety, and a third nucleotide sequence encoding the D1 domain and the D2 domain of VWF fused to a first heterologous moiety via a VWF linker, and wherein the XTEN sequence is present in the first strand or the second strand. In this embodiment, the D1 domain and the D2 domain are expressed separately (and not linked to the D 'D3 domain of the VWF protein) to allow proper disulfide bond formation and folding of the D' D3 domain. D1D2 domain expression may be cis or trans.

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

The expression vectors of the invention will include polynucleotides encoding the chimeric molecules.

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

A gene expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates efficient transcription and translation of the coding nucleic acid to which it is operably linked. The gene expression control sequence may be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters of the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters from the Long Terminal Repeat (LTR) of Cytomegalovirus (CMV), simian viruses (e.g., SV40), papilloma viruses, adenoviruses, Human Immunodeficiency Virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus, and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. Promoters useful as gene expression sequences in the present invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to facilitate transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

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

Viral vectors include, but are not limited to, nucleic acid sequences from: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV 40-type virus; a polyoma virus; Epstein-Barr virus (Epstein-Barr virus); papilloma virus; herpes virus; vaccinia virus; poliovirus; and RNA viruses, such as retroviruses. Other carriers well known in the art can be readily employed. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with target genes. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those replication-defective retroviruses (i.e., capable of directing the synthesis of a desired protein, but incapable of producing infectious particles). These genetically altered retroviral expression vectors have general utility for efficient transduction of genes in vivo. Standard protocols for the production of replication-defective retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus by packaging cell line, collecting virions from tissue culture medium, and infecting target cells with the virions) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman Co., New York (1990) and Murry, E.J., Methods in Molecular Biology, Vol.7, Humana Press, Inc., Clifton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-defective and capable of infecting a wide variety of cell types and species. It further has advantages such as thermal and lipid solvent stability; high transduction frequency in cells of different lineages (including hematopoietic cells); and lack of dual infection inhibition, thus allowing multiple series of transduction. It has been reported that adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the likelihood of insertional mutagenesis and variability in the expression profile of inserted genes for retroviral infections. In addition, wild-type adeno-associated virus infection has been passaged more than 100 times in tissue culture in the absence of selective pressure, indicating that integration of the adeno-associated virus genome is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

Other vectors include plasmid vectors. Plasmid vectors are widely described in the art and are well known to those skilled in the art. See, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo, since they are unable to replicate in and integrate into the host genome. However, these plasmids with promoters compatible with the host cell may allow for expression of the peptide from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40 and pBluescript. Other examples of specific plasmids include pcdna3.1, catalog No. V79020; pcDNA3.1/hygro, Cat No. V87020; pcDNA4/myc-His, Cat No. V86320; and pbudce4.1, catalog No. V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be custom designed using standard molecular biology techniques to remove and/or add specific DNA fragments.

In one insect expression system that can be used to produce the proteins of the present invention, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus was grown in Spodoptera frugiperda (Spodoptera frugiperda) cells. Coding sequences can be cloned into non-essential regions of the virus (e.g., polyhedral genes) and placed under the control of ACNPV promoters (e.g., polyhedral promoters). Successful insertion of the coding sequence will result in inactivation of the polyhedral gene and production of non-occluded recombinant viruses (i.e., viruses that lack the protein coat encoded by the polyhedral gene). These recombinant viruses are then used to infect spodoptera frugiperda cells that have expressed the inserted gene. (see, e.g., Smith et al, (1983) J Virol 46: 584; U.S. Pat. No. 4,215,051). Other examples of such expression systems can be found in Ausubel et al, (1989) Current Protocols in Molecular Biology, Vol.2, Greene publishing. Assoc. & Wiley Interscience.

Another system that can be used to express the proteins of the invention is the glutamine synthetase gene expression system, also known as the "GS expression system" (Lonza Biologics PLC, Berkshire UK). Such an expression system is described in detail in U.S. patent No. 5,981,216.

In mammalian host cells, a variety of viral-based expression systems are available. In the case of using an adenovirus as an expression vector, the coding sequence can be ligated to an adenovirus transcription/translation control complex, e.g., a late promoter and a triple leader sequence. Such chimeric genes can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the peptide in an infected host. See, e.g., Logan and Shenk (1984) Proc Natl Acad Sci USA 81: 3655). Alternatively, the vaccinia 7.5K promoter may be used. See, e.g., 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 increase production efficiency, polynucleotides can be designed to encode multiple units of a protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with an appropriate enzyme) to recover the polypeptide units. This may increase the yield of polypeptide driven by a single promoter. When used in an appropriate viral expression system, internally directs the translation of each polypeptide encoded by the mRNA in the transcript; for example, via an internal ribosome entry site IRES. Thus, the polycistronic construct directs the transcription of a single large polycistronic mRNA, which in turn directs the translation of multiple 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.

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

The selectable markers may be introduced into the cells on another plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (e.g., U.S. patent No. 4,713,339).

The expression vector may encode a tag that allows for easy purification of the recombinantly produced protein. Examples include, but are not limited to, the vector pUR278(Ruther et al, (1983) EMBO J2: 1791) in which the coding sequence for the protein to be expressed can be ligated into a vector in-frame with the lac z coding region in order to generate a tagged fusion protein; pGEX vectors can be used to express proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are generally soluble and can be easily purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vector includes a cleavage site (thrombin or factor Xa PROTEASE or PRESCISSION PROTEASE) for easy removal of the tag after purification^TM(Pharmacia,Peapack,N.J.))。

One or more expression vectors are then transfected or co-transfected into a suitable target cell, which will express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al, (1978) Cell 14: 725), electroporation (Neumann et al, (1982) EMBO J1: 841), and liposome-based agents. A variety of host-expression vector systems can be used to express the proteins described herein, including prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (e.g., E.coli) transformed with recombinant phage DNA or plasmid DNA expression vectors containing appropriate coding sequences; yeast or filamentous fungi transformed with recombinant yeast or fungal expression vectors containing appropriate coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing appropriate coding sequences; plant cell systems infected with recombinant viral expression vectors containing appropriate coding sequences (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors containing appropriate coding sequences (e.g., Ti plasmid); or animal cell systems, including mammalian cells (e.g., HEK293, CHO, Cos, HeLa, HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell with a defined nucleus. Eukaryotic animal cells include vertebrate (e.g., mammalian) cells, and invertebrate (e.g., insect) cells. Eukaryotic plant cells may specifically include, but are not limited to, yeast cells. Eukaryotic cells are distinct from prokaryotic cells (e.g., bacteria).

In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK293 cell, which is a human embryonic kidney cell line. HEK293 cells can be obtained from American Type Culture Collection, Manassas, VA at CRL-1533 and from Invitrogen (Carlsbad, Calif.) at 293-H cells (catalog No. 11631-017) or 293-F cells (catalog No. 11625-019). In some embodiments, the mammalian cell isA cell which is a human cell line derived from the retina.Cells can be obtained from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a human hamster ovary (CHO) cell. CHO cells are available from American Type Culture Collection, Manassas, Va (e.g., CHO-K1; CCL-61). In other embodiments, the mammalian cellsThe cells were Baby Hamster Kidney (BHK) cells. BHK cells are available from American Type Culture Collection, Manassas, Va (e.g., CRL-1632). In some embodiments, the mammalian cell is an HKB11 cell that is a hybrid cell line of HEK293 cells and human B cell lines. Mei et al, mol.biotechnol.34 (2): 165-78(2006).

In one embodiment, the plasmid encoding the VWF protein, VWF linker, heterologous moiety or chimeric protein of the invention further comprises a selectable marker, e.g., bleomycin resistance, and is transfected into HEK293 cells for production of the chimeric protein.

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

Host cells containing the DNA construct of the protein are grown in an appropriate growth medium. As used herein, the term "suitable growth medium" refers to a medium that comprises nutrients required for cell growth. Nutrients required for cell growth may include carbon sources, nitrogen sources, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the culture medium may contain bovine serum or Fetal Calf Serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium will generally be selected for cells containing the DNA construct by, for example, drug selection or lack thereof in essential nutrients supplemented by or co-transfected with a selectable marker on the DNA construct. The mammalian cells being cultured are typically grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM/F12). In one embodiment, the medium is CD293(Invitrogen, Carlsbad, CA.). In another embodiment, the culture medium is CD17(Invitrogen, Carlsbad, CA.). The selection of a medium appropriate for the particular cell line used is within the skill level of one skilled in the art.

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

The cell culture conditions are also selected to allow association of the first strand with the second strand in the chimeric molecule. Conditions which allow expression of the chimeric molecule may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK293 cells are cultured in CD293 media (Invitrogen, Carlsbad, CA) or OptiCHO media (Invitrogen, Carlsbad, CA) supplemented with 4mM glutamine.

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

In other embodiments, the protein product containing the chimeric molecule is secreted into the culture medium. The culture medium is separated from the cells, concentrated, filtered and then passed through two or three affinity columns, e.g., a protein a column and one or two anion exchange columns.

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

In vitro production allows for scale-up to obtain large quantities of the desired variant polypeptides of the invention. Techniques for mammalian cell culture 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 reactor; or immobilized or embedded cell cultures, e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. The solution of the polypeptide can be purified by a conventional chromatographic method, such as gel filtration, ion exchange chromatography, Hydrophobic Interaction Chromatography (HIC), DEAE-cellulose chromatography or affinity chromatography, as necessary and/or desired.

The invention also includes a method of improving FVIII activity of a chimeric FVIII protein comprising a VWF protein fused to a first heterologous moiety and an XTEN sequence and a FVIII protein fused to a second heterologous moiety, the method comprising inserting a VWF linker between the VWF protein and the first heterologous moiety, wherein the VWF linker comprises a polypeptide selected from the group consisting of: (i) the a2 region from factor viii (fviii); (ii) from the a1 region of FVIII; (iii) from the a3 region of FVIII; (iv) a thrombin cleavage site comprising X-V-P-R (SEQ ID NO: 3) wherein X is an aliphatic amino acid, and a PAR1 exosite interacting motif; or (v) any combination thereof. In some embodiments, FVIII activity is measured by an aPTT assay or ROTEM assay.

Pharmaceutical compositions

Compositions comprising the chimeric molecules of the invention may contain a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and/or auxiliaries which facilitate processing of the active compounds into preparations designed for delivery to the site of action.

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

Formulations suitable for parenteral administration also include aqueous solutions of the active compounds in water-soluble form (e.g., water-soluble salts). In addition, suspensions of the active compounds in the form of suitable oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example sesame oil, or synthetic fatty acid esters, for example ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and dextran. Optionally, the suspension may also contain a stabilizer. Liposomes can also be used to encapsulate the molecules of the invention for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, 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 comprises an isotonic agent, for example, a sugar, a polyol such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions comprise pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering agents which enhance the shelf life or effectiveness of the active ingredient.

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

The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition a delayed absorption agent (e.g., monostearate salts and gelatin).

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

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

Supplementary active compounds may be incorporated into the compositions. In one embodiment, the chimeric molecule of the invention is formulated with another coagulation factor, or a variant, fragment, analog or derivative thereof. For example, blood coagulation factors include, but are not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, wenweber's factor, or recombinant soluble tissue factor (rsTF), or an activated form of any of the foregoing. The coagulation factors of the hemostatic agent may also include antifibrinolytics, e.g., epsilon-amino-hexanoic acid, tranexamic acid.

The dosage regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as dictated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, the parenteral compositions are preferably formulated in unit dosage form. See, e.g., Remington's Pharmaceutical Sciences (Mack pub. Co., Easton, Pa.1980).

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

Non-limiting examples of suitable Pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences of e.w. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain a pH buffering agent, and a wetting or emulsifying agent.

For oral administration, the pharmaceutical composition may take the form of tablets or capsules prepared by conventional means. The composition may also be prepared in liquid form, such as a syrup or suspension. The liquid may include suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils), and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). The formulations may also include flavoring agents, coloring agents and sweetening agents. Alternatively, the composition may be presented in the form of an anhydrous product for constitution with water or another suitable vehicle.

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

For administration by inhalation, the compounds for use according to the invention are preferably delivered in the form of a spray aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or a nebulizer, optionally with a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the unit of measure may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical compositions may also be formulated for rectal administration, for example, in the form of suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides.

Gene therapy

Chimeric molecules of the invention can be produced in vivo in a mammal (e.g., a human patient) using gene therapy methods for treating a bleeding disease or disorder selected from bleeding coagulation disorders, hemarthrosis, muscle bleeding, oral bleeding, hemorrhage into a muscle, oral hemorrhage, trauma, traumatic tinea capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intra-thoracic hemorrhage, bone fractures, central nervous system hemorrhage, postpharyngeal interstitial hemorrhage, retroperitoneal interstitial hemorrhage, or bleeding in the musculus sheath of the hip and lumbar, which would be therapeutically beneficial. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia a. This involves administering a nucleic acid encoding a suitable chimeric molecule operably linked to a suitable expression control sequence. In certain embodiments, these sequences are incorporated into a viral vector. Viral vectors suitable for such gene therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, epstein-barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral 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 E1 gene or E3 gene. When using adenoviral vectors, the mammal may not be exposed to nucleic acid encoding a selectable marker gene. In other embodiments, the sequence is incorporated into a non-viral vector known to those skilled in the art.

Methods of use of chimeric proteins

The invention also provides methods of using the chimeric molecules of the invention to reduce the frequency or extent of bleeding events in a subject in need thereof. An exemplary method comprises administering to the subject in need thereof a therapeutically effective amount of a chimeric molecule of the invention. In other aspects, the invention includes methods of preventing the occurrence of a bleeding event in a subject in need thereof using the chimeric molecules of the invention. In other aspects, a composition comprising a DNA encoding a recombinant protein of the invention can be administered to a subject in need thereof. In certain aspects of the invention, a cell expressing a chimeric molecule of the invention can be administered to a subject in need thereof. In certain aspects of the invention, a pharmaceutical composition comprises (i) a chimeric molecule, (ii) an isolated nucleic acid encoding a chimeric molecule, (iii) a vector comprising a nucleic acid encoding a chimeric molecule, (iv) a cell comprising an isolated nucleic acid encoding a chimeric molecule and/or a vector comprising a nucleic acid encoding a chimeric molecule, or (v) a combination thereof, and further comprises an acceptable excipient or carrier.

A bleeding event may be caused or obtained by a coagulation disorder. Coagulation disorders may also be referred to as coagulation dysfunction. In one example, the blood coagulation disorder treatable with the pharmaceutical composition of the present disclosure is hemophilia or von Willebrand disease (vWD). In another example, the blood coagulation disorder treatable with a pharmaceutical composition of the present disclosure is hemophilia a.

In some embodiments, the type of bleeding associated with the hemorrhagic condition is selected from the group consisting of hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, oral hemorrhage, trauma, traumatic tinea capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system hemorrhage, postpharyngeal interstitial hemorrhage, retroperitoneal interstitial hemorrhage, bleeding in the hip-lumbar sheath, or any combination thereof.

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

In another example, the subject is concomitantly treated with factor IX. Since the compounds of the invention are capable of activating FIXa, they can be used to pre-activate FIXa polypeptides prior to administration of FIXa to a subject.

The methods of the invention can be practiced to a subject in need of prophylactic or on-demand treatment.

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

The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injections, and any similar injection or infusion technique. The composition may also be, for example, a suspension, an emulsion, a sustained release formulation, a cream, a gel, or a powder. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides.

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

Examples

Throughout the examples, the following materials and methods were used unless otherwise indicated.

Materials and methods

In general, the practice of the present invention employs, unless otherwise indicated, conventional chemistry, biophysics, molecular biology, recombinant DNA techniques, immunology techniques (such as antibody techniques, among others), and standard electrophoresis techniques. See, e.g., Sambrook, Fritsch and manitis, 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), coded by McCafferty, Irl Pr (1996); antibodies: a Laboratory Manual, Harlow et al, CS.H.L.Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons (1992).

Example 1 evaluation of thrombin-mediated D' D3 Release from various VWF constructs

This example evaluates the kinetics of thrombin-mediated D' D3 release at 37 ℃ for the various VWF constructs mentioned in figure 2. Biocore experiments were performed with VWF-Fc constructs containing different thrombin-cleavable linkers between the D' D3 domain of VWF and Fc. The final objective was to apply the information collected from the VWF-Fc thrombin digestion to the FVIII-VWF heterodimer as described herein. All VWF-D' D3 constructs were operated on-chip to achieve protein capture densities in the range of 100-700 RU. After capturing the VWF construct on the chip, 5U/ml thrombin was injected on the surface for 5 minutes. Fc remains bound to the chip while D' D3 in the cleavable construct is released. The rate (RU/s) versus capture density (RU) was plotted as shown in fig. 3 and 4. The rate of clearance is directly proportional to the initial capture density, and the slope provides a measure of the susceptibility to thrombin cleavage for each construct.

Figure 3 shows that VWF-052 (which does not have a thrombin cleavage site in the linker region) as expected is not cleaved by thrombin. The rate of VWF-039 (LVPR with PAR1 site) was comparable to the FVIII cleavage rate (data not shown). Therefore, VWF-039 served as a benchmark for complete release of D' D3 from Fc. The thrombin cleavage efficiency was determined using the ratio of the slope of the various VWF-Fc constructs relative to VWF-039. VWF-039 (LVPR with PAR1 site) was cleaved with thrombin at a rate about 70-80 times higher than VWF-031 (LVPR). The cleavage rate of VWF-51(ALRPRVV) was 1.8 times that of VWF-031 (LVPR). VWF-034 containing 288XTEN along the LVPR site showed slower cleavage compared to VWF-031.

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

Fig. 5 shows the slope values of the thrombin cleavage curves for the different VWF constructs. According to these results, the acidic region 2(a2) of FVIII appears to be a highly potent thrombin cleavage site and is incorporated into the FVIII-VWF heterodimer as described herein.

Example 2 evaluation of hemostatic efficacy of FVIII/VWFD' D3 heterodimer Using a HemA patient Whole blood ROTEM assay

The hemostatic efficacy of FVIII/VWFD' D3 heterodimers containing different thrombin cleavable linkers was evaluated in a HemA donor whole blood rotational thromboelastometry (ROTEM) assay. Whole blood samples were collected from donors with severe hemophilia a bleeding disorder using sodium citrate as anticoagulant. FVIII/VWFD' D3 heterodimer variants containing different thrombin cleavable linkers, FVIII155/VWF031(48aa, LVPR site), FVIII155/VWF039(26aa, LVPR + PAR1 site), FVIII155/VWF055(34aa, a2 from FVIII), were diluted in whole blood samples to final concentrations of 100%, 30%, 10% and 3% of normal values, as measured by FVIII chromogenic assay, 40 minutes after blood sample collection. Shortly after addition of FVIII/VWFD' D3 heterodimer, by addition of CaCl₂To initiate the ROTEM reaction. Clotting time (time from start of test to reach 2mm amplitude) was recorded by the instrument and plotted against FVIII concentration in the sample (figure 6). It is hypothesized that a more potent FVIII/VWFD 'D3 heterodimer will induce a faster clotting process, thus resulting in a shorter clotting time compared to a less potent FVIII/VWFD' D3 heterodimer. As shown in fig. 6, the sample with FVIII/VWF039 heterodimer added had the shortest clotting time at all concentrations that had been tested, and the sample with FVIII/VWF031 heterodimer added had the longest clotting time at all concentrations. The clotting time of the samples with added FVIII155/VWF055 heterodimer was centered. Thus, the hemostatic efficacy rating is FVIII155/VWF039>FVIII155/VWF055>FVIII155/VWF 031. Since the only difference between the three molecules is that the thrombin-cleavable linker between the VWF protein and the Fc region, the results indicate that the linker containing the LVPR site and the PAR1 exosite interaction motif and the a2 region of FVIII functions better than the linker containing the LVPR site alone.

Example 3 evaluation of the Activity of FVIII/VWF heterodimers

FVIII-XTEN/VWF heterodimer constructs were transfected into HEK293F cells using three plasmids: first expression FVIII-XTEN-Fc, second expression VWF-XTEN-Fc and third expression PACE. Transfection was performed using a Polyethyleneimine (PEI) standard protocol and 5 days after transfection, tissue culture medium was collected. Purifying various combinations of FVIII-VWF heterodimers from the culture medium. The activity of the purified protein was tested in the chromogenic (two-stage) and aPTT (one-stage) coagulation assays using standard protocols. Introduction of acidic region 2(a2) of FVIII between FVIII and Fc or D' D3 and Fc as shown in table 7A and figure 7 can improve aPTT activity of FVIII-VWF heterodimer as shown in table 7C. For example, FVIII169/VWF059 heterodimer has an a2 thrombin cleavage site in the D 'D3-Fc linker region and has better aPTT activity than FVIII169/VWF057, which contains an LVPR thrombin site in the D' D3Fc linker, as shown in table 7C.

Similarly, incorporation of the a2 region between FVIII and Fc can increase the one-stage clotting activity of the heterodimer, as shown by the improved color development/aPTT ratios of FVIII286/VWF059 and FVIII286/VWF062 shown in table 7B.

TABLE 7A

Serial number	Construct	Linker length between FVIII and Fc (aa)	Thrombin site in linker
				1	FVIII169	-	Is free of
2	FVIII286	32	FVIII-a2
Serial number	Construct	Linker length between D' D3 and Fc (aa)	Thrombin site in linker
				1	VWF057	144AE XTEN+35+LVPR	LVPR
2	VWF059	144AEXTEN+32	FVIII-a2
				3	VWF062	144AE XTEN	Is free of

TABLE 7B

TABLE 7C

Example 4: acute efficacy of FVIII-XTEN-Fc/D' D3-XTEN-Fc heterodimer in the model of tail snip bleeding in HemA mice

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

Male HemA mice 8-12 weeks old were randomly assigned to 5 treatment groups and treated with a single intravenous administration of SQ BDD-FVIII, rFVIII169/VWF034, rFVIII169/VWF057, rFVIII169/VWF059, or vehicle solution, respectively. To mimic the situational treatment of FVIII (at reconstituted 50-100% normal FVIII plasma levels), the therapeutic dose of FVIII selected was 75IU/kg as measured by FVIII aPTT activity. At this dose level, all tested FVIII variants will reconstitute about 70% of normal murine plasma FVIII activity 5 minutes post-dose.

The tail cutting procedure was performed as follows. Briefly, mice were anesthetized with a 50mg/kg ketamine/0.5 mg/kg dexmedetomidine cocktail prior to tail injury and placed on a 37 ℃ heating pad to help maintain body temperature. The tail of the mouse was then immersed in saline at 37 ℃ for 10 minutes to dilate the lateral vein. After venous expansion, FVIII variant or vehicle solution was injected via tail vein and then 5mm of tail end was cut off 5 min post dose using a straight side #11 scalpel. The shed blood was collected in 13ml of 37 ℃ saline for 30 minutes and the blood loss was determined by the weight change of the blood collection tube: blood loss (final weight of collection tube-initial weight +0.10) ml. Statistical analysis was performed using the t-test (Kolmogorov-Smirnov test) and one-way ANOVA (KRUSKAL-Wallis test, post-test: Dunns multiple comparison test).

The blood loss for each individual animal in the study is plotted in figure 8. A significant reduction in blood loss was observed in all FVIII treated groups compared to vehicle treated animals (p < 0.05, table 8). Similar reductions in blood loss compared to BDD-FVIII treatment were observed from all heterodimer treatment groups (p > 0.5, table 8), indicating that the heterodimer molecule could potentially be as effective as SQ BDD-FVIII for on-demand treatment.

Table 8: p value of Kolmogorov-Smirnov test

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

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

pSYN VWF057 protein sequence (VWF D' D3-Fc with LVPR thrombin site in linker): in bold face The underlined region shows the thrombin cleavable LVPR (SEQ ID NO:80) containing the linker region

Nucleotide sequence of pSYN VWF059 (VWF D' D3 having an acidic region 2(a2) thrombin site in the linker) Fc)(SEQ ID NO：81)

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

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

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

pSYN FVIII286 nucleotide sequence (FVIII-Fc with another a2 region between FVIII and Fc) (SEQ ID NO: SEQ ID NO) ID NO：85)

pSYN FVIII286 protein sequence (FVIII-Fc with another a2 region between FVIII and Fc; crude Body and underlined) (SEQ ID NO:86)

FVIII169 nucleotide sequence (SEQ ID NO:87)

FVIII169 protein sequence (SEQ ID NO:88)

VWF034 nucleotide sequence (SEQ ID NO: 91)

VWF034 protein sequence (SEQ ID NO: 92)

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation and without departing from the general concept of the present invention. Therefore, 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 presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

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

All patents and publications cited herein are incorporated by reference in their entirety.

This application claims priority from U.S. provisional patent application No. 61/840,872 filed on 28/6/2013. The contents of the above-mentioned application are incorporated herein by reference in their entirety.

Sequence listing

<110> Biobelati therapeutic Co

<120> Thrombin cleavable linker with XTEN and uses thereof

<130> 2159.420PC01

<140> to be assigned

<141> 2014-06-27

<150> 61/840,872

<151> 2013-06-28

<160> 92

<170> PatentIn 3.5 edition

<210> 1

<211> 8442

<212> DNA

<213> Intelligent people

<400> 1

atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60

ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120

gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180

ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240

agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300

accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360

gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420

gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480

ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540

acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600

gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660

ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720

gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780

ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840

gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900

tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960

tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020

ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080

acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140

aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa gagctttgac 1200

aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260

cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320

acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380

ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa aggtgacctc 1440

cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500

gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560

tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620

ctggcrgagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680

gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740

gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800

ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860

tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920

gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980

tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040

gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100

tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160

atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220

agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280

agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340

ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400

agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460

tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520

acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580

catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640

ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700

aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760

tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820

gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880

tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940

tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000

ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060

tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120

tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180

agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240

ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgcctgcttc 3300

tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360

aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420

gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480

gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540

aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600

gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660

cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720

ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct gtatgtggag 3780

gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga cctggtcttc 3840

ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa ggcctttgtg 3900

gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc cgtggtggag 3960

taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc gtcagagctg 4020

cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac cagcgaggtc 4080

ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc ctcccgcatc 4140

gccctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt tgtccgctac 4200

gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg gccccatgcc 4260

aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc cttcgtgctg 4320

agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct ctgtgacctt 4380

gcccctgaag cccctcctcc tactctgccc cccgacatgg cacaagtcac tgtgggcccg 4440

gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct ggatgtggcg 4500

ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag caaggagttc 4560

atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt cacggtgctg 4620

cagtactcct acatggtgac cgtggagtac cccttcagcg aggcacagtc caaaggggac 4680

atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa cactgggctg 4740

gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg ggagcaggcg 4800

cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa gaggctgcct 4860

ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca ggagctggag 4920

aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct cccccgagag 4980

gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat ccccaccctc 5040

tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga tggctcctcc 5100

agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt catttcaaaa 5160

gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag catcaccacc 5220

attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct tgtggacgtc 5280

atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc tgtgcgatac 5340

ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt catcctggtc 5400

acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc caacagagtg 5460

acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg gatcttggca 5520

ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct ccctaccatg 5580

gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag gatttgcatg 5640

gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga ccagtgccac 5700

accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt caactgtgac 5760

cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga agagacctgt 5820

ggctgccgct ggacctgccc ctgygtgtgc acaggcagct ccactcggca catcgtgacc 5880

tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt tcaaaacaag 5940

gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc aaggcagggc 6000

tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagstgca cagtgacatg 6060

gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa catggaagtc 6120

aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca catcttcaca 6180

ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt tgcttcaaag 6240

acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat gctgagggat 6300

ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca gcggccaggg 6360

cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc ccactgccag 6420

gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc cacattctat 6480

gccatctgcc agcaggacag ttgccaccag gagcaagtgt gtgaggtgat cgcctcttat 6540

gcccacctct gtcggaccaa cggggtctgc gttgactgga ggacacctga tttctgtgct 6600

atgtcatgcc caccatctct ggtctacaac cactgtgagc atggctgtcc ccggcactgt 6660

gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg ccctccagat 6720

aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg cattggtgag 6780

gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc ctgtcagatc 6840

tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc cacggccaaa 6900

gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga ccagtgctgc 6960

cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt gcctcactgt 7020

gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa cttcacctgc 7080

gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc gcaccgtttg 7140

cccacccttc ggaagaccca gtgctgtgat gagtatgagt gtgcctgcaa ctgtgtcaac 7200

tccacagtga gctgtcccct tgggtacttg gcctcaaccg ccaccaatga ctgtggctgt 7260

accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat ctaccctgtg 7320

ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga ggatgccgtg 7380

atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg tcggtcgggc 7440

ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc tgcctgtgag 7500

gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt cggctcccag 7560

tgggcctccc cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa ggaggaggtc 7620

tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg cccctcgggc 7680

tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga gcgcatggag 7740

gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat cgatgtgtgc 7800

acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct ggagtgcagg 7860

aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac aggtgaatgt 7920

tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca gatcatgaca 7980

ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa ggtcaatgag 8040

agaggagagt acttctggga gaagagggtc acaggctgcc caccctttga tgaacacaag 8100

tgtcttgctg agggaggtaa aattatgaaa attccaggca cctgctgtga cacatgtgag 8160

gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaaggtggg aagctgtaag 8220

tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa agccatgtac 8280

tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac acggacggag 8340

cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga ggttctcaat 8400

gccatggagt gcaaatgctc ccccaggaag tgcagcaagt ga 8442

<210> 2

<211> 2813

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (2016)..(2016)

<223> wherein Xaa may be any amino acid except cysteine

<400> 2

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr

1235 1240 1245

Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser

1250 1255 1260

Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu

1265 1270 1275

Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe

1280 1285 1290

Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg

1295 1300 1305

Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp

1310 1315 1320

Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser

1325 1330 1335

Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln

1340 1345 1350

Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile

1355 1360 1365

Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu Leu

1370 1375 1380

Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val

1385 1390 1395

Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro

1400 1405 1410

Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile

1415 1420 1425

Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val

1430 1435 1440

Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys

1445 1450 1455

Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met

1460 1465 1470

Ala Gln Val Thr Val Gly Pro Gly Leu Leu Gly Val Ser Thr Leu

1475 1480 1485

Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val Ala Phe Val Leu

1490 1495 1500

Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn Arg Ser Lys

1505 1510 1515

Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly Gln Asp

1520 1525 1530

Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr Val

1535 1540 1545

Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln

1550 1555 1560

Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr

1565 1570 1575

Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser

1580 1585 1590

Gln Gly Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr

1595 1600 1605

Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile

1610 1615 1620

Gln Val Val Pro Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu

1625 1630 1635

Leu Glu Arg Ile Gly Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp

1640 1645 1650

Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Arg

1655 1660 1665

Cys Cys Ser Gly Glu Gly Leu Gln Ile Pro Thr Leu Ser Pro Ala

1670 1675 1680

Pro Asp Cys Ser Gln Pro Leu Asp Val Ile Leu Leu Leu Asp Gly

1685 1690 1695

Ser Ser Ser Phe Pro Ala Ser Tyr Phe Asp Glu Met Lys Ser Phe

1700 1705 1710

Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile Gly Pro Arg Leu Thr

1715 1720 1725

Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr Thr Ile Asp Val

1730 1735 1740

Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu Ser Leu Val

1745 1750 1755

Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly Asp Ala

1760 1765 1770

Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His Gly Ala

1775 1780 1785

Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr Asp Val

1790 1795 1800

Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg Ser Asn

1805 1810 1815

Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr Asp Ala

1820 1825 1830

Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser Asn Val

1835 1840 1845

Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val Thr Leu

1850 1855 1860

Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val Arg Ile

1865 1870 1875

Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp Val Trp

1880 1885 1890

Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro Asp Gly

1895 1900 1905

Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg Gly Leu

1910 1915 1920

Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val Glu Glu

1925 1930 1935

Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr Gly Ser

1940 1945 1950

Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe Lys Leu

1955 1960 1965

Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu Gln Asp

1970 1975 1980

Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly Ala Arg

1985 1990 1995

Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala Leu Ser

2000 2005 2010

Val Glu Xaa His Ser Asp Met Glu Val Thr Val Asn Gly Arg Leu

2015 2020 2025

Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn Val Tyr

2030 2035 2040

Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly His Ile

2045 2050 2055

Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln Leu Ser

2060 2065 2070

Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly Ile Cys

2075 2080 2085

Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly Thr Val

2090 2095 2100

Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val Gln Arg

2105 2110 2115

Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys Leu Val

2120 2125 2130

Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu Phe Ala

2135 2140 2145

Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala Ile Cys

2150 2155 2160

Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val Ile Ala

2165 2170 2175

Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val Cys Val Asp Trp

2180 2185 2190

Arg Thr Pro Asp Phe Cys Ala Met Ser Cys Pro Pro Ser Leu Val

2195 2200 2205

Tyr Asn His Cys Glu His Gly Cys Pro Arg His Cys Asp Gly Asn

2210 2215 2220

Val Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe Cys Pro

2225 2230 2235

Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu Glu Ala

2240 2245 2250

Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln Phe Leu

2255 2260 2265

Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys Thr Cys

2270 2275 2280

Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys Pro Thr

2285 2290 2295

Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg Leu Arg

2300 2305 2310

Gln Asn Ala Asp Gln Cys Cys Pro Glu Tyr Glu Cys Val Cys Asp

2315 2320 2325

Pro Val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu Arg Gly

2330 2335 2340

Leu Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro Asn Phe

2345 2350 2355

Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser Pro Pro

2360 2365 2370

Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr Gln Cys

2375 2380 2385

Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser Thr Val

2390 2395 2400

Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn Asp Cys

2405 2410 2415

Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val Cys Val His

2420 2425 2430

Arg Ser Thr Ile Tyr Pro Val Gly Gln Phe Trp Glu Glu Gly Cys

2435 2440 2445

Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala Val Met Gly Leu

2450 2455 2460

Arg Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser Cys Arg

2465 2470 2475

Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys Gly Arg

2480 2485 2490

Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro Arg Gly

2495 2500 2505

Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp Ala Ser

2510 2515 2520

Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val Lys Glu

2525 2530 2535

Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro Gln Leu Glu

2540 2545 2550

Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys Lys Thr Ser

2555 2560 2565

Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala Cys Met

2570 2575 2580

Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met Ile Asp

2585 2590 2595

Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val Ile Ser

2600 2605 2610

Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro Cys Pro

2615 2620 2625

Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys Gly Arg

2630 2635 2640

Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly Gln Ile

2645 2650 2655

Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly Cys Asp Thr

2660 2665 2670

His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys

2675 2680 2685

Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys Leu Ala

2690 2695 2700

Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys Asp Thr

2705 2710 2715

Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu Gln Tyr

2720 2725 2730

Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp Ile His

2735 2740 2745

Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser Ile Asp

2750 2755 2760

Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro Thr Arg

2765 2770 2775

Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly Ser Val

2780 2785 2790

Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys Ser Pro

2795 2800 2805

Arg Lys Cys Ser Lys

2810

<210> 3

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> wherein X is an aliphatic amino acid

<400> 3

Xaa Val Pro Arg

1

<210> 4

<211> 34

<212> PRT

<213> Artificial sequence

<220>

<223> area a2

<400> 4

Ile Ser Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp

1 5 10 15

Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser

20 25 30

Phe Ser

<210> 5

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> area a1

<400> 5

Ile Ser Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu

1 5 10 15

Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro

20 25 30

Ser Phe Ile Gln Ile Arg Ser Val

35 40

<210> 6

<211> 46

<212> PRT

<213> Artificial sequence

<220>

<223> area a3

<400> 6

Ile Ser Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile

1 5 10 15

Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp

20 25 30

Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln

35 40 45

<210> 7

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 7

Ser Phe Leu Leu Arg Asn

1 5

<210> 8

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 8

Pro Asn Asp Lys

1

<210> 9

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 9

Pro Asn Asp Lys Tyr

1 5

<210> 10

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 10

Pro Asn Asp Lys Tyr Glu

1 5

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 11

Pro Asn Asp Lys Tyr Glu Pro

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 12

Pro Asn Asp Lys Tyr Glu Pro Phe

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 13

Pro Asn Asp Lys Tyr Glu Pro Phe Trp

1 5

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 14

Pro Asn Asp Lys Tyr Glu Pro Phe Trp Glu

1 5 10

<210> 15

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 15

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser

<210> 16

<211> 2332

<212> PRT

<213> Intelligent people

<400> 16

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr

1 5 10 15

Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro

20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys

35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro

50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val

65 70 75 80

Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val

85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala

100 105 110

Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val

115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn

130 135 140

Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser

145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu

165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu

180 185 190

His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp

195 200 205

His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser

210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg

225 230 235 240

Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His

245 250 255

Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu

260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile

275 280 285

Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly

290 295 300

Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met

305 310 315 320

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg

325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp

340 345 350

Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe

355 360 365

Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His

370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu

385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro

405 410 415

Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr

420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile

435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile

450 455 460

Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile

465 470 475 480

Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys

485 490 495

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys

500 505 510

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys

515 520 525

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala

530 535 540

Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp

545 550 555 560

Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe

565 570 575

Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln

580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe

595 600 605

Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser

610 615 620

Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu

625 630 635 640

Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr

645 650 655

Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro

660 665 670

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp

675 680 685

Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala

690 695 700

Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu

705 710 715 720

Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala

725 730 735

Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg

740 745 750

Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys

755 760 765

Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn

770 775 780

Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro

785 790 795 800

His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe

805 810 815

Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser

820 825 830

Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val

835 840 845

Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly

850 855 860

Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser

865 870 875 880

Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala

885 890 895

Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His

900 905 910

Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro

915 920 925

Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp

930 935 940

Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp

945 950 955 960

Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys

965 970 975

Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys

980 985 990

Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala

995 1000 1005

Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu

1010 1015 1020

Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu

1025 1030 1035

Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp

1040 1045 1050

Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr

1055 1060 1065

Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly

1070 1075 1080

Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys

1085 1090 1095

Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His

1100 1105 1110

Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln

1115 1120 1125

Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe

1130 1135 1140

Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr

1145 1150 1155

Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn

1160 1165 1170

Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His

1175 1180 1185

Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr

1190 1195 1200

Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr

1205 1210 1215

Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg

1220 1225 1230

Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu

1235 1240 1245

Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys

1250 1255 1260

His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu

1265 1270 1275

Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys

1280 1285 1290

Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr

1295 1300 1305

Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu

1310 1315 1320

Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr

1325 1330 1335

Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr

1340 1345 1350

Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser

1355 1360 1365

Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala

1370 1375 1380

Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser

1385 1390 1395

Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser

1400 1405 1410

Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val

1415 1420 1425

Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu

1430 1435 1440

Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu

1445 1450 1455

Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys

1460 1465 1470

Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr

1475 1480 1485

Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys

1490 1495 1500

Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu

1505 1510 1515

Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile

1520 1525 1530

Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg

1535 1540 1545

Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp

1550 1555 1560

Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu

1565 1570 1575

Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys

1580 1585 1590

Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His

1595 1600 1605

Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu

1610 1615 1620

Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln

1625 1630 1635

Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr

1640 1645 1650

Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile

1655 1660 1665

Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp

1670 1675 1680

Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr

1685 1690 1695

Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser

1700 1705 1710

Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro

1715 1720 1725

Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe

1730 1735 1740

Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

1745 1750 1755

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val

1760 1765 1770

Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser

1775 1780 1785

Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

1790 1795 1800

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys

1805 1810 1815

Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys

1820 1825 1830

Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His

1835 1840 1845

Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu

1850 1855 1860

Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu

1865 1870 1875

Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu

1880 1885 1890

Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu

1895 1900 1905

Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly

1910 1915 1920

Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln

1925 1930 1935

Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile

1940 1945 1950

His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys

1955 1960 1965

Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe

1970 1975 1980

Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val

1985 1990 1995

Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu

2000 2005 2010

Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala

2015 2020 2025

Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr

2030 2035 2040

Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser

2045 2050 2055

Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val

2060 2065 2070

Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly

2075 2080 2085

Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile

2090 2095 2100

Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn

2105 2110 2115

Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser

2120 2125 2130

Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr

2135 2140 2145

Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg

2150 2155 2160

Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu

2165 2170 2175

Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser

2180 2185 2190

Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala

2195 2200 2205

Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val

2210 2215 2220

Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met

2225 2230 2235

Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr

2240 2245 2250

Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly

2255 2260 2265

His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe

2270 2275 2280

Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp

2285 2290 2295

Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp

2300 2305 2310

Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala

2315 2320 2325

Gln Asp Leu Tyr

2330

<210> 17

<211> 7053

<212> DNA

<213> Intelligent people

<400> 17

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctcccaga attcaagaca ccctagcact aggcaaaagc aatttaatgc caccacaatt 2340

ccagaaaatg acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa 2400

atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc tactccacat 2460

gggctatcct tatctgatct ccaagaagcc aaatatgaga ctttttctga tgatccatca 2520

cctggagcaa tagacagtaa taacagcctg tctgaaatga cacacttcag gccacagctc 2580

catcacagtg gggacatggt atttacccct gagtcaggcc tccaattaag attaaatgag 2640

aaactgggga caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca 2700

tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac tgataataca 2760

agttccttag gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta 2820

tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt gagtgaagaa 2880

aataatgatt caaagttgtt agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940

aaaaatgtat cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000

gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt aaagacaaac 3060

aaaacttcca ataattcagc aactaataga aagactcaca ttgatggccc atcattatta 3120

attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180

gtgacacctt tgattcatga cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240

aatcatatgt caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300

gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360

ttgccagaat cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg 3420

caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480

aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540

ggactcaaag agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat 3600

ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660

gaaacattaa tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag 3720

aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg ttcatatgac 3780

ggggcatatg ctccagtact tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840

aagaaacaca cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900

aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc tcctaataca 3960

agccagcaga attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca 4020

ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac ccagtggtcc 4080

aaaaacatga aacatttgac cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140

aaaggggcca ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct 4200

caagcaaata gatctccatt acccattgca aaggtatcat catttccatc tattagacct 4260

atatatctga ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat 4320

agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380

aacctttctt tagccattct aaccttggag atgactggtg atcaaagaga ggttggctcc 4440

ctggggacaa gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500

aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560

cagaaggacc tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620

gaagggagcc ttcttcaggg aacagaggga gcgattaagt ggaatgaagc aaacagacct 4680

ggaaaagttc cctttctgag agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740

ttggatcctc ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa 4800

tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860

aacgcttgtg aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920

atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980

gtcttgaaac gccatcaacg ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040

attgactatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 5100

gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160

gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 5220

gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 5280

tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340

tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 5400

ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 5460

cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 5520

catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580

gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 5640

acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 5700

atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760

ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 5820

aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 5880

tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 5940

gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 6000

gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060

attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 6120

cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 6180

caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 6240

tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 6300

cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360

atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 6420

accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 6480

cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 6540

cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 6600

agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 6660

acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720

caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780

ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840

atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900

gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 6960

ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 7020

gaggttctgg gctgcgaggc acaggacctc tac 7053

<210> 18

<211> 1438

<212> PRT

<213> Artificial sequence

<220>

<223> B Domain deleted FVIII

<400> 18

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr

1 5 10 15

Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro

20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys

35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro

50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val

65 70 75 80

Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val

85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala

100 105 110

Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val

115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn

130 135 140

Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser

145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu

165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu

180 185 190

His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp

195 200 205

His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser

210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg

225 230 235 240

Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His

245 250 255

Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu

260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile

275 280 285

Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly

290 295 300

Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met

305 310 315 320

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg

325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp

340 345 350

Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe

355 360 365

Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His

370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu

385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro

405 410 415

Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr

420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile

435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile

450 455 460

Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile

465 470 475 480

Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys

485 490 495

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys

500 505 510

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys

515 520 525

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala

530 535 540

Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp

545 550 555 560

Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe

565 570 575

Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln

580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe

595 600 605

Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser

610 615 620

Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu

625 630 635 640

Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr

645 650 655

Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro

660 665 670

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp

675 680 685

Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala

690 695 700

Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu

705 710 715 720

Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala

725 730 735

Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His

740 745 750

Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile

755 760 765

Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp

770 775 780

Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

785 790 795 800

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly

805 810 815

Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser

820 825 830

Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser

835 840 845

Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

850 855 860

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr

865 870 875 880

Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile

885 890 895

Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe

900 905 910

Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His

915 920 925

Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe

930 935 940

Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro

945 950 955 960

Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln

965 970 975

Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr

980 985 990

Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro

995 1000 1005

Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg

1010 1015 1020

Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu

1025 1030 1035

Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met

1040 1045 1050

Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val

1055 1060 1065

Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn

1070 1075 1080

Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys

1085 1090 1095

Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His

1100 1105 1110

Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln

1115 1120 1125

Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile

1130 1135 1140

Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg

1145 1150 1155

Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro

1160 1165 1170

Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His

1175 1180 1185

Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr

1190 1195 1200

Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp

1205 1210 1215

Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe

1220 1225 1230

Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro

1235 1240 1245

Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser

1250 1255 1260

Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn

1265 1270 1275

Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp

1280 1285 1290

Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr

1295 1300 1305

Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn

1310 1315 1320

Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val

1325 1330 1335

Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly

1340 1345 1350

Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile

1355 1360 1365

Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn

1370 1375 1380

Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro

1385 1390 1395

Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg

1400 1405 1410

Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu

1415 1420 1425

Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1430 1435

<210> 19

<211> 4371

<212> DNA

<213> Artificial sequence

<220>

<223> B Domain deleted FVIII

<400> 19

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctctcaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340

cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400

gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460

cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520

catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580

caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640

ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700

agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760

cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820

ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880

gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940

ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000

tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060

gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120

tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180

caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240

attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300

tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360

tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420

gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480

cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540

tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600

ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660

ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720

cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780

aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840

tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900

atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960

tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020

agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080

aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140

tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200

cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260

tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320

cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta c 4371

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 20

Pro Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp

1 5 10

<210> 21

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 21

Pro Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp Glu

1 5 10

<210> 22

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 22

Pro Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp Glu Glu

1 5 10

<210> 23

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> PAR1 exosite interacting motif

<400> 23

Pro Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp Glu Glu Ser

1 5 10

<210> 24

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> VWF linker

<400> 24

Gly Gly Leu Val Pro Arg Ser Phe Leu Leu Arg Asn Pro Asn Asp Lys

1 5 10 15

Tyr Glu Pro Phe Trp Glu Asp Glu Glu Ser

20 25

<210> 25

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<400> 25

Leu Val Pro Arg

1

<210> 26

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<400> 26

Ala Leu Arg Pro Arg Val Val

1 5

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> FXIa cleavage site

<400> 27

Thr Gln Ser Phe Asn Asp Phe Thr Arg

1 5

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> FXIa cleavage site

<400> 28

Ser Val Ser Gln Thr Ser Lys Leu Thr Arg

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<400> 29

Asp Phe Leu Ala Glu Gly Gly Gly Val Arg

1 5 10

<210> 30

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<400> 30

Thr Thr Lys Ile Lys Pro Arg

1 5

<210> 31

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<400> 31

Leu Val Pro Arg Gly

1 5

<210> 32

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 32

Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro

1 5 10 15

Ser Ala Pro Ala

20

<210> 33

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 33

Ala Ala Pro Ala Ser Pro Ala Pro Ala Ala Pro Ser Ala Pro Ala Pro

1 5 10 15

Ala Ala Pro Ser

20

<210> 34

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 34

Ala Pro Ser Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala

1 5 10 15

Ser Pro Ser Ser

20

<210> 35

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 35

Ala Pro Ser Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala

1 5 10 15

Ser Pro Ser

<210> 36

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 36

Ser Ser Pro Ser Ala Pro Ser Pro Ser Ser Pro Ala Ser Pro Ser Pro

1 5 10 15

Ser Ser Pro Ala

20

<210> 37

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 37

Ala Ala Ser Pro Ala Ala Pro Ser Ala Pro Pro Ala Ala Ala Ser Pro

1 5 10 15

Ala Ala Pro Ser Ala Pro Pro Ala

20

<210> 38

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> PAS sequence

<400> 38

Ala Ser Ala Ala Ala Pro Ala Ala Ala Ser Ala Ala Ala Ser Ala Pro

1 5 10 15

Ser Ala Ala Ala

20

<210> 39

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AE42

<400> 39

Gly Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly

1 5 10 15

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala

20 25 30

Thr Ser Gly Ser Glu Thr Pro Ala Ser Ser

35 40

<210> 40

<211> 78

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AE72

<400> 40

Gly Ala Pro Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser

1 5 10 15

Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala

20 25 30

Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu

35 40 45

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr

50 55 60

Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ala Ser Ser

65 70 75

<210> 41

<211> 143

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AE144

<400> 41

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu

1 5 10 15

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly

20 25 30

Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

35 40 45

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro

50 55 60

Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly

65 70 75 80

Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

85 90 95

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu

100 105 110

Ser Ala Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser

115 120 125

Glu Thr Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

130 135 140

<210> 42

<211> 144

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AG144

<400> 42

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr

1 5 10 15

Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr

20 25 30

Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro

35 40 45

Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro

50 55 60

Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala

65 70 75 80

Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro

85 90 95

Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro

100 105 110

Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala

115 120 125

Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro

130 135 140

<210> 43

<211> 288

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AE288

<400> 43

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro

1 5 10 15

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

20 25 30

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

35 40 45

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

50 55 60

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr

65 70 75 80

Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

85 90 95

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu

100 105 110

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr

115 120 125

Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

130 135 140

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu

145 150 155 160

Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro

165 170 175

Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

180 185 190

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro

195 200 205

Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr

210 215 220

Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

225 230 235 240

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro

245 250 255

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

260 265 270

Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

275 280 285

<210> 44

<211> 288

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AG288

<400> 44

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser

1 5 10 15

Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr

20 25 30

Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser

35 40 45

Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser

50 55 60

Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr

65 70 75 80

Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser

85 90 95

Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser

100 105 110

Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser

115 120 125

Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

130 135 140

Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser

145 150 155 160

Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser

165 170 175

Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly

180 185 190

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser

195 200 205

Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly

210 215 220

Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly

225 230 235 240

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser

245 250 255

Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr

260 265 270

Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser

275 280 285

<210> 45

<211> 576

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AE576

<400> 45

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu

1 5 10 15

Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu

20 25 30

Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

35 40 45

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

50 55 60

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

65 70 75 80

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

85 90 95

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala

100 105 110

Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro

115 120 125

Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

130 135 140

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala

145 150 155 160

Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu

165 170 175

Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

180 185 190

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

195 200 205

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

210 215 220

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

225 230 235 240

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

245 250 255

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

260 265 270

Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

275 280 285

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu

290 295 300

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly

305 310 315 320

Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

325 330 335

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

340 345 350

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu

355 360 365

Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

370 375 380

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

385 390 395 400

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr

405 410 415

Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

420 425 430

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro

435 440 445

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

450 455 460

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

465 470 475 480

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

485 490 495

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

500 505 510

Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

515 520 525

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala

530 535 540

Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro

545 550 555 560

Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

565 570 575

<210> 46

<211> 576

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AG576

<400> 46

Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser

1 5 10 15

Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser

20 25 30

Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly

35 40 45

Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser

50 55 60

Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser

65 70 75 80

Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

85 90 95

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro

100 105 110

Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser

115 120 125

Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

130 135 140

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser

145 150 155 160

Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr

165 170 175

Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

180 185 190

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser

195 200 205

Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly

210 215 220

Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser

225 230 235 240

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro

245 250 255

Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly

260 265 270

Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser

275 280 285

Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser

290 295 300

Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser

305 310 315 320

Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

325 330 335

Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser

340 345 350

Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser

355 360 365

Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

370 375 380

Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro

385 390 395 400

Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly

405 410 415

Ala Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser

420 425 430

Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro

435 440 445

Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly

450 455 460

Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser

465 470 475 480

Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser

485 490 495

Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser

500 505 510

Ser Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser

515 520 525

Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser

530 535 540

Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser

545 550 555 560

Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser

565 570 575

<210> 47

<211> 864

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AE864

<400> 47

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu

1 5 10 15

Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu

20 25 30

Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

35 40 45

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

50 55 60

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

65 70 75 80

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

85 90 95

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala

100 105 110

Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro

115 120 125

Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

130 135 140

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala

145 150 155 160

Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu

165 170 175

Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

180 185 190

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

195 200 205

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

210 215 220

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

225 230 235 240

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

245 250 255

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

260 265 270

Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

275 280 285

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu

290 295 300

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly

305 310 315 320

Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

325 330 335

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

340 345 350

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu

355 360 365

Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

370 375 380

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

385 390 395 400

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr

405 410 415

Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

420 425 430

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro

435 440 445

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

450 455 460

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

465 470 475 480

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

485 490 495

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

500 505 510

Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

515 520 525

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala

530 535 540

Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro

545 550 555 560

Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

565 570 575

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro

580 585 590

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

595 600 605

Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

610 615 620

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

625 630 635 640

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr

645 650 655

Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

660 665 670

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu

675 680 685

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr

690 695 700

Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

705 710 715 720

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu

725 730 735

Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro

740 745 750

Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

755 760 765

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro

770 775 780

Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr

785 790 795 800

Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

805 810 815

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro

820 825 830

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

835 840 845

Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

850 855 860

<210> 48

<211> 864

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN AG864

<400> 48

Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Pro

1 5 10 15

Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr

20 25 30

Gly Thr Gly Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro

35 40 45

Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro

50 55 60

Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser

65 70 75 80

Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro

85 90 95

Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly

100 105 110

Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

115 120 125

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

130 135 140

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr

145 150 155 160

Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

165 170 175

Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro

180 185 190

Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro

195 200 205

Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro Ser Ala Ser Thr

210 215 220

Gly Thr Gly Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro

225 230 235 240

Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ala Ser Pro

245 250 255

Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

260 265 270

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

275 280 285

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro

290 295 300

Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

305 310 315 320

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

325 330 335

Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Thr Pro Gly

340 345 350

Ser Gly Thr Ala Ser Ser Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

355 360 365

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

370 375 380

Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ser Ser Thr

385 390 395 400

Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala

405 410 415

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

420 425 430

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr

435 440 445

Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala

450 455 460

Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro

465 470 475 480

Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro

485 490 495

Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

500 505 510

Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro

515 520 525

Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro

530 535 540

Gly Thr Ser Ser Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser

545 550 555 560

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

565 570 575

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr

580 585 590

Pro Ser Gly Ala Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala

595 600 605

Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro

610 615 620

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr

625 630 635 640

Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala

645 650 655

Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro

660 665 670

Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro

675 680 685

Gly Thr Ser Ser Thr Gly Ser Pro Gly Thr Pro Gly Ser Gly Thr Ala

690 695 700

Ser Ser Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro

705 710 715 720

Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro Gly Ser Ser Pro

725 730 735

Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser

740 745 750

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

755 760 765

Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Pro

770 775 780

Ser Ala Ser Thr Gly Thr Gly Pro Gly Ala Ser Pro Gly Thr Ser Ser

785 790 795 800

Thr Gly Ser Pro Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro

805 810 815

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro Gly Ser Ser Thr

820 825 830

Pro Ser Gly Ala Thr Gly Ser Pro Gly Ser Ser Thr Pro Ser Gly Ala

835 840 845

Thr Gly Ser Pro Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

850 855 860

<210> 49

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AD

<400> 49

Gly Glu Ser Pro Gly Gly Ser Ser Gly Ser Glu Ser

1 5 10

<210> 50

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AD

<400> 50

Gly Ser Glu Gly Ser Ser Gly Pro Gly Glu Ser Ser

1 5 10

<210> 51

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AD

<400> 51

Gly Ser Ser Glu Ser Gly Ser Ser Glu Gly Gly Pro

1 5 10

<210> 52

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AD

<400> 52

Gly Ser Gly Gly Glu Pro Ser Glu Ser Gly Ser Ser

1 5 10

<210> 53

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AE, AM

<400> 53

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

1 5 10

<210> 54

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AE, AM, AQ

<400> 54

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

1 5 10

<210> 55

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AE, AM, AQ

<400> 55

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

1 5 10

<210> 56

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AE, AM, AQ

<400> 56

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

1 5 10

<210> 57

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif families AF, AM

<400> 57

Gly Ser Thr Ser Glu Ser Pro Ser Gly Thr Ala Pro

1 5 10

<210> 58

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif families AF, AM

<400> 58

Gly Thr Ser Thr Pro Glu Ser Gly Ser Ala Ser Pro

1 5 10

<210> 59

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif families AF, AM

<400> 59

Gly Thr Ser Pro Ser Gly Glu Ser Ser Thr Ala Pro

1 5 10

<210> 60

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif families AF, AM

<400> 60

Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro

1 5 10

<210> 61

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AG, AM

<400> 61

Gly Thr Pro Gly Ser Gly Thr Ala Ser Ser Ser Pro

1 5 10

<210> 62

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AG, AM

<400> 62

Gly Ser Ser Thr Pro Ser Gly Ala Thr Gly Ser Pro

1 5 10

<210> 63

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AG, AM

<400> 63

Gly Ser Ser Pro Ser Ala Ser Thr Gly Thr Gly Pro

1 5 10

<210> 64

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AG, AM

<400> 64

Gly Ala Ser Pro Gly Thr Ser Ser Thr Gly Ser Pro

1 5 10

<210> 65

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AQ

<400> 65

Gly Glu Pro Ala Gly Ser Pro Thr Ser Thr Ser Glu

1 5 10

<210> 66

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AQ

<400> 66

Gly Thr Gly Glu Pro Ser Ser Thr Pro Ala Ser Glu

1 5 10

<210> 67

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AQ

<400> 67

Gly Ser Gly Pro Ser Thr Glu Ser Ala Pro Thr Glu

1 5 10

<210> 68

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AQ

<400> 68

Gly Ser Glu Thr Pro Ser Gly Pro Ser Glu Thr Ala

1 5 10

<210> 69

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AQ

<400> 69

Gly Pro Ser Glu Thr Ser Thr Ser Glu Pro Gly Ala

1 5 10

<210> 70

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family AQ

<400> 70

Gly Ser Pro Ser Glu Pro Thr Glu Gly Thr Ser Ala

1 5 10

<210> 71

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BC

<400> 71

Gly Ser Gly Ala Ser Glu Pro Thr Ser Thr Glu Pro

1 5 10

<210> 72

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BC

<400> 72

Gly Ser Glu Pro Ala Thr Ser Gly Thr Glu Pro Ser

1 5 10

<210> 73

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BC

<400> 73

Gly Thr Ser Glu Pro Ser Thr Ser Glu Pro Gly Ala

1 5 10

<210> 74

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BC

<400> 74

Gly Thr Ser Thr Glu Pro Ser Glu Pro Gly Ser Ala

1 5 10

<210> 75

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BD

<400> 75

Gly Ser Thr Ala Gly Ser Glu Thr Ser Thr Glu Ala

1 5 10

<210> 76

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BD

<400> 76

Gly Ser Glu Thr Ala Thr Ser Gly Ser Glu Thr Ala

1 5 10

<210> 77

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BD

<400> 77

Gly Thr Ser Glu Ser Ala Thr Ser Glu Ser Gly Ala

1 5 10

<210> 78

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> XTEN motif family BD

<400> 78

Gly Thr Ser Thr Glu Ala Ser Glu Gly Ser Ala Ser

1 5 10

<210> 79

<211> 4974

<212> DNA

<213> Artificial sequence

<220>

<223> VWF057 (VWF D' D3-Fc with LVPR thrombin site in linker)

<400> 79

atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60

ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120

gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180

ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240

agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300

accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360

gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420

gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480

ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540

acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600

gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660

ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720

gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780

ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840

gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900

tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960

tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020

ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080

acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140

aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa gagctttgac 1200

aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260

cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320

acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380

ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa aggtgacctc 1440

cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500

gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560

tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620

ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680

gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740

gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800

ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860

tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920

gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980

tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040

gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100

tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160

atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220

agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280

agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340

ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400

agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460

tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520

acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580

catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640

ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700

aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760

tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820

gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880

tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940

tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000

ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060

tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120

tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180

agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240

ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgccgcattc 3300

tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360

aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420

gaggctgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480

gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540

aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600

gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660

cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720

atatcgggcg cgccaacatc agagagcgcc acccctgaaa gtggtcccgg gagcgagcca 3780

gccacatctg ggtcggaaac gccaggcaca agtgagtctg caactcccga gtccggacct 3840

ggctccgagc ctgccactag cggctccgag actccgggaa cttccgagag cgctacacca 3900

gaaagcggac ccggaaccag taccgaacct agcgagggct ctgctccggg cagcccagcc 3960

ggctctccta catccacgga ggagggcact tccgaatccg ccaccccgga gtcagggcca 4020

ggatctgaac ccgctacctc aggcagtgag acgccaggaa cgagcgagtc cgctacaccg 4080

gagagtgggc cagggagccc tgctggatct cctacgtcca ctgaggaagg gtcaccagcg 4140

ggctcgccca ccagcactga agaaggtgcc tcgagcggcg gtggaggatc cggtggcggg 4200

ggatccggtg gcgggggatc cggtggcggg ggatccggtg gcgggggatc cggtggcggg 4260

ggatccctgg tcccccgggg cagcggaggc gacaaaactc acacatgccc accgtgccca 4320

gctccagaac tcctgggcgg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 4380

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 4440

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 4500

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 4560

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 4620

cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 4680

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 4740

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 4800

tacaagacca cgcctcccgt gttggactcc gacggctcct tcttcctcta cagcaagctc 4860

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 4920

gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga 4974

<210> 80

<211> 1662

<212> PRT

<213> Artificial sequence

<220>

<223> VWF057 (VWF D' D3-Fc with LVPR thrombin site in linker)

<400> 80

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Ala Pro Thr Ser Glu

1235 1240 1245

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser

1250 1255 1260

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1265 1270 1275

Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

1280 1285 1290

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

1295 1300 1305

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro

1310 1315 1320

Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1325 1330 1335

Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

1340 1345 1350

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala

1355 1360 1365

Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro

1370 1375 1380

Thr Ser Thr Glu Glu Gly Ala Ser Ser Gly Gly Gly Gly Ser Gly

1385 1390 1395

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1400 1405 1410

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu

1415 1420 1425

Val Pro Arg Gly Ser Gly Gly Asp Lys Thr His Thr Cys Pro Pro

1430 1435 1440

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

1445 1450 1455

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

1460 1465 1470

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

1475 1480 1485

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

1490 1495 1500

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

1505 1510 1515

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

1520 1525 1530

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

1535 1540 1545

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

1550 1555 1560

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

1565 1570 1575

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

1580 1585 1590

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

1595 1600 1605

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

1610 1615 1620

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

1625 1630 1635

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

1640 1645 1650

Lys Ser Leu Ser Leu Ser Pro Gly Lys

1655 1660

<210> 81

<211> 4959

<212> DNA

<213> Artificial sequence

<220>

<223> VWF059 (VWF D' D3-Fc having an acidic region thrombin site in the linker)

<400> 81

atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60

ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120

gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180

ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240

agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300

accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360

gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420

gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480

ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540

acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600

gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660

ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720

gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780

ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840

gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900

tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960

tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020

ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080

acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140

aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa gagctttgac 1200

aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260

cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320

acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380

ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa aggtgacctc 1440

cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500

gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560

tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620

ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680

gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740

gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800

ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860

tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920

gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980

tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040

gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100

tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160

atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220

agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280

agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340

ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400

agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460

tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520

acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580

catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640

ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700

aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760

tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820

gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880

tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940

tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000

ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060

tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120

tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180

agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240

ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgccgcattc 3300

tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360

aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420

gaggctgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480

gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540

aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600

gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660

cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720

atatcgggcg cgccaacatc agagagcgcc acccctgaaa gtggtcccgg gagcgagcca 3780

gccacatctg ggtcggaaac gccaggcaca agtgagtctg caactcccga gtccggacct 3840

ggctccgagc ctgccactag cggctccgag actccgggaa cttccgagag cgctacacca 3900

gaaagcggac ccggaaccag taccgaacct agcgagggct ctgctccggg cagcccagcc 3960

ggctctccta catccacgga ggagggcact tccgaatccg ccaccccgga gtcagggcca 4020

ggatctgaac ccgctacctc aggcagtgag acgccaggaa cgagcgagtc cgctacaccg 4080

gagagtgggc cagggagccc tgctggatct cctacgtcca ctgaggaagg gtcaccagcg 4140

ggctcgccca ccagcactga agaaggtgcc tcgatatctg acaagaacac tggtgattat 4200

tacgaggaca gttatgaaga tatttcagca tacttgctga gtaaaaacaa tgccattgaa 4260

ccaagaagct tctctgacaa aactcacaca tgcccaccgt gcccagctcc agaactcctg 4320

ggcggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 4380

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 4440

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 4500

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 4560

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 4620

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 4680

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 4740

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 4800

cccgtgttgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 4860

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 4920

tacacgcaga agagcctctc cctgtctccg ggtaaatga 4959

<210> 82

<211> 1652

<212> PRT

<213> Artificial sequence

<220>

<223> VWF059 (VWF D' D3-Fc with LVPR thrombin site in linker)

<400> 82

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Ala Pro Thr Ser Glu

1235 1240 1245

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser

1250 1255 1260

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1265 1270 1275

Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

1280 1285 1290

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

1295 1300 1305

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro

1310 1315 1320

Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1325 1330 1335

Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

1340 1345 1350

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala

1355 1360 1365

Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro

1370 1375 1380

Thr Ser Thr Glu Glu Gly Ala Ser Ile Ser Asp Lys Asn Thr Gly

1385 1390 1395

Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu

1400 1405 1410

Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Asp Lys Thr

1415 1420 1425

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

1430 1435 1440

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

1445 1450 1455

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

1460 1465 1470

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

1475 1480 1485

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

1490 1495 1500

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

1505 1510 1515

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

1520 1525 1530

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

1535 1540 1545

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

1550 1555 1560

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

1565 1570 1575

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

1580 1585 1590

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

1595 1600 1605

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

1610 1615 1620

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

1625 1630 1635

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

1640 1645 1650

<210> 83

<211> 4860

<212> DNA

<213> Artificial sequence

<220>

<223> VWF062 (VWF D' D3-Fc having no thrombin site in the linker)

<400> 83

atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60

ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120

gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180

ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240

agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300

accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360

gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420

gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480

ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540

acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600

gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660

ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720

gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780

ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840

gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900

tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960

tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020

ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080

acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140

aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa gagctttgac 1200

aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260

cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320

acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380

ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa aggtgacctc 1440

cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500

gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560

tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620

ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680

gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740

gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800

ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860

tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920

gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980

tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040

gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100

tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160

atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220

agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280

agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340

ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400

agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460

tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520

acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580

catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640

ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700

aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760

tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820

gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880

tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940

tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000

ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060

tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120

tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180

agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240

ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgccgcattc 3300

tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360

aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420

gaggctgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480

gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540

aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600

gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660

cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720

atatcgggcg cgccaacatc agagagcgcc acccctgaaa gtggtcccgg gagcgagcca 3780

gccacatctg ggtcggaaac gccaggcaca agtgagtctg caactcccga gtccggacct 3840

ggctccgagc ctgccactag cggctccgag actccgggaa cttccgagag cgctacacca 3900

gaaagcggac ccggaaccag taccgaacct agcgagggct ctgctccggg cagcccagcc 3960

ggctctccta catccacgga ggagggcact tccgaatccg ccaccccgga gtcagggcca 4020

ggatctgaac ccgctacctc aggcagtgag acgccaggaa cgagcgagtc cgctacaccg 4080

gagagtgggc cagggagccc tgctggatct cctacgtcca ctgaggaagg gtcaccagcg 4140

ggctcgccca ccagcactga agaaggtgcc tcgagcgaca aaactcacac atgcccaccg 4200

tgcccagctc cagaactcct gggcggaccg tcagtcttcc tcttcccccc aaaacccaag 4260

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 4320

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 4380

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 4440

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 4500

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 4560

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 4620

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 4680

aacaactaca agaccacgcc tcccgtgttg gactccgacg gctccttctt cctctacagc 4740

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 4800

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 4860

<210> 84

<211> 1619

<212> PRT

<213> Artificial sequence

<220>

<223> VWF062 (VWF D' D3-Fc having no thrombin site in the linker)

<400> 84

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Ala Pro Thr Ser Glu

1235 1240 1245

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser

1250 1255 1260

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1265 1270 1275

Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

1280 1285 1290

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr

1295 1300 1305

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro

1310 1315 1320

Thr Ser Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1325 1330 1335

Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

1340 1345 1350

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala

1355 1360 1365

Gly Ser Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro

1370 1375 1380

Thr Ser Thr Glu Glu Gly Ala Ser Ser Asp Lys Thr His Thr Cys

1385 1390 1395

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

1400 1405 1410

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

1415 1420 1425

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

1430 1435 1440

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

1445 1450 1455

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

1460 1465 1470

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

1475 1480 1485

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

1490 1495 1500

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

1505 1510 1515

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

1520 1525 1530

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

1535 1540 1545

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

1550 1555 1560

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

1565 1570 1575

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

1580 1585 1590

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

1595 1600 1605

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

1610 1615

<210> 85

<211> 6033

<212> DNA

<213> Artificial sequence

<220>

<223> FVIII286 (FVIII-Fc with another a2 region between FVIII and Fc)

<400> 85

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcta gcttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctctcaaa acggcgcgcc aggtacctca gagtctgcta cccccgagtc agggccagga 2340

tcagagccag ccacctccgg gtctgagaca cccgggactt ccgagagtgc cacccctgag 2400

tccggacccg ggtccgagcc cgccacttcc ggctccgaaa ctcccggcac aagcgagagc 2460

gctaccccag agtcaggacc aggaacatct acagagccct ctgaaggctc cgctccaggg 2520

tccccagccg gcagtcccac tagcaccgag gagggaacct ctgaaagcgc cacacccgaa 2580

tcagggccag ggtctgagcc tgctaccagc ggcagcgaga caccaggcac ctctgagtcc 2640

gccacaccag agtccggacc cggatctccc gctgggagcc ccacctccac tgaggaggga 2700

tctcctgctg gctctccaac atctactgag gaaggtacct caaccgagcc atccgaggga 2760

tcagctcccg gcacctcaga gtcggcaacc ccggagtctg gacccggaac ttccgaaagt 2820

gccacaccag agtccggtcc cgggacttca gaatcagcaa cacccgagtc cggccctggg 2880

tctgaacccg ccacaagtgg tagtgagaca ccaggatcag aacctgctac ctcagggtca 2940

gagacacccg gatctccggc aggctcacca acctccactg aggagggcac cagcacagaa 3000

ccaagcgagg gctccgcacc cggaacaagc actgaaccca gtgagggttc agcacccggc 3060

tctgagccgg ccacaagtgg cagtgagaca cccggcactt cagagagtgc cacccccgag 3120

agtggcccag gcactagtac cgagccctct gaaggcagtg cgccagcctc gagcccacca 3180

gtcttgaaac gccatcaagc tgaaataact cgtactactc ttcagtcaga tcaagaggaa 3240

atcgattatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 3300

gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 3360

gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 3420

gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 3480

tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 3540

tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 3600

ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 3660

cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 3720

catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 3780

gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 3840

acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 3900

atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 3960

ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 4020

aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 4080

tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 4140

gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 4200

gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 4260

attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 4320

cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 4380

caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 4440

tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 4500

cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 4560

atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 4620

accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 4680

cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 4740

cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 4800

agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 4860

acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 4920

caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 4980

ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 5040

atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 5100

gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 5160

ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 5220

gaggttctgg gctgcgaggc acaggacctc tacgacaaga acactggtga ttattacgag 5280

gacagttatg aagatatttc agcatacttg ctgagtaaaa acaatgccat tgaaccaaga 5340

agcttctctg acaaaactca cacatgccca ccgtgcccag ctccagaact cctgggcgga 5400

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 5460

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 5520

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 5580

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 5640

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 5700

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 5760

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 5820

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 5880

ttggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 5940

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 6000

cagaagagcc tctccctgtc tccgggtaaa tga 6033

<210> 86

<211> 1991

<212> PRT

<213> Artificial sequence

<220>

<223> FVIII286 (FVIII-Fc with another a2 region between FVIII and Fc)

<400> 86

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr

1 5 10 15

Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro

20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys

35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro

50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val

65 70 75 80

Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val

85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala

100 105 110

Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val

115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn

130 135 140

Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser

145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu

165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu

180 185 190

His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp

195 200 205

His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser

210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg

225 230 235 240

Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His

245 250 255

Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu

260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile

275 280 285

Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly

290 295 300

Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met

305 310 315 320

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg

325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp

340 345 350

Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe

355 360 365

Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His

370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu

385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro

405 410 415

Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr

420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile

435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile

450 455 460

Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile

465 470 475 480

Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys

485 490 495

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys

500 505 510

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys

515 520 525

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala

530 535 540

Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp

545 550 555 560

Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe

565 570 575

Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln

580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe

595 600 605

Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser

610 615 620

Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu

625 630 635 640

Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr

645 650 655

Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro

660 665 670

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp

675 680 685

Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala

690 695 700

Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu

705 710 715 720

Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala

725 730 735

Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly Thr Ser Glu

740 745 750

Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly

755 760 765

Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

770 775 780

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu

785 790 795 800

Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu

805 810 815

Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

820 825 830

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro

835 840 845

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro

850 855 860

Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

865 870 875 880

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr

885 890 895

Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr Pro

900 905 910

Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

915 920 925

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro

930 935 940

Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly

945 950 955 960

Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu

965 970 975

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr

980 985 990

Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser Gly

995 1000 1005

Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly

1010 1015 1020

Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Ala Ser

1025 1030 1035

Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu Ile Thr Arg Thr

1040 1045 1050

Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile

1055 1060 1065

Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp

1070 1075 1080

Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr

1085 1090 1095

Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser

1100 1105 1110

Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro

1115 1120 1125

Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe

1130 1135 1140

Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

1145 1150 1155

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val

1160 1165 1170

Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser

1175 1180 1185

Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

1190 1195 1200

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys

1205 1210 1215

Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys

1220 1225 1230

Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His

1235 1240 1245

Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu

1250 1255 1260

Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu

1265 1270 1275

Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu

1280 1285 1290

Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu

1295 1300 1305

Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly

1310 1315 1320

Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln

1325 1330 1335

Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile

1340 1345 1350

His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys

1355 1360 1365

Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe

1370 1375 1380

Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val

1385 1390 1395

Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu

1400 1405 1410

Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala

1415 1420 1425

Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr

1430 1435 1440

Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser

1445 1450 1455

Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val

1460 1465 1470

Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly

1475 1480 1485

Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile

1490 1495 1500

Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn

1505 1510 1515

Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser

1520 1525 1530

Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr

1535 1540 1545

Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg

1550 1555 1560

Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu

1565 1570 1575

Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser

1580 1585 1590

Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala

1595 1600 1605

Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val

1610 1615 1620

Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met

1625 1630 1635

Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr

1640 1645 1650

Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly

1655 1660 1665

His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe

1670 1675 1680

Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp

1685 1690 1695

Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp

1700 1705 1710

Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala

1715 1720 1725

Gln Asp Leu Tyr Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser

1730 1735 1740

Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile

1745 1750 1755

Glu Pro Arg Ser Phe Ser Asp Lys Thr His Thr Cys Pro Pro Cys

1760 1765 1770

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

1775 1780 1785

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

1790 1795 1800

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

1805 1810 1815

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

1820 1825 1830

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

1835 1840 1845

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

1850 1855 1860

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

1865 1870 1875

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

1880 1885 1890

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

1895 1900 1905

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

1910 1915 1920

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

1925 1930 1935

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

1940 1945 1950

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

1955 1960 1965

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

1970 1975 1980

Ser Leu Ser Leu Ser Pro Gly Lys

1985 1990

<210> 87

<211> 5937

<212> DNA

<213> Artificial sequence

<220>

<223> FVIII 169

<400> 87

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcta gcttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctctcaaa acggcgcgcc aggtacctca gagtctgcta cccccgagtc agggccagga 2340

tcagagccag ccacctccgg gtctgagaca cccgggactt ccgagagtgc cacccctgag 2400

tccggacccg ggtccgagcc cgccacttcc ggctccgaaa ctcccggcac aagcgagagc 2460

gctaccccag agtcaggacc aggaacatct acagagccct ctgaaggctc cgctccaggg 2520

tccccagccg gcagtcccac tagcaccgag gagggaacct ctgaaagcgc cacacccgaa 2580

tcagggccag ggtctgagcc tgctaccagc ggcagcgaga caccaggcac ctctgagtcc 2640

gccacaccag agtccggacc cggatctccc gctgggagcc ccacctccac tgaggaggga 2700

tctcctgctg gctctccaac atctactgag gaaggtacct caaccgagcc atccgaggga 2760

tcagctcccg gcacctcaga gtcggcaacc ccggagtctg gacccggaac ttccgaaagt 2820

gccacaccag agtccggtcc cgggacttca gaatcagcaa cacccgagtc cggccctggg 2880

tctgaacccg ccacaagtgg tagtgagaca ccaggatcag aacctgctac ctcagggtca 2940

gagacacccg gatctccggc aggctcacca acctccactg aggagggcac cagcacagaa 3000

ccaagcgagg gctccgcacc cggaacaagc actgaaccca gtgagggttc agcacccggc 3060

tctgagccgg ccacaagtgg cagtgagaca cccggcactt cagagagtgc cacccccgag 3120

agtggcccag gcactagtac cgagccctct gaaggcagtg cgccagcctc gagcccacca 3180

gtcttgaaac gccatcaagc tgaaataact cgtactactc ttcagtcaga tcaagaggaa 3240

atcgattatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 3300

gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 3360

gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 3420

gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 3480

tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 3540

tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 3600

ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 3660

cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 3720

catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 3780

gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 3840

acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 3900

atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 3960

ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 4020

aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 4080

tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 4140

gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 4200

gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 4260

attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 4320

cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 4380

caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 4440

tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 4500

cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 4560

atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 4620

accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 4680

cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 4740

cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 4800

agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 4860

acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 4920

caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 4980

ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 5040

atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 5100

gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 5160

ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 5220

gaggttctgg gctgcgaggc acaggacctc tacgacaaaa ctcacacatg cccaccgtgc 5280

ccagctccag aactcctggg cggaccgtca gtcttcctct tccccccaaa acccaaggac 5340

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 5400

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 5460

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 5520

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 5580

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 5640

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 5700

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 5760

aactacaaga ccacgcctcc cgtgttggac tccgacggct ccttcttcct ctacagcaag 5820

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 5880

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 5937

<210> 88

<211> 1978

<212> PRT

<213> Artificial sequence

<220>

<223> FVIII 169

<400> 88

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Gly Ala Pro Gly

755 760 765

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala

770 775 780

Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu

785 790 795 800

Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly

805 810 815

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu

820 825 830

Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser

835 840 845

Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly

850 855 860

Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser

865 870 875 880

Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser

885 890 895

Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly

900 905 910

Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser

915 920 925

Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu

930 935 940

Ser Gly Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly

945 950 955 960

Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Ser Glu Pro Ala

965 970 975

Thr Ser Gly Ser Glu Thr Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser

980 985 990

Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly

995 1000 1005

Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro

1010 1015 1020

Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr

1025 1030 1035

Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser

1040 1045 1050

Ala Pro Ala Ser Ser Pro Pro Val Leu Lys Arg His Gln Ala Glu

1055 1060 1065

Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr

1070 1075 1080

Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile

1085 1090 1095

Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

1100 1105 1110

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr

1115 1120 1125

Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser

1130 1135 1140

Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

1145 1150 1155

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu

1160 1165 1170

His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp

1175 1180 1185

Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser

1190 1195 1200

Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly

1205 1210 1215

Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr

1220 1225 1230

Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu

1235 1240 1245

Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu

1250 1255 1260

Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His

1265 1270 1275

Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln

1280 1285 1290

Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp

1295 1300 1305

Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn

1310 1315 1320

Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His

1325 1330 1335

Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met

1340 1345 1350

Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser

1355 1360 1365

Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr

1370 1375 1380

Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr

1385 1390 1395

Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly

1400 1405 1410

Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly

1415 1420 1425

Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro

1430 1435 1440

Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala

1445 1450 1455

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His

1460 1465 1470

Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser

1475 1480 1485

Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile

1490 1495 1500

Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser

1505 1510 1515

Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr

1520 1525 1530

Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn

1535 1540 1545

Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile

1550 1555 1560

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg

1565 1570 1575

Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys

1580 1585 1590

Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln

1595 1600 1605

Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser

1610 1615 1620

Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp

1625 1630 1635

Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe

1640 1645 1650

Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys

1655 1660 1665

Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser

1670 1675 1680

Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys

1685 1690 1695

Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val

1700 1705 1710

Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His

1715 1720 1725

Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu

1730 1735 1740

Gly Cys Glu Ala Gln Asp Leu Tyr Asp Lys Thr His Thr Cys Pro

1745 1750 1755

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

1760 1765 1770

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

1775 1780 1785

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

1790 1795 1800

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

1805 1810 1815

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

1820 1825 1830

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

1835 1840 1845

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

1850 1855 1860

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

1865 1870 1875

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

1880 1885 1890

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

1895 1900 1905

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

1910 1915 1920

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

1925 1930 1935

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

1940 1945 1950

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

1955 1960 1965

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

1970 1975

<210> 89

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Gly/Ser linker

<400> 89

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 90

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Gly/Ser linker

<400> 90

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 91

<211> 5379

<212> DNA

<213> Artificial sequence

<220>

<223> VWF034

<400> 91

atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60

ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120

gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180

ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240

agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300

accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360

gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420

gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480

ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540

acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600

gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660

ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720

gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780

ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840

gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900

tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960

tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020

ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080

acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140

aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa gagctttgac 1200

aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260

cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320

acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380

ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa aggtgacctc 1440

cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500

gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560

tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620

ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680

gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740

gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800

ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860

tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920

gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980

tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040

gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100

tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160

atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220

agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280

agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340

ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400

agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460

tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520

acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580

catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640

ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700

aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760

tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820

gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880

tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940

tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000

ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060

tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120

tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180

agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240

ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgccgcattc 3300

tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360

aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420

gaggctgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480

gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540

aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600

gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660

cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720

atatcgggta cctcagagtc tgctaccccc gagtcagggc caggatcaga gccagccacc 3780

tccgggtctg agacacccgg gacttccgag agtgccaccc ctgagtccgg acccgggtcc 3840

gagcccgcca cttccggctc cgaaactccc ggcacaagcg agagcgctac cccagagtca 3900

ggaccaggaa catctacaga gccctctgaa ggctccgctc cagggtcccc agccggcagt 3960

cccactagca ccgaggaggg aacctctgaa agcgccacac ccgaatcagg gccagggtct 4020

gagcctgcta ccagcggcag cgagacacca ggcacctctg agtccgccac accagagtcc 4080

ggacccggat ctcccgctgg gagccccacc tccactgagg agggatctcc tgctggctct 4140

ccaacatcta ctgaggaagg tacctcaacc gagccatccg agggatcagc tcccggcacc 4200

tcagagtcgg caaccccgga gtctggaccc ggaacttccg aaagtgccac accagagtcc 4260

ggtcccggga cttcagaatc agcaacaccc gagtccggcc ctgggtctga acccgccaca 4320

agtggtagtg agacaccagg atcagaacct gctacctcag ggtcagagac acccggatct 4380

ccggcaggct caccaacctc cactgaggag ggcaccagca cagaaccaag cgagggctcc 4440

gcacccggaa caagcactga acccagtgag ggttcagcac ccggctctga gccggccaca 4500

agtggcagtg agacacccgg cacttcagag agtgccaccc ccgagagtgg cccaggcact 4560

agtaccgagc cctctgaagg cagtgcgcca gattctggcg gtggaggttc cggtggcggg 4620

ggatccggtg gcgggggatc cggtggcggg ggatccggtg gcgggggatc cctggtcccc 4680

cggggcagcg gaggcgacaa aactcacaca tgcccaccgt gcccagctcc agaactcctg 4740

ggcggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 4800

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 4860

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 4920

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 4980

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 5040

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 5100

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 5160

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 5220

cccgtgttgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 5280

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 5340

tacacgcaga agagcctctc cctgtctccg ggtaaatga 5379

<210> 92

<211> 1778

<212> PRT

<213> Artificial sequence

<220>

<223> VWF034

<400> 92

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Ala Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Ala Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Ile Ser Gly Thr Ser Glu Ser Ala

1235 1240 1245

Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser

1250 1255 1260

Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

1265 1270 1275

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser

1280 1285 1290

Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro

1295 1300 1305

Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser

1310 1315 1320

Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

1325 1330 1335

Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro Gly Thr Ser

1340 1345 1350

Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Pro Ala Gly Ser

1355 1360 1365

Pro Thr Ser Thr Glu Glu Gly Ser Pro Ala Gly Ser Pro Thr Ser

1370 1375 1380

Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro

1385 1390 1395

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser

1400 1405 1410

Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Glu Ser Ala

1415 1420 1425

Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser

1430 1435 1440

Glu Thr Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser Glu Thr Pro

1445 1450 1455

Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser

1460 1465 1470

Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Thr Glu Pro

1475 1480 1485

Ser Glu Gly Ser Ala Pro Gly Ser Glu Pro Ala Thr Ser Gly Ser

1490 1495 1500

Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro

1505 1510 1515

Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Asp Ile Gly

1520 1525 1530

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Val Pro Arg Gly

1535 1540 1545

Ser Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1550 1555 1560

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

1565 1570 1575

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

1580 1585 1590

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

1595 1600 1605

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

1610 1615 1620

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

1625 1630 1635

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

1640 1645 1650

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

1655 1660 1665

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

1670 1675 1680

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

1685 1690 1695

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

1700 1705 1710

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

1715 1720 1725

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

1730 1735 1740

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

1745 1750 1755

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

1760 1765 1770

Leu Ser Pro Gly Lys

1775