US20210355473A1

US20210355473A1 - Chimeric clotting factors

Info

Publication number: US20210355473A1
Application number: US17/222,636
Authority: US
Inventors: Joe Salas; Robert Peters; Alan Bitonti
Original assignee: Bioverativ Therapeutics Inc
Current assignee: Bioverativ Therapeutics Inc
Priority date: 2010-07-09
Filing date: 2021-04-05
Publication date: 2021-11-18
Also published as: JP2013534427A; LT2591006T; EA201291482A1; EP3560962A1; PT2591006T; PL2591006T3; ES2739503T3; AU2011274423B2; US20210292729A1; NZ605400A; US9856468B2; AU2011274423A1; KR20140002601A; US20130216513A1; WO2012006633A1; MX2013000301A; WO2012006635A1; TW201217526A; CY1122044T1; EP2591006B1

Abstract

Chimeric clotting factors which localize the therapeutic to sites of coagulation (e.g., by being targeted to platelets or being activatable at sites of coagulation), have reduced clearance rates, have improved manufacturability, have reduced thrombogenicity, have enhanced activity, or have more than one of these characteristics are described as are methods for making chimeric clotting factors and methods for improving hemostasis using these clotting factors.

Description

REFERENCE TO EARLIER FILED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/809,289, which is the national phase application of International Application No. PCT/US2011/043597, filed Jul. 11, 2011 and published as WO 2012/006633, which claims the benefit of U.S. Provisional Application Nos. 61/363,183, filed Jul. 9, 2010; 61/363,186, filed Jul. 9, 2010; 61/442,055, filed Feb. 11, 2011; 61/442,150, filed Feb. 11, 2011; 61/442,029, filed Feb. 11, 2011; 61/467,880, filed Mar. 25, 2011; and 61/491,762, filed May 31, 2011, all of which are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name: 2159_3750008_SEQUENCE_LISTING.txt, Size: 603,490 bytes; and Date of Creation: Jul. 27, 2016) was originally submitted in the International Application No. PCT/US2011/043597 and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Initiation of the extrinsic clotting pathway is mediated by the formation of a complex between tissue factor, which is exposed as a result of injury to a vessel wall, and Factor VIIa. This complex then converts Factors IX and X to their active forms. Factor Xa converts limited amounts of prothrombin to thrombin on the tissue factor-bearing cell. This resulting thrombin is then able to diffuse away from the tissue-factor bearing cell and activate platelets, and Factors V and VIII, making Factors Va and VIIIa. During the propagation phase of coagulation, Factor Xa is generated by Factor IXa (in complex with factor VIIIa) on the surface of activated platelets. Factor Xa, in complex with the cofactor Factor Va, activates prothrombin into thrombin, generating a thrombin burst. The cascade culminates in the conversion of fibrinogen to fibrin by thrombin, which results in the formation of a fibrin clot. Factor VII and tissue factor are key players in the initiation of blood coagulation.
Factor VII is a plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive. Although single-chain Factor VII may be converted to two-chain Factor VIIa by a variety of factors in vitro, Factor Xa is an important physiological activator of Factor VII. The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of the peptide bond linking the Arginine residue at amino acid position 152 and the Ile residue at amino acid position 153. In the presence of tissue factor, phospholipids and calcium ions, the two-chain Factor VIIa activates Factor X or Factor IX. Factor VIIa is thought to be the physiologic initiator of the clotting cascade by acting at the surface of a TF-bearing cell, typically a damaged endothelial cell, and generating the initial amount of thrombin that then diffuses to platelets to activate them and prime them for the propagation phase of thrombin generation. Therapeutically, recombinant FVIIa acts by activating Factor X on the surface of activated platelets, bypassing the need for FIXa or FVIIIa to generate a thrombin burst during the propagation phase of coagulation. Since FVIIa has relatively low affinity for platelets, recombinant FVIIa is dosed at supra-physiological levels. This process is thought be tissue factor-independent.
Human factor IX circulates as a single-chain glycoprotein (mol wt 57,000). It is present in plasma as a zymogen and is converted to a serine protease, Factor IXaβ (more commonly referred to as FIXa), by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXaβ (mol wt approximately equal to 46,000). Factor IXalβ is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond.
Factor X is also synthesized as a single-chain polypeptide containing the light and heavy chains connected by an Arg-Lys-Arg tripeptide. The single-chain molecule is then converted to the light and heavy chains by cleavage of two (or more) internal peptide bonds.
In plasma, these two chains are linked together by a disulfide bond, forming Factor X. Activated Factor X, Factor Xa, participates in the final common pathway whereby prothrombin is converted to thrombin, which in turn converts fibrinogen to fibrin.
Clotting factors have been administered to patients to improve hemostasis for some time. The advent of recombinant DNA technology has significantly improved treatment for patients with clotting disorders, allowing for the development of safe and consistent protein therapeutics. For example, recombinant activated factor VII has become widely used for the treatment of major bleeding, such as that which occurs in patients having haemophilia A or B, deficiency of coagulation Factors XI or VII, defective platelet function, thrombocytopenia, or von Willebrand's disease. Recombinant factor IX is therapeutically useful as well.
Although such recombinant molecules are effective, there is a need for improved versions which localize the therapeutic to sites of coagulation, have improved pharmacokinetic properties, have reduced clearance rates, have improved manufacturability, have reduced thrombogenicity, or have enhanced activity, or more than one of these characteristics.

SUMMARY OF THE INVENTION

The instant invention relates to chimeric clotting factors which have enhanced activity. The present invention features inter alia methods for making chimeric clotting factors, the chimeric clotting factors made using these methods, and methods for improving hemostasis using these clotting factors. The chimeric clotting factors of the invention possess enhanced pharmacokinetic properties, have reduced clearance rates, have improved manufacturability, have reduced thrombogenicity, have enhanced activity, or more than one of these characteristics. In one embodiment, improved clotting factors of the invention have increased activity where needed, e.g., by targeting the clotting factor to platelets or by being present in a subject in an activatable form (a non-naturally occurring activatable form) that is activated at the site of clot formation.
In one aspect, the invention pertains to a chimeric clotting factor which comprises a clotting factor selected from the group consisting of FVII, FIX and FX and a targeting moiety which binds to platelets and optionally a spacer moiety between the clotting factor and the targeting moiety.
In one embodiment, the clotting factor comprises a structure represented by the formula A B C, wherein A is the clotting factor; wherein B is a spacer moiety; and wherein C is at least one targeting moiety which binds to platelets.
In one embodiment, the clotting factor comprises a structure from amino terminus to carboxy terminus represented by a formula selected from the group consisting of: A B C; C B A
In one embodiment, the clotting factor exhibits increased generation of thrombin in the presence of platelets as compared to an appropriate control lacking the at least one targeting moiety.
In one embodiment, the clotting factor comprises a scaffold moiety and, optionally, a second spacer moiety.
In one embodiment, the clotting factor further comprises D and E, wherein D is a spacer moiety; and E is a scaffold moiety and wherein the chimeric clotting factor comprises a structure from amino terminus to carboxy terminus represented by a formula selected from the group consisting of: A B C D E; A D E B C; E D A B C; C B A D E; E D C B A; and C B E D A.
In one embodiment, E is a dimeric Fc region comprising a first Fc moiety, F1 and a second Fc moiety, F2.
In one embodiment, the clotting factor is expressed as a polypeptide comprising a cleavable scFc (cscFc) linker interposed between two Fc moieties, wherein the cscFc linker is adjacent to at least one enzymatic cleavage site which results in cleavage of the cscFc polypeptide linker.
In one embodiment, the cscFc linker is adjacent to at least one enzymatic cleavage site which results in cleavage of the cscFc linker.
In one embodiment, the chimeric clotting factor of claim 9, wherein the at least one enzymatic cleavage site is an intracellular processing site.
In one embodiment, wherein the polypeptide linker is flanked by two enzymatic cleavage sites which are recognized by the same or by different enzymes.
In one embodiment, the polypeptide linker has a length of about 10 to about 50 amino acids.
In one embodiment, the polypeptide linker has a length of about 20 to about 30 amino acids.
In one embodiment, the polypeptide linker comprises a gly/ser peptide.
In one embodiment, the gly/ser peptide is of the formula (Gly₄Ser)n, or Ser(Gly₄Ser)n wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In one embodiment, the (Gly₄Ser)n linker is selected from the group consisting of (Gly₄Ser)6, Ser(Gly₄Ser)6, (Gly₄Ser)4 and Ser(Gly₄Ser)4.
In one embodiment, the clotting factor comprises two polypeptide chains.
In one embodiment, the chimeric clotting factor has a structure selected from the group consisting of: A linked to F1 via a spacer moiety and C linked to F2; A linked to F1 via a spacer moiety and C linked to F2 via a spacer moiety; A linked to F1 and C is linked to F2 via a spacer moiety; A linked to F1 via a spacer moiety and C is linked to F2 via a spacer moiety.
In one embodiment, a chimeric clotting factor comprises two polypeptides wherein the first polypeptide comprises the moieties A B F1; A B F1; A B F1; or A B F1 D C and the second polypeptide comprises the moieties C F2; C D F2; F2 D C; or F2 D C, wherein the two polypeptide chains form an Fc region.
In one embodiment, the targeting moiety is fused to at least one of the polypeptide chains of the Fc region. In one embodiment, the targeting moiety is fused to at least one of F1 and F2 directly. In one embodiment, the targeting moiety is fused to at least one of F1 and F2 via a spacer moiety. In one embodiment, the targeting moiety is fused to at least one of F1 and F2 via a cleavable linker. In one embodiment, the targeting moiety is selected from the group consisting of: an antibody molecule, an antigen binding fragment of an antibody molecule, an scFv molecule, a receptor binding portion of a receptor, a peptide. In one embodiment, wherein the targeting moiety binds to resting platelets. In one embodiment, the targeting moiety selectively binds to activated platelets. In one embodiment, the targeting moiety selectively binds to a target selected from the group consisting of: GPIba, GPVI, and the nonactive form of GPIIb/IIIa. In one embodiment, the targeting moiety selectively binds to a target selected from the group consisting of: the active form of GPIIb/IIIa, P selectin, GMP-33, LAMP-1, LAMP-2, CD40L, and LOX-1. In one embodiment, the targeting moiety binds to the GPIb complex In one embodiment, the targeting moiety is a peptide selected from the group consisting of: PS4, OS1, and OS2. In one embodiment, the targeting moiety comprises an antibody variable regions from an antibody selected from the group consisting of: SCE5, MB9, and AP3.
In one embodiment, wherein the clotting factor is Factor VII.
In one embodiment, the clotting factor is a high specific activity variant of Factor VII. In one embodiment, the clotting factor is Factor IX. In one embodiment, the clotting factor is a high specific activity variant of Factor IX. In one embodiment, the clotting factor is Factor X. In one embodiment, clotting factor is a high specific activity variant of Factor X.
In one embodiment, the clotting factor is secreted by a cell in active form. In one embodiment, the clotting factor is activated in vivo.
In one embodiment, the chimeric clotting factor comprises a heterologous enzymatic cleavage site not naturally present in the clotting factor.
In one embodiment, the enzymatic cleavage site is genetically fused to the amino terminus of the heavy chain moiety of the clotting factor.
In one embodiment, the clotting factor comprises a scaffold moiety is a protein molecule which increases the hydrodynamic radius of the chimeric clotting factor. In one embodiment, the scaffold moiety, if present, is selected from the group consisting of albumin and XTEN®
In another aspect, the invention pertains to a polypeptide comprising FVII, which
FVII comprises a heterologous enzymatic cleavage site activatable by a component of the clotting cascade.
In one embodiment, the polypeptide comprises a scaffold moiety and, optionally, a spacer moiety.
In one embodiment, the scaffold moiety is a dimeric Fc region comprising a first Fc moiety, F1 and a second Fc moiety, F2.
In one embodiment, the clotting factor comprises two polypeptide chains.
In one embodiment, the chimeric clotting factor has a structure selected from the group consisting of: the clotting factor linked to the first Fc moiety via a spacer moiety; the clotting factor linked to the second Fc moiety via a spacer moiety; the clotting factor is directly linked to F1; and the clotting factor is directly linked to F2.
In one embodiment, the chimeric clotting factor further comprises a targeting moiety.
In one embodiment, the chimeric clotting factor is synthesized as a single polypeptide chain comprising a cscFc linker. In one embodiment, the cscFc linker is linked to (e.g., directly linked or adjacent to) at least one enzymatic cleavage site which results in cleavage of the linker.
In one embodiment, the at least one enzymatic cleavage site is an intracellular processing site. In one embodiment, the cscFc linker is flanked by two enzymatic cleavage sites which are recognized by the same or by different enzymes. In one embodiment, the cscFc linker has a length of about 10 to about 50 amino acids. In one embodiment, the cscFc linker has a length of about 20 to about 30 amino acids.
In one embodiment, the cscFc linker comprises a gly/ser peptide.
In one embodiment, wherein the gly/ser peptide is of the formula (Gly₄Ser)n, or Ser(Gly₄Ser)n wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In one embodiment, the (Gly₄Ser)n linker is selected from the group consisting of (Gly₄Ser)6, Ser(Gly₄Ser)6, (Gly₄Ser)4 and Ser(Gly₄Ser)4.
In one embodiment, the clotting factor is a high specific activity variant of Factor VII. In one embodiment, the heterologous enzymatic cleavage site present in the chimeric clotting factor is cleaved at the site of clot formation. In one embodiment, the cleavage site is selected from the group consisting of: a factor XIa cleavage site, a factor Xa cleavage site, and a thrombin cleavage site. In one embodiment, the enzymatic cleavage site is genetically fused to the amino terminus of the heavy chain moiety of the clotting factor.
In one embodiment, the targeting moiety binds to resting platelets. In one embodiment, the targeting moiety selectively binds to activated platelets.
In one embodiment, wherein the targeting moiety selectively binds to a target selected from the group consisting of: GPIba, GPVI, and the nonactive form of GPIIb/IIIa. In one embodiment, the targeting moiety selectively binds to a target selected from the group consisting of: the active form of GPIIb/IIIa, P selectin, GMP-33, LAMP-1, LAMP-2, CD40L, and LOX-1.
In one embodiment, the scaffold moiety is a protein molecule which increases the hydrodynamic radius of the chimeric clotting factor. In one embodiment, the scaffold moiety, if present, is selected from the group consisting of albumin and XTEN®
In one aspect the invention pertains to a linear sequence of moieties from amino terminus to carboxy terminus selected from the group consisting of: A B C; C B A; A B C D E; A D E B C, E D A B C, C B A D E, E D C B A, C B E D A, wherein A an activatable clotting factor, B is absent or is a linker, C is a targeting moiety, D is absent or is a linker, and E is a scaffold moiety.
In one embodiment, the clotting factor comprises a light and heavy chain of a clotting factor and each of the light and heavy chains are expressed as separate polypeptide chains.
In one embodiment, the invention pertains to a nucleic acid molecule encoding a chimeric clotting factor of the invention. In one embodiment, the nucleic acid molecule is present in a vector. In one embodiment, the vector further comprises a nucleotide sequence encoding an enzyme which cleaves at least one of the enzymatic cleavage sites.
In one embodiment, the invention pertains to a host cell comprising the expression vector of the invention. In one embodiment, the host cell expresses an enzyme capable of intracellular processing. In one embodiment, the enzyme is endogenous to the cell. In one embodiment, the enzyme is heterologous to the cell.
In another embodiment, the invention pertains to a method for producing a chimeric clotting factor comprising culturing the host cell in culture and recovering the chimeric clotting factor from the medium.
In another embodiment, the invention pertains to a processed, heterodimeric polypeptide comprising two polypeptide chains, wherein said processed, heterodimeric polypeptide is made by expressing the vector in a cell cultured in cell culture medium and isolating the mature, heterodimeric polypeptide from the culture medium.
In one embodiment, the invention pertains to a composition comprising a chimeric clotting factor and a pharmaceutically acceptable carrier.
In another embodiment, the invention pertains to a composition comprising the nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
In another embodiment, the invention pertains to a method for improving hemostasis in a subject, comprising administering the composition of the invention.
in one aspect, the invention pertains to an chimeric clotting factor which comprises a light chain moiety and a heavy chain moiety of a clotting factor, and at least one targeting moiety, wherein said targeting moiety (i) specifically binds to platelets, (ii) is not interposed between the light and heavy chains of the clotting factor, and wherein said chimeric clotting factor exhibits increased generation of thrombin in the presence of platelets as compared to an appropriate control lacking the at least one targeting moiety.
In another aspect, the invention pertains to an chimeric clotting factor, which comprises the moieties A-B-C-D-E in linear sequence wherein A is a clotting factor, an activatable clotting factor, or an activated clotting factor; B is absent or is a linker; C is a targeting moiety; D is absent or is a linker; and E is absent or is a scaffold moiety.
In still another aspect, the invention pertains to an chimeric clotting factor, which comprises a linear sequence of moieties from amino terminus to carboxy terminus selected from the group consisting of: ABC; ABCDE; ADEBC, EDABC, CBADE, EDCBA, CBEDA, wherein A is a clotting factor, an activatable clotting factor or an activated clotting factor, B is absent or is a linker, C is a targeting moiety, D is absent or is a linker, and E is a scaffold moiety.
In yet another aspect, the invention pertains to an chimeric clotting factor, which comprises a linear sequence of moieties from amino terminus to carboxy terminus selected from the group consisting of: ABF1:F2; ABF1:CDF2; ABF1:F2DC, ABF1DC:F2DC, wherein A is a clotting factor, an activatable clotting factor or an activated clotting factor, B is absent or is a linker, C is a targeting moiety, D is absent or is a linker, and F1 and F2 are each an Fc moiety, and: represents dimerization mediated by the F1 and F2 chains of two polypeptide chains.
In still another aspect, the invention pertains to an chimeric clotting factor which comprises a light chain moiety and a heavy chain moiety of a clotting factor, and at least one targeting moiety, wherein said targeting moiety specifically binds to platelets, wherein the chimeric clotting factor comprises a disulfide linked Fc region which comprises two polypeptide chains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates exemplary chimeric clotting factor constructs comprising a targeting domain. These exemplary constructs comprise an Fc region.

FIG. 2 illustrates exemplary chimeric clotting factor constructs comprising a targeting domain. These exemplary constructs comprise a cleavable single chain Fc (cscFc) in which the scFc linker is processed by a cell in which it is expressed to form a two chain Fc region.

FIG. 3 illustrates exemplary chimeric clotting factor constructs which comprise a targeting domain and wherein the clotting factor moiety lacks a Gla domain. These exemplary constructs comprise an Fc region.

FIG. 4 illustrates exemplary chimeric clotting factor constructs which comprise a targeting domain and wherein the clotting factor moiety lacks a Gla domain. These exemplary constructs comprise a cleavable single chain Fc (cscFc) in which the scFc linker is processed by a cell in which it is expressed to form a two chain Fc region.

FIG. 5 illustrates exemplary chimeric clotting factor constructs which are activated (e.g., after activation in vitro or by separate expression of the light and heavy chains of the clotting factor) or which are activatable, i.e., comprise a moiety which is cleavable in vivo at the site of a clot (see panels D and E). These exemplary constructs comprise an Fc region. Constructs A, B, and C did not express well in early experiments.

FIG. 6 illustrates exemplary chimeric clotting factor constructs which are activated (e.g., after activation in vitro or by separate expression of the light and heavy chains of the clotting factor) or which are activatable, i.e., comprise a moiety which is cleavable in vivo at the site of a clot. These exemplary constructs comprise a cleavable single chain Fc (cscFc) in which the scFc linker is processed by a cell in which it is expressed to form a two chain Fc region.

FIG. 7 illustrates exemplary chimeric clotting factor constructs comprising a targeting domain.

FIG. 8 illustrates exemplary chimeric clotting factor constructs which comprise a targeting domain and wherein the clotting factor moiety lacks a Gla domain.

FIG. 9 illustrates exemplary chimeric clotting factor constructs which are activated (e.g., after activation in vitro or by separate expression of the light and heavy chains of the clotting factor) or which are activatable, i.e., comprise a moiety which is cleavable in vivo at the site of a clot.

FIG. 10 shows SDS PAGE for purification and activation of FVII-011.

FIG. 11 shows SDS PAGE for purification of active FVII-053.

FIG. 12A shows schematics of FVII-011 and FVII-102, and FIG. 12B shows binding of FVIII-011 and FVII-027 to activated platelets determined by FACS.

FIGS. 13A, 13B, 13C, and 13D show thrombin generation assays to measure activity of FVII-027, FVII-011 and NOVOSEVEN® in the presence of activated platelets.

FIG. 14A shows that PAC-1 eliminates increased binding to platelets associated with FVII-027, and FIG. 14B shows that PAC-1 eliminates increased rates of thrombin generation associated with FVII-027.

FIG. 15 shows constructs used in a thrombin generation assay to measure activity of FVII-037 and NOVOSEVEN® in the presence of activated platelets.

FIGS. 16A, 16B, and 16C show thrombin generation assays to measure activity of FVII-037 and NOVOSEVEN® in the presence of activated platelets.

FIG. 17 shows the constructs used in the thrombin generation assay to measure activity of FVII-044, FVII-045, FVII-046, FVII-011 and NOVOSEVEN® in the presence of activated platelets shown in FIGS. 18A, 18B, 18C, and 18D.

FIGS. 18A, 18B, 18C, and 18D show thrombin generation assays to measure activity of FVII-044, FVII-045, FVII-046, FVII-011 and NOVOSEVEN® in the presence of activated platelets.

FIG. 19 shows the constructs used in the thrombin generation assay to measure activity of FVII-047, FVII-048, FVII-049, FVII-011 and NOVOSEVEN® in the presence of activated platelets shown in FIGS. 20A, 20B, 20C, and 20D.

FIGS. 20A, 20B, 20C, and 20D show thrombin generation assays to measure activity of FVII-047, FVII-048, FVII-049, FVII-011 and NOVOSEVEN® in the presence of activated platelets.

FIG. 21 shows the construct used in the thrombin generation assay to measure activity of FVII-053 and FVII-011 in the presence of activated platelets shown in FIGS. 22A, 22B, 22C, and 22D.

FIGS. 22A, 22B, 22C, and 22D show thrombin generation assays to measure activity of FVII-053 and FVII-011 in the presence of activated platelets.

FIGS. 23A and 23B show that PAC-1 eliminates increased rate of thrombin generation associated with FVII-053.

FIG. 24 shows the constructs used in the Western blot analysis of FVIIFc species following transient transfection of HEK 293 cells and protein A pulldown shown in FIG. 25.

FIG. 25 shows Western blot analysis of FVIIFc species following transient transfection of HEK 293 cells and protein A pulldown.

FIG. 26 shows Western blot of protein A immunoprecipitation following transient transfection of pSYN-FVII-024 with or without pSYN-PC5-003. Lane 1, pSYN-FVII-024, non reducing; lane 2, pSYN-FVII-024, non reducing; lane 3, pSYN-FVII-024, reducing; lane 4, pSYN-FVII-024, reducing.

FIG. 27 shows Western blot analysis (Fc western) of FVIIFc species following transient transfection of HEK 293 cells and protein A pulldown.

FIG. 28 shows FVII-039 and FVII-040 treatment by FXIa.

FIG. 29 shows that an FVIIaFc variant targeted to active form of GPIIbIlla shows an increased rate of thrombin generation.

FIG. 30 shows a Rotation Thromoboelastometry (ROTEM) assay to measure the activity of FVII-088 and wild type recombinant FVIIaFc in hemophilia A human blood. Clotting Time, Clot Forming Time and Alpha Angle parameters are shown.

FIG. 31 shows exemplary cleavage sites and illustrative positions of such cleavage sites in activatable clotting factor constructs. In this Figure FVII is used as an example.

FIG. 32 shows cleavage of the constructs illustrated in FIG. 31.

FIG. 33 shows additional activatable constructs and a Western blot illustrating their cleavage.

FIG. 34 shows the results of a thrombin generation assay using the FVII-062 and -090 constructs. FVII-062 is a negative control which lacks a thrombin cleavage site, so the construct cannot be activated. FVII-090 contains the ALRPR cleavage site and so is activatable by thrombin.

FIG. 35 illustrates the cleavage of high specific activity FVII variants. FVII heavy chain-Fc and light chain Fc collapse in 1 band because the heavy chain loses a glycosylation site after insertion of the trypsin 170 loop and becomes smaller.

FIG. 36 illustrates the results of a thrombin generation assay using FVII-090 and

FVII-100.

FIG. 37 illustrates the results of a thrombin generation assay using FVII-090 and

FVII-115.

FIG. 38 illustrates amidolytic activity of activatable FVIIFC activated with thrombin.

Amidolytic activity of the activatable variants can be measured following thrombin activation and there is increased amidolytic activity for the high specific activity variants as compared to FVII-090. In these assays, after activation of the activatable molecule by thrombin, hirudin is added to inhibit thrombin cleavage of the chromogenic substrate. In this manner, the thrombin does not interfere with the ability to detect FVIIa activity.

FIG. 39 illustrates the results of an assay measuring activation of FX by FVIIa using substrate S2765, which is not cleaved by FVIIa. In this assay, 10 uM of FX was incubated with FVIIaFc for 15 minutes at 37° C. The reaction was quenched with EDTA and substrate was added. FIG. 39 shows the results of the control experiment which demonstrates that FX activation by FVIIaFc can be detected.

FIG. 40 shows FXa generation activity by “activatable FVIIFc.” The experiment shown in FIG. 40 shows that there is an increase in FX activation activity for the high specific activity variants. In this experiment, FVIIFc (100 nM) was activated with thrombin (100 nM) Hirudin was added to inhibit the thrombin. FX (10 uM) was added, followed by EDTA to inhibit the reaction. The activity of FX was measured by detecting the FXa substrate.

FIG. 41 illustrates exemplary activatable construct formats, including an activatable monomer structure used in FVII-118, FVII-119, and FVII-127.

FIG. 42 illustrates the efficiency of thrombin cleavage of activatable constructs, specifically monomeric (FVII-118 and -119) as compared to the heterodimeric (FVII-090).

FIG. 43 illustrates the results of a thrombin generation assay to compare wild type activatable FVIIFc (FVII-118) to the high specific activity variant (FVII-127).

FIG. 44A illustrates several targeted constructs. In this instance, an SCE5 scFv which binds to the active conformation of GPIIbIIIa was included at various sites in the construct.

FIG. 44B illustrates the results of thrombin generation assays in platelet-rich FVIII-deficient plasma using these constructs. N7 is the NOVOSEVEN® control. FIG. 44C illustrates the binding of recombinant FVIIaFc variants to platelets by FACs.

FIG. 45A illustrates several targeted FVIIa constructs which include AP3, an scFv against GPIIbIIIa present on resting and activated platelets. FIG. 45B shows the results of thrombin generation assays in platelet-rich FVIII-deficient plasma. FIG. 45C shows the results of binding of rFVIIaFc variants to platelets by FACS.

FIG. 46A shows several targeted FVIIa constructs that target GPIb-alpha using peptides that bind to that molecule, specifically, the PS4, OS1, and OS2 peptides. FIG. 46B shows the results of thrombin generation assays in platelet-rich FVIII-deficient plasma using the C terminal peptide constructs shown in FIG. 46A.

FIG. 47A shows the results of thrombin generation assays in platelet-rich FVIII-deficient plasma using the N terminal peptide constructs shown in FIG. 46A. FIG. 47B shows a direct comparison of FVII-045 and FVII-048.

FIG. 48 shows the binding of FVII-045 and FVII-048 and wild type FVIIaFc to platelets as determined by FACS. The figure also shows the affinity of the targeting peptides as reported in Bernard et al. Biochemistry 2008, 47:4674-4682.

FIG. 49A shows an exemplary targeted FVIII construct. FIG. 49B shows the results of a thrombin generation assay in FVIII deficient platelet-rich plasma. In this experiment, the assay was activated with tissue factor (top panel) or by platelet activation (bottom panel).

FIG. 50 shows results of an experiment measuring half life of a targeted FVII construct comprising a g1a domain (FVII-011) and lacking a g1a domain (FVII-028).

FIG. 51A shows several FIX construct comprising targeting moieties, in this case SCE5 scFv. FIG. 51B shows the results of thrombin generation assays in platelet-rich FIX-deficient plasma using the constructs of FIG. 51A. FIG. 51C illustrates that both FIX-068 and FIX-088 have at least 4 times more activity than FIX-042 as measured by Thrombin generation.

FIG. 52A shows the results of a thrombin generation assay comparing FIX-090 and BENEFIX®. FIG. 52B shows that the activity of FIX-090 is almost 4 times that of BENEFIX®.

FIG. 53A shows a targeted FIX construct comprising a peptide that binds to GPIb, present on resting and activated platelets. FIG. 53B shows the results of thrombin generation assays in platelet-rich FIX deficient plasma. FIG. 53C demonstrates that FIX-089 is roughly 4-times stronger than FIX-042 as measured by thrombin generation, while having a lower specific activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to chimeric clotting factors. The present invention is based, at least in part, on the development of novel ways to enhance the efficacy, pharmacokinetic properties, and/or manufacturability of clotting factors. In one embodiment, improved clotting factors of the invention have increased activity where needed, e.g., by targeting the clotting factor to platelets or by being present in a subject in an activatable form (a non-naturally occurring activatable form) that is activated at the site of clot formation. This can be accomplished, e.g., by targeting the clotting factors or by making them in an activatable form.
In one embodiment, the subject clotting factors are targeted to the site of coagulation. By incorporating a targeting moiety which targets the clotting factor to resting or activated platelets, the activity of a clotting factor can be enhanced. For example, in the case of factor VII, unlike endogenous FVII that is likely activated by tissue factor (TF) at endothelial cell surfaces to generate activated factor X (FXa), exogenous FVIIa likely generates FXa/FIXa in a TF independent manner, most effective at the surface of activated platelets where other clotting factors are localized. However, physiologically FVIIa acts at the surface of a TF-bearing cell, such as an endothelial cell, and has low affinity for platelets. It has been hypothesized that therapeutic recombinant FVIIa acts by converting Factor X into Factor Xa on the surface of activated platelets. To overcome low platelet affinity and be effective at treating bleeds, recombinant FVIIa is dosed at supra-physiological levels. Therefore, in the case of FVIIa, targeting to platelet surfaces could significantly increase the efficacy of this molecule. Although other clotting factors (e.g. FIX, FVIII, FX) have higher affinity to platelets, these too may exhibit enhanced activity by incorporating platelet targeting moieties. In addition, FVIIa has a relatively short half-life (˜2.3 hours) in humans. This short half-life likely contributes to the need to dose recombinant FVIIa multiple times to control a bleed. Thus, targeting clotting factors, and in particular FVIIa, to platelets improves efficiency.
The targeting moiety can be positioned at a number of places in a chimeric doting factor. Exemplary structures of targeted chimeric clotting factors are set forth, e.g., in FIGS. 1-4, 7, 8, 17, 19, 21, 44, 46, 49, 51, and 53.
In another embodiment, a chimeric clotting factor of the invention is made in a form that is activatable at the cite of coagulation. For use in bypass therapy exogenous clotting factors are only efficacious when given in the activated form. However, such activated clotting factors are rapidly inactivated by endogenous pathways (e.g. antithrombin III, TFPI), leading to clearance of the active form and a short effective half life. Giving higher doses does not solve this problem as it can result in thrombogenic effects. Thus, in one embodiment, the invention pertains to an “activatable” chimeric clotting factor constructs which comprise a heterologous enzymatic cleavage site not normally present in the clotting factor. These molecules circulate as enhanced zymogens and have a longer half life due to the lack of inactivation upon dosing, but can readily be activated at the site of clotting by cleavage by an enzyme. In one embodiment, such a heterologous enzymatic cleavage site is one for an enzyme produced during the clotting cascade. For example, in one embodiment, the heterologous cleavage site of an activatable construct comprises a Factor XIa, Xa, or thrombin cleavage site. Exemplary FXIa cleavage sites include, e.g.: TQSFNDFTR and SVSQTSKLTR. Exemplary thrombin cleavage sites include, e.g.: DFLAEGGGVR, TTKIKPR, and ALRPR. In one embodiment, a heterologous cleavage site is interposed between the light and heavy chains of the clotting factor. In another embodiment, a heterologous cleavage site is not interposed between the two chains of the clotting factor. In one embodiment, the heterologous cleavage site is amino terminal to the heavy chain of the clotting factor.
The heterologous cleavage site is present in a cleavable linker can be positioned at a number of places in a chimeric doting factor. Exemplary structures of activatable chimeric clotting factors are set forth, e.g., in FIGS. 5, 6, 9, 29, 27, 31, and 41. Exemplary such constructs are activated in the presence of clot formation and are described in more detail below.
In one embodiment, a chimeric clotting factor of the invention comprises a scaffold, e.g., to enhance the hydrodynamic radius of the molecule. For example, a chimeric clotting factor of the invention may be a fusion protein. Exemplary scaffolds include, e.g., FcRn binding moieties (e.g., complete Fc regions or portions thereof which bind to FcRn), single chain Fc regions (ScFc regions, e.g., as described in US 2008/0260738, WO 2008/012543, or WO 2008/1439545), cleaveable scFc regions (comprising a cscFc regions as described herein), less complicated proteins or portions thereof, e.g., XTen polypeptides®, or albumin.
In one embodiment, a chimeric clotting factor of the invention employs an Fc region or an FcRn binding portion thereof as a scaffold moiety. In one embodiment, the Fc moiety to which the chimeric clotting factor is fused is a naturally occurring (or wild type (WT)) Fc moiety. In another embodiment, the Fc moiety comprises one or more variations in sequence.
In another embodiment, the Fc moiety is a scFc moiety (e.g., comprising a non-cleavable or a cscFc linker). In a construct comprising a cscFc linker an unprocessed molecule comprises a cleavable single chain Fc region in which the component Fc moieties are genetically-fused in a single polypeptide chain forming a functional, single chain, dimeric Fc region. The cscFc linker can link the Fc moieties that will comprise the dimeric Fc region of the polypeptide in tandem or may link one Fc moiety to a non-Fc moiety of the construct, e.g., a clotting factor or targeting moiety, which is, in turn, linked to a second Fc moiety. The cscFc linker is interposed between the Fc moieties that comprise the scFc region and is flanked by at least one enzymatic cleavage site, e.g., an intracellular enzymatic processing sites. In one embodiment, the scFc linker is flanked by two enzymatic cleavage sites resulting in the excision of the linker (e.g., all or substantially all of the linker) when the protein encoded by the nucleic acid molecule is processed in a cell, In another embodiment, the scFc linker is adjacent to at least one enzymatic cleaveage site that allows for excision of the linker in vitro after the polypeptide has been secreted by a cell or comprises at least one enzymatic cleaveage site that allows for cleavage of the linker in vivo after the construct is administered to a subject. Thus, in one embodiment, although the such a polypeptide comprises scFc region(s) encoded in a single open reading frame (ORF) as part of one contiguous nucleotide sequence in unprocessed form, the cscFc linker is enzymatically cleaved (e.g., prior to administration or in vivo after administration), resulting in a polypeptide which is a heterodimeric molecule comprising an Fc region which is not fused in a single amino acid chain, i.e., the resulting processed construct has a Fc region which comprises two polypeptide chains. In such embodiments, all or substantially all of the linker is excised, while in some embodiments, a portion of the cleavage site may remain, e.g., four arginines of the RRRR cleavage site.
In one embodiment, the scFc linker is flanked by two processing sites for cleavage.
The two processing sites can be the same or different. In one embodiment, at least one processing site is a cluster of basic amino acid residues as recognized by arginine kex2/furin enzymes. Such enzymes cleave immediately C-terminal to an arginine residue. In another embodiment, at least one cleavage site is one that may be cleaved in vivo, for example a cleavage site recognized by thrombin.
In one embodiment, a chimeric clotting factor of the invention is manufactured in an activated form in the context of an scFc molecule comprising a csFc linker. For example, Factor VII, is generally produced recombinantly as a zymogen, and requires activation during manufacturing to produce the active form for administration. In one embodiment, a chimeric clotting factor of the invention is secreted from the cell in which it is expressed in active form to improve manufacturability. As is set forth in more detail below, such clotting factors can be produced by incorporating a single chain Fc region into the molecule. Single chain Fc regions are formed by dimerization of Fc moieties which are present in a single polypeptide chain. In one embodiment, such a construct comprises an scFc polypeptide linker linking the two Fc moieties of the scFc which is adjacent to at least one intracellular processing site. Cleavage of such a construct is delayed until late in the secretory pathway, e.g., when the protein colocalizes with active processing enzymes in the trans-Golgi apparatus.
In one embodiment, a cell expressing a construct encoding a polypeptide of the invention endogenously expresses an enzyme which cleaves the scFc linker at one or more processing sites resulting in a dimeric molecule comprising two polypeptide chains. In another embodiment, a cell expressing a construct encoding a polypeptide of the invention exogenously expresses an enzyme which cleaves the scFc linker at one or more processing sites.
In one embodiment, a chimeric clotting factor of the invention can combine two or more of these features to create an optimized construct e.g. targeting an activatable fusion protein construct to resting platelets, such that it can be activated efficiently as well as at a higher local concentration at the site of active coagulation. Exemplary such combination constructs include chimeric clotting factors that are both targeted and comprise an scFc linker for enhanced processing. In another embodiment, a construct of the invention is targeted and activatable.
Exemplary constructs of the invention are illustrated in the accompanying Figures and sequence listing. In one embodiment, the invention pertains to a polypeptide having the structure as set forth in the Figures. In another embodiment, the invention pertains to a polypeptide having the sequence set forth in the accompanying sequence listing or the the nucleic acid molecule encoding such polypeptides. In one embodiment, the invention pertains to a mature form of a polypeptide having the sequence set forth in the accompanying sequence listing. It will be understood that these constructs and nucleic acid molecules encoding them can be used to improve hemostasis in a subject.
In order to provide a clear understanding of the specification and claims, the following definitions are provided below.

I. Definitions

As used herein, the term “protein” or “polypeptide” refers to a polymer of two or more of the natural amino acids or non-natural amino acids.
The term “amino acid” includes alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional amino acids are also within the scope of the invention and include norleucine, omithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. Introduction of the non-traditional amino acid can also be achieved using peptide chemistries known in the art. As used herein, the term “polar amino acid” includes amino acids that have net zero charge, but have non-zero partial charges in different portions of their side chains (e.g. M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic interactions and electrostatic interactions. As used herein, the term “charged amino acid” include amino acids that can have non-zero net charge on their side chains (e.g. R, K, H, E, D). These amino acids can participate in hydrophobic interactions and electrostatic interactions.
An “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different “replacement” amino acid residue. An “amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present larger “peptide insertions”, can be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
Polypeptides may be either monomers or multimers. For example, in one embodiment, a protein of the invention is a dimer. A dimeric polypeptide of the invention may comprise two polypeptide chains or may consist of one polypeptide chain (e.g., in the case of an scFc molecule). In one embodiment, the dimers of the invention are homodimers, comprising two identical monomeric subunits or polypeptides (e.g., two identical Fc moieties or two identical biologically active moieties). In another embodiment, the dimers of the invention are heterodimers, comprising two non-identical monomeric subunits or polypeptides (e.g., comprising two different clotting factors or portions thereof or one clotting factor only). See, e.g., U.S. Pat. No. 7,404,956, incorporated herein by reference.
As used herein, the term “polypeptide linkers” refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) which connects two domains in a linear amino acid sequence of a polypeptide chain. In one embodiment, the polypeptides of invention are encoded by nucleic acid molecules that encode polypeptide linkers which either directly or indirectly connect the two Fc moieties which make up the construct. These linkers are referred to herein as “scFc linkers” and the scFc linker is interposed between the two Fc moieties of a polypeptide which comprises it. If the scFc linker connects two Fc moieties contiguously in the linear polypeptide sequence, it is a “direct” linkage. In contract, the scFc linkers may link the first Fc moiety to a binding moiety which is, in turn, linked to the second Fc moiety, thereby forming an indirect linkage. These scFc linkers permit the formation of a single chain genetic construct. In one embodiment, the polypeptides also comprise enzymatic cleavage sites which result in the scFc linker being cleavable (a cscFc linker) and, in one embodiment, substantially excised (e.g., during processing by a cell). Thus, the resulting processed polypeptide is a dimeric molecule comprising at least two amino acid chains and substantially lacking extraneous linker amino acid sequences. In some embodiments, all or substantially all of the linker is excised, while in some embodiments, a portion of the cleavage site may remain, e.g., four arginines of the RRRR cleavage site.
In another embodiment, another type of polypeptide linker, herein referred to as a “spacer” may be used to connect different moieties, e.g., a clotting factor or targeting moiety to an Fc moiety on the polypeptide. This type of linker may provide flexibility to the polypeptide molecule. Spacers are not typically cleaved; however in certain embodiments, such cleavage may be desirable. Exemplary positions of spacers are shown in the accompanying drawings. Spacers can be located between the clotting factors, targeting moieties, and/or scaffolds, e.g., at the N or C terminus of these moieties. In one embodiment, these linkers are not removed during processing.
A third type of linker which may be present in a chimeric clotting factor of the invention is herein referred to as a “cleavable linker” which comprises a heterologous cleavage site (e.g., a factor XIa, Xa, or thrombin cleavage site) and which may include additional spacer linkers on either the N terminal of C terminal or both sides of the cleavage site. Exemplary locations for such sites are shown in the accompanying drawings and include, e.g., placement adjacent to targeting moieties. In another embodiment, such linkers may be adjacent to a clotting factor or portion thereof. For example, in one embodiment, a cleavable linker may be fused to the N terminus of the heavy chain of a clotting factor to make an activatable form of the clotting factor. In such cases, the cleavable linker may include additional spacer linkers at the N terminus of the cleavage site, but requires direct fusion at the C-terminus of the cleavage site to the amino terminus of the heavy chain of the clotting factor.
As used herein, the term “gly-ser polypeptide linker” refers to a peptide that consists of glycine and serine residues. An exemplary gly/ser polypeptide linker comprises the amino acid sequence (Gly₄Ser)_n. (SEQ ID NO:4) Another exemplary gly/ser polypeptide linker comprises the amino acid sequence S(Gly₄Ser)_n.
In one embodiment, n=1. In one embodiment, n=2. In another embodiment, n=3, i.e., (Gly₄Ser)₃. In another embodiment, n=4, i.e., (Gly₄Ser)₄(SEQ ID NO:6). In another embodiment, n=5. In yet another embodiment, n=6. In another embodiment, n=7. In yet another embodiment, n=8. In another embodiment, n=9. In yet another embodiment, n=10. Another exemplary gly/ser polypeptide linker comprises the amino acid sequence Ser(Gly₄Ser)_n(SEQ ID NO:26). In one embodiment, n=1. In one embodiment, n=2. In a preferred embodiment, n=3. In another embodiment, n=4. In another embodiment, n=5. In yet another embodiment, n=6.
A polypeptide or amino acid sequence “derived from” a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence.
Polypeptides derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. Preferably, the polypeptide comprises an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting antibody. In a preferred embodiment, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule. In one embodiment, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e. same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
Preferred polypeptides of the invention comprise an amino acid sequence (e.g., at least one clotting factor or Fc moiety or domain) derived from a human protein sequence. However, polypeptides may comprise one or more amino acids from another mammalian species. For example, a clotting factor, Fc domain, or targeting moiety may be derived from a non-human species and included in the subject polypeptides. Alternatively, one or more amino acids may be present in a polypeptide which are derived from a non-human species. Preferred polypeptides of the invention are not immunogenic.
It will also be understood by one of ordinary skill in the art that the polypeptides of the invention may be altered such that they vary in amino acid sequence from the naturally occurring or native polypeptides from which they were derived, while retaining the desirable activity of the native polypeptides. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made. An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an Fc domain, moiety, or antigen binding site) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
The polypeptides of the invention may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a polypeptide may be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. Alternatively, in another embodiment, mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into polypeptides of the invention and screened for their ability to bind to the desired target.
In the context of polypeptides, a “linear sequence” or a “sequence” is the order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
As used herein, the terms “linked,” “fused”, or “fusion” refer to linkage via a peptide bonds (e.g., genetic fusion), chemical conjugation or other means. For example, one way in which molecules or moieties can be linked employs polypeptide linkers which link the molecules or moieties via peptide bonds. The terms “genetically fused,” “genetically linked” or “genetic fusion” are used interchangeably and refer to the co-linear, covalent linkage or attachment of two or more proteins, polypeptides, or fragments thereof via their individual peptide backbones, through genetic expression of a single polynucleotide molecule encoding those proteins, polypeptides, or fragments. Such genetic fusion results in the expression of a single contiguous genetic sequence. Preferred genetic fusions are in frame, i.e., two or more open reading frames (ORFs) are fused to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single polypeptide containing two or more protein segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). In this case, the single polypeptide is cleaved during processing to yield dimeric molecules comprising two polypeptide chains.
As used herein, the term “Fc region” is defined as the portion of a polypeptide which corresponds to the Fc region of native immunoglobulin, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region is homodimeric and comprises two polypeptide chains. In contrast, the term “genetically-fused Fc region” or “single-chain Fc region” (scFc region), as used herein, refers to a synthetic dimeric Fc region comprised of Fc domains genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence).
As used herein, the term “Fc domain” refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.
As used herein, the term “Fc domain portion” or “Fc moiety” includes an amino acid sequence of an Fc domain or derived from an Fc domain. In certain embodiments, an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc moiety comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, a Fc moiety comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In another embodiment, an Fc moiety comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In another embodiment, an Fc moiety consists of a CH3 domain or portion thereof. In another embodiment, an Fc moiety consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In another embodiment, a Fc moiety consists of a CH2 domain (or portion thereof) and a CH3 domain. In another embodiment, a Fc moiety consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In one embodiment, an Fc moiety lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
As used herein, the term “half-life” refers to a biological half-life of a particular polypeptide in vivo. Half-life may be represented by the time required for half the quantity administered to a subject to be cleared from the circulation and/or other tissues in the animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid α-phase and longer β-phase. The α-phase typically represents an equilibration of the administered Fc polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide. The β-phase typically represents the catabolism of the polypeptide in the intravascular space. Therefore, in a preferred embodiment, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical β phase half-life of a human antibody in humans is 21 days.
As used herein the term “moiety” refers to a component part or constituent of a chimeric polypeptide.
As used herein, the term “targeting moiety” refers to a molecule, fragment thereof or a component of a polypeptide which localizes or directs the polypeptides of the invention to a desired site or cell. In one embodiment, a construct of the invention comprises a “targeting moiety” which enhances the activity of the polypeptide, e.g., by localizing the molecule to a desired site. Such a moiety may be, e.g., an antibody or variant thereof (e.g., and scFv) or a peptide. In another embodiment, such a targeting moiety may be a polypeptide, a receptor binding portion of a ligand, or a ligand binding portion of a receptor which is linked to a polypeptide of the invention and binds to the desired target, e.g., on a cell or tissue. The targeting moiety may be genetically fused to a construct, chemically conjugated to the construct or linked to the construct via a spacer. For example, targeting moieties may be attached to a construct of the invention by formation of a bond between the targeting moiety and an Fc moiety of a construct, where the targeting moiety comprises a first functional group and the Fc moiety comprises a second functional group, and where the first and second functional groups are capable of reacting with each other to form a chemical bond (see, e.g., U.S. Pat. No. 7,381,408). In one embodiment, a targeting moiety binds to platelets. Exemplary targeting moieties are described in more detail below.
In one embodiment a targeting moiety for use in a construct of the invention comprises an antibody variant. The term “antibody variant” or “modified antibody” includes an antibody which does not occur in nature and which has an amino acid sequence or amino acid side chain chemistry which differs from that of a naturally-derived antibody by at least one amino acid or amino acid modification as described herein. As used herein, the term “antibody variant” includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules; single-chain antibodies; diabodies; triabodies; and antibodies with altered effector function and the like.
As used herein the term “scFv molecule” includes binding molecules which consist of one light chain variable domain (VL) or portion thereof, and one heavy chain variable domain (VH) or portion thereof, wherein each variable domain (or portion thereof) is derived from the same or different antibodies. scFv molecules preferably comprise an scFv linker interposed between the VH domain and the VL domain. ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019, Ho et al. 1989. Gene 77:51; Bird et al. 1988 Science 242:423; Pantoliano et al. 1991. Biochemistry 30:10117; Milenic et al. 1991. Cancer Research 51:6363; Takkinen et al. 1991. Protein Engineering 4:837.
A “scFv linker” as used herein refers to a moiety interposed between the VL and VH domains of the scFv. scFv linkers preferably maintain the scFv molecule in a antigen binding conformation. In one embodiment, a scFv linker comprises or consists of an scFv linker peptide. In certain embodiments, a scFv linker peptide comprises or consists of a glyser polypeptide linker. In other embodiments, a scFv linker comprises a disulfide bond.
The term “glycosylation” refers to the covalent linking of one or more carbohydrates to a polypeptide. Typically, glycosylation is a posttranslational event which can occur within the intracellular milieu of a cell or extract therefrom. The term glycosylation includes, for example, N-linked glycosylation (where one or more sugars are linked to an asparagine residue) and/or O-linked glycosylation (where one or more sugars are linked to an amino acid residue having a hydroxyl group (e.g., serine or threonine). In one embodiment, a molecule of the invention is glycosylated. In another embodiment, a molecule of the invention is glycosylated. In yet another embodiment, a molecule of the invention has reduced glycosylation as compared to that in a wild type Fc region.
As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by native disulfide bonds and the two heavy chains are linked by two native disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
The term “vector” or “expression vector” is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired polynucleotide in a cell. As known to those skilled in the art, such vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
Numerous expression vector systems may be employed to produce the chimeric clotting factors of the invention. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. In one embodiment, an inducible expression system can be employed. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In one embodiment, a secretion signal, e.g., any one of several well characterized bacterial leader peptides (e.g., pelB, phoA, or ompA), can be fused in-frame to the N terminus of a polypeptide of the invention to obtain optimal secretion of the polypeptide. (Lei et al. (1988), Nature, 331:543; Better et al. (1988) Science, 240:1041; Mullinax et al., (1990). PNAS, 87:8095).
The term “host cell” refers to a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of proteins from recombinant hosts, the terms “cell” and “cell culture” are used interchangeably to denote the source of protein unless it is clearly specified otherwise. In other words, recovery of protein from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells. The host cell line used for protein expression is most preferably of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), PerC6 cells), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature. The polypeptides of the invention can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the polypeptides typically become part of inclusion bodies. The polypeptides must be isolated, purified and then assembled into functional molecules.
In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available including Pichia pastoris. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., (1979), Nature, 282:39; Kingsman et al., (1979), Gene, 7:141; Tschemper et al., (1980), Gene, 10:157) is commonly used. This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, (1977), Genetics, 85:12). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
As used herein the term “endogenous” refers to molecules (e.g. nucleic acid and/or protein molecules) that are naturally present in a cell. In contrast, the term “exogenous” or “heterologous” refers to such molecules that are not normally found in a given context, e.g., in a cell or in a polypeptide. For example, an exogenous or heterologous molecule may be introduced into a cell and are only present after manipulation of the cell, e.g., by transfection or other forms of genetic engineering or a heterologous amino acid sequence may be present in a protein in which it is not naturally found.
As used herein, the term “cleavage site” or “enzymatic cleavage site” refers to a site recognized by an enzyme. Certain enzymatic cleavage sites comprise an intracellular processing site. In one embodiment, a polypeptide has an enzymatic cleavage site cleaved by an enzyme that is activated during the clotting cascade, such that cleavage of such sites occurs at the site of clot formation. Exemplary such sites include e.g., those recognized by thrombin, Factor XIa or Factor Xa. Exemplary FXIa cleavage sites include, e.g, TQSFNDFTR and SVSQTSKLTR. Exemplary thrombin cleavage sites include, e.g, DFLAEGGGVR, TTKIKPR, LVPRG SEQ ID NO:35) and ALRPR. Other enzymatic cleavage sites are known in the art.
As used herein, the term “processing site” or “intracellular processing site” refers to a type of enzymatic cleavage site in a polypeptide which is the target for enzymes that function after translation of the polypeptide. In one embodiment, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides prior to secretion of the protein from the cell. Examples of such processing sites include, e.g., those targeted by the PACE/furin (where PACE is an acronym for Paired basic Amino acid Cleaving Enzyme) family of endopeptidases. These enzymes are localized to the Golgi membrane and cleave proteins on the carboxyterminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein the “furin” family of enzymes includes, e.g., furin, PC2, PC1/Pc3, PC4, PACE4, PC5/PC6, and LPC/PC7/PC8/SPC7. Other processing sites are known in the art.
In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.
In vitro production allows scale-up to give large amounts of the desired altered polypeptides of the invention. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC, chromatography over DEAE-cellulose or affinity chromatography.
As used herein, the phrase “subject that would benefit from administration of a polypeptide” includes subjects, such as mammalian subjects, that would benefit from administration of polypeptides of the invention, e.g., to improve hemostasis.
A “chimeric protein” or “fusion protein”, as used herein, refers to any protein comprised of a first amino acid sequence derived from a first source, bonded, covalently or non-covalently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same. A first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein can include for example, a protein derived from at least 2 different biological sources. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog, as further described herein, of any of the above). A synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences). A chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein may also comprise a first amino acid sequence derived from a first source, covalently or non-covalently linked to a nucleic acid, derived from any source or a small organic or inorganic molecule derived from any source. The chimeric protein may comprise a linker molecule between the first and second amino acid sequence or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.
As used herein, the term “clotting factor,” refers to molecules, or analogs thereof, naturally occurring or recombinantly produced which prevent or decrease the duration of a bleeding episode in a subject. In other words, it means molecules having pro-clotting activity, i.e., are responsible for the conversion of fibrinogen into a mesh of insoluble fibrin causing the blood to coagulate or clot.
Clotting activity, as used herein, means the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and/or reduces the severity, duration or frequency of hemorrhage or bleeding episode.
Hemostasis, as used herein, means the stopping or slowing of bleeding or hemorrhage; or the stopping or slowing of blood flow through a blood vessel or body part.
Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot. Examples of such disorders include the hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or “Christmas disease”) and hemophilia C (factor XI deficiency, mild bleeding tendency), Von Willebrand disease, factor Xi deficiency (PTA deficiency), Factor XII deficiency, deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X or factor XIII, Bernard-Soulier syndrome is a defect or deficiency in GPIb. GPIb, the receptor for vWF, can be defective and lead to lack of primary clot formation (primary hemostasis) and increased bleeding tendency), and thrombasthenia of Glanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this may increase bleeding risk.
The chimeric molecules of the invention can be used prophylacticly. As used herein the term “prophylactic treatment” refers to the administration of a molecule prior to a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to or after surgery as a prophylactic. The chimeric protein of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, or stem cell transplantation
On-demand treatment includes treatment for a bleeding episode, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (head trauma), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. The subject may be in need of surgical prophylaxis, peri-operative management, or treatment for surgery. Such surgeries include, e.g., minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.
As used herein the term “acute bleeding” refers to a bleeding episode regardless of the underlying cause. For example, a subject may have trauma, uremia, a hereditary bleeding disorder (e.g., factor VII deficiency) a platelet disorder, or resistance owing to the development of antibodies to clotting factors.
Treat, treatment, treating, as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, the prophylaxis of one or more symptoms associated with a disease or condition.

II. Clotting Factors

In particular, the invention pertains to improved versions of factors VII, IX, and X. These factors are all structurally related in that in each the amino terminal end of the light chain is not amenable to the incorporation of additional moieties. Similarly, the amino terminal end of the heavy chain of these three clotting factors is not amenable to the incorporation of additional moieties, with the exception of cleaveable moieties, i.e., moieties linked via a cleavage site or moieties which consist of a cleaveage site. The chimeric clotting factor constructs of the invention were designed based on these shared properties and it will be understood that although factor VII is often shown to illustrate exemplary embodiments of the invention, the subject constructs may be made using factor VII, IX, or X. For example, one of skill in the art would understand that the FVII portion of a construct of the invention could be substituted with a FVIII, FIX or FX portion to make an enhanced version of one of these clotting factors.
Exemplary chimeric clotting factor constructs of the invention are set forth in the accompanying Figures. Although the Figures generally illustrate the clotting Factor as a single chain (in its zymogen form) it will be understood that the clotting factor may also be present in its active form in a construct of the invention, e.g. as a two chain, disulfide bonded form.
In one embodiment, a chimeric clotting factor of the invention is expressed by a cell in active form. In another embodiment, a chimeric clotting factor is expressed in inactive form and is subsequently activated under appropriate conditions in vitro such that the active form of the clotting factor is present in the construct. In another embodiment, a chimeric clotting factor of the invention comprises a clotting factor in inactive form and the clotting factor is activated in vivo after administration.
In one embodiment, an scFc scaffold can be used to produce an active form of a molecule. Certain clotting factors are produced recombinantly as zymogens and, therefore, require activation during manufacturing. Active forms of Factors VII, IX, and X are comprised of dimeric molecules in which the heavy and light chain are linked only by a disulfide bond.
In one embodiment, a chimeric clotting factor is activated prior to administration to a subject to improve hemostasis. Methods for activating clotting factors are known in the art. For example, in one embodiment, a chimeric clotting factor of the invention is contacted with media containing CaCl₂at a concentration of approximately 5 mM.
In another embodiment, a chimeric clotting factor of the invention is secreted in active form by a cell in which it is expressed. In one embodiment, an active chimeric clotting factor is made by expressing the heavy and light chain of a clotting factor as separate polypeptides.
In another embodiment, the N-terminus of the heavy chain of the clotting factor is modified to comprise an intracellular processing site which delays the activation of the clotting factor during synthesis until later in the secretory pathway, (i.e. until protein colocalizes with active processing enzymes in the trans-Golgi network), leading to greater productivity. Exemplary such intracellular processing sites include those recognized by furin. Exemplary cleavage sites for this family of enzymes include an amino acid sequence comprising the motif Arg-Xaa-Lys/Arg-Arg.
In a preferred embodiment, an active construct of the invention is made in the context of an Fc fusion protein, e.g., using an scFc linker (e.g., a cscfc linker).
Exemplary constructs are shown in the accompanying figures.
In one embodiment, the invention pertains to processed (e.g., mature) polypeptides in which the at least one cleavage site adjacent to an scFc polypeptide linker has been cleaved such that the molecule is no longer a single polypeptide chain such that the polypeptide is comprised of at least two polypeptide chains (owing to cleavage at the enzymatic cleavage site(s) P1 and/or P2).
In one embodiment, such processed polypeptides comprise a clotting factor or portion thereof linked to the second Fc moiety (i.e., the second Fc moiety when counting from the amino terminus to the carboxy terminus prior to cleavage of the polypeptide linker) which has a free amino terminus after cleavage of the polypeptide linker.
In one embodiment, a clotting factor attached to the N-terminus of the second Fc moiety is catalytically active, e.g., has enzymatic activity. In another embodiment, a clotting factor attached to the N-terminus of the second Fc moiety is secreted by a cell as a zymogen requiring further enzymatic processing of the clotting factor in order to be fully activated.
In one embodiment, the invention pertains to clotting factors which are secreted from cells in active or activated form without the need for further activation during processing. For example, Factor VII is generally produced recombinantly as a zymogen and requires activation during manufacturing to produce the active form for administration. In one embodiment, a polypeptide of the invention is secreted from the cell in which it is expressed in active form to improve manufacturability. As is set forth in more detail below, such clotting factors can be produced by expressing the light chain of a clotting factor and the heavy chain of a clotting factor separately in the context of an scFc molecule comprising a cscFc linker. Activation of such a construct is delayed until late in the secretory pathway during processing, e.g., when the protein colocalizes with active processing enzymes in the trans-Golgi apparatus.
In one embodiment, a clotting factor of the invention is a mature form of Factor VII or a variant thereof. Factor VII (FVII, F7; also referred to as Factor 7, coagulation factor VII, serum factor VII, serum prothrombin conversion accelerator, SPCA, proconvertin and eptacog alpha) is a serine protease that is part of the coagulation cascade. FVII includes a Gla domain, two EGF domains (EGF-1 and EGF-2), and a serine protease domain (or peptidase S1 domain) that is highly conserved among all members of the peptidase S1 family of serine proteases, such as for example with chymotrypsin. FVII occurs as a single chain zymogen, an activated zymogen-like two-chain polypeptide and a fully activated two-chain form. As used herein, a “zymogen-like” protein or polypeptide refers to a protein that has been activated by proteolytic cleavage, but still exhibits properties that are associated with a zymogen, such as, for example, low or no activity, or a conformation that resembles the conformation of the zymogen form of the protein. For example, when it is not bound to tissue factor, the two-chain activated form of FVII is a zymogen-like protein; it retains a conformation similar to the uncleaved FVII zymogen, and, thus, exhibits very low activity. Upon binding to tissue factor, the two-chain activated form of FVII undergoes conformational change and acquires its full activity as a coagulation factor.
Exemplary FVII variants include those with increased specific activity, e.g., mutations that increase the activity of FVII by increasing its enzymatic activity (Kcat or Km). Such variants have been described in the art and include, e.g., mutant forms of the molecule as described for example in Persson et al. 2001. PNAS 98:13583; Petrovan and Ruf. 2001. J. Biol. Chem. 276:6616; Persson et al. 2001 J. Biol. Chem. 276:29195; Soejima et al. 2001. J. Biol. Chem. 276:17229; Soejima et al. 2002. J. Biol. Chem. 247:49027. In one embodiment, a variant form of FVII includes the mutations Exemplary mutations include V158D-E296V-M298Q. In another embodiment, a variant form of FVII includes a replacement of amino acids 608-619 (LQQSRKVGDSPN, corresponding to the 170-loop) from the FVII mature sequence with amino acids EASYPGK from the 170-loop of trypsin. High specific activity variants of FIX are also known in the art. Fir example, Simioni et al. (2009 N. E. Journal of Medicine 361:1671) describe an R338L mutation. Chang et al. (1988 JBC 273:12089) and Pierri et al. (2009 Human Gene Therapy 20:479) describe an R338A mutation. Other mutations are known in the art and include those described, e.g., in Zogg and Brandstetter. 2009 Structure 17:1669; Sichler et al. 2003. J. Biol. Chem. 278:4121; and Sturzebecher et al. 1997. FEBS Lett 412:295. The contents of these references are incorporated herein by reference.
Full activation, which occurs upon conformational change from a zymogen-like form, occurs upon binding to is co-factor tissue factor. Also, mutations can be introduced that result in the conformation change in the absence of tissue factor. Hence, reference to FVIIa includes both two-chain forms thereof, the zymogen-like form and the fully activated two-chain form.
In one embodiment, a clotting factor of the invention is a mature form of Factor VIII or a variant thereof. FVIII functions in the intrinsic pathway of blood coagulation as a cofactor to accelerate the activation of factor X by factor IXa, a reaction that occurs on a negatively charged phospholipid surface in the presence of calcium ions. FVIII is synthesized as a 2351 amino acid single-chain polypeptide having the domain structure A1-A2-B-A3-C1-C2. Wehar, G. A. et al., Nature 312:337-342 (1984) and Toole, J. J. et al., Nature 312:342-347 (1984). The domain structure of FVIII is identical to that of the homologous coagulation factor, factor V (FV). Kane, W. H. et al., PNAS (USA) 83:6800-6804 (1986) and Jenny, R. J. et al., PNAS (USA) 84:4846-4850 (1987). The FVIII A-domains are 330 amino acids and have 40% amino acid identity with each other and to the A-domain of FV and the plasma copper-binding protein ceruloplasmin. Takahashi, N. et al., PNAS (USA) 81:390-394 (1984). Each C-domain is 150 amino acids and exhibits 40% identity to the C-domains of FV, and to proteins that bind glycoconjugates and negatively charged phospholipids. Stubbs, J. D. et al., PNAS (USA) 87:8417-8421 (1990). The FVIII B-domain is encoded by a single exon and exhibits little homology to any known protein including FV B-domain. Gitschier, J. et al., Nature 312:326-330 (1984) and Cripe, L. D. et al., Biochemistry 31:3777-3785 (1992).
FVIII is secreted into plasma as a heterodimer of a heavy chain (domains A1-A2-B) and a light chain (domains A3-C1-C2) associated through a noncovalent divalent metal ion linkage between the A1- and A3-domains. In plasma, FVIII is stabilized by binding to von Willebrand factor. More specifically, the FVIII light chain is bound by noncovalent interactions to a primary binding site in the amino terminus of von Willebrand factor. Upon proteolytic activation by thrombin, FVIII is activated to a heterotrimer of 2 heavy chain fragments (A1, a 50 kDa fragment, and A2, a 43 kDa fragment) and the light chain (A3-C1-C2, a 73 kDa chain). The active form of FVIII (FVIIIa) thus consists of an A1-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit associated with the A1 domain through an ion association. Eaton, D. et al., Biochemistry 25: 505 (1986); Lollar, P. et al., J. Biol. Chem. 266: 12481 (1991); and Fay, P. J. et al., J. Biol. Chem. 266: 8957 (1991). This FVIIIa heterotrimer is unstable and subject to rapid inactivation through dissociation of the A2 subunit under physiological conditions.
In one embodiment, a clotting factor comprises a B-domain deleted version of factor VIII. “B-domain” of Factor VIII, as used herein, is the same as the B-domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741-Arg1648 of full-length human Factor VIII. The other human Factor VIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the a3 acidic region. The locations of the boundaries for all of the domains, including the B-domains, for porcine, mouse and canine Factor VIII are also known in the art. In one embodiment, the B domain of Factor VIII is deleted (“B-domain-deleted factor VIII” or “BDD FVIII”). An example of a BDD FVIII is REFACTO® (recombinant BDD FVIII with 5743/Q1638 fusion), which is known in the art.
A “B-domain-deleted Factor VIII” may have the full or partial deletions disclosed in U.S. Pat. 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, each of which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted Factor VIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No. 6,346,513). In another embodiment, a B-domain deleted Factor VIII is the 5743/Q1638 B-domain deleted Factor VIII (SQ version Factor VIII) (e.g., Factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII having amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 6, i.e., SEQ ID NO: 2). In some embodiments, a B-domain-deleted Factor VIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. Nos. 6,060,447, 5,595,886, and 6,228,620). In some embodiments, a B-domain-deleted Factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments, a B-domain-deleted Factor VIII has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91/09122, which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted Factor VIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. A B-domain-deleted Factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of Factor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety. Additional B domain deletions that are part of the invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 though 1648 (Pasek (PCT application No. 88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions may be made in any Factor VIII sequence. In one embodiment, the invention pertains to a targeted version of FVIII, wherein the targeting (i) specifically binds to platelets, (ii) is not interposed between the light and heavy chains of the clotting factor, and wherein said chimeric clotting factor exhibits increased generation of thrombin in the presence of platelets as compared to an appropriate control lacking the at least one targeting moiety.
In one embodiment, a clotting factor of the invention is a mature form of Factor IX or a variant thereof. Factor IX circulates as a 415 amino acid, single chain plasma zymogen (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993)). The zymogen of FIX is activated by FXIa or by the tissue factor/FVIIa complex. Specific cleavages between arginine-alanine 145-146 and arginine-valine 180-181 result in a light chain and a heavy chain linked by a single disulfide bond between cysteine 132 and cysteine 289 (S. Bajaj et al., Biochemistry 22, 4047 (1983)). The structural organization of FIX is similar to that of the vitamin K-dependent blood clotting proteins FVII, FX and protein C (B. Furie and B. Furie, supra). The approximately 45 amino acids of the amino terminus comprise the gamma-carboxyglutamic acid, or gla, domain. This is followed by two epidermal growth factor homology domains (EGF), an activation peptide and the catalytic “heavy chain” which is a member of the serine protease family (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993); S. Spitzer et al., Biochemical Journal 265, 219 (1990); H. Brandstetter et al., Proc. Natl. Acad Sci. USA 92, 9796 (1995)).
In one embodiment, a clotting factor of the invention is a mature form of Factor X. Factor X is a vitamin-K dependent glycoprotein of a molecular weight of 58.5 kDa, which is secreted from liver cells into the plasma as a zymogen. Initially factor X is produced as a prepropeptide with a signal peptide consisting in total of 488 amino acids. The signal peptide is cleaved off by signal peptidase during export into the endoplasmatic reticulum, the propeptide sequence is cleaved off after gamma carboxylation took place at the first 11 glutamic acid residues at the N-terminus of the mature N-terminal chain. A further processing step occurs by cleavage between Arg182 and Ser183. This processing step also leads concomitantly to the deletion of the tripeptide Arg180-Lys181-Arg182. The resulting secreted factor X zymogen consists of an N-terminal light chain of 139 amino acids (M, 16,200) and a C-terminal heavy chain of 306 amino acids (M, 42,000) which are covalently linked via a disulfide bridge between Cys172 and Cys342. Further posttranslational processing steps include the .beta.-hydroxylation of Asp103 as well as N- and O-type glycosylation.
It will be understood that in addition to wild type (WT) versions of these clotting factors or biologically active portions thereof, the present invention may also employ precursor truncated forms thereof that have activity, allelic variants and species variants, variants encoded by splice variants, and other variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the mature form of the clotting factor and which retain the ability to promote clot formation. For example, modified FVII polypeptides and variants thereof which retain at least one activity of a FVII, such as TF binding, factor X binding, phospholipid binding, and/or coagulant activity of a FVII may be employed. By retaining activity, the activity can be altered, such as reduced or increased, as compared to a wild-type clotting factor so long as the level of activity retained is sufficient to yield a detectable effect. Exemplary sequences of clotting factors that can be used in the constructs of the invention are found in the accompanying sequence listing.
Exemplary modified polypeptides include, but are not limited to, tissue-specific isoforms and allelic variants thereof, synthetic molecules prepared by translation of nucleic acids, proteins generated by chemical synthesis, such as syntheses that include ligation of shorter polypeptides, through recombinant methods, proteins isolated from human and non-human tissue and cells, chimeric polypeptides and modified forms thereof. The instant clotting factors may also consist of fragments or portions of WT molecules that are of sufficient length or include appropriate regions to retain at least one activity (upon activation if needed) of a full-length mature polypeptide. Exemplary clotting factor variants are known in the art.
As used herein, the term “Gla domain” refers to the conserved membrane binding motif which is present In vitamin K-dependent proteins, such as prothrombin, coagulation factors VII, IX and X, proteins C, S, and Z. These proteins require vitamin K for the posttranslational synthesis of g-carboxyglutamic acid, an amino acid clustered in the N-terminal Gla domain of these proteins. All glutamic residues present in the domain are potential carboxylation sites and many of them are therefore modified by carboxylation. In the presence of calcium ions, the Gla domain interacts with phospholipid membranes that include phosphatidylserine. The Gla domain also plays a role in binding to the FVIIa cofactor, tissue factor (TF). Complexed with TF, the Gla domain of FVIIa is loaded with seven Ca2+ ions, projects three hydrophobic side chains in the direction of the cell membrane for interaction with phospholipids on the cell surface, and has significant contact with the C-terminal domain of TF.
The Gla domain of factor VII comprises the uncommon amino acid—carboxyglutamic acid (Gla), which plays a vital role in the binding of clotting factors to negatively charged phospholipid surfaces.
The GLA domain is responsible for the high-affinity binding of calcium ions. It starts at the N-terminal extremity of the mature form of proteins and ends with a conserved aromatic residue. A conserved Gla-x(3)-Gla-x-Cys motif is found in the middle of the domain which seems to be important for substrate recognition by the carboxylase.
Using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, the Gla domain has been found to be important in the sequence of events whereby the protease domain of FVIIa initiates contact with sTF (Biochemical and Biophysical Research Communications. 2005. 337:1276). In addition, clearance of clotting factors may be significantly mediated through Gla interactions, e.g., on liver cells and clearance receptors, e.g., EPCR.
In one embodiment, targeted clotting factors are modified to lack a Gla domain. The Gla domain is responsible for mediating clearance of clotting factors via multiple pathways, such as binding to liver cells, clearance receptors such as EPCR, etc. Thus, eliminating the Gla domain has beneficial effects on half life of clotting factors. Though Gla domain is also generally required for activity by localizing clotting factors to sites of coagulation, the inclusion of a platelet targeting domain moiety targets the Gla deleted clotting factor to platelets. In one embodiment, a clotting factor of the invention comprises a targeting moiety and lacks a Gla domain. For example, in the case of Factor VII, the Gla domain is present at the amino terminus of the light chain and consists of amino acids 1-35. The Gla domains of exemplary clotting factors are indicated in the accompanying sequence listing. This domain can be removed using standard molecular biology techniques, replaced with a targeting domain, and the modified light chain incorporated into a construct of the invention. In one embodiment, a cleavage site may be introduced into constructs lacking a Gla domain to facilitate activation of the molecule. For example, in one embodiment, such a cleavage site may be introduced between the amino acids that are cleaved when the clotting factor is activated (e.g., between amino acids 152 and 153 in the case of Factor VII). Exemplary clotting factors lacking a Gla domain are shown in the accompanying figures
In one embodiment, a cleavage site may be introduced into constructs lacking a Gla domain to facilitate activation of the molecule. For example, in one embodiment, such a cleavage site may be introduced between the amino acids that are cleaved when the clotting factor is activated (e.g., between amino acids 152 and 153 in the case of Factor VII). Exemplary clotting factors lacking a Gla domain are shown in the accompanying figures.
Exemplary clotting factors are those of mammalian, e.g., human, origin. The sequences of exemplary clotting factors are presented in the accompanying sequence listing, e.g., alone or in the context of a chimeric clotting factor construct.

III. Targeting Moieties

In one embodiment, a clotting factor of the invention is targeted to platelets to enhance its efficacy by localizing the clotting factor to the site of coagulation using a “targeting moiety” which binds to a target molecule expressed on platelets. Preferably the targeted molecules are not expressed on cells or tissues other than platelets, i.e., the targeting moieties specifically bind to platelets.
In one embodiment, receptors/conformations found on resting platelets are targeted. By doing so, sites for coagulation could be primed for enhanced efficacy. Targeting such molecule may also extend half life of the clotting factor and/or prevent clearance. Examples of such targets include GpIb of the GpIb/V/IX complex, and GpVI and nonactive form of GPIIb/IIIa.
In one embodiment, receptors/conformations only found on activated platelets are targeted in order to localize the clotting factor to site of active coagulation. Examples of such targets include, e.g., the active form of GpIIb/IIIa as well as CD62P.
In one embodiment, a polypeptide of the invention comprises a “targeting moiety” which has affinity for and binds to platelets. For example, in one embodiment, a targeting moiety binds to the GPIb complex, e.g, GPIb-alpha. Examples of such targeting moieties include the peptides PS4, OS1, and OS2 which bind to both active and nonactive platelets (Benard et al. 2008 Biochemistry 47:4674); In another embodiment, a targeting moiety binds to the active conformation of GPIIbIIIa. Examples of such targeting moieties inlcude SCE5 and MB9 variable regions which bind active platelets only (Schwarz et al. 2004 FASEB Journal express article 10.1096/fj.04-1513fje; Schwarz et al. 2006 Circulation Research. 99:25-33; U.S. Patent publication 20070218067). In another embodiment, a targeting moiety binds to both the active/nonactive conformation of GPIIbIIIa. An example of such a targeting moiety is the variable region of the AP3 antibody (Peterson et al. 2003. Hemostasis, Thrombosis, and Vascular Biology 101:937; WO 2010115866). Other targets and targeting moieties are known in the art. Another version of factor IX (the triple mutant V86A/E277A/R338A) with augmented clotting activities has been described by Lin et al. 2010. Journal of Thrombosis and Hemostasis 8: 1773). The contents of these references are incorporated herein by this reference.
The chimeric clotting factors of the invention can comprise one or more than one targeting moiety. Exemplary configurations are set forth in the accompanying Figures. Additionally, two or more targeting moieties may be linked to each other (e.g., via a spacer) in series, and the tandem array operably linked to a construct of the invention. When two or more targeting moieties are present in a chimeric clotting factor of the invention, the moieties may be the same or different.
In one embodiment, a targeting moiety is fused to a chimeric clotting factor of the invention by a cleaveable linker which may be cleaved to remove the targeting moiety at the site of a clot. In another embodiment, a targeting moiety is not attached via a cleaveable linker and, therefore, is not cleaved at the site of a clot.
In one embodiment, the targeting moiety is located on the N- or C-terminus of factor VIII. In another embodiment, a targeting moiety is located on the C-terminus of FVII, FIX, FX, or the C-terminus of either or both chains of FVIIa, FIXa, of FXa. In embodiments in which an Fc region or portion thereof is employed, the targeting moiety may be positioned at the N or C terminus of the second Fc chain, or the C-terminus of either or both Fc chains.
In one embodiment, a targeting moiety is not genetically fused directly to a construct, but rather is linked via a spacer or a chemical bond to the construct. For example, targeting moieties may be attached to a construct of the invention by formation of a bond between the targeting moiety and an Fc moiety of a construct, where the targeting moiety comprises a first functional group and the Fc moiety comprises a second functional group, and where the first and second functional groups are capable of reacting with each other to form a chemical bond (see, e.g., U.S. Pat. No. 7,381,408).
In one embodiment, a polypeptide of the invention comprises at least one of an antigen binding site (e.g., an antigen binding site of an antibody, antibody variant, or antibody fragment), a polypeptide, a receptor binding portion of ligand, or a ligand binding portion of a receptor which specifically binds to platelets, e.g., resting or activated platelets. Exemplary targeting moieties include scFv molecules or peptides which bind to molecules to be targeted. Examples of targeting moieties are found in the instant examples and Figures. Other molecules useful as targeting moieties can readily be selected by one of skill in the art based upon the teaching herein.

A. Antigen Binding Sites Which Bind to Platelets

In certain embodiments, a polypeptide of the invention comprises at least one antigen binding portion (e.g., binding site) of an antibody. In one embodiment, the antigen binding portion targets the polypeptide to platelets
In other embodiments, a polypeptide of the invention may comprise an antigen binding portion. The term “antigen-binding portion” refers to a polypeptide fragment of an immunoglobulin, antibody, or antibody variant which binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). For example, said antigen binding portions can be derived from any of the antibodies or antibody variants described supra. Antigen binding portions can be produced by recombinant or biochemical methods that are well known in the art. Exemplary antigen-binding portions include Fv, Fab, Fab′, and (Fab′)₂as well as scFv molecules.
In other embodiments, a chimeric clotting factor of the invention may comprise a binding site from single chain binding molecule (e.g., a single chain variable region or scFv). Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be adapted to produce single chain binding molecules. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain antibody. Techniques for the assembly of functional Fv fragments in E coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
In certain embodiments, a polypeptide of the invention comprises one or more binding sites or regions comprising or consisting of a single chain variable region sequence (scFv). Single chain variable region sequences comprise a single polypeptide having one or more antigen binding sites, e.g., a V_Ldomain linked by a flexible linker to a V_Hdomain. The VL and/or VH domains may be derived from any of the antibodies or antibody variants described supra. ScFv molecules can be constructed in a V_H-linker-V_Lorientation or V_L-linker-V_Horientation. The flexible linker that links the V_Land V_Hdomains that make up the antigen binding site preferably comprises from about 10 to about 50 amino acid residues. In one embodiment, the polypeptide linker is a gly-ser polypeptide linker. An exemplary gly/ser polypeptide linker is of the formula (Gly4Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6). Other polypeptide linkers are known in the art. Antibodies having single chain variable region sequences (e.g. single chain Fv antibodies) and methods of making said single chain antibodies are well-known in the art (see e.g., Ho et al. 1989. Gene 77:51; Bird et al. 1988 Science 242:423; Pantoliano et al. 1991. Biochemistry 30:10117; Milenic et al. 1991. Cancer Research 51:6363; Takkinen et al. 1991. Protein Engineering 4:837).
In certain embodiments, a scFv molecule employed in a polypeptide of the invention is a stabilized scFv molecule. In one embodiment, the stabilized cFv molecule may comprise a scFv linker interposed between a V_Hdomain and a V_Ldomain, wherein the V_Hand V_Ldomains are linked by a disulfide bond between an amino acid in the V_Hand an amino acid in the V_Ldomain. In other embodiments, the stabilized scFv molecule may comprise a scFv linker having an optimized length or composition. In yet other embodiments, the stabilized scFv molecule may comprise a V_Hor V_Ldomain having at least one stabilizing amino acid substitution(s). In yet another embodiment, a stabilized scFv molecule may have at least two of the above listed stabilizing features.
Stabilized scFv molecules have improved protein stability or impart improved protein stability to the polypeptide to which it is operably linked. Preferred scFv linkers of the invention improve the thermal stability of a polypeptide of the invention by at least about 2° C. or 3° C. as compared to a conventional polypeptide Comparisons can be made, for example, between the scFv molecules of the invention. In certain preferred embodiments, the stabilized scFv molecule comprises a (Gly₄Ser)₄scFv linker and a disulfide bond which links V_Hamino acid 44 and V_Lamino acid 100. Other exemplary stabilized scFv molecules which may be employed in the polypeptides of the invention are described in U.S. Provisional Patent Application No. 60/873,996, filed on Dec. 8, 2006 or U.S. patent application Ser. No. 11/725,970, filed on Mar. 19, 2007, each of which is incorporated herein by reference in its entirety.
Polypeptides of the invention may comprise a variable region or portion thereof (e.g. a VL and/or VH domain) derived from an antibody using art recognized protocols. For example, the variable domain may be derived from antibody produced in a non-human mammal, e.g., murine, guinea pig, primate, rabbit or rat, by immunizing the mammal with the antigen or a fragment thereof. See Harlow & Lane, supra, incorporated by reference for all purposes. The immunoglobulin may be generated by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., purified tumor associated antigens or cells or cellular extracts comprising such antigens) and an adjuvant. This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes.
While the variable region may be derived from polyclonal antibodies harvested from the serum of an immunized mammal, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs) from which the desired variable region is derived. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for making monoclonal antibodies. Monoclonal antibodies can be prepared against a fragment by injecting an antigen fragment into a mouse, preparing “hybridomas” and screening the hybridomas for an antibody that specifically binds to the antigen. In this well known process (Kohler et al., (1975), Nature, 256:495) the relatively short-lived, or mortal, lymphocytes from the mouse which has been injected with the antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas” which are both immortal and capable of producing the antibody genetically encoded by the B cell. The resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed “monoclonal”.
Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established. Generally, culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)). It will further be appreciated that the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, affinity chromatography (e.g., protein-A, protein-G, or protein-L affinity chromatography), hydroxylapatite chromatography, gel electrophoresis, or dialysis.
Optionally, antibodies may be screened for binding to platelets of a specific activation state or to a specific region or desired fragment of the antigen without binding to other nonoverlapping fragments of the antigen. The latter screening can be accomplished by determining binding of an antibody to a collection of deletion mutants of the antigen and determining which deletion mutants bind to the antibody. Binding can be assessed, for example, by Western blot or ELISA. The smallest fragment to show specific binding to the antibody defines the epitope of the antibody. Alternatively, epitope specificity can be determined by a competition assay is which a test and reference antibody compete for binding to the antigen. If the test and reference antibodies compete, then they bind to the same epitope or epitopes sufficiently proximal such that binding of one antibody interferes with binding of the other.
DNA encoding the desired monoclonal antibody or binding site thereof may be readily isolated and sequenced using any of the conventional procedures described supra for the isolation of constant region domain sequences (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. More particularly, the isolated DNA (which may be synthetic as described herein) may be used to clone the desired variable region sequences for incorporation in the polypeptides of the invention.
In other embodiments, the binding site is derived from a fully human antibody. Human or substantially human antibodies may be generated in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369, each of which is incorporated herein by reference). For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge. Another preferred means of generating human antibodies using SCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies may also be isolated and manipulated as described herein.
In other aspects, the polypeptides of the invention may comprise antigen binding sites, or portions thereof, derived from modified forms of antibodies. Exemplary such forms include, e.g., minibodies, diabodies, triabodies, nanobodies, camelids, Dabs, tetravalent antibodies, intradiabodies (e.g., Jendreyko et al. 2003. J. Biol. Chem. 278:47813), fusion proteins (e.g., antibody cytokine fusion proteins, proteins fused to at least a portion of an Fc receptor), and bispecific antibodies. Other modified antibodies are described, for example in U.S. Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Kohler et al., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res. 41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984); Morrison, Science 229:1202 (1985); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559. Reassorted immunoglobulin chains also are known. See, for example, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references cited therein.
In another embodiment, a chimeric clotting factor of the invention comprises an antigen binding site or region which is a diabody or an antigen binding site derived therefrom. Diabodies are dimeric, tetravalent molecules each having a polypeptide similar to scFv molecules, but usually having a short (e.g., less than 10 and preferably 1-5) amino acid residue linker connecting both variable domains, such that the V_Land V_Hdomains on the same polypeptide chain cannot interact. Instead, the V_Land V_Hdomain of one polypeptide chain interact with the V_Hand V_Ldomain (respectively) on a second polypeptide chain (see, for example, WO 02/02781). In one embodiment, a polypeptide of the invention comprises a diabody which is operably linked to the N-terminus and/or C-terminus of at least one genetically-fused Fc region (i.e., scFc region).
In certain embodiments, a polypeptide of the invention comprises a single domain binding molecule (e.g. a single domain antibody) as a targeting moiety. Exemplary single domain molecules include an isolated heavy chain variable domain (V_H) of an antibody, i.e., a heavy chain variable domain, without a light chain variable domain, and an isolated light chain variable domain (V_L) of an antibody, i.e., a light chain variable domain, without a heavy chain variable domain. Exemplary single-domain antibodies employed in the binding molecules of the invention include, for example, the Camelid heavy chain variable domain (about 118 to 136 amino acid residues) as described in Hamers-Casterman, et al., Nature 363:446-448 (1993), and Dumoulin, et al., Protein Science 11:500-515 (2002). Other exemplary single domain antibodies include single VH or VL domains, also known as Dabs® (Domantis Ltd., Cambridge, UK). Yet other single domain antibodies include shark antibodies (e.g., shark Ig-NARs). Shark Ig-NARs comprise a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR), wherein diversity is concentrated in an elongated CDR3 region varying from 5 to 23 residues in length. In camelid species (e.g., llamas), the heavy chain variable region, referred to as VHH, forms the entire antigen-binding domain. The main differences between camelid VHH variable regions and those derived from conventional antibodies (VH) include (a) more hydrophobic amino acids in the light chain contact surface of VH as compared to the corresponding region in VHH, (b) a longer CDR3 in VHH, and (c) the frequent occurrence of a disulfide bond between CDR1 and CDR3 in VHH. Methods for making single domain binding molecules are described in U.S. Pat. Nos. 6,005,079 and 6,765,087, both of which are incorporated herein by reference. Exemplary single domain antibodies comprising VHH domains include Nanobodies® (Ablynx NV, Ghent, Belgium).
Exemplary antibodies from which binding sites can be derived for use in the binding molecules of the invention are known in the art. Examples of such targeting moieties inlcude SCE5 and MB9 variable regions which bind active platelets only (Schwarz et al. 2004 FASEB Journal express article 10.1096/fj.04-1513fje; Schwarz et al. 2006 Circulation Research. 99:25-33; U.S. Patent publication 20070218067). In another embodiment, a targeting moiety binds to both the active/nonactive conformation of GPIIbIIIa. An example of such a targeting moiety is the variable region of the AP3 antibody (Peterson et al. 2003. Hemostasis, Thrombosis, and Vascular Biology 101:937; WO 2010115866).

B. Non-Immunoglobulin Platelet Binding Molecules

In certain other embodiments, the polypeptides of the invention comprise one or more platelet binding sites derived from a non-immunoglobulin binding molecule. As used herein, the term “non-immunoglobulin binding molecules” are binding molecules whose binding sites comprise a portion (e.g., a scaffold or framework) which is derived from a polypeptide other than an immunoglobulin, but which may be engineered (e.g., mutagenized) to confer a desired binding specificity to a platelet target Other examples of binding molecules comprising binding sites not derived from antibody molecules include receptor binding sites and ligand binding sites which bind to platelets.
Non-immunoglobulin binding molecules may be identified by selection or isolation of a target-binding variant from a library of binding molecules having artificially diversified binding sites. Diversified libraries can be generated using completely random approaches (e.g., error-prone PCR, exon shuffling, or directed evolution) or aided by art-recognized design strategies. For example, amino acid positions that are usually involved when the binding site interacts with its cognate target molecule can be randomized by insertion of degenerate codons, trinucleotides, random peptides, or entire loops at corresponding positions within the nucleic acid which encodes the binding site (see e.g., U.S. Pub. No. 20040132028). The location of the amino acid positions can be identified by investigation of the crystal structure of the binding site in complex with the target molecule. Candidate positions for randomization include loops, flat surfaces, helices, and binding cavities of the binding site. In certain embodiments, amino acids within the binding site that are likely candidates for diversification can be identified using techniques known in the art. Following randomization, the diversified library may then be subjected to a selection or screening procedure to obtain binding molecules with the desired binding characteristics, e.g. specific binding platelets using methods known in the art. Selection can be achieved by art-recognized methods such as phage display, yeast display, or ribosome display. In one embodiment, molecules known in the art to bind to platelets may be employed in the constructs of the invention. For example, peptides which bind to GPIba as described in the art (e.g., PS4, 0S1, or 0S2) may be used (Benard et al. 2008. Biochemistry 47:4674-4682).

IV. Activatable Clotting Factors

Clotting factors given for bypass therapy are efficacious when given in the activated form, since exogenous clotting factors are often not activated with sufficient kinetics to be effective. However, they are also rapidly inactivated by endogenous pathways (e.g., by antithrombin III or TFPI), leading to clearance of the active form and a short effective half life. In one embodiment, a chimeric clotting factor of the invention is “activatable.” Such activatable constructs circulate as an enhanced zymogen with a longer half life, but can be readily cleaved at the site of clotting when necessary.
In one embodiment, an activatable construct of the invention comprises a cleavable linker comprising, e.g., a factor XIa, Xa, or thrombin cleavage site (which is cleaved by factor XIa, Xa, or thrombin, respectively) leading to formation of the active form of the clotting factor at the site of a clot. Exemplary factor FXIa cleavage sites include, e.g., TQSFNDFTR and SVSQTSKLTR. Exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR, TTKIKPR, and a sequence comprising or consisting of ALRPR (e.g. ALRPRVVGGA)).
In one embodiment, the cleavable linker may be flanked on one or more sides (upstream, downstream or both) by a spacer moiety.
In one embodiment, the cleavable linker is interposed between the light chain and heavy chain of the clotting factor. In another embodiment, the cleavable linker is not interposed between the light chain and heavy chain of the clotting factor. In one embodiment, the cleavable linker is located amino terminal to the heavy chain.
Exemplary activatable constructs are shown in the accompanying Figures and following Examples.

V. Scaffold Moieties

Some embodiments of the invention comprise a scaffold moiety, which can be selected from, e.g., a protein moiety, cscFc region, a Fc moiety, albumin, XTEN, etc.

A. Protein Moieties

In one embodiment, the scaffold is a protein moiety. Such a moiety may comprise a complete protein or a portion thereof, or a synthetic molecule. Preferred protein moieties are of a sufficient molecular size that they improve the half life of a chimeric clotting factor of the invention when incorporated into a construct. For example, in one embodiment, an artificial protein, XTEN, may be included in a construct as a scaffold (Schellenberger et al. 2009. 27:1186). In another embodiment, albumin (e.g., human serum albumin) may be included in a construct of the invention. For example, as known in the art, serum albumin (for example, HSA) can be used as a protein scaffold. In particular various domains and sub-domains of HSA, have a structure that is quite amenable to mutation or randomization for the generation of serum albumin scaffold-based protein libraries. Examples of albumin, e.g., fragments thereof, that may be used in the present invention are known. e.g., U.S. Pat. Nos. 7,592,010; 6,686,179; and Schulte, Thrombosis Res. 124 Suppl. 2:S6-S8 (2009), each of which is incorporated herein by reference in its entirety.
B. scFc Regions
In one embodiment, the invention provides for polypeptides comprising at least one genetically fused Fc region or portion thereof within a single polypeptide chain (i.e., polypeptides comprising a single-chain Fc (scFc) region) in one embodiment, comprising a cscFc.
In one embodiment, a chimeric clotting factor which comprises a clotting factor selected from the group consisting of FVII, FIX and FX and a targeting moiety which binds to platelets and optionally a spacer moiety between the clotting factor and the targeting moiety. In another embodiment, polypeptide comprising FVII, which FVII comprises a heterologous enzymatic cleavage site activatable by a component of the clotting cascade.
In one embodiment, the invention provides unprocessed polypeptides in which at least two Fc moieties or domains (e.g., 2, 3, 4, 5, 6, or more Fc moieties or domains) within the same linear polypeptide chain that are capable of folding (e.g., intramolecularly or intermolecularly folding) to form one functional scFc region which is linked by an Fc polypeptide linker. For example, in one preferred embodiment, a polypeptide of the invention is capable of binding, via its scFc region, to at least one Fc receptor (e.g. an FcRn, an FcγR receptor (e.g., FcγRIII), or a complement protein (e.g. C1q)) in order to improve half life or trigger an immune effector function (e.g., antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC) and/or to improve manufacturability).
A variety of polypeptides of alternative designs are within the scope of the invention.
For example, in one embodiment, a polypeptide comprises the moieties:
A-F1-P1-L-P2-B-F2 (I)
in linear sequence from the amino to carboxy terminus wherein A, if present, is a clotting factor or portion thereof, F1 is a first Fc moiety or domain, P1 is an enzymatic cleavage site, L is an ScFc linker, P2 is an enzymatic cleavage site B, if present, is a clotting factor or portion thereof, F2 is a second Fc moiety or domain and “-” represents a peptide bond. Formula (I) comprises at least an A or B and optionally both. A and B, if both present, can be the corresponding heavy and light chains of a clotting factor. Formula (I) comprises at least a P1 or P2 and optionally both. P1 and P2, if both present, can be the same or different. Formula (I) comprises at least a F1 and F2. F1 and F2, if both present, can be the same or different.
Exemplary polypeptides according to formula I include: A-F1-P1-L-P2-F2; F1-P1-L-P2-B-F2; A-F1-P1-L-F2; F1-P1-L-B-F2; A-F1-L-P2-F2; and F1-L-P2-B-F2.
In one embodiment, F1 and F2 each comprise a CH2 and CH3 moiety.
In one embodiment, after cleavage and substantial excision of the cscFc linker (L), a polypeptide of the invention comprises two polypeptide chains where the first polypeptide chain comprises A linked to a first Fc moiety and where the second polypeptide chain comprises B linked to a second Fc moiety, where F1 and F2 dimerize to form an Fc region. In one embodiment, A and B are optionally present and are clotting factors or portions thereof.
In one embodiment, A is the light chain of a clotting factor and B is the heavy chain of a clotting factor. In one embodiment, B is the light chain of a clotting factor and A is the heavy chain of a clotting factor. In one embodiment, when A and B associate in the polypeptide, the polypeptide then forms a functional clotting factor, e.g., FVII, FIX or FX. In one embodiment, such a polypeptide is enzymatically active upon secretion from a cell.
i) Fc Moieties or Domains
Fc moieties useful as F1 and F2 for producing the polypeptides of the present invention may be obtained from a number of different sources. In preferred embodiments, an Fc moiety of the polypeptide is derived from a human immunoglobulin. It is understood, however, that the Fc moiety may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover, the polypeptide Fc domain or portion thereof may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the human isotype IgG1 is used.
A variety of Fc moiety gene sequences (e.g. human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains comprising an Fc moiety sequence can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc moiety sequences (e.g. hinge, CH2, and/or CH3 sequences, or portions thereof) can be derived from these sequences using art recognized techniques. The genetic material obtained using any of the foregoing methods may then be altered or synthesized to obtain polypeptides of the present invention. It will further be appreciated that the scope of this invention encompasses alleles, variants and mutations of constant region DNA sequences.
Fc moiety sequences can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. To clone an Fc moiety sequence from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes 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 in, e.g., “PCR Protocols: A Guide to Methods and Applications” Innis et al. eds., Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplification of antibody genes are known in the art (e.g., 5′ primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). The cloning of antibody sequences is further described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by reference herein.
The polypeptides of the invention may comprise two or more Fc moieties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc moieties). These two or more Fc moieties can form a Fc region. In one embodiment, the Fc moieties may be of different types. In one embodiment, at least one Fc moiety present in the polypeptide comprises a hinge domain or portion thereof. In another embodiment, the polypeptide of the invention comprises at least one Fc moiety which comprises at least one CH2 domain or portion thereof. In another embodiment, the polypeptide of the invention comprises at least one Fc moiety which comprises at least one CH3 domain or portion thereof. In another embodiment, the polypeptide of the invention comprises at least one Fc moiety which comprises at least one CH4 domain or portion thereof. In another embodiment, the polypeptide of the invention comprises at least one Fc moiety which comprises at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g, in the hinge-CH2 orientation). In another embodiment, the polypeptide of the invention comprises at least one Fc moiety which comprises at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g, in the CH2-CH3 orientation). In another embodiment, the polypeptide of the invention comprises at least one Fc moiety comprising at least one hinge domain or portion thereof, at least one CH2 domain or portion thereof, and least one CH3 domain or portion thereof, for example in the orientation hinge-CH2-CH3, hinge-CH3-CH2, or CH2-CH3-hinge.
In certain embodiments, the polypeptide comprises at least one complete Fc region derived from one or more immunoglobulin heavy chains (e.g., an Fc domain including hinge, CH2, and CH3 domains, although these need not be derived from the same antibody). In other embodiments, the polypeptide comprises at least two complete Fc regions derived from one or more immunoglobulin heavy chains. In preferred embodiments, the complete Fc moiety is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG1).
In another embodiment, a polypeptide of the invention comprises at least one Fc moiety comprising a complete CH3 domain (about amino acids 341-438 of an antibody Fc region according to EU numbering). In another embodiment, a polypeptide of the invention comprises at least one Fc moiety comprising a complete CH2 domain (about amino acids 231-340 of an antibody Fc region according to EU numbering). In another embodiment, a polypeptide of the invention comprises at least one Fc moiety comprising at least a CH3 domain, and at least one of a hinge region (about amino acids 216-230 of an antibody Fc region according to EU numbering), and a CH2 domain. In one embodiment, a polypeptide of the invention comprises at least one Fc moiety comprising a hinge and a CH3 domain. In another embodiment, a polypeptide of the invention comprises at least one Fc moiety comprising a hinge, a CH₂, and a CH₃domain. In preferred embodiments, the Fc moiety is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG1). In one embodiment, an Fc moiety comprises or consists of amino acids corresponding to EU numbers 221 to 447.
In another embodiment, a polypeptide of the invention comprises at least one Fc moiety comprising an FcRn binding partner. An FcRn binding partner is a molecule or portion thereof that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. Specifically bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 10⁶M⁻¹, or more preferably higher than 10⁸M⁻¹. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.
The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, monkey FcRn rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834; U52003-0235536A1) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.
FcRn binding partners of the present invention encompass molecules that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.
The Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example, the following single amino acid residues in human IgG1 Fc (Fc yl) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wildtype proline substituted by alanine at position number 238. As an example, one specific embodiment, incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids may be substituted for the wildtype amino acids at the positions specified above. Mutations may be introduced singly into Fc giving rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more FcRn binding partners. Moreover, one of the FcRn binding partners of a construct of the invention may be mutated and the other FcRn binding partner not mutated at all, or they both may be mutated but with different mutations. Any of the mutations described herein, including N297A, may be used to modify Fc, regardless of the biologically active molecule (e.g., EPO, IFN, Factor VII, Factor IX, T20).
Certain of the above mutations may confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased “on” rate, a decreased “off” rate or both an increased “on” rate and a decreased “off” rate. Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).
Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG1 “ELLG” to the corresponding sequence from IgG2 “PVA” (with one amino acid deletion). It has been shown that FcγRI, FcγRII, and FcγRIII, which mediate various effector functions will not bind to IgG1 when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.
In one embodiment, the FcRn binding partner is a polypeptide including the sequence PKNSSMISNTP (SEQ ID NO: 12) and optionally further including a sequence selected from HQSLGTQ (SEQ ID NO: 13), HQNLSDGK (SEQ ID NO: 14), HQNISDGK (SEQ ID NO: 24), or VISSHLGQ (SEQ ID NO: 25) (U.S. Pat. No. 5,739,277).
Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginally, as an aerosol administered nasally or via a pulmonary route, or via an ocular route. In another embodiment, the chimeric protein can be administered invasively, e.g., subcutaneously, intravenously.
The constant region domains or portions thereof making up an Fc moiety of a polypeptide of the invention may be derived from different immunoglobulin molecules. For example, a polypeptide of the invention may comprise a CH2 domain or portion thereof derived from an IgG1 molecule and a CH3 region or portion thereof derived from an IgG3 molecule. In another example, a polypeptide can comprise an Fc moiety comprising a hinge domain derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. As set forth herein, it will be understood by one of ordinary skill in the art that an Fc moiety may be altered such that it varies in amino acid sequence from a naturally occurring antibody molecule.
In another embodiment, a polypeptide of the invention comprises an scFc region comprising one or more truncated Fc moieties that are nonetheless sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of an Fc domain that binds to FcRn (i.e., the FcRn binding portion) comprises from about amino acids 282-438 of IgG1, EU numbering (with the primary contact sites being amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus, an Fc moiety of a polypeptide of the invention may comprise or consist of an FcRn binding portion. FcRn binding portions may be derived from heavy chains of any isotype, including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn binding portion from an antibody of the human isotype IgG1 is used. In another embodiment, an FcRn binding portion from an antibody of the human isotype IgG4 is used.
In one embodiment, a polypeptide of the invention lacks one or more constant region domains of a complete Fc region, i.e., they are partially or entirely deleted. In a certain embodiments polypeptides of the invention will lack an entire CH2 domain (ΔCH2 constructs). Those skilled in the art will appreciate that such constructs may be preferred due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody. In certain embodiments, polypeptides of the invention comprise CH2 domain-deleted Fc regions derived from a vector (e.g., from IDEC Pharmaceuticals, San Diego) encoding an IgG₁human constant region domain (see, e.g., WO 02/060955A2 and WO02/096948A2). This exemplary vector is engineered to delete the CH2 domain and provide a synthetic vector expressing a domain-deleted IgG₁constant region. It will be noted that these exemplary constructs are preferably engineered to fuse a binding CH3 domain directly to a hinge region of the respective Fc domain.
In other constructs it may be desirable to provide a spacer moiety between one or more constituent Fc moieties. For example, a spacer moiety may be placed between a hinge region and a CH2 domain and/or between a CH2 and a CH3 domains. For example, compatible constructs could be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (synthetic or unsynthetic) is joined to the hinge region with a 5-20 amino acid spacer moiety. Such a spacer moiety may be added, for instance, to ensure that the regulatory elements of the constant region domain remain free and accessible or that the hinge region remains flexible. Preferably, any linker peptide compatible with the instant invention will be relatively non-immunogenic and not prevent proper folding of the scFc region.
In certain embodiments, the polypeptides of the invention may comprise a dimeric Fc region comprising Fc moieties of the same, or substantially the same, sequence composition (herein termed a “homodimeric Fc region”). In other embodiments, the polypeptides of the invention may comprise a dimeric Fc region comprising at least two Fc moieties which are of different sequence composition (i.e., herein termed a “heterodimeric Fc region”). In one exemplary embodiment, the heterodimeric Fc region comprises an amino acid substitution in a first Fc moiety (e.g., an amino acid substitution of Asparagine at EU position 297), but not in a second Fc moiety.
In certain embodiments, the Fc region is hemi-glycosylated. For example, the heteromeric scFc region may comprise a first, glycosylated, Fc moiety (e.g., a glycosylated CH2 region) and a second, aglycosylated, Fc moiety (e.g., an aglycosylated CH2 region), wherein a linker is interposed between the glycosylated and aglycosylated Fc moieties. In other embodiments, the Fc region is fully glycosylated, i.e., all of the Fc moieties are glycosylated. In still further embodiments, the Fc region may be aglycosylated, i.e., none of the Fc moieties are glycosylated.
In certain embodiments, an Fc moiety employed in a polypeptide of the invention is altered, e.g., by amino acid mutation (e.g., addition, deletion, or substitution). For example, in one embodiment, an Fc moiety has at least one amino acid substitution as compared to the wild-type Fc from which the Fc moiety is derived. For example, wherein the Fc moiety is derived from a human IgG1 antibody, a variant comprises at least one amino acid mutation (e.g., substitution) as compared to a wild type amino acid at the corresponding position of the human IgG1 Fc region.
The amino acid substitution(s) of an Fc variant may be located at a position within the Fc moiety referred to as corresponding to the position number that that residue would be given in an Fc region in an antibody (as set forth using the EU numbering convention). One of skill in the art can readily generate alignments to determine what the EU number corresponding to a position in an Fc moiety would be.
In one embodiment, the Fc variant comprises a substitution at an amino acid position located in a hinge domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH2 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH3 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH4 domain or portion thereof.
In certain embodiments, the polypeptides of the invention comprise an Fc variant comprising more than one amino acid substitution. The polypeptides of the invention may comprise, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. Preferably, the amino acid substitutions are spatially positioned from each other by an interval of at least 1 amino acid position or more, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions or more. More preferably, the engineered amino acids are spatially positioned apart from each other by an interval of at least 5, 10, 15, 20, or 25 amino acid positions or more.
In certain embodiments, the Fc variant confers a change in at least one effector function imparted by an Fc region comprising said wild-type Fc domain (e.g., an improvement or reduction in the ability of the Fc region to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) or complement proteins (e.g. Clq), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine residue
The polypeptides of the invention may employ art-recognized Fc variants which is known to impart a change (e.g., an enhancement or reduction) in effector function and/or FcR or FcRn binding. Specifically, a binding molecule of the invention may include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, and WO06/085967A2; US Patent Publication Nos. US2007/0231329, US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767, US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; and 7,317,091, each of which is incorporated by reference herein. In one embodiment, the specific change (e.g., the specific substitution of one or more amino acids disclosed in the art) may be made at one or more of the disclosed amino acid positions. In another embodiment, a different change at one or more of the disclosed amino acid positions (e.g., the different substitution of one or more amino acid position disclosed in the art) may be made.
In certain embodiments, a polypeptide of the invention comprises an amino acid substitution to an Fc moiety which alters the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody.
Such polypeptides exhibit either increased or decreased binding to FcRn when compared to polypeptides lacking these substitutions and, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered polypeptide is desired, e.g., to treat a chronic disease or disorder (see, e.g, U.S. Pat. Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations where the starting polypeptide has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be desired include those applications in which localization the brain, kidney, and/or liver is desired. In one exemplary embodiment, the polypeptides of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the polypeptides of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, a polypeptide with altered FcRn binding comprises at least one Fc moiety (e.g, one or two Fc moieties) having one or more amino acid substitutions within the “FcRn binding loop” of an Fc moiety. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering) of a wild-type, full-length, Fc moiety. In other embodiments, a polypeptide of the invention having altered FcRn binding affinity comprises at least one Fc moiety (e.g, one or two Fc moieties) having one or more amino acid substitutions within the 15 Å FcRn “contact zone.” As used herein, the term 15 Å FcRn “contact zone” includes residues at the following positions of a wild-type, full-length Fc moiety: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering). In preferred embodiments, a polypeptide of the invention having altered FcRn binding affinity comprises at least one Fc moiety (e.g, one or two Fc moieties) having one or more amino acid substitutions at an amino acid position corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein.
A polypeptide of the invention may also comprise an art recognized amino acid substitution which alters the glycosylation of the polypeptide. For example, the scFc region of the binding polypeptide may comprise an Fc moiety having a mutation leading to reduced glycosylation (e.g., N- or O-linked glycosylation) or may comprise an altered glycoform of the wild-type Fc moiety (e.g., a low fucose or fucose-free glycan).
In other embodiments, a polypeptide of the invention comprises at least one Fc moiety having engineered cysteine residue or analog thereof which is located at the solvent-exposed surface. Preferably the engineered cysteine residue or analog thereof does not interfere with an effector function conferred by the scFc region. More preferably, the alteration does not interfere with the ability of the scFc region to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) or complement proteins (e.g. Clq), or to trigger immune effector function (e.g., antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)).
In one embodiment, an unprocessed polypeptide of the invention may comprise a genetically fused Fc region (i.e., scFc region) having two or more of its constituent Fc moieties independently selected from the Fc moieties described herein. In one embodiment, the Fc moieties of a dimeric Fc region are the same. In another embodiment, at least two of the Fc moieties are different. For example, the Fc moieties of the polypeptides of the invention comprise the same number of amino acid residues or they may differ in length by one or more amino acid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc moieties of the polypeptides of the invention may differ in sequence at one or more amino acid positions. For example, at least two of the Fc moieties may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).

VI. Polypeptide Linkers

As used herein, the term “polypeptide linkers” refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) which connects two domains in a linear amino acid sequence of a polypeptide chain. The polypeptides of invention are encoded by nucleic acid molecules that encode polypeptide linkers which either directly or indirectly connect the two Fc moieties which make up the construct. These linkers are referred to herein as “scFc linkers”. If the scFc linker connects two Fc moieties contiguously in the linear polypeptide sequence, it is a “direct” linkage. In contract, the scFc linkers may link the first Fc moiety to a binding moiety which is, in turn, linked to the second Fc moiety, thereby forming an indirect linkage. These scFc linkers (L) result in the formation of a single chain genetic construct. However, in one embodiment, the scFc polypeptides also comprise enzymatic cleavage sites which result in the scFc linker being cleavable (an cscFc linker) and, in one embodiment, substantially excised (e.g., during processing by a cell). Thus, the processed molecule is a dimeric molecule comprising at least two amino acid chains and substantially lacking extraneous linker amino acid sequences. In some embodiments, all or substantially all of the linker is excised, while in some embodiments, a portion of the cleavage site may remain, e.g., four arginines of the RRRR cleavage site.
In another embodiment, another type of polypeptide linker, herein referred to as a “spacer” may be used to connect different moieties, e.g., a clotting factor or a targeting moiety to an Fc moiety. This type of polypeptide linker may provide flexibility to the polypeptide molecule. Spacers are not typically cleaved, however such cleavage may be desirable. Exemplary positions of spacers are shown in the accompanying drawings. Spacers can be located between the clotting factors, targeting moieties, and/or scaffolds, e.g., at the N or C terminus of these moieties. In one embodiment, these linkers are not removed during processing.
A third type of linker which may be present in a chimeric clotting factor of the invention is a cleavable linker which comprises a cleavage site (e.g., a factor XIa, Xa, or thrombin cleavage site) and which may include additional spacer linkers on either the N terminal of C terminal or both sides of the cleavage site. These cleavable linkers when incorporated into a clotting factor result in a chimeric molecule having a heterologous cleavage site. Exemplary locations for such sites are shown in the accompanying drawings and include, e.g., adjacent to targeting moieties, In another embodiment, such linkers may be adjacent to a clotting factor or portion thereof. For example, in one embodiment, a cleavable linker may be fused to the N terminus of the heavy chain of a clotting factor to make an activatable form of the clotting factor. In such cases, the cleavable linker may include additional spacer linkers at the N terminus of the cleavage site, but require direct fusion at the C-terminus of the cleavage site to the amino terminus of the heavy chain of the clotting factor.
In one embodiment, an unprocessed polypeptide of the instant invention comprises two or more Fc domains or moieties linked via a cscFc linker to form an Fc region comprised in a single polypeptide chain. The cscFc linker is flanked by at least one enzymatic cleavage site, e.g., a site for processing by an intracellular enzyme. Cleavage of the polypeptide at the at least one enzymatic cleavage site results in a polypeptide which comprises at least two polypeptide chains. In one embodiment, an cscFc linker links F1 or F2 to, e.g., a clotting factor, optionally via a cleavage site.
As is set forth above, other polypeptide linkers may optionally be used in a construct of the invention, e.g., to connect a clotting factor or targeting moiety to an Fc moiety. One type of pollypeptide linker is referred to here as spacers. Some exemplary locations of spacers that can be used in connection with the invention include, e.g., polypeptides comprising GlySer amino acids such as those set forth in the accompanying figures and described in more detail below. In one embodiment, a spacer may be adjacent to one or more moieties each independently selected from clotting factor, scaffold moiety, e.g., Fc, cleavage site, and a targeting moiety.
In one embodiment, the polypeptide linker is synthetic, i.e., non-naturally occurring. In one embodiment, a polypeptide linker includes peptides (or polypeptides) (which may or may not be naturally occurring) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature. For example, in one embodiment the polypeptide linker may comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion). In another embodiment, the polypeptide linker may comprise non-naturally occurring amino acids. In another embodiment, the polypeptide linker may comprise naturally occurring amino acids occurring in a linear sequence that does not occur in nature. In still another embodiment, the polypeptide linker may comprise a naturally occurring polypeptide sequence.
For example, in certain embodiments, a polypeptide linker can be used to fuse identical Fc moieties, thereby forming a homomeric scFc region. In other embodiments, a polypeptide linker can be used to fuse different Fc moieties (e.g. a wild-type Fc moiety and an Fc moiety variant), thereby forming a heteromeric scFc region.
In another embodiment, a polypeptide linker comprises or consists of a gly-ser linker.
In one embodiment, an scFc or cscFc linker comprises at least a portion of an immunoglobulin hinge and a gly-ser linker. As used herein, the term “gly-ser linker” refers to a peptide that consists of glycine and serine residues. An exemplary gly/ser linker comprises an amino acid sequence of the formula (Gly₄Ser)n (SEQ ID NO: 4), wherein is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). A preferred gly/ser linker is (Gly₄Ser)₂(SEQ ID NO:29), (Gly₄Ser)₄(SEQ ID NO:6), or (Gly₄Ser)₆. (SEQ ID NO: 5) Another exemplary gly-ser linker is GGGSSGGGSG (SEQ ID NO: 30). In certain embodiments, said gly-ser linker may be inserted between two other sequences of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In other embodiments, a gly-ser linker is attached at one or both ends of another sequence of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In yet other embodiments, two or more gly-ser linker are incorporated in series in a polypeptide linker. In one embodiment, a polypeptide linker of the invention comprises at least a portion of an upper hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule), at least a portion of a middle hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of gly/ser amino acid residues (e.g., a gly/ser linker such as (Gly₄Ser)n) (SEQ ID NO:4)).
Polypeptide linkers of the invention are at least one amino acid in length and can be of varying lengths. In one embodiment, a polypeptide linker of the invention is from about 1 to about 50 amino acids in length. As used in this context, the term “about” indicates+/−two amino acid residues. Since linker length must be a positive interger, the length of from about 1 to about 50 amino acids in length, means a length of from 1-3 to 48-52 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 10-20 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 15 to about 50 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 20 to about 45 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 15 to about 35 or about 20 to about 30 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, or 60 amino acids in length. In one embodiment, a peptide linker of the invention is 20 or 30 amino acids in length.
Polypeptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced.

VIII. Enzymatic Cleavage Sites

In one embodiment, one or more enzymatic cleavage site(s) is linked to e.g., flanks or is adjacent to, a cscFc linker (L) of an unprocessed polypeptide of the invention. Such cleavage sites can be upstream or downstream of the cscFc liner or both. For example, in one embodiment of a construct encoding a polypeptide of the invention, a cleavage site is linked (e.g., directly or indirectly) to one or both ends of a cscFc linker (L).
For example, in one embodiment, a nucleic acid molecule of the invention specifies a polypeptide represented by the formula:
A-F1-P1-L-P2-B-F2 (I)
in linear sequence from the amino to carboxy terminus wherein A, if present, is a clotting factor or portion thereof, F1 is a first Fc moiety or domain, P1 is an enzymatic cleavage site, L is a cscFc linker, P2 is an enzymatic cleavage site B, if present, is a clotting factor or portion thereof, F2 is a second Fc moiety or domain and “-” represents a peptide bond. Formula (I) comprises at least an A or B and optionally both. A and B, if both present, can be the corresponding heavy and light chains of a clotting factor. Formula (I) comprises at least a P1 or P2 and optionally both. P1 and P2, if both present, can be the same or different. Formula (I) comprises at least a F1 and F2. F1 and F2, if both present, can be the same or different.
In another embodiment, a Factor XIa or Xa cleavage site may be incorporated into a construct of the invention, e.g., in a cleavable linker. Exemplary FXIa cleavage sites include, e.g, TQSFNDFTR and SVSQTSKLTR. Exemplary thrombin cleavage sites include, e.g, DFLAEGGGVR, TTKIKPR, LVPRG SEQ ID NO:35) and ALRPRVVGGA Other useful cleavage sites are known in the art.
In one embodiment, some portion of the linker may remain after cleavage at the at least one enzymatic cleavage site. In order to minimize the presence of extraneous amino acid sequences, two cleavage sites may be included in a polypeptide of the invention. In some embodiments, all or substantially all of the linker is excised, while in some embodiments, a portion of the cleavage site may remain, e.g., four arginines of the RRRR cleavage site.
Preparation of Polypeptides
A variety of methods are available for recombinantly producing a chimeric clotting factor of the invention. In one embodiment, the invention relates to a nucleic acid construct comprising a nucleic acid sequence encoding the chimeric proteins of the invention. It will be understood that because of the degeneracy of the code, a variety of nucleic acid sequences will encode the amino acid sequence of the polypeptide. The desired polynucleotide can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared polynucleotide.
Oligonucleotide-mediated mutagenesis is one method for preparing a substitution, in-frame insertion, or alteration (e.g., altered codon) to introduce a codon encoding an amino acid substitution (e.g., into an Fc variant moiety). For example, the starting polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer. In one embodiment, genetic engineering, e.g., primer-based PCR mutagenesis, is sufficient to incorporate an alteration, as defined herein, for producing a polynucleotide encoding a polypeptide of the invention.
For recombinant production, a polynucleotide sequence encoding the chimeric protein is inserted into an appropriate expression vehicle, i. e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
The nucleic acid encoding the chimeric protein is inserted into the vector in proper reading frame. The expression vector is then 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) and electroporation (Neumann et al. 1982, EMBO, J. 1: 841). A variety of host-expression vector systems may be utilized to express the chimeric proteins described herein in eukaryotic cells. In one embodiment, the eukaryotic cell is an animal cell, including mammalian cells (e. g. 293 cells, PerC6, CHO, BHK, Cos, HeLa cells). When the chimeric protein is expressed in a eukaryotic cell the DNA encoding the chimeric protein may also code for a signal sequence that will permit the chimeric protein to be secreted. One skilled in the art will understand that while the protein is translated the signal sequence is cleaved by the cell to form the mature chimeric protein. Various signal sequences are known in the art e. g., native factor V11 signal sequence, native factor IX signal sequence and the mouse IgK light chain signal sequence. Alternatively, where a signal sequence is not included the chimeric protein can be recovered by lysing the cells.
The chimeric protein of the invention can be synthesized in a transgenic animal, such as a rodent, goat, sheep, pig, or cow. The term “transgenic animals” refers to non-human animals that have incorporated a foreign gene into their genome. Because this gene is present in germline tissues, it is passed from parent to offspring. Exogenous genes are introduced into single-celled embryos (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82: 4438). Methods of producing transgenic animals are known in the art. including transgenics that produce immunoglobulin molecules (Wagner et al. 1981, Proc. Natl. Acad. Sci. USA 78: 6376; McKnight et al. 1983, Cell 34: 335; Brinster et al. 1983, Nature 306: 332; Ritchie et al. 1984, Nature 312: 517; Baldassarre et al. 2003, Theriogenology 59: 831; Robl et al. 2003, Theriogenology 59: 107; Malassagne et al. 2003, Xenotransplantation 10 (3): 267).
The expression vectors can encode for tags that permit for easy purification or identification of the recombinantly produced protein. Examples include, but are not limited to, vector pUR278 (Ruther et al. 1983, EMBO J. 2: 1791) in which the chimeric protein described herein coding sequence may be ligated into the vector in frame with the lac z coding region so that a hybrid protein is produced; pGEX vectors may be used to express proteins with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (e. g. PreCission Protease (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.
For the purposes of this invention, numerous expression vector systems may be employed. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors may include expression control sequences including, but not limited to, promoters (e.g., naturally-associated or heterologous promoters), enhancers, signal sequences, splice signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Expression vectors may also utilize DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), cytomegalovirus (CMV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites.
Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362). Cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation.
A preferred expression vector is NEOSPLA (U.S. Pat. No. 6,159,730). This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in cells, followed by selection in G418 containing medium and methotrexate amplification. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., >30 pg/cell/day. Other exemplary vector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.
In other embodiments the polypeptides of the invention of the instant invention may be expressed using polycistronic constructs. In these expression systems, multiple gene products of interest such as multiple polypeptides of multimer binding protein may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides of the invention in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.
More generally, once the vector or DNA sequence encoding a polypeptide has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably, plasmid introduction into the host is via electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or flourescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
As used herein, the term “transformation” shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
Along those same lines, “host cells” refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of polypeptides from recombinant hosts, the terms “cell” and “cell culture” are used interchangeably to denote the source of polypeptide unless it is clearly specified otherwise. In other words, recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
The host cell line used for protein expression is most preferably of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte), PerC6, and 293 (human kidney). Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
In one embodiment, a host cell endogenously expresses an enzyme (or the enzymes) necessary to cleave an scFc linker (e.g., if such a linker is present and contains intracellular processing site(s)) during processing to form the mature polyptide. During this processing, the scFc linker may be substantially removed to reduce the presence of extraneous amino acids. In another embodiment of the invention, a host cell is transformed to express one or more enzymes which are exogenous to the cell such that processing of an scFc linker occurs or is improved.
In one embodiment an enzyme which may be endogenously or exogenously expressed by a cell is a member of the furin family of enzymes. Complete cDNA and amino acid sequences of human furin (i.e., PACE) were published in 1990. Van den Ouweland A M et al. (1990) Nucleic Acids Res. 18:664; Erratum in: Nucleic Acids Res. 18:1332 (1990).
U.S. Pat. No. 5,460,950, issued to Barr et al., describes recombinant PACE and the coexpression of PACE with a substrate precursor polypeptide of a heterologous protein to improve expression of active, mature heterologous protein.
U.S. Pat. No. 5,935,815, issued to van de Ven et al., likewise describes recombinant human furin (i.e., PACE) and the coexpression of furin with a substrate precursor polypeptide of a heterologous protein to improve expression of active, mature heterologous protein. Possible substrate precursors disclosed in this patent include a precursor of Factor IX. Other family members in the mammalian furin/subtilisin/Kex2p-like proprotein convertase (PC) family in addition to PACE are reported to include PC1/PC3, PC2, PC4, PC5/6 (hereinafter referred to simply as PC5), PACE4, and LPC/PC7/PC8/SPC7. While these various members share certain conserved overall structural features, they differ in their tissue distribution, subcellular localization, cleavage specificities, and preferred substrates. For a review, see Nakayama K (1997) Biochem J. 327:625-35. Similar to PACE, these proprotein convertases generally include, beginning from the amino terminus, a signal peptide, a propeptide (that may be autocatalytically cleaved), a subtilisin-like catalytic domain characterized by Asp, His, Ser, and Asn/Asp residues, and a Homo B domain that is also essential for catalytic activity and characterized by an Arg-Gly-Asp (RGD) sequence. PACE, PACE4, and PC5 also include a Cys-rich domain, the function of which is unknown. In addition, PC5 has isoforms with and without a transmembrane domain; these different isoforms are known as PCSB and PCSA, respectively. Comparison between the amino acid sequence of the catalytic domain of PACE and the amino acid sequences of the catalytic domains of other members of this family of proprotein convertases reveals the following degrees of identity: 70 percent for PC4; 65 percent for PACE4 and PC5; 61 percent for PC1/PC3; 54 percent for PC2; and 51 percent for LPC/PC7/PC8/SPC7. Nakayama K (1997) Biochem J. 327:625-35.
PACE and PACE4 have been reported to have partially overlapping but distinct substrates. In particular, PACE4, in striking contrast to PACE, has been reported to be incapable of processing the precursor polypeptide of FIX. Wasley L C et al. (1993) J Biol Chem. 268:8458-65; Rehemtulla A et al. (1993) Biochemistry. 32:11586-90.
U.S. Pat. No. 5,840,529, issued to Seidah et al., discloses nucleotide and amino acid sequences for human PC7 and the notable ability of PC7, as compared to other PC family members, to cleave HIV gp160 to gp120 and gp41.
Nucleotide and amino acid sequences of rodent PC5 were first described as PC5 by Lusson J et al. (1993) Proc Natl Acad Sci USA 90:6691-5 and as PC6 by Nakagawa T et al. (1993) J Biochem (Tokyo) 113:132-5. U.S. Pat. No. 6,380,171, issued to Day et al., discloses nucleotide and amino acid sequences for human PCSA, the isoform without the transmembrane domain. The sequences of these enzymes and method of cloning them are known in the art.
Genes encoding the polypeptides of the invention can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e., those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the polypeptides typically become part of inclusion bodies. The polypeptides must be isolated, purified and then assembled into functional molecules.
In addition to prokaryates, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Other yeast hosts such Pichia may also be employed. Yeast expression vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.
Alternatively, polypeptide-coding nucleotide sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489, and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for polypeptides in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.
In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-) affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein. An affinity tag sequence (e.g. a His(6) tag) may optionally be attached or included within the polypeptide sequence to facilitate downstream purification.
In one embodiment, a host cell of the invention comprises a genetic construct encoding a polypeptide comprising an scFc scaffold and one or more enzymes that can process a cscFc linker. The construct and the enzyme(s) can be expressed using a single vector or two vectors.
In one embodiment, the invention pertains to nucleic acid molecules which encode a polypeptide of the invention. In one embodiment, the nucleic acid molecule encodes a chimeric clotting factor selected from the group consisting of FVII, FIX and FX and which comprises a targeting moiety which binds to platelets and optionally a spacer moiety between the clotting factor and the targeting moiety.
In another embodiment, the invention pertains to a nucleic acid molecule encoding a polypeptide comprising FVII, which FVII which comprises a heterologous enzymatic cleavage site activatable by a component of the clotting cascade.
Once expressed, the chimeric clotting factor can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity column chromatography, HPLC purification, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)) and see specifically the methods used in the instant Examples. Substantially pure proteins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
IX. Methods of Administering Polypeptides of the Invention
In another embodiment, the invention relates to a method of treating a subject with a hemostatic disorder comprising administering a therapeutically effective amount of an enhanced clotting factor of the Invention.
Compositions for administration to a subject include nucleic acid molecules which comprise a nucleotide sequence encoding a chimeric clotting factor of the invention (for gene therapy applications) as well as polypeptide molecules.
In one embodiment, an enhanced doting factor composition of the invention is administered in combination with at least one other agent that promotes hemostasis. Said other agent that promotes hemostasis Is a therapeutic with demonstrated clotting activity. As an example, but not as a limitation, hemostatic agent can include Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, prothrombin, or fibrinogen or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamic acid.
In one embodiment of the invention, the composition (e.g., the polypeptide or nucleic acid molecule encoding the polypeptide) is one in which the clotting factor is present in active form when administered to a subject. Such an activated molecule may be expressed by a cell in active form or may be activated in vitro prior to administration to a subject. In another embodiment, the composition is one in which the clotting factor is present in activatable form and the clotting factor is activated in vivo at the site of clotting after administration to a subject.
The chimeric clotting factor of the invention can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, sublingually, nasally, rectally, vaginally or via pulmonary route. The chimeric protein can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the desired site.
For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can 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, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.
For buccal and sublingual administration the composition may take the form of tablets, lozenges or fast dissolving films according to conventional protocols.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer (e.g. in PBS), with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
In one embodiment, the route of administration of the polypeptides of the invention is parenteral. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous form of parenteral administration is preferred. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, the polypeptides can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity 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 dispersion and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a polypeptide by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated 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 an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to clotting disorders.
The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
Effective doses of the compositions of the present invention, for the treatment of conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
In one embodiment, the dose of a biologically active moiety (e.g., comprising FIX) can range from about 25 to 100 IU/kg, e.g., 0.417 mg/kg to 1.67 mg/kg. In another embodiment, the dose of a biologically active moiety (e.g., comprising FVIII) can range from about 25 to 65 IU/kg, e.g., 0.003125 mg/kg to 0.008125 mg/kg. In another embodiment, the dose of a biologically active moiety (e.g., comprising FVII), can range from about 90 to 270 ug/kg or 0.090 to 0.270 mg/kg.
Dosages can range from 1000 ug/kg to 0.1 ng/kg body weight. In one embodiment, the dosing range is 1 ug/kg to 100 ug/kg. The protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e. g., STA-CLOT Vlla-rTF clotting assay. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models, e. g., a hemophiliac dog (Mount et al. 2002, Blood 99 (8): 2670).
Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. In some methods, two or more polypeptides may be administered simultaneously, in which case the dosage of each polypeptide administered falls within the ranges indicated.
Polypeptides of the invention can be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of modified polypeptide or antigen in the patient. Alternatively, polypeptides can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the polypeptides of the invention or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance or minimize effects of disease. Such an amount is defined to be a “prophylactic effective dose.” A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
Polypeptides of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).
As used herein, the administration of polypeptides of the invention in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed polypeptides. Those skilled in the art will appreciate that the administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment. A skilled artisan (e.g. a physician) would be readily be able to discern effective combined therapeutic regimens without undue experimentation based on the selected adjunct therapy and the teachings of the instant specification.
It will further be appreciated that the polypeptides of the instant invention may be used in conjunction or combination with an agent or agents (e.g. to provide a combined therapeutic regimen). Exemplary agents with which a polypeptide of the invention may be combined include agents that represent the current standard of care for a particular disorder being treated. Such agents may be chemical or biologic in nature. The term “biologic” or “biologic agent” refers to any pharmaceutically active agent made from living organisms and/or their products which is intended for use as a therapeutic.
The amount of agent to be used in combination with the polypeptides of the instant invention may vary by subject or may be administered according to what is known in the art. See for example, Bruce A Chabner et al., Antineoplastic Agents, in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Hardman et al., eds., 9^thed. 1996). In another embodiment, an amount of such an agent consistent with the standard of care is administered.
As previously discussed, the polypeptides of the present invention, may be administered in a pharmaceutically effective amount for the in vivo treatment of clotting disorders. In this regard, it will be appreciated that the polypeptides of the invention can be formulated to facilitate administration and promote stability of the active agent. Preferably, pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. Of course, the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.
In one embodiment, a chimeric clotting factor of the invention can be administered as a nucleic acid molecule. Nucleic acid molecules can be administered using techniques known in the art, including via vector, plasmid, liposome, DNA injection, electroporation, gene gun, intravenously injection or hepatic artery infusion. Vectors for use in gene therapy embodiments are known in the art.
In keeping with the scope of the present disclosure, the chimeric clotting factors of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect.
The chimeric proteins of the invention have many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject with a disease or condition. The disease or condition can include, but is are not limited to, hemostatic disorders.
In one embodiment, the invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric clotting factor of the invention.
The chimeric clotting factors of the invention treat or prevent a hemostatic disorder by promoting the formation of a fibrin clot. The chimeric clotting factor of the invention can activate any member of a coagulation cascade. The clotting factor can be a participant in the extrinsic pathway, the intrinsic pathway or both.
A chimeric clotting factor of the invention can be used to treat hemostatic disorders, e.g., those known to be treatable with the particular clotting factor present in the chimeric clotting factor. The hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X, or Factor XIII
In one embodiment, the hemostatic disorder is an inherited disorder. In one embodiment, the subject has hemophilia A, and the chimeric protein comprises Factor VII or Factor VIIIa. In another embodiment, the subject has hemophilia A and the chimeric clotting factor comprises Factor VII or Factor VIIa. In another embodiment, the subject has hemophilia B and the chimeric clotting factor comprises Factor IX or Factor IXa. In another embodiment, the subject has hemophilia B and the chimeric protein comprises Factor VII or Factor VIIa. In another embodiment, the subject has inhibitory antibodies to Factor VII or Factor VIIIa and the chimeric clotting factor comprises Factor VII or Factor VIIa. In yet another embodiment, the subject has inhibitory antibodies against Factor IX or Factor IXa and the chimeric protein comprises Factor VII or Factor VIIa.
The chimeric clotting factor of the invention can be used to prophylactically treat a subject with a hemostatic disorder. The chimeric clotting factor of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder.
In one embodiment, the hemostatic disorder is the result of a deficiency in a clotting factor, e.g., Factor IX, Factor VIII. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor.
In another embodiment, the hemostatic disorder can be an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an autoimmune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).
The invention also relates to methods of treating a subject that does not have a hemostatic disorder or a secondary disease or condition resulting in acquisition of a hemostatic disorder. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of at least one chimeric clotting factor of the invention. For example, in one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric clotting factor of the invention can be administered prior to or after surgery as a prophylactic. The chimeric clotting factor of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, or stem cell transplantation.
In another embodiment, the chimeric clotting factor of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

EXAMPLES

Throughout the examples, the following materials and methods were used unless otherwise stated.
General Materials and Methods
In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in electrophoresis. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C S. H. L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Example 1

Heterodimeric Constructs Comprising FVII-Fc and MB9-Fc at the Amino Terminus of the Second Fc Chain
Cloning of pSYN-FVII-027
The FVII-027 construct comprises cscFc for cleavage when processed during manufacture in a cell. The construct comprises a targeting moiety, a scFv moiety that binds to GPIIbIIIa, MB9.
Plasmid (pSYN-FVII-027) was generated for the expression FVII-Fc and MB9-Fc heterodimer, where MB9 is a scFv previously shown to bind to receptor GPIIb/IIIa on activated platelets. Protein from pSYN-FVII-027 is expressed in the cell as a single polypeptide where the C-terminus of the FVII-Fc subunit is linked to the N-terminus of the MB9-Fc subunit by a (GGGGS)_6xpolypeptide linker. Furthermore, RRRRS and RKRRKR sequences were inserted at the 5′ and 3′ end of the polypeptide linker, respectively, for intracellular cleavage by proprotein convertases following the last Arg at each sequence. Consequently, cells will express a 2 chain FVII-Fc/MB9-Fc heterodimer where the FVII-Fc chain has a RRRRS sequence at the C-terminus, but the remainder of the linker and the RKRRKR sequence have otherwise been removed.
As a first step a series of intermediate plasmid were generated using the following primers:

HindIII-SalI-BpsEI-Fc-F

AGTCAAGCTTGTCGACTCCGGAACTCCTGGGCGGACC

BamHI-linker-Fc-R

CATCGGATCCCCCGCCACCGGAACCTCCACCGCCTGATCCACCCCCACCT

GATCCGCCGCCACCTTTACCCGGAGACAGGGAGAGG

BclI-Fc-F

CAGTCTTGATCAGACAAAACTCACACATGCCCACC

scFc-EcoRI-R

ACTGACGAATTCTCATTTACCCGGAGACAGGGAG

HindIII-Kozak-FVII-F:

CGACAAGCTTGCCGCCACCATGGTCTCCCAGGCCCTCAGG

FVII-HC-BspEI-R:

AGGAGTTCCGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGGATCC

CCCGCCACCGGAACCTCCACCGCCTGATCCACCCCCACCTGATCCGCCGC

CACCGGACCCACCTCCGCCGGAGCCACCGCCACCGGGAAATGGGGCTCGC

AGGAGG

A 50 ul PCR reaction was carried out with 25 pmol of HindIII-SalI-BpEI-Fc-F and BamHI-linker-Fc-R and template pSYN-Fc-001 using the following cycle: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 54° C. 30 seconds, 72° C. 1 minute). The expected sized band (˜700 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned into the HindIII and BamHI restriction sites of pBUDCE4 (Invitrogen, Carlsbad, Calif.) to generate intermediate pSYN-FVII-007. Primers Hindlll-SalI-BpEI-Fc-F and BamHI-linker-Fc-R amplify the Fc region starting at amino acid 221 (EU numbering) and add a HindIII and a SalI restriction enzyme site immediately upstream of site Fc region, as well as a DNA fragment encoding a (GGGGS)_4xlinker followed by a BamHI site immediately downstream of the Fc coding region. Next, a 50 ul reaction was carried out with 25 pmol of Bc1I-Fc-F and scFc-EcoRI-R, and template pSYN-Fc-011 using the same cycles as above. The expected sized band (˜700 bp) was gel purified as above, cut with restriction enzymes BamHI and EcoRI, and cloned in the Bc1I/EcoRI restriction sites of pSYN-FVII-007 to generate the intermediate plasmid pSYN-FVII-008. The primer pair Bc1I-Fc-F and scFc-EcoRI-R amplifies the Fc region while adding a Ben and EcoRI restriction sites immediately upstream and downstream of the Fc coding region, respectively. To generate the last intermediate plasmid, a 50 ul PCR reaction was carried out with 25 pmol of HindIII-Kozak-FVII-F and FVII—HC-BspEI-R and template pSYN-FVII-001 using the following cycle: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 55° C. 30 seconds, 72° C. 90 seconds). The primer pair amplifies the FVII coding region while adding a DNA fragment at the 3′ end of the molecule encoding a (GGGGS)_6xpolypeptide linker followed by a fragment of the Fc region ending at amino acid 221 (EU numbering). Primer HindIII-Kozak-FVII-F generates a HindIII restriction site at the 5′ of the molecule followed by a Kozak sequence directly upstream of the FVII coding region. The FVII—HC-BspEI-R primer introduces DNA encoding the polypeptide linker as well as the Fc portion. The expected sized band (1500 bp) was gel purified as above and cloned into the HindIII/BspEI sites of pSYN-FVII-008 to generate pSYN-FVII-011.
Next, 2 DNA fragments were synthesized: Genescript-FVII-027-1 and Genescript-FVII-026-2. Genescript-FVII-027-1 consists of a DNA fragment encoding a portion of the Fc region (starting at nucleotide 1306, EU numbering) followed by the sequence RRRRS-(GGGGS)_6x-RKRRKR followed by a portion of the MB9 scFv (residues 1-142). An EcoRI site was introduced in the coding sequence of MB9 using the degeneracy of the genetic code to preserve the proper amino acid sequence and overlaps the last 6 bases of Genescript-FVII-027-1. In addition, the first 6 bases at the 5′ include a SapI site found within the Fc region. Genescript-FVII-026-2 consists of a DNA fragment encoding a portion of the MB9 (residues 143-273) followed by a (GGGGS)_6xpolypeptide linker followed by the Fc region and an EcoRI site. An EcoRI site was introduced in the coding sequence of MB9 using the degeneracy of the genetic code to preserve the proper amino acid sequence and overlaps the first 6 bases of Genescript-FVII-026-2.
Genescript-FVII-027-1 was cloned into the SapI and EcoRI sites of pSYN-FVII-011 to generate pSYN-FVII-036. Next, Genescript-FVII-026-2 was cloned into the EcoRI site of pSYN-FVII-036 to generate pSYN-FVII-027. Correct orientation of the last cloning step was confirmed by restriction enzyme analysis and DNA sequencing.

Example 2

Heterodimeric Constructs Comprising FVII-Fc and MB9-Fc, MB9 at the Carboxy Terminus of the Second Fc Chain
Cloning of FVII-037
The FVII-037 construct is made using an scFc scaffold which is not cleaved during processing. In this construct the targeting moiety, again the MB9 scFv which binds to GPIIbIIIa is attached to the c-terminus of the second Fc moiety.
Synthesis of DNA fragment Genescript-FVII-037 was outsourced (Genescript) This fragment comprises a portion of the Fc region (residues 434 to 447, EU numbering) followed by a (GGGGS)4x polypeptide linker and the MB9 scFv. A SaI/PEcoRI fragment of Genescript-FVII-037 was subcloned into the SapI/EcoRI of pSYN-FVII-011 (refer to P0830) to generate and intermediate construct. A SapI fragment from pSYN-FVII-011 was subcloned into the SapI sites of the intermediate construct to generate pSYN-FVII-037.

Example 3

Heterodimeric Constructs Comprising FVII-Fc and a Peptide Against GPIb at the Carboxy Terminus of the Second Fc Chain
Cloning of THE pSYN-FVII-041 intermediate construct.
In order to make this construct, the FVII-041 construct was first made as an intermediate. Synthesis of DNA molecule Genescript-FVII-041 was outsourced (Genescript). This fragment was digested with SapI and cloned into SapI sites of pSYN-FVII-011 to generate pSYN-FVII-041. This process introduces a unique SalI site (residues 412-413 EU numbering, GTG GAC to GTC GAC) in the second Fc.
Cloning of pSYN-FVII-044-, -045 and -046.
The FVII-041 construct was used as the starting material to generate several constructs that comprise targeting moieties which are peptides that bind to GPIb. The PS4 peptide is used in the -044 construct, the OS1 peptide in the -045 construct, ad the OS2 peptide in the -046 construct. In these constructs an scFc scaffold is used and the peptides are attached via a linker to the C-terminus of the second Fc moiety.
Synthesis of Genescript-FVII-044, -045 and -046 was outsourced (Genescript). These DNA fragments were cleaved with SalI/EcoRI and subcloned into the SalI/EcoRI sites of pSYN-FVII-041 to generate pSYN-FVII-044, -045 and -046.

Example 4

Heterodimeric Constructs Comprising FVII-Fc and a Peptide Against GPIb at the Amino Terminus of the Second Fc Chain
Cloning of the pSYN-FVII-043 intermediate.
In order to make this construct, the FVII-043 construct was first made as an intermediate. Synthesis of DNA fragment Genescript-FVII-043 was outsourced (Genescript). This fragment comprises a DNA molecule encoding a portion of the Fc region (residues 232 to 447, EU numbering) followed by a (GGGGS)4x polypeptide linker and another portion of the Fc region (residues 221 to 238, EU numbering). This DNA fragment was digested with BspEI and RsrII and subcloned into the BspEI/RsrII sites of pSYN-FVII-042 to generate pSYN-FVII-050. This process introduces a unique SalI site (residues 412-413 EU numbering, GTG GAC to GTC GAC) in the first Fc. A HindIII/EcoRI fragment of pSYNFVII-050 was subcloned into the HindIII/EcoRI sites of pSYN-FVII-011 to generate p SYN-FVII-043.
Cloning of pSYN-FVII-047, -048 and -049.
The FVII-043 construct was used as the starting material to generate several constructs that comprise targeting moieties which are peptides that bind to GPIb. The PS4 peptide is used in the -047 construct, the 0S1 peptide in the -048 construct, and the 0S2 peptide in the -049 construct. In these constructs an scFc scaffold is used and the peptides are interposed between the scFc linker and a linker which is attached to the N-terminus of the second Fc moiety.
Synthesis of DNA molecules Genescript-FVII-047, -048 and -049 was outsourced (Genescript). A Sal/RsrII fragment from Genescript-FVII-047, -048 and -049 was subcloned into SalI/RsrII sites of pSYN-FVII-043 to generate pSYN-FVII-047, -048 and 049, respectively.

Example 5

Heterodimeric Constructs Comprising a Gla-Deleted FVII-Fc and a Targeting Molecule
Cloning of the FVII-028 intermediate
In order to make this construct, the FVII-028 construct was first made as an intermediate. Synthesis of DNA fragment Genescript-FVII-028 was outsourced (Genescript). This fragment was cut with HindIII/XbaI and subcloned into pSYN-FVII-011 to generate p SYN-FVII-028.
Cloning of FVII-053
The FVII-028 construct was used as the starting material to generate a construct that comprises a targeting moiety and employs a clotting factor that lacks a Gla domain. For this construct, amino acids 1-35 were removed from FVII and an RKRRKR insertion was added after residue R152 (WT FVII numbering) to facilitate intracellular activation. The MB9 scFv served as the targeting moiety.
DNA molecule Genescript-FVII-025 was outsourced and an XbaI/BsiWI fragment from this molecule was subcloned into XbaI/BsiWI sites of pSYN-FVII-028 to generate p SYN-FVII-053.

Example 6

Heterodimeric Constructs Comprising a Factor VII Heavy and Light Chains as Two Separate Polypeptides.
Cloning of pSYN-FVII-024 intermediate construct
The FVII-024 construct is one in which the heavy and light chains of factor FVII are not contiguous in a single chain molecule. The construct employs cscFc such that the cscFc linker is cleaved by proteases in the trans-Golgi network This cleavage results in linker removal as well as activation of FVII, resulting in the expression of activated FVIIaFc.
The coding sequence of FVII was obtained by reverse transcription coupled to polymerase chain reaction from a human liver mRNA library (Ambion, Austin, Tex.) using the following primers:

	FVII-F1
	GGGAATGTCAACAGGCAGGG

	FVII-R1
	CTTGGCTTTCTCTCCACAGGC

A 50 μl reaction was carried out with 10 pmol of each primer using the Superscript One-step RT-PCR with Platinum Taq system (Invitrogen, Carlsbad, Calif.) according to the manufacturer's standard protocol in a MJ thermocycler. The cycle used was 50° C. for 30 minutes for the reverse transcription followed by denaturing at 94° C. for 2 minutes and 30 cycles of (94° C. 30 seconds, 53° C. 30 seconds, 72° C. 90 seconds) followed by 10 minutes at 72° C. The expected sized band (˜1400 bp) was gel-purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned in pCR2.1 TOPO using the TOPO TA Cloning kit (Invitrogen, Carlsbad, Calif.) to produce the intermediate plasmid pSYN-FVII-001. To construct a plasmid for the expression of a two-chain FVII-Fc and Fc heterodimer, the FVII coding sequence was PCR-amplified using the following primers:

HindIII-Kozak-FVII-F

CGACAAGCTTGCCGCCACCATGGTCTCCCAGGCCCTCAGG

BspeI-Fc-FVII-R

CGACTCCGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGGGAAATG

GGGCTCGCAGG

The forward primer HindIII-Kozak-FVII-F adds a Hindlll restriction site followed by a Kozak sequence immediately upstream of the FVII coding region. The reverse primer BspeI-Fc-FVII-R adds a fragment of the constant region of IgG1 (the Fc region) comprising amino acids 221-233 (EU numbering). This process also incorporates a BspEI restriction site at amino acids 231-233 using the degeneracy of the genetic code to preserve the correct amino acid sequence (EU numbering). A 50 ul reaction was carried out with 15 pmol of each primer and template pSYN-FVII-001 using Platinum Pfx DNA Polymerase system according to manufacturer's protocol in a MJ Thermocycler using the following cycles: 95° C. 2 minutes; 30 cycles of (95° C. 15 seconds, 49° C. 30 seconds, 68° C. 90 seconds); 68° C. 10 minutes. Plasmid pSYN-FIX-027 (pBUD FIXFc/Fc) was digested with Hindlll and BspEI and the expected sized band for the vector (approximately 5800 bp) was purified away from the FIX insert (expected size band approximately 1480 bp) with a Gel Extraction kit (Qiagen, Valencia, Calif.). Next, the PCR-amplified FVII sequence was subcloned into HindIII and EcoRI sites of the vector derived from pSYN-FIX-027 after removing the FIX insert. This generated pSYN-FVII-002 (pBUD FVIIFc/Fc). Next, A (GGGGS)_6xpolypeptide linker was added between FVII and the Fc region coding sequences in pSYN-FVII-002 using the following primers:

FVII-linker-F:

CATCCCCAGCACGTACGTCC

FVII-Linker-R:

GGGCATGTGTGAGTTTTGTCTGATCCCCCGCCACCGGAACCTCCACCGCC

TGATCCACCCCCACCTGATCCGCCGCCACCGGACCCACCTCCGCCGGAGC

CACCGCCACCGGGAAATGGGGCTCGCAGGAGG

Fc-linker-F:

GACAAAACTCACACATGCCCACC

Fc-linker-R:

GCAGAATTCTCATTTACCCGGAG

Two 12 μl PCR reactions were carried out with either 12 pmol of FVII-linker-F and FVII-Linker-R (reaction 1) or Fc-linker-F and Fc-linker-R (reaction 2) using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, Ind.) according to manufacturer's standard protocol in a MJ Thermocycler. The first and second reactions were carried out with 1 μg of pSYN-FVII-002 as template using the following cycle: 94° C. 2 minutes; 14 cycles of (94° C. 30 seconds, 55° C. 30 seconds, 72° C. 2 minutes); 72° C. 10 minutes. The expected sized bands (532 bp for reaction 1 and 670 bp for reaction 2) were gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.), then combined in a PCR reaction with 25 pmol of FVII-linker-F and Fc-linker-R as before, but with 30 rounds of amplification. The expected sized band (1200 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and digested with restriction enzymes KpnI and EcoRI. The expected sized band (920 bp) was gel purified as before and cloned into the KpnI/EcoRI sites of pSYN-FVII-002 to generate pSYN-FVII-003 (pBUD FVIIFc/6x(GGGGS)/Fc).
Cloning of pSYN-FVII-024 to express a two-chain heterodimer
Plasmid (pSYN-FVII-024) was generated for the expression of a two-chain heterodimer where one chain consists of the FVII light chain (residues 1-152) followed by a (GGGGS)_6xlinker followed by the Fc region, while the other chain contains a FVII heavy chain (residues 153 to 406) followed by a (GGGGS)_6xlinker followed by the Fc region. The plasmid is designed to express the heterodimer as a single polypeptide where the C-terminus of the FVII heavy chain-linker-Fc chain is connected to the N-terminus of the heavy chain-linker-Fc chain by the following polypeptide sequence: RRRRS-(GGGGS)_6x-RKRRKR, where the RRRRS and RKRRKR sequences are proprotein convertase cleavage sites. Intracellular cleavage by proprotein convertases following the last Arg at each cleavage site can result in removal of the polypeptide linker. Consequently, cells will express a 2 chain heterodimer where the FVII light chain-linker-Fc chain has a RRRRS sequence at the C-terminus, but the remainder of the linker and the RKRRKR sequence have otherwise been removed. Construction of the pSYN-FVII-024 and several intermediate plasmids required the use of the following primers:

HindIII-SalI-BpEI-Fc-F

AGTCAAGCTTGTCGACTCCGGAACTCCTGGGCGGACC

BamHI-linker(PACE1)-Fc-R

CATCGGATCCCCCGCCACCGGAACCTCCACCGCCTGATCCACCCCCACCT

GATCCGCCGCCACCGCTCCGGCGGCGCCGTTTACCCGGAGACAGGGAGAG

G

HindIII-Kozak-FVII-F

CGACAAGCTTGCCGCCACCATGGTCTCCCAGGCCCTCAGG

BspEI-Fc-linker-FVIILC-R

GAGTTCCGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCTGATCCCC

CGCCACCGGAACCTCCACCGCCTGATCCACCCCCACCTGATCCGCCGCCA

CCGGACCCACCTCCGCCGGAGCCACCGCCACCTCGGCCTTGGGGTTTGCT

GG

BamHI-2xlink-pace-HC-F

CAGTCTGGATCCGGCGGTGGAGGTTCCGGTGGGGGTGGATCAAGGAAGAG

GAGGAAGAGGATTGTGGGGGGCAAGGTGTGCC

Fc-EcoRI-R

ATGTCTGAATTCTCATTTACCCGGAGACAGGGAGAGG

To generate the first intermediate plasmid, a PCR reaction was performed with 25 pmol of primers Hindlll-SalI-BpEI-Fc-F and BamHI-linker(PACE1)-Fc-R and template pSYN-Fc-001 using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, Ind.) according to manufacturer's standard protocol in a MJ Thermocycler. The following cycles were used: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 58° C. 30 seconds, and 72° C. 1 minute); 72° C. 10 minutes. The correct sized band (approximately 730 bp) was gel purified as above and cloned into the HindIII/BamHI sites of pBUDCE4 vector (Invitrogen, Carlsbad, Calif.), generating pSYN-FVII-014. PCR amplification with primers HindIII-SalI-BpEI-Fc-F and BamHI-linker(PACE1)-Fc-R generated a DNA fragment encoding a portion of the Fc region (Amino A X-Y) followed by an RRRRS sequence and (GGGGS)_2xpolypeptide linker. Primer HindIII-SalI-BpEI-Fc-F introduces a HindIII and SalI restriction site at the 5′ end of the molecule, while primer BamHI-linker(PACE1)-Fc-R introduces a BamHI at the 3′ end that overlaps the codons encoding the last 2 residues of the GGGGS linker (residues GS with codons GGA TCC). Next, another PCR reaction was performed as above with primers HindIII-Kozak-FVII-F and BspEI-Fc-linker-FVIILC-R and template pSYN-FVII-002 using the same conditions described for cloning of pSYN-FVII-014, but with an annealing temperature of 57° C. The expected sized band (approximately 700 bp) was gel purified and cloned into the Hindlll and BspEI sites of pSYN-FVII-014 to generate pSYN-FVII-023. Primers Hindlll-Kozak-FVII-F and BspEI-Fc-linker-FVIILC-R amplified a DNA fragment encoding the FVII light chain followed by a (GGGGS)_6xpolypeptide linker and a portion of the Fc region up to amino acid 232 (EU numbering). Primer HindIII-Kozak-FVII-F introduces a HindIII restriction site at the 5′ end of the molecule followed by a Kozak sequence while primer BspEI-Fc-linker-FVIILC-R adds a BspeI site at the 3′ end of the molecule.
In the final step a PCR reaction was carried out as above with primers BamHI-2×link-pace-HC-F and Fc-EcoRI-R and template pSYN-FVII-003 with the following cycles: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 55° C. 30 seconds, and 72° C. 2 minute); 72° C. 7 minutes. This PCR reaction generated a DNA molecule encoding a (GGGGS)_2xpolypeptide linker followed by a RKRRKR sequence followed by the FVII heavy chain. Primers BamHI-2×link-pace-HC-F and Fc-EcoRI-R introduce a BamHI site and an EcoRI site at the 5′ and 3′ end of the molecule, respectively. The expected sized band (approximately 1600 bp) was cloned into the BamHI and EcoRI sites of pSYN-FVII-023 to generate pSYN-FVII-024.
Cloning of intermediate pSYN-FVII-073
A silent mutation was introduced in the first Fc moiety of FVII-024 by PCR-based site-directed mutagenesis methods, resulting in the generation of a SalI site at DNA region encoding amino acids in position 412 and 413 (EU numbering). This generated the intermediate construct FVII-073.
Cloning of pSYN-FVII-057
The synthesis of the DNA sequence comprising nucleotides from the SalI to BsiWI sites of pSYN-FVII-057 was outsourced. This DNA was subcloned into the SalI/BsiWI sites of pSYN-FVII-073 to generate pSYN-FVII-057.
Cloning of pSYN-FVII-058, pSYN-FVII-059, pSYN-FVII-060, pSYN-FVII-061 and pSYN-FVII-062
These constructs were cloned as described for pSYN-FVII-057 (outsourced synthesis of DNA from SalI to BsiWI and subcloned into pSYN-FVII-073).
Cloning of pSYN-FVII-066
The synthesis of the DNA sequence comprising nucleotides from the SalI to RsrII sites of pSYN-FVII-066 was outsourced. This DNA was subcloned into the SalI/RsrII sites of pSYN-FVII-043 to generate pSYN-FVII-066.
Cloning of pSYN-FVII-067
The synthesis of the DNA sequence comprising nucleotides from the SalI to EcoRI sites of pSYN-FVII-067 was outsourced. This DNA was subcloned into the SalI/EcoRI sites of pSYN-FVII-041 to generate pSYN-FVII-067.
Cloning of pSYN-FVII-090
The synthesis of the DNA sequence comprising nucleotides from the BamHI to BsiWI sites of pSYN-FVII-090 was outsourced. This DNA was subcloned into pSYN-FVII-061 by 3-way ligation (where the outsourced DNA was cut with BamHI/BsiWI and pSYN-FVII-061 with BamHI/BsiWI/NotI) to generate pSYN-FVII-090.
Cloning of pSYN-FVII-100
A portion (amino acids 311 to 322 of the FVII mature sequence) of the 170 loop of FVII was replaced with the 170 loop of trypsin (amino acids EASYPGK). This mutation was introduced by standard overlapping PCR methods using the pSYN-FVII-090 as template and backbone structure to generate pSYN-FVII-100.
Cloning of pSYN-FVII-115
A triple point mutation (V158D, E296V and M298Q; mature FVII sequence numbering) was introduced into the FVII coding region of pSYN-FVII-090 by PCR-based site-directed mutagenesis to generate pSYN-FVII-115.
Cloning of pSYN-FVII-118
The synthesis of the DNA sequence comprising nucleotides from the XbaI to BsiWI sites of pSYN-FVII-118 was outsourced. This DNA was subcloned into the XbaI/BsiWI sites of pSYN-FVII-011 to generate pSYN-FVII-118.
Cloning of pSYN-FVII-119
The synthesis of the DNA sequence comprising nucleotides from the XbaI to BsiWI sites of pSYN-FVII-119 was outsourced. This DNA was subcloned into the XbaI/BsiWI sites of pSYN-FVII-011 to generate pSYN-FVII-119.
Cloning of pSYN-FVII-127
A DNA fragment comprising the 170 loop of trypsin was generated by PCR using pSYN-FVII-100 as template. This PCR reaction generated BsiWI and BspEI restriction sites at the 5′ and 3′, respectively. The DNA fragment was subcloned into the BsiWI/BspEI sites of pSYN-FVII-118 to generate pSYN-FVII-127.
Cloning of pSYN-FIX-042
A HindIII/BspEI fragment from pSYN-FIX-030 (as described in U.S. Pat. No. 7,566,565) was subcloned into the HindIII/BspEI sites of pSYN-FVII-011 to generate pSYN-FIX-042.
Cloning of pSYN-FIX-068
A HindIII/BspEI fragment from pSYN-FIX-030 (plasmid described in full in U.S. Pat. No. 7,566,565) was subcloned into the HindIII/BspEI sites of pSYN-FVII-066 to generate pSYN-FIX-068.
Cloning of pSYN-FIX-088
A BspEI-EcoRI fragment from pSYN-FIX-067 was subcloned into BspEI-EcoRI sites of pSYN-FIX-053 to generate pSYN-FIX-088.
Cloning of pSYN-FIX-089
A BspEI-EcoRI fragment from pSYN-FIX-048 was subcloned into BspEI-EcoRI sites of pSYN-FIX-053 to generate pSYN-FIX-089
Cloning of pSYN-FIX-090
A DNA fragment comprising the FIX coding region from the XbaI site to the C-terminus of the protein followed by a 6x(GGGGGS) linker, the SCE5 coding sequence and an EcoRI site was outsourced for synthesis and subcloned into the XbancoRI sites of pSYN-FIX-053 to generate pSYN-FIX-090. The SCE5 sequence is set forth below:

AQVQLQESGGGLVQPGGSLRLSCAASGEMESRYAMSWVRQAPGKGPEW

VSGISGSGGSTYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYY

CARGATYTSRSDVPDQTSFDYWGQGTLVTVSSGSASAPKLEEGEFSEA

RVSELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKPGQAPTLVI

YGLSKRPSGIPDRFSASSSGNTASLTITGAQAEDEADYYCLLYYGGGQ

QGVFGGGTKLTVLRQPKAAPSVTLFPPSSAA

Cloning of pSYN-FVII-094
A DNA fragment comprising a sequence encoding a 6x(GGGGS) linker followed by the SCE5 coding sequence was synthesized (outsourced) and cloned into the EcoRV/EcoRI sites of a pSYN-FVII-011 variant that had been previously modified to generate an EcoRV site at the C-terminus of the FVII coding region.
Cloning of pSYN-FVII-088
The synthesis of the DNA sequence comprising nucleotides from the SalI to RsrII sites of pSYN-FVII-088 was outsourced. This DNA was subcloned into the SalI/RsrII sites of pSYN-FVII-066 to generate pSYN-FVII-088.
Cloning of pSYN-FVII-125
A DNA fragment was PCR amplified from pSYN-FVII-088, comprising the AP3 region and part of the linker. This PCR reaction generated BamHI and EcoRI sites at the 5′ and 3′ of the DNA fragment, respectively. This DNA fragment was subcloned into the BamHI/EcoRI sites of pSYN-FVII-011 to generate pSYN-FVII-125.
Cloning of pSYN-FVIII-041
The coding sequence of human recombinant B-domain deleted FVIII was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human liver poly A RNA (Clontech) using FVIII-specific primers. The FVIII sequence includes the native signal sequence for FVIII. The B-domain deletion starts after serine 743 (S743; 2287 bp) and ends before glutamine 1638 (Q1638; 4969 bp) for a total deletion of 2682 bp (SQ version).
The coding sequence for human recombinant Fc was obtained by RT-PCR from a human leukocyte cDNA library (Clontech) using Fc specific primers. Primers were designed such that the B-domain deleted FVIII sequence was fused directly to the N-terminus of the Fc sequence with no intervening linker. The FVIIIFc DNA sequence was cloned into the mammalian dual expression vector pBUDCE4.1 (Invitrogen) under control of the CMV promoter.
A second identical Fc sequence including the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of the second promoter, EF1α, in the expression vector pBUDCE4.1. This final construct was designated pSYN-FVIII-013.
A second plasmid was created from similar constructs using PCR and standard molecular biology techniques, in order to express rFVIIIBDD-Fc-Fc in which the rFVIIIBDDFc coding sequence was fused to the second Fc sequence with a (GGGGS)4 linker, allowing for production of only the rFVIIIBDD-Fc monomer-dimer hybrid in transient transfection. This construct was designated pSYN-FVIII-041.
Cloning of pSYN-FVIII-049
Generated intermediate pSYN-FVIII-048 by cloning NheI/XhoI fragment from pBUD-CE4.1 into pSYN-FVIII-013. The synthesis of a DNA fragment comprising the region from RsrII to XbaI sites of pSYN-FVIII-049 was outsourced. This fragment was subcloned into the RsrII/XbaI sites of pSYN-FVIII-048 to generate p SYN-FVIII-049.
Cloning of pSYN-FVIII-108
A SalI/RsrII fragment from pSYN-FVII-066 was subcloned into pSYN-FVIII-049 to generate pSYN-FVIII-108.

Example 7

Additional Attempts at Expression of Activated Constructs
Several other constructs were made with the goal of expressing activated FVII.
However, these constructs did not successfully express activated molecules. By Western blot it was demonstrated that that the FVII heavy chain cannot be expressed with a free N terminus using a common method of fusing a heterologous signal peptide to the N-terminus of the heavy chain.
Cloning of pSYN-FVII-010
The FVII-010 construct is one in which the heavy chain of factor VII was expressed in the context of an scFc scaffold and the light chain was expressed separately.
PCR-amplify with primer pairs FVII-HC-Hind3-IggKss-F/FVII—HC-BspEI-R, using pSYN-FVII-001 (see supra.). Clone in BspEI/HindIII sites of pSYN-FVII-008 (see supra), generating pSYN-FVII-009.
PCR amplify FVII light chain from pSYN-FVII-003 (refer to P0830) with primers FVII-LC-NotI-F/FVII-LC-XhoI-R and clone in pSYN-FVII-009 to generate pSYN-FVII-010.

Primers

FVII-HC-BspEI-R

AGGAGTTCCGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGGAT

CCCCCGCCACCGGAACCTCCACCGCCTGATCCACCCCCACCTGATCCG

CCGCCACCGGACCCACCTCCGCCGGAGCCACCGCCACCGGGAAATGGG

GCTCGCAGGAGG

FVII-HC-Hind3-IggKss-f

ACTGACAAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGG

GTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTATTGTGGGGGGCAAG

GTGTGC

FVII-LC-NotI-F

ACTGACGCGGCCGCGCCGCCACCATGGTCTCCCAGG

FVII-LC-XhoI-R

ACTGACCTCGAGTTATCGGCCTTGGGGTTTGCTGG

Cloning of pSYN-FVII-013
The FVII-013 construct is one in which the light chain was expressed in the context of an scFc scaffold and the heavy chain was expressed separately.
PCR-amplify with primer pair FVII-LC-linker-BamHI-R/HindIII-Kozak-FVII-F from pSYN-FVII-001 (refer to P0830) and clone in BamHI/HindIII sites of pSYN-FVII-011, generating pSYN-FVII-012. PCR-amplify FVII-HC from pSYN-FVII-009 using primer pair FVII-HC-NotI-F/FVII-HC-XhoI-R ad subclone in pSYN-FVII-012 to generate pSYN-FVII-013.

Primers

FVII-LC-6xlinker-BamHI

RACTGACGGATCCCCCGCCACCGGAACCTCCACCGCCTGATCCACCCC

CACCTGATCCGCCGCCACCGGACCCACCTCCGCCGGAGCCACCGCCAC

CTCGGCCTTGGGGTTTGCTGGC

HindIII-Kozak-FVII-F

CGACAAGCTTGCCGCCACCATGGTCTCCCAGGCCCTCAGG

FVII-HC-NotI-F

ACTGACGCGGCCGCGCCGCCACCATGGAGACAGAC

FVII-HC-XhoI-R

ACTGACCTCGAGTTAGGGAAATGGGGCTCGCAGGAG

Cloning of pSYN-FVII-018
For the FVII-018 construct, the heavy chain of FVII was expressed as an Fc fusion protein and the light chain of FVII was separately expressed as a separate Fc fusion protein.
Primers FVII-HC-Hind3-IggKss-F/scFc-EcoRI-R were used to PCR amplify HCFVII-linker-Fc, using pSYN-FVII-010 as template. Subclone in HindIII/EcoRI sites of pBUDCE4. This makes pSYN-FVII-017. Next, PCR-amplify from pSYN-FVII-013 with primers FVII-LC-NotI-F/FC-XHOI-R and subclone in XhoI/NotI sites of FVII-017. This makes PSYN-FVII-018.

	Primers
	scFc-EcoRI-R
	ACTGACGAATTCTCATTTACCCGGAGACAGGGAG

	Fc-XhoI-R
	AGCTCTCGAGTCATTTACCCGGAGACAGGG

Example 8

Attempts at Expression of Activatable Constructs
Cloning of FVII-039, -040
Several constructs were made in an attempt to generate constructs in which Factor VII can be activated in vivo at the site of clotting using an appropriate cleavage site, in this case the DFTR Factor XIa cleavage site.
The 039 construct was made in the context of an scFc scaffold. The construct included the FVII light chain, the FXIa cleavage site, and the FVII heavy chain with a I153V mutation in linear sequence attached to the N-terminus of the first Fc moiety.
The 040 construct was also made in the context of an scFc scaffold. The construct included the FVII light chain with an R152 deletion, the FXIa cleavage site, and the FVII heavy chain with an I153V mutation in linear sequence attached to the N-terminus of the first Fc moiety. The DFTR cleavage sequence is a natural FXIa sequence found in FIX. In FIX, the DFTR sequence is followed by a valine, so an I152V mutation was introduced in pSYN-FVII-039, -040 to increase FXIa cleavage efficiency.
Synthesis of DNA molecule Genescript-FVII-039 and -040 was outsourced (Genescript). An XbaI/BsiWI fragment from Genescript-FVII-039 and -040 was subcloned into XbaI/BsiWI sites of pSYN-FVII-011 to generate pSYN-FVII-039 and -040, respectively.

Example 9

Transient Transfection of Constructs
For expression of constructs, HEK-293-F cells were grown in suspension in Freestyle media (Invitrogen) supplemented with vitamin K3 (For FVII and FIX transfections only) (Sigma Aldrich, St. Louis, Mo.) to 2 μg/liter (growth media) as suspension cells at 37° C./10% CO₂. Cells we subcultured every three to four days by seeding at cell density of 5×10⁵cell s/ml.
Twenty-four hours prior to transfection cells were seeded at a density of 7×10⁵cells/ml in growth media supplemented with LONG™ R3IGF-1 (Sigma Aldrich, St. Louis, Mo.) to 20 μg/liter (transfection media). On the day of transfection, a transfection solution was made with a volume equal to 5% of the total volume of the cell culture to be transfected. In the transfection solution DNA was added (final concentration 20 mg/L) to a freshly made solution of PEI (60 mg/L) in transfection media. The solution was swirled for 30 seconds and incubated for five minutes at room temperature before adding directly to the cell culture. Four hours later a volume equal to the cell culture volume of OptiCHO (Invitrogen) supplemented with vitamin K3, LONG™ R3IGF-1 and 200 mM L-glutamine was added to the cells. The cell culture was allowed to grow as shown above and daily media samples were taken to assess protein expression. On the day of harvest, the cells were spun down and the media filtered in preparation for protein purification or protein analysis by protein A pulldown/western blot.

Example 10

Protein Purification of FVIIFc Molecules (except FVII-028 and FVII-053) and FIXFc Molecules
FVIIFc molecules were purified from conditioned media using the following columns: 1) Anion exchange chromatography with pseudo-affinity elution (e.g. Q sepharose 4FF (GE Healthcare) followed by elution with varying levels of CaCl₂to selectively elute the most active species), followed by 2) shFcRn (soluble human FcRn) affinity (NHS-coupled shFcRn with sepharose 4FF beads) chromatography, binding Fc-containing proteins at low pH (e.g. pH 6.2) and eluting at neutral pH (e.g. pH 8.0). In some cases, an additional step was included utilizing cation exchange chromatography with NaCl elution. These purification steps utilized standard methods known to those in the art to generate purified proteins of >95% purity by SEC analysis and SDS-PAGE. FIXFc proteins were purified as previously described in U.S. Pat. No. 7,566,565.

Example 11

Protein Purification of FVII-028 and FVII-053
FVII-028 and -053 were purified from conditioned media using the following columns: 1) Hydrophobic interaction chromatography (e.g. Phenyl FF (high sub) (GE Healthcare)), followed by 2) Anion/cation exchange chromatography with salt elution. These purification steps utilized standard methods known to those in the art to generate purified proteins of >95% purity by SEC analysis and SDS-PAGE.

Example 12

Purification of FIX-090
FIX-090 was purified through a 2-step chromatography process, first using an immunoaffinity chromatography step with an anti-GLA domain antibody, followed by anion exchange chromatography using pseudoaffinity elution similar to FIXFc proteins described above. These purification steps utilized standard methods known to those in the art to generate purified proteins of >95% purity by SEC analysis and SDS-PAGE.

Example 13

Purification of FVIIIFc proteins
FVIIIFc proteins were purified from clarified and chemically defined harvest media using a two or three column purification process, including a FVIII-specific affinity purification step (McCue 2009) followed by a combination of anion exchange with standard NaCl elution and/or shFcRn (soluble human FcRn) affinity (NHS-coupled shFcRn with sepharose 4FF beads) chromatography, binding Fc-containing proteins at low pH (e.g. pH 6.2) and eluting at neutral pH (e.g. pH 8.0). These purification steps utilized standard methods known to those in the art to generate purified proteins of >95% purity by SEC analysis and SDS-PAGE.

Example 14

Activation of FVII Constructs
Fractions eluted from the FcRn column containing FVIIFc were pooled, and total protein was concentrated to 4 mg/ml. The CaCl₂concentration was raised to 5 mM and the sample was incubated at 4° C. for 24 to 48 hours until at least 80% of FVIIFc was activated. The extent of activation was assessed by SDS PAGE (FIG. 10).

Example 15

FVIIa Activity Assays, Soluble Tissue Factor Method
Specific activity of the FVIIaFc variants was determined by the soluble tissue factor method. Unlike lipidated full length tissue factor, soluble tissue factor (extracellular portion of tissue factor) can't activate FVII into FVIIa, but it acts as an activator of the conversion of factor X into factor Xa by FVIIa. To determine the specific activity of FVIIaFc variants, A STACLOT® FVII-rTF kit (Diagnostica Stago, Asnieres, France) was used following manufacturer's recommendations. Table 1 summarizes the data and shows comparable specific activity for all variants.

TABLE 1

Specific activity of FVIIaFc variants based
on the soluble tissue factor method

	FVIIaFc	IU/nM

	FVII-011	991
	FVII-024	929
	FVII-027	790
	FVII-037	1131
	FVII-044	1300
	FVII-045	906
	FVII-046	1145
	FVII-047	924
	FVII-048	973
	FVII-049	1130
	FVII-053	929

Example 16

FACs Assays to Study Binding of FVIIaFc and Platelets
In this example, the following reagents and methods were used:
Reagents
ADP: Sigma Aldrich, cat #A2754, stock 1 mM, working concentration 10 uM
SFLLRN peptide: in-house synthesis, stock concentration 5 mg/ml (6.7 mM), working concentration 50 ug/ml (67 uM)
FVII antibody-FITC-labeled: Affinity Biologicals SAFVII-APFTC
Platelet buffer: 15 mM HEPES, 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5 mM CaCl2, 5.5 mM dextrose and mg/ml BSA, pH 7.4
Method
Count platelets
Add 20 ul of ˜2-4×10⁸cells/ml gel-purified platelets to 1 ml of platelet buffer
Make 100 ul aliquot for each sample
Add agonist and FVIIaFc (to desired concentration) as needed Incubate at 37C for 15 minutes
Add equal volume of HBS/5 mM CaCl₂/1.5% formaldehyde, incubate 20′ at RT
Spin 15′ at 3000 g
Wash in HBS/5 mM CaCl2/1 mg/ml BSA, spin again and resuspend in 100 ul of platelet buffer.
Add 2.5 ul of FVII antibody-FITC-labeled and incubate for 30 at room temperature.
Analyze by FACs

Example 17

Thrombin Generation Assay
In this example, the following reagents and methods were used:
Reagents
FV: HTI, cat #HCV-0100, lot #Z0413, 5.1 mg/ml
Prothrombin: HTI, cat #HCP-0010, lot #Z0128, 4.8 mg/ml
FX: HTI, cat #HCX-0050, lot #X0401, 5.4 mg/ml
ATIII: HTI, cat #HCATIII-0120, lot #Y0401, 8.2 mg/ml
TFPI: American Diagnostica, cat #4900PC, lot #081031, 100 ug/ml
Reader: Fluorskan, Thermo Electron Fluorometer
Thrombin Calibrator: Thrombinoscope, cat #TS20.00
Fluca: Thrombinoscope, cat #TS50.00
Platelet buffer: 15 mM Hepes pH 7.4, 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.5 mM Dextrose, supplemented with 1 mg/ml BSA before using
ADP: Sigma Aldrich, cat #A2754, stock 1 mM, working concentration 10 uM
SFLLRN peptide: in-house synthesis, stock concentration 5 mg/ml (6.7 mM), working concentration 50 ug/ml (67 uM)


Primary stock	Working solution	[final]
(mg/ml)	(ug/ml)	ug/ml

FV	5.1	105.6	4.4
FII	4.8	1200	54
FX	5.4	120	5
ATIII	8.2	1800	75
TFPI	0.1	1.44	0.06
Platelet	2-10E8	1.74E8	0.6E8
FVIIaFc
1 mg/ml (10 uM)	1200 nM	200 nM
FVIIaFc
1 mg/ml (10 uM)	200 nM	62.5 nM
FVIIaFc
1 mg/ml (10 uM)	62.5 nM	12.5 nM

Method

- Set up software according to manufacturer's recommendations
- Prewarm water and Fluca buffer
- Make clotting factor mix. Dilute stock concentration of FV, FII, FX, ATIII and TFPI to make working solution. Need 5 ul/well, so for a 30 well assay prepare 180 ul of each Mix all the clotting factor solutions in a single bulk solution
- Premake FVIIaFc dilutions. Make 1200 nM solution (12 ul into 88 ul of buffer) in 100 ul and dilute 4-fold twice (25 ul into 75 ul of buffer) to obtain 200 nM and 62.5 nM solutions
- Make calibrator solution (1 ml of warm water in calibrator vial)
- Add 20 ul of buffer or calibrator to the wells
- Add 25 ul of clotting factor mix to the wells (or 25 ul of buffer to calibrator wells)
- Add 20 ul of FVIIaFc to the wells (or buffer to calibrator wells)
- Add 35 ul of platelets (previously add ADP and SFLLRN). Add platelets to calibrator well
- Put plate in instrument, prepare Fluca buffer and start reaction (add 20 ul Fluca/well) 5 minutes after putting plate into instrument.

Example 18

Analysis of Protein Generated from Transient Transfections
For analysis of protein from transient transfections, conditioned media from transfections of pSYN-FVII-010, 011, -013, -018, -003, -019-020 and -024 were subjected to protein A immunoprecipitation. Briefly, cell culture supernatant was mixed with approximately 50 ul of protein A-Sepharose 50% slurry and incubated at 4° C. with rocking for 1 hour, then centrifuged to pellet the protein A beads. Beads were washed twice by resuspending in 1 ml of PBS, spinning and aspirating. The beads were resuspended with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions, heated for 5 minutes at 100° C., spun down and loaded on SDS-PAGE gels and run according to standard protocols. Gels were transferred to nitrocellulose membranes and Western blots were performed to detect the Fc region or the FVII light chain. For Fc detection, the antibody used was a goat anti-human IgG (Fc specific)-horseradish peroxidase conjugate (Pierce ImmunoPure antibody, catalog #31413). For FVII light chain detection an anti light chain monoclonal antibody was used (Green Mountain, clone 6MA-219). The antibodies were diluted 1:15,000 (for Fc detection) or 1:200 (for light chain detection) in PBST (PBS with 0.1% Tween-20) with 5% nonfat dry milk and incubated with the membrane for 1 hour at room temperature. The membrane was then washed in PBST 3 times for 10 minutes and signal was detected by a chemiluminescent method for Fc detection. For FVII light chain detection, the membrane was further incubated for one hour in a solution containing HRP-labeled goat anti-mouse antibody (Southern Biotech, #1010-05) diluted 1:5000 in PBST. The membrane was also washed in PBST 3 times for 10 minutes and the signal was detected by a chemiluminescent method. Chemiluminescent detection was performed using ECL Plus Western Blotting Detection System (Amersham Biosciences catalog #RPN2132) according to manufacturer's protocol. Signal was visualized in a Storm 840 Phosphorimager (Molecular Devices).
The effect of PC5 on the processing of the proprotein convertase cleavage sites in the FVII-024 linker was tested as shown in FIG. 26. Under nonreducing conditions the effect of PC5 on cleavage site processing can not be detected because the FVII light chain-Fc and FVII heavy chain-Fc subunits remain linked via 2 disulfide bonds in the Fc region (lanes 2 and 3). Under reducing conditions we observed partial processing of FVII-024 generated from cells not cotransfected with PC5 (lane 4), but full processing when the cells were cotransfected with PC5 (lane 5). Full processing of the linker results in secretion of activated FVII (FVIIa), since a free N-terminus of the heavy chain is required and sufficient to activate the protein

Example 19

Cleavage of FVII-039 and FVII-040 by FXIa
The activation FVII-039 and FVII-040 by FXIa, as a result of the FXIa cleavage site inserted immediately upstream of the FVII light chain in these proteins, was characterized in vitro. A 1.5 μM solution of FVII-039, FVII-040 or FVII-011 (non activated) containing 15 nM FXIa in 50 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl₂, pH 7.4 was incubated for 5 or 20 minutes, and cleavage of FVIIFc was determined by SDS PAGE under reducing conditions, followed by SYPRO Ruby staining (Invitrogen). FXIa failed to cleave FVII-039, FVII-040 and nonactivated FVII-011, as shown in FIG. 28.

Example 20

Alternative Activatable FVIIFc Constructs
Failure of FXIa to cleave FVII-039 and FVII-040 may have been caused by inaccessibility of the protease to the cleavage site by steric hindrance. To improve FXIa or thrombin cleavage site accessibility, the sites will be placed upstream of the heavy chain in a structure where the heavy chain is not preceded by the light chain (Light chain-linker-Fc-scFclinker-FXIa/thrombin cleavage site-heavy chain-linker-Fc). In some embodiment, the heavy chain will comprise the I152V mutation. Once the best cleavage site is determined, a cscFc will be introduced so that the cell secretes a heterodimeric protein with the following structure: light chain-linker-Fc which is disulfide bonded to a second chain: FXIa/thrombin cleavage site-heavy chain-linker Fc.
In order to improve the level of activation observed with the constructs shown in FIG. 28, a second generation of activatable variants illustrated in FIG. 31 (similar in structure to those shown in FIG. 6E) was used to increase accessibility of the cleavage site. In this example, FXIa and thrombin cleavage sites were used for these constructs (See FIG. 31). Constructs were transiently transfected as previously described. FVIIFc was captured from media with protein A. FVIIFc bound to the beads was put in buffer and FXIa or thrombin was added and incubated. FVIIFc was eluted from beads with SDS PAGE loading buffer at 100 C for 5 minutes. The gel was loaded and western blot performed to detect Fc as previously described and the results are shown in FIG. 32. As shown in FIG. 32, both the thrombin and factor XIa cleavage sites could be cleaved to yield FVII heavy and light chain molecules in the presence of the appropriate enzyme. Best cleavage was observed for constructs FVII-060 and FVII-061, while no cleavage was observed for the negative control (FVII-062) in the presence of thrombin.

Example 21

A Factor VII Activatable Construct
The constructs depicted in FIG. 33 were (FVII-090, FVII-089 and FVII-062) were cloned, expressed and purificatied as previously described (these proteins do not require activation). Due to a cloning error a “VVGGA” sequence was inserted after the ALRPR thrombin cleavage sequence of FVII-060 and FVII-061, but while this insertion would be expected to affect the activity, it would not be predicted to affect the assessment of cleavage by thrombin in SDS-PAGE based assays. This sequence was removed in FVII-089 and FVII-090. To 125 nM of FVII-090, FVII-089, FVII-062, or plasma-derived FVII (FVII) increasing concentrations of thrombin were added and incubated for 10 minutes at 37° C. The mixture was run on SDS-PAGE gel to determine cleavage by thrombin (FIG. 33).
Generation of FVII light chain-Fc and FVII heavy chain-Fc was observed for FVII-089 and FVII-090 after incubation with thrombin. The fact that there was no cleavage of plasma-derived FVII or the FVII-062 negative control by thrombin shows specificity. No significant difference in cleavage efficiency was observed for FVII-089 and FVII-090.
Thrombin generation assays were used to measure activity of activatable variant FVII-090. A thrombin generation assays in FVIII-deficient platelet-rich plasma was performed as previously described, but replacing clotting factors and platelets with FVIII-deficient platelet-rich plasma. The results depicted in FIG. 34 are from an assay in which thrombin generation was activated with 50 nM of FVIIaFc. As shown in FIG. 34, thrombin is generated by 50 nM of FVIIaFc. The addition of 200 nM FVII-090 (not FVII-062, the negative control) to 50 nM of FVIIaFc results in a significant increase in thrombin generation, suggesting that FVI-090 becomes activated by thrombin generated by FVIIaFc. FVII-090 in the absence of any FVIIaFc activation also shows increased thrombin generation relative to FVII-062 in the absence of activation. This could be caused by activation of FVII-090 from small amounts of thrombin generated by residual levels of tissue factor or contact pathway activation.

Example 22

A High Specific Activity Factor VII Activatable Construct
To make the high specific activity version of Factor VII, FVII-100, amino acids 311 to 322 of the FVII mature sequence (LQQSRKVGDSPN, corresponding to the 170-loop) from FVII-090, were replaced with amino acids EASYPGK from the 170-loop of trypsin. This substitution has been shown to confer high specific activity.
An additional high specific activity version of Factor VII, FVII-115, was constructed. In this version, the 170 loop is wild type, but there are three point mutations in the heavy chain of FVII, V158D, E296V and M298Q. FVII-100 and FVII-115 are illustrated in FIG. 41.
In a soluble tissue factor assay, the specific activity of FVII-011(wild type FVIIaFc) is 10,000 IU/mg. FVII-090 has a specific activity of 0.32 IU/mg, FVII-100 has a specific activity of 0.25 IU/mg, and FVII-115 has a specific activity of 14 IU/mg. Thus, each of the activatable forms (prior to activation by the appropriate enzyme) is essentially inactive in this assay.
In the context of activated FVII, such high specific activity variants have the potential to be more efficacious, but also to be more susceptible to inhibition by proteins such as antithrombin. This inhibition depends on FVIIa being active; therefore, high specific activity variants which are activatable (dosed as nonactive proteins) should be more resistant to antithrombin inhibition while having the potential to have high specific activity once activated at the site of injury.
The cleavage of purified FVII-090 and FVII-100 and 115 (high specific activity variants) by thrombin was tested as previously described. The results are shown in FIG. 35. SDS PAGE analysis showed how the 3 proteins were cleaved by thrombin with comparable efficiency. For FVII-100, FVII heavy chain-Fc and light chain Fc collapse in 1 band because a glycosylation site is removed from the heavy chain after insertion of the trypsin 170 loop, reducing the mass of the FVII HC-Fc band which therefore migrates faster on the gel and comigrates with the FVII LC-Fc band. Thrombin generation assays were used to measure activity of activatable variant FVII-090 and high specific activity variant FVII-100. As set forth previously herein, thrombin generation was tested in a reconstituted system with human platelets, Factor X, Factor V, prothrombin, antithrombin and tissue-factor pathway inhibitor. Activity was measured with or without 5 nM thrombin activation.
As shown in FIG. 36, activity of FVII-090 is enhanced in the presence of thrombin, suggesting activation of FVII-090 by thrombin. However, activity is significantly increased in the context of the high specific activity variant FVII-100 with thrombin activation. High activity with longer initiation time for FVII-100 in the absence of thrombin, suggests that residual levels of tissue factor, thrombin or contact pathway activation can generate enough thrombin to activate FVII-100 without exogenous addition of thrombin. FIG. 37 shows that similar results were obtained for the other high specific activity variant, FVII-115.

Example 23

Confirmation of Activity of Activatable Variants Using Various Assays
In this example, chromogenic assays were used to measure FVII activity. One of the assays used measures the amidolytic activity of FVIIaFc by measuring the cleavage of a chromogenic substrate by FVIIa. Another of these measures the FX activation activity by measuring the ability of FVIIa to activate FX, as determined by measuring levels of a chromogenic substrate that is cleaved by activated FX (FXa).
In amidolytic assays, the chromogenic substrate Chromozyme t-PA was used. FVIIa cleaves this substrate in a dose dependent manner. The substrate is also cleaved by thrombin, but the cleavage by thrombin can be inhibited by hirudin (data not shown).
As shown in FIG. 38, amidolytic activity of the activatable variants can be measured following thrombin activation and there is increased amidolytic activity for the high specific activity variants as compared to FVII-090. In these assays, after activation of the activatable molecule by thrombin, hirudin is added to inhibit thrombin cleavage of the chromogenic substrate. In this manner, the thrombin does not interfere with the ability to detect FVIIa activity.
The activation of FX by FVIIa is also enhanced in the high specific activity FVII activatable variants. To measure activation of FX by FVIIa, substrate S2765 was used. This chromogenic substrate is also recognized by FX. In the assay, 10 uM of FX was incubated with FVIIaFc for 15 minutes and the reaction was quenched with EDTA. FIG. 39 shows the results of the control experiment which demonstrates that FX activation by FVIIaFc can be detected.
The experiment shown in FIG. 40 shows that there is an increase in FX activation activity for the high specific activity activatable variant FVII-100. In this experiment, FVIIFc (100 nM) was activated with thrombin (100 nM) for 20 minutes at 37C. Hirudin was added to inhibit the thrombin. FX (10 uM) was added and incubated for 15 minutes at 37C, followed by EDTA to inhibit the reaction. S2765 substrate was added and FXa generation was detected by monitoring substrate cleavage.

Example 24

Monomeric Fc Molecules can Also be Synthesized in Activatable Form
Three monomeric constructs were made as shown in FIG. 41. In FVII-118, an ALRPR cleavage site was inserted between the light chain and heavy chain. In FVII-119, the sequence GGGGS-ALRPR was inserted between the light chain and heavy chain. For FVII-127, the construct was made like FVII-118, but with the same high specific activity mutation used in FVII-100. The specific activity of the non-activated purified forms of these constructs was tested in a soluble tissue factor assay and compared to FVII-011 (wild type FVIIaFc which had an activity of 10,000 IU/mg). FVII-118 had an activity of 4.5 IU/mg and FVII-127 had an activity of 1.8 IU/mg, demonstrating that these molecules had essentially no activity in their activatable form.
Thrombin cleavage reactions of FVII-118, FVII-119 and FVII-090 followed by SDS
PAGE analysis were performed as previously described. As shown in FIG. 42, the cleavage site in an activatable construct can be cleaved in the context of both the monomer and heterodimer Fc molecules. In the figure, the decrease in the intensity of the nonactivated FVIIFc band with increasing thrombin concentration is similar for the FVII-118, FVII-119, and FVII-090 constructs.
Another activatable monomeric construct, FVII-127, was made and tested. FVII-127 has the backbone of FVII-118, but the same 170 loop substitution used in FVII-100 to confer high specific activity. As shown in FIG. 43, the activity of FVII-127 is significantly increased as compared to FVII-118 lacking the high specific activity amino acid substitution. High activity with longer initiation time for FVII-127 in the absence of thrombin, suggests that residual levels of tissue factor, thrombin or contact pathway activation can generate enough thrombin to activate FVII-127 without exogenous addition of thrombin. FVII-127 activity is accelerated by thrombin.

Example 25

FVIIaFc Variants Targeted to the Active Form of GPIIbIIIa
In this example the constructs illustrated in FIG. 44A were cloned, transiently expressed, purified and activated as previously described. FVII-066 was cotrasfected with PC5 to fully process the cscFc linker, described in the protein sequence, connecting the first Fc moiety to the platelet targeting moiety. These constructs employed the targeting moiety SCE5, a scFv against the active conformation of GPIIbIIIa. SCE5 has been shown to crossreact with mouse and human receptor. The SCE5 targeting moiety was placed at the N-terminus (FVII-066) or C terminus (FVII-067) of the second Fc moiety of FVIIaFc. In addition, the SCE5 was placed at the C-terminus of FVIIa (FVII-094). FVIIaFc (FVII-011) and NOVOSEVEN® were used as controls. As shown in FIG. 44B, these proteins were tested by thrombin generation assays in FVIII-deficient human plasma as previously described. These experiments revealed increased rates of thrombin generation for all the proteins containing the SCE5 targeting moiety relative to the controls. The highest rates of thrombin generation were observed for FVII-066, followed by FVII-094 and FVII-067, suggesting that the placement of the SCE5 targeting moiety can have a significant effect on the activity of the protein. Binding of these proteins to activated human platelets was determined by FACS assays as previously described (FIG. 44C). All the FVIIa proteins containing the SCE5 targeting moiety showed increased binding to platelets relative to the FVIIaFc control. This shows that attaching the SCE5 targeting moiety to FVIIa can increase its affininity from platelets. Since the SCE5 targeting moiety has been shown to interact with the mouse GPIIbIIIa receptor, FVII-066 was tested in thrombin generation assays using mouse FVIII-deficient platelet rich pasma, as well as in a reconstituted system using human purified components and platelets, as previously described (FIG. 29). We observed increased rates of thrombin generation for FVII-066 relative to the controls in both systems.

Example 26

Additional FVIIaFc Variants Targeted to the Active Form of GPIIbIIIa
In this example, construct FVII-027 illustrated in FIG. 12A was cloned, expressed (with PC5 cotransfection to fully process the cscFc linker, described in the protein sequence, connecting the first Fc moiety to the platelet targeting moiety), purified and activated as previously described. This construct employs the targeting moiety MB9, a scFv that has been shown to bind to the active conformation of GPIIbIlla. FACS assays were performed as previously described to assess binding to activated platelets, and FVII-027 was shown to bind to activated platelets with higher affinity than the FVII-011 control (FIG. 12B). Thrombin generation assays were performed with reconstituted purified human proteins and platelets as previously described (FIGS. 13A, 13B and 13C). FVII-027 showed increased rates of thrombin generation relative to the controls. FIG. 13D illustrates that FVII-027 has four times more activity than FVII-011 or NOVOSEVEN®, based on thrombin generation assays. FIGS. 14A and 14B illustrate that the enhanced platelet binding and thrombin generation activity of FVII-027 were abrogated by PAC1, an antibody that competes with MB9 for binding to the activated form of GPIIbIIIa, demonstrating the effects are mediated by the interaction of MB9 with the activated form of GPIIbIlla. The MB9 targeting moiety was also placed at the C-terminus of the second Fc moiety of FVIIaFc to generate FVII-037 illustrated in FIG. 15. Thombin generation assays in a FVIII-deficient reconstituted system with platelets revealed increased rates of thrombin generation for FVII-037 relative to the NOVOSEVEN® control (FIGS. 16A, 16B, and 16C).

Example 27

Factor VII constructs targeted to both activated and nonactivated platelets
In this example, the constructs illustrated in FIG. 45A were cloned, expressed, purified and activated as previously described. FVII-088 was cotransfected with PC5 to fully process the cscFc linker, described in the protein sequence, connecting the first Fc moiety to the platelet targeting moiety. FVII-088 and FVII-125 employed the AP3 targeting moiety, a scFv that binds to both active and nonactive conformations of human GPIIbIIIa. The results in FIG. 45B show thrombin generation assays in FVIII-deficient platelet-rich plasma, and both FVII-088 and FVII-125 showed increased rates of thrombin generation relative to the controls, demonstrating that targeting FVIIaFc or FVIIa to the active and nonactive conformation of GPIIbIlla results in increased activity. Binding of FVII-088 and FVIIaFc to activated human platelets was tested by FACS (FIG. 45C). These data reveal that FVII-088 binds to platelets with higher affinity than FVIIaFc (FVII-011), showing that the AP3 targeting moiety can increase the affinity of FVIIaFc for platelets.
Rotation Thromboelastometry (ROTEM®, Pentapharm GmbH, Munich, Germany) is another method to evaluate platelet-targeted FVIIa constructs, since it allows for the characterization of several coagulation parameters in whole blood (in the presence of platelets). The ability of FVII-088 and wild type recombinant FVIIaFc (rFVIIaFc) to form firm and stable clots was evaluated by ROTEM with Calcium Chloride as activator (NATEM) following manufacturer's recommendations. Hemophilia A blood from a human donor was spiked with FVIIFc to a final concentration of 100, 30 or 10 nM. The NATEM reaction was initiated by the addition of CaCl₂. Coagulation parameters, including Clotting Time (relates to coagulation initiation time), Clot Formation Time (relates to rates of coagulation) and Alpha Angle (relates to rates of coagulation) were assessed as shown in FIG. 30. FVII-088 showed a significant reduction in the Clotting Time and Clot Forming Time and an increase in the alpha angle relative to wild type rFVIIaFc, consistent with enhanced coagulation kinetics for FVII-088. These data demonstrate that FVII-088 has enhanced activity relative to wild type FVIIaFc in agreement with the thrombin generation assay data.

Example 28

Use of Peptides for Targeting FVIIa to Platelets
The constructs illustrated in FIG. 46A were cloned, expressed, purified and activated as previously described. These proteins were made using peptides that bind to platelet receptor GPIb-alpha (found in both activated and nonactivated platelets), specifically PS4, OS1, and OS2 as platelet targeting moieties. In making these molecules the peptide was attached to either the N or the C terminus of the second Fc moiety of the construct. The FVII-044 construct employed the PS4 peptide attached to the C terminus of the second Fc moiety of the construct; FVII-045 employed the OS1 peptide attached to the C terminus of the second Fc moiety of the construct; and the FVII-046 construct employed the 0S2 molecule attached to the C terminus of the second Fc moiety of the construct. In contrast, the FVII-047 construct employed the PS4 peptide attached to the N terminus of the second Fc moiety of the construct; the FVII-048 molecule employed the 0S1 peptide attached to the N-terminus of the second Fc moiety of the construct; and the FVII-049 molecule employed the OS2 peptide attached to the N-terminus of the second Fc moiety of the construct. Thrombin generation assays were performed using FVIII-deficient platelet rich plasma as previously described. As shown in FIG. 46B, when the assay was performed with limiting concentrations of FVIIa, each of the FVII-044, FVII-045, and FVII-046 C-terminal fusion constructs exhibited enhanced thrombin generation as compared to the NOVOSEVEN® control. A similar result is shown in FIG. 47A for the N-terminal fusion constructs. FIG. 47B shows that the FVII-045 construct may be marginally better than the FVII-048 construct in this assay, but that again both the N and C terminal fusions are better than the NOVOSEVEN® control. In addition, there is a correlation between the published GPIb-alpha affinity for each peptide (FIG. 48) and the increase in the activity associated with that peptide when recombinantly fused to FVIIaFc. FIG. 48 shows the binding of FVII-049, FVII-048 and wild type FVIIaFc (FVII-011 control) to activated platelets as determined by FACS as well as the affinity for the targeting peptides reported in Bernard et al. Biochemistry 2008. 47:4674-4682. FACS data revealed increased affinity of FVII-045 and FVII-048 for platelets relative to the FVII-011 control

Example 29

An FVIIIFc Variant Targeted to the Active Form of GPIIbIIIa
The constructs illustrated in FIG. 49A were made as previously described. FVIII-041 is wild type FVIIIFc, while FVIII-108 has a SCE5 platelet targeting moiety at the N-terminus of the second Fc moiety. For expression, FVIII-108 was cotransfected with PC5 to fully process the cscFc linker, described in the protein sequence, connecting the first Fc moiety to the platelet targeting moiety. These proteins were tested in thrombin generation assays using FVIII deficient platelet rich plasma as previously described, but in addition the thrombin generation assay was also activated with tissue factor As shown in FIG. 49B, no significant improvement was seen using the targeted version of FVIIIFc. It is noteworthy that the thrombin generation assays described herein measure thrombin generation on the surface of platelets and, therefore, are an accurate measure of activity.

Example 30

Making and Testing a Version of FVII Targeted to Platelets that Lacks a Gla domain
In this example a version of FVIIaFc illustrated in FIG. 21 was generated. This protein has the MB9 scFv at the N-terminus and a deletion that removes the Gla domain. FVII-053 contains an RKRRKR sequence inserted between the light and heavy chain for intracellular activation. FVII-053 was transiently expressed (cotransfected with PC5 for processing of the RKRRKR sequence which results in activation of the protein) and purified as previously described. Thrombin generation assays with purified components and platelets reveal that FVII-053 has some activity (FIGS. 22A, 22B, 22C, and 22D), even though this activity is compromised relative to the FVII-011 control (FIG. 22D). Data in FIGS. 23A and 23B show how the PAC1 antibody, which competes with MB9 for GPIIbIIIa binding, inhibits thrombin generation activity associated with FVII-053, suggesting that platelet targeting is important for activity. Another construct identical to FVII-053, but without the RKRRKR insertion was generated (FVII-028) and tested in a Pk study in the nonactivated together with nonactivated FVII-011. As shown in FIG. 50, the terminal half-life of the targeted, Gla-less FVII-028 molecule was nearly three times longer (20.3 hours) than that of the FVII-011 control (7.1 hours), suggesting that removing the Gla domain increases the terminal half-life of FVIIFc.

Example 31. Platelet Targeted FIX Molecules

In this example, the FIX constructs illustrated in FIG. 51A were made and tested.
FIX-068 was cotransfected with PC5 to fully process the cscFc linker, described in the protein sequence, connecting the first Fc moiety to the platelet targeting moiety. FIX-068 has the SCE5 platelet targeting moiety at the N-terminus of the second Fc moiety, while FIX-088 has SCE5 at the C-terminus of the second Fc moiety. FIX-090 is a FIX construct without an Fc domain and has the SCE5 moiety attached at the C-terminus of the FIX protein. FIX-042 is a FIXFc as a single chain Fc without a targeting moiety and was made as a control. BENEFIX® (Pfizer) was also used as a control. To remove trace amounts of activated FIX (FIXa) from the BENEFIX® sample that cause misleading results in thrombin generation assays, the BENEFIX® sample was treated with the irreversible active site inhibitor glutamyl-glycyl-arginyl-chloromethylketone (Hematologic Technologies). BENEFIX® was incubated with an excess amount of the inhibitor for 180 minutes at room temperature. The sample was then dialyzed to remove unbound inhibitor. The treated BENEFIX® is hereafter referred to as BENEFIX®. The specific activities of the molecules made were FIX-042, 6 IU/nmol; FIX068, 5.1 IU/nmol; FIX-088, 3.5 IU/nmol; FIX-090, 13.8 IU/nmol, and BENEFIX®, 12 IU/nmol. When these constructs (FIX-068, FIX-088 and FIX-042) were tested in a thrombin generation assay in platelet-rich FIX-deficient plasma as shown in FIG. 51B, each of the targeted molecules had a higher activity than the FIX-042 control. FIG. 51C illustrates that both FIX-068 and FIX-088 have at least 4 times more activity than FIX-042 as measured by thrombin generation. Since the specific activity of FIX-042 is higher than FIX-068 and FIX-088, the increased activity observed in the thrombin generation assays may be underestimated, and therefore the increased activity by platelet targeting may be greater than 4-fold.
As shown in FIG. 52A, FIX-090 (which lacks an Fc) also shows increased activity relative to BENEFIX®, suggesting that targeting FIX to platelets in the absence of Fc also increases activity. FIG. 52B shows that the activity of FIX-090 is at least 4 times that of BENEFIX®. Since both FIX-090 and BENEFIX® have similar specific activities, the 4-fold increase in activity in thrombin generations assays must be caused by the platelet targeting effect.

Example 32

Use of Peptides for Targeting FIX to Platelets
In this example, the FIX-089 construct illustrated in FIG. 53A was cloned, transiently expressed and purified as previously described. The molecule comprises the 0S1 peptide, which binds to GPIb-alpha receptor, attached to the N-terminus of the second Fc moiety of the construct. The specific activity of the FIX-089 construct was 2.4 IU/nmol as compared to 6 IU/nmol for the control FIX-042 molecule.
As shown in FIG. 53B, the FIX-089 molecule is more active than the FIX-042 control in thrombin generation assays with FIX-platelet rich plasma; this is particularly evident at limiting concentrations of FIX. FIG. 53C demonstrates that FIX-089 is roughly 4-times stronger than FIX-042 as measured by thrombin generation, while having a lower specific activity. This further suggests that targeting to GPIb increases the activity of FIXFc.
Draft Sequence Listing
FVII-027 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or MB9 to Fc region is underlined, and linker with proprotein convertase processing sites is shown in bold


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGQ TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYVFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKEN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KRRRRSGGGG SGGGGSGGGG

SGGGGSGGGG SGGGGSRKRR KRAEVQLVQS GAEVNKPGAS VKVSCKASGY TFTGYYMHWV

RQAPGQGLEW MGWINPNSGG TNYAQKFQGW VTMTRDTSIS TAYMELSRLR SDDTAVYYCA

RGRALYNRND RSPNWFDPWG QGTLVTVSSG SASAPTLKLE EGEFSEARVQ AVLTQPPSVS

VAPGQTARIT CGGNNIGSKS VQWYQQKPGQ APVLVVYDDS DRPSGIPERF SGSNSGNMAT

LTISRVEAGD EADYYCQVWD SSSDHVVFGG GTKLTVLGQP KAAPSVTLFP PSAAAGGGGS

GGGGSGGGGS GGGGSGGGGS GGGGSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTIMISR

TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN

GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR KELTKNQVSL TCLVKGFYPS

DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH

YTQKSLSLSP GK

DNA sequence of FVII-027
atggtctccc aggccctcag gctcctctgc cttctgcttg ggcttcaggg ctgcctggct

gcagtcttcg taacccagga ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

gcgttcctgg aggagctgcg gccgggctcc ctggagaggg agtgcaagga ggagcagtgc

tccttcgagg aggcccggga gatcttcaag gacgcggaga ggacgaagct gttctggatt

tcttacagtg atggggacca gtgtgcctca agtccatgcc agaatggggg ctcctgcaag

gaccagctcc agtcctatat ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

acgcacaagg atgaccagct gatctgtgtg aacgagaacg gcggctgtga gcagtactgc

agtgaccaca cgggcaccaa gcgctcctgt cggtgccacg aggggtactc tctgctggca

gacggggtgt cctgcacacc cacagttgaa tatccatgtg gaaaaatacc tattctagaa

aaaagaaatg ccagcaaacc ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

gagtgtccat ggcaggtcct gttgttggtg aatggagctc agttgtgtgg ggggaccctg

atcaacacca tctgggtggt ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

aacctgatcg cggtgctggg cgagcacgac ctcagcgagc acgacgggga tgagcagagc

cggcgggtgg cgcaggtcat catccccagc acgtacgtcc cgggcaccac caaccacgac

atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg accatgtggt gcccctctgc

ctgcccgaac ggacgttctc tgagaggacg ctggccttcg tgcgcttctc attggtcagc

ggctggggcc agctgctgga ccgtggcgcc acggccctgg agctcatggt cctcaacgtg

ccccggctga tgacccagga ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

atcacggagt acatgttctg tgccggctac tcggatggca gcaaggactc ctgcaagggg

gacagtggag gcccacatgc cacccactac cggggcacgt ggtacctgac gggcatcgtc

agctggggcc agggctgcgc aaccgtgggc cactttgggg tgtacaccag ggtctcccag

tacatcgagt ggctgcaaaa gctcatgcgc tcagagccac gcccaggagt cctcctgcga

gccccatttc ccggtggcgg tggctccggc ggaggtgggt ccggtggcgg cggatcaggt

gggggtggat caggcggtgg aggttccggt ggcgggggat ccgacaaaac tcacacatgc

ccaccgtgcc cagctccgga actcctgggc ggaccgtcag tcttcctctt ccccccaaaa

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag

ccggagaaca actacaagac cacgcctccc gtgttggact ccgacggctc cttcttcctc

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt

aaacggcgcc gccggagcgg tggcggcgga tcaggtgggg gtggatcagg cggtggaggt

tccggtggcg ggggatccgg cggtggaggt tccggtgggg gtggatcaag gaagaggagg

aagagggcgg aagtgcagct ggtgcagtct ggagctgagg tgaataagcc tggggcctca

gtgaaggtct cctgcaaggc ttctggatac accttcaccg gctactatat gcactgggtg

cgacaggccc ctggacaagg gcttgagtgg atgggatgga tcaaccctaa cagtggtggc

acaaactatg cacagaagtt tcagggctgg gtcaccatga ccagggacac gtccatcagc

accgcctaca tggagctgag caggctgaga tctgacgaca cggccgtgta ttactgtgcg

agaggccgtg ctttgtataa ccggaacgac cggtccccca actggttcga cccctggggc

cagggaaccc tggtcaccgt ctcctcaggg agtgcatccg ccccaaccct taagcttgaa

gaaggtgaat tctcagaagc acgcgtacag gctgtgctga ctcagccgcc ctcggtgtca

gtggccccag gacagacggc caggattacc tgtgggggaa acaacattgg aagtaaaagt

gtgcagtggt accagcagaa gccaggccag gcccctgtgc tggtcgtcta tgatgatagc

gaccggccct cagggatccc tgagcgattc tctggctcca actctgggaa catggccacc

ctgaccatca gcagggtcga agccggggat gaggccgact attactgtca ggtgtgggat

agtagtagtg atcatgtggt attcggcgga gggaccaagc tgaccgtcct aggtcagccc

aaggctgccc cctcggtcac tctgttcccg ccgtccgcgg ccgctggtgg cggtggctcc

ggcggaggtg ggtccggtgg cggcggatca ggtgggggtg gatcaggcgg tggaggttcc

ggtggcgggg gatcagacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg

ggaggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgc

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct

cccgtgttgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac

tacacgcaga agagcctctc cctgtctccg ggtaaatga

Genscript-FVII-027-1 DNA sequence
gaagagcctc tccctgtctc cgggtaaacg gcgccgccgg agcggtggcg gcggatcagg

tgggggtgga tcaggcggtg gaggttccgg tggcggggga tccggcggtg gaggttccgg

tgggggtgga tcaaggaaga ggaggaagag ggcggaagtg cagctggtgc agtctggagc

tgaggtgaat aagcctgggg cctcagtgaa ggtctcctgc aaggcttctg gatacacctt

caccggctac tatatgcact gggtgcgaca ggcccctgga caagggcttg agtggatggg

atggatcaac cctaacagtg gtggcacaaa ctatgcacag aagtttcagg gctgggtcac

catgaccagg gacacgtcca tcagcaccgc ctacatggag ctgagcaggc tgagatctga

cgacacggcc gtgtattact gtgcgagagg ccgtgctttg tataaccgga acgaccggtc

ccccaactgg ttcgacccct ggggccaggg aaccctggtc accgtctcct cagggagtgc

atccgcccca acccttaagc ttgaagaagg tgaattc

Genscript-FVII-026-2 DNA sequence
gaattctcag aagcacgcgt acaggctgtg ctgactcagc cgccctcggt gtcagtggcc

ccaggacaga cggccaggat tacctgtggg ggaaacaaca ttggaagtaa aagtgtgcag

tggtaccagc agaagccagg ccaggcccct gtgctggtcg tctatgatga tagcgaccgg

ccctcaggga tccctgagcg attctctggc tccaactctg ggaacatggc caccctgacc

atcagcaggg tcgaagccgg ggatgaggcc gactattact gtcaggtgtg ggatagtagt

agtgatcatg tggtattcgg cggagggacc aagctgaccg tcctaggtca gcccaaggct

gccccctcgg tcactctgtt cccgccgtcc gcggccgctg gtggcggtgg ctccggcgga

ggtgggtccg gtggcggcgg atcaggtggg ggtggatcag gcggtggagg ttccggtggc

gggggatcag acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggagga

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgcgatgag

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg

ttggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

cagaagagcc tctccctgtc tccgggtaaa tgagaattc

FVII-037 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or MB9 to Fc region is underlined, and linker connecting both Fcs sites is shown in bold


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

HKDDQLICV NENGGCEDQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMLLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV

DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA

KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG

GGSGGGGSGG GGSGGGGSAE VQLVQSGAEV NKPGASVKVS CKASGYTFTG YYMHWVRQAP

GQGLEWMGWI NPNSGGTNYA QKFQGWVTMT RDTSISTAYM ELSRLRSDDT AVYYCARGRA

LYNRNDRSPN WFDPWGQGTL VTVSSGSASA PTLKLEEGEF SEARVQAVLT QPPSVSVAPG

ATARITCGGN NIGSKSVQWY QQKPGQAPVL VVYDDSDRPS GIPERFSGSN SGNMATLTIS

RVEAGDEADY YCQVWDSSSD HVVFGGGTKL TVLGQPKAAP SVTLFPPSAA A

	FIX-037 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctgggc ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtgga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggtggcg gcggatcagg

	tgggggtgga tcaggcggtg gaggttccgg tggcggggga

	tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg

	aactcctggg aggaccgtca gtcttcctct tccccccaaa

	acccaaggac accctcatga tctcccggac ccctgaggtc

	acatgcgtgg tggtggacgt gagccacgaa gaccctgagg

	tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa

	tgccaagaca aagccgcggg aggagcagta caacagcacg

	taccgtgtgg tcagcgtcct caccgtcctg caccaggact

	ggctgaatgg caaggagtac aagtgcaagg tctccaacaa

	agccctccca gcccccatcg agaaaaccat ctccaaagcc

	aaagggcagc cccgagaacc acaggtgtac accctgcccc

	catcccgcga tgagctgacc aagaaccagg tcagcctgac

	ctgcctggtc aaaggcttct atcccagcga catcgccgtg

	gagtgggaga gcaatgggca gccggagaac aactacaaga

	ccacgcctcc cgtgttggac tccgacggct ccttcttcct

	ctacagcaag ctcaccgtcg acaagagcag gtggcagcag

	gggaacgtct tctcatgctc cgtgatgcat gaggctctgc

	acaaccacta cacgcagaag agcctctccc tgtctccggg

	taaaggtggc ggtggctccg gcggaggtgg gtccggtggc

	ggcggatcag gtgggggtgg atcaggcggt ggaggttccg

	gtggcggggg atcagcggaa gtgcagctgg tgcagtctgg

	agctgaggtg aataagcctg gggcctcagt gaaggtctcc

	tgcaaggctt ctggatacac cttcaccggc tactatatgc

	actgggtgcg acaggcccct ggacaagggc ttgagtggat

	gggatggatc aaccctaaca gtggtggcac aaactatgca

	cagaagtttc agggctgggt caccatgacc agggacacgt

	ccatcagcac cgcctacatg gagctgagca ggctgagatc

	tgacgacacg gccgtgtatt actgtgcgag aggccgtgct

	ttgtataacc ggaacgaccg gtcccccaac tggttcgacc

	cctggggcca gggaaccctg gtcaccgtct cctcagggag

	tgcatccgcc ccaaccctta agcttgaaga aggtgaattt

	tcagaagcac gcgtacaggc tgtgctgact cagccgccct

	cggtgtcagt ggccccagga cagacggcca ggattacctg

	tgggggaaac aacattggaa gtaaaagtgt gcagtggtac

	cagcagaagc caggccaggc ccctgtgctg gtcgtctatg

	atgatagcga ccggccctca gggatccctg agcgattctc

	tggctccaac tctgggaaca tggccaccct gaccatcagc

	agggtcgaag ccggggatga ggccgactat tactgtcagg

	tgtgggatag tagtagtgat catgtggtat tcggcggagg

	gaccaagctg accgtcctag gtcagcccaa ggctgccccc

	tcggtcactc tgttcccgcc gtccgcggcc gcttga

FVII-053 amino acid sequence. Signal sequence is shown in dotted underline, linker region connecting FVII to Fc region is underlined, linker connecting both Fcs sites is shown in bold, and MB9 is italicized


APGQGLEWMG WINPNSGGTN YAQKFQGWVT MTRDTSISTA YMELSRLRSD DTAVYYCARG

RALYNRNDRS PNWFDPWGQG TLVTVSSGSA SAPTLKLEEG EFSEARVQAV LTQPPSVSVA

PGQTARITCG GNNIGSKSVQ WYQQKPGQAP VLVVYDDSDR PSGIPERFSG SNSGNMATLT

ISRVEAGDEA DYYCQVWDSS SDHVVFGGGT KLTVLGQPKA APSVTLFPPS AAARTKLFWI

SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE THKDDQLICV NENGGCEQYC

SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE KRNASKPQGR RKRRKRIVGG

KVCPKGECPW QVLLLVNGAQ LCGGTLINTI WVVSAAHCFD KIKNWRNLIA VLGEHDLSEH

DGDEQSRRVA QVIIPSTYVP GTTNHDIALL RLHQPVVLTD HVVPLCLPER TFSERTLAFV

RFSLVSGWGQ LLDRGATALE LMVLNVPRLM TQDCLQQSRK VGDSPNITEY MFCAGYSDGS

KDSCKGDSGG PHATHYRGTW YLTGIVSWGQ GCATVGHFGV YTRVSQYIEW LQKLMRSEPR

PGVLLRAPFP GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSDKT HTCPPCPAPE

LLGGPSVFVF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE

EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

KSRWQQGNVF SCSVMHEALTH NHYTQKSLSL SPGK

	FVII-053 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcggaagtgc agctggtgca

	gtctggagct gaggtgaata agcctggggc ctcagtgaag

	gtctcctgca aggcttctgg atacaccttc accggctact

	atatgcactg ggtgcgacag gcccctggac aagggcttga

	gtggatggga tggatcaacc ctaacagtgg tggcacaaac

	tatgcacaga agtttcaggg ctgggtcacc atgaccaggg

	acacgtccat cagcaccgcc tacatggagc tgagcaggct

	gagatctgac gacacggccg tgtattactg tgcgagaggc

	cgtgctttgt ataaccggaa cgaccggtcc cccaactggt

	tcgacccctg gggccaggga accctggtca ccgtctcctc

	agggagtgca tccgccccaa cccttaaact tgaagaaggt

	gaattttcag aagcacgcgt acaggctgtg ctgactcagc

	cgccctcggt gtcagtggcc ccaggacaga cggccaggat

	tacctgtggg ggaaacaaca ttggaagtaa aagtgtgcag

	tggtaccagc agaagccagg ccaggcccct gtgctggtcg

	tctatgatga tagcgaccgg ccctcaggga tccctgagcg

	attctctggc tccaactctg ggaacatggc caccctgacc

	atcagcaggg tcgaagccgg ggatgaggcc gactattact

	gtcaggtgtg ggatagtagt agtgatcatg tggtattcgg

	cggagggacc aagctgaccg tcctaggtca gcccaaggct

	gccccctcgg tcactctgtt cccgccgtcc gcggccgcta

	ggacgaagct gttctggatt tcttacagtg atggggacca

	gtgtgcctca agtccatgcc agaatggggg ctcctgcaag

	gaccagctcc agtcctatat ctgcttctgc ctccctgcct

	tcgagggccg gaactgtgag acgcacaagg atgaccagct

	gatctgtgtg aacgagaacg gcggctgtga gcagtactgc

	agtgaccaca cgggcaccaa gcgctcctgt cggtgccacg

	aggggtactc tctgctggca gacggggtgt cctgcacacc

	cacagttgaa tatccatgtg gaaaaatacc tattctagaa

	aaaagaaatg ccagcaaacc ccaaggccga aggaagagga

	ggaagaggat tgtggggggc aaggtgtgcc ccaaagggga

	gtgtccatgg caggtcctgt tgttggtgaa tggagctcag

	ttgtgtgggg ggaccctgat caacaccatc tgggtggtct

	ccgcggccca ctgtttcgac aaaatcaaga actggaggaa

	cctgatcgcg gtgctgggcg agcacgacct cagcgagcac

	gacggggatg agcagagccg gcgggtggcg caggtcatca

	tccccagcac gtacgtcccg ggcaccacca accacgacat

	cgcgctgctc cgcctgcacc agcccgtggt cctcactgac

	catgtggtgc ccctctgcct gcccgaacgg acgttctctg

	agaggacgct ggccttcgtg cgcttctcat tggtcagcgg

	ctggggccag ctgctggacc gtggcgccac ggccctggag

	ctcatggtcc tcaacgtgcc ccggctgatg acccaggact

	gcctgcagca gtcacggaag gtgggagact ccccaaatat

	cacggagtac atgttctgtg ccggctactc ggatggcagc

	aaggactcct gcaaggggga cagtggaggc ccacatgcca

	cccactaccg gggcacgtgg tacctgacgg gcatcgtcag

	ctggggccag ggctgcgcaa ccgtgggcca ctttggggtg

	tacaccaggg tctcccagta catcgagtgg ctgcaaaagc

	tcatgcgctc agagccacgc ccaggagtcc tcctgcgagc

	cccatttccc ggtggcggtg gctccggcgg aggtgggtcc

	ggtggcggcg gatcaggtgg gggtggatca ggcggtggag

	gttccggtgg cgggggatcc gacaaaactc acacatgccc

	accgtgccca gctccggaac tcctgggcgg accgtcagtc

	ttcctcttcc ccccaaaacc caaggacacc ctcatgatct

	cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag

	ccacgaagac cctgaggtca agttcaactg gtacgtggac

	ggcgtggagg tgcataatgc caagacaaag ccgcgggagg

	agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac

	cgtcctgcac caggactggc tgaatggcaa ggagtacaag

	tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga

	aaaccatctc caaagccaaa gggcagcccc gagaaccaca

	ggtgtacacc ctgcccccat cccgggatga gctgaccaag

	aaccaggtca gcctgacctg cctggtcaaa ggcttctatc

	ccagcgacat cgccgtggag tgggagagca atgggcagcc

	ggagaacaac tacaagacca cgcctcccgt gttggactcc

	gacggctcct tcttcctcta cagcaagctc accgtggaca

	agagcaggtg gcagcagggg aacgtcttct catgctccgt

	gatgcatgag gctctgcaca accactacac gcagaagagc

	ctctccctgt ctccgggtaa aggtggcggc ggatcaggtg

	ggggtggatc aggcggtgga ggttccggtg gcgggggatc

	cgacaaaact cacacatgcc caccgtgccc agcacctgaa

	ctcctgggag gaccgtcagt cttcctcttc cccccaaaac

	ccaaggacac cctcatgatc tcccggaccc ctgaggtcac

	atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc

	aagttcaact ggtacgtgga cggcgtggag gtgcataatg

	ccaagacaaa gccgcgggag gagcagtaca acagcacgta

	ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg

	ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag

	ccctcccagc ccccatcgag aaaaccatct ccaaagccaa

	agggcagccc cgagaaccac aggtgtacac cctgccccca

	tcccgcgatg agctgaccaa gaaccaggtc agcctgacct

	gcctggtcaa aggcttctat cccagcgaca tcgccgtgga

	gtgggagagc aatgggcagc cggagaacaa ctacaagacc

	acgcctcccg tgttggactc cgacggctcc ttcttcctct

	acagcaagct caccgtggac aagagcaggt ggcagcaggg

	gaacgtcttc tcatgctccg tgatgcatga ggctctgcac

	aaccactaca cgcagaagag cctctccctg tctccgggta

	aatga

FVII-044 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or PS4 to Fc region is underlined, linker connecting both Fcs sites is shown in bold, and PS4 peptide is italicized


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWLYTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV

DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA

KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG

GGSGGGGS AC TERWALHNLC GG

	FVII-044 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctgggc ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtgga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggtggcg gcggatcagg

	tgggggtgga tcaggcggtg gaggttccgg tggcggggga

	tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg

	aactcctggg aggaccgtca gtcttcctct tccccccaaa

	acccaaggac accctcatga tctcccggac ccctgaggtc

	acatgcgtgg tggtggacgt gagccacgaa gaccctgagg

	tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa

	tgccaagaca aagccgcggg aggagcagta caacagcacg

	taccgtgtgg tcagcgtcct caccgtcctg caccaggact

	ggctgaatgg caaggagtac aagtgcaagg tctccaacaa

	agccctccca gcccccatcg agaaaaccat ctccaaagcc

	aaagggcagc cccgagaacc acaggtgtac accctgcccc

	catcccgcga tgagctgacc aagaaccagg tcagcctgac

	ctgcctggtc aaaggcttct atcccagcga catcgccgtg

	gagtgggaga gcaatgggca gccggagaac aactacaaga

	ccacgcctcc cgtgttggac tccgacggct ccttcttcct

	ctacagcaag ctcaccgtcg acaagagcag gtggcagcag

	gggaacgtct tctcatgctc cgtgatgcat gaggctctgc

	acaaccacta cacgcagaag agcctctccc tgtctccggg

	taaaggtggc ggcggatcag gtgggggtgg atcaggcggt

	ggaggttccg gtggcggggg atcagcctgc accgagcggt

	gggccctgca caacctgtgc ggcgggtga

FVII-045 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or OS1 to Fc region is underlined, linker connecting both Fcs sites is shown in bold, and OS1 peptide is italicized


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK KQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV

DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSVNKALP APIEKTISKA

KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG

GGSGGGGSAC TERMALHNLC GG

	FVII-045 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctgggc ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtgga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggtggcg gcggatcagg

	tgggggtgga tcaggcggtg gaggttccgg tggcggggga

	tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg

	aactcctggg aggaccgtca gtcttcctct tccccccaaa

	acccaaggac accctcatga tctcccggac ccctgaggtc

	acatgcgtgg tggtggacgt gagccacgaa gaccctgagg

	tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa

	tgccaagaca aagccgcggg aggagcagta caacagcacg

	taccgtgtgg tcagcgtcct caccgtcctg caccaggact

	ggctgaatgg caaggagtac aagtgcaagg tctccaacaa

	agccctccca gcccccatcg agaaaaccat ctccaaagcc

	aaagggcagc cccgagaacc acaggtgtac accctgcccc

	catcccgcga tgagctgacc aagaaccagg tcagcctgac

	ctgcctggtc aaaggcttct atcccagcga catcgccgtg

	gagtgggaga gcaatgggca gccggagaac aactacaaga

	ccacgcctcc cgtgttggac tccgacggct ccttcttcct

	ctacagcaag ctcaccgtcg acaagagcag gtggcagcag

	gggaacgtct tctcatgctc cgtgatgcat gaggctctgc

	acaaccacta cacgcagaag agcctctccc tgtctccggg

	taaaggtggc ggcggatcag gtgggggtgg atcaggcggt

	ggaggttccg gtggcggggg atcagcctgc accgagcgga

	tggccctgca caacctgtgc ggcgggtga

FVII-046 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or OS2 to Fc region is underlined, linker connecting both Fcs sites is shown in bold, and OS2 peptide is italicized


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV

DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA

KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG

GGSGGGGS AC TERDALHNLC GG

	FVII-046 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctgggc ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtgga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggtggcg gcggatcagg

	tgggggtgga tcaggcggtg gaggttccgg tggcggggga

	tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg

	aactcctggg aggaccgtca gtcttcctct tccccccaaa

	acccaaggac accctcatga tctcccggac ccctgaggtc

	acatgcgtgg tggtggacgt gagccacgaa gaccctgagg

	tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa

	tgccaagaca aagccgcggg aggagcagta caacagcacg

	taccgtgtgg tcagcgtcct caccgtcctg caccaggact

	ggctgaatgg caaggagtac aagtgcaagg tctccaacaa

	agccctccca gcccccatcg agaaaaccat ctccaaagcc

	aaagggcagc cccgagaacc acaggtgtac accctgcccc

	catcccgcga tgagctgacc aagaaccagg tcagcctgac

	ctgcctggtc aaaggcttct atcccagcga catcgccgtg

	gagtgggaga gcaatgggca gccggagaac aactacaaga

	ccacgcctcc cgtgttggac tccgacggct ccttcttcct

	ctacagcaag ctcaccgtcg acaagagcag gtggcagcag

	gggaacgtct tctcatgctc cgtgatgcat gaggctctgc

	acaaccacta cacgcagaag agcctctccc tgtctccggg

	taaaggtggc ggcggatcag gtgggggtgg atcaggcggt

	ggaggttccg gtggcggggg atcagcctgc accgagcggg

	acgccctgca caacctgtgc ggcgggtga

FVII-047 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or PS4 to Fc regions is underlined, and PS4 peptide is italicized


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SGGGGSGGGG S ACTERWALH NLC GGGGSGG GGSGGGGSGG GGSGGGGSGG GGSDKTHTCP

PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA

KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

	FVII-047 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctggga ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtcga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggcggtg gcggttcagg

	tggaggaggg tcaggcggtg gtggatccgg cgggggcgga

	tccggtggcg gagggtcagg cggtggcgga tcagcctgca

	ccgagcggtg ggccctgcac aacctgtgcg gtggcggtgg

	ctccggcgga ggtgggtccg gtggcggcgg atcaggtggg

	ggtggatcag gcggtggagg ttccggtggc gggggatccg

	acaaaactca cacatgccca ccgtgcccag caccggaact

	cctgggcgga ccgtcagtct tcctcttccc cccaaaaccc

	aaggacaccc tcatgatctc ccggacccct gaggtcacat

	gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa

	gttcaactgg tacgtggacg gcgtggaggt gcataatgcc

	aagacaaagc cgcgggagga gcagtacaac agcacgtacc

	gtgtggtcag cgtcctcacc gtcctgcacc aggactggct

	gaatggcaag gagtacaagt gcaaggtctc caacaaagcc

	ctcccagccc ccatcgagaa aaccatctcc aaagccaaag

	ggcagccccg agaaccacag gtgtacaccc tgcccccatc

	ccgggatgag ctgaccaaga accaggtcag cctgacctgc

	ctggtcaaag gcttctatcc cagcgacatc gccgtggagt

	gggagagcaa tgggcagccg gagaacaact acaagaccac

	gcctcccgtg ttggactccg acggctcctt cttcctctac

	agcaagctca ccgtggacaa gagcaggtgg cagcagggga

	acgtcttctc atgctccgtg atgcatgagg ctctgcacaa

	ccactacacg cagaagagcc tctccctgtc tccgggtaaa

	tga

FVII-048 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or OS1 to Fc regions is underlined, and OS1 peptide is italicized


SFEEAREIFK KAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SGGGGSGGGG S ACTERMALH NLC GGGGSGG GGSGGGGSGG GGSGGGGSGG GGSDKTHTCP

PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA

KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

SKLTVDKSRW QQGNFVSCSV MHEALHNHYT QKSLSLSPGK

	DNA sequence of FVII-048
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctggga ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtcga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggcggtg gcggttcagg

	tggaggaggg tcaggcggtg gtggatccgg cgggggcgga

	tccggtggcg gagggtcagg cggtggcgga tcagcctgca

	ccgagcggat ggccctgcac aacctgtgcg gtggcggtgg

	ctccggcgga ggtgggtccg gtggcggcgg atcaggtggg

	ggtggatcag gcggtggagg ttccggtggc gggggatccg

	acaaaactca cacatgccca ccgtgcccag caccggaact

	cctgggcgga ccgtcagtct tcctcttccc cccaaaaccc

	aaggacaccc tcatgatctc ccggacccct gaggtcacat

	gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa

	gttcaactgg tacgtggacg gcgtggaggt gcataatgcc

	aagacaaagc cgcgggagga gcagtacaac agcacgtacc

	gtgtggtcag cgtcctcacc gtcctgcacc aggactggct

	gaatggcaag gagtacaagt gcaaggtctc caacaaagcc

	ctcccagccc ccatcgagaa aaccatctcc aaagccaaag

	ggcagccccg agaaccacag gtgtacaccc tgcccccatc

	ccgggatgag ctgaccaaga accaggtcag cctgacctgc

	ctggtcaaag gcttctatcc cagcgacatc gccgtggagt

	gggagagcaa tgggcagccg gagaacaact acaagaccac

	gcctcccgtg ttggactccg acggctcctt cttcctctac

	agcaagctca ccgtggacaa gagcaggtgg cagcagggga

	acgtcttctc atgctccgtg atgcatgagg ctctgcacaa

	ccactacacg cagaagagcc tctccctgtc tccgggtaaa

	tga

FVII-049 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or OS2 to Fc regions is underlined, and OS2 peptide is italicized


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SGGGGSGGGG S ACTERDALH NLC GGGGSGG GGSGGGGSGG GGSGGGGSGG GGSDKTHTCP

PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA

KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

	DNA sequence of FVII-049
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctggga ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtcga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggcggtg gcggttcagg

	tggaggaggg tcaggcggtg gtggatccgg cgggggcgga

	tccggtggcg gagggtcagg cggtggcgga tcagcctgca

	ccgagcggga cgccctgcac aacctgtgcg gtggcggtgg

	ctccggcgga ggtgggtccg gtggcggcgg atcaggtggg

	ggtggatcag gcggtggagg ttccggtggc gggggatccg

	acaaaactca cacatgccca ccgtgcccag caccggaact

	cctgggcgga ccgtcagtct tcctcttccc cccaaaaccc

	aaggacaccc tcatgatctc ccggacccct gaggtcacat

	gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa

	gttcaactgg tacgtggacg gcgtggaggt gcataatgcc

	aagacaaagc cgcgggagga gcagtacaac agcacgtacc

	gtgtggtcag cgtcctcacc gtcctgcacc aggactggct

	gaatggcaag gagtacaagt gcaaggtctc caacaaagcc

	ctcccagccc ccatcgagaa aaccatctcc aaagccaaag

	ggcagccccg agaaccacag gtgtacaccc tgcccccatc

	ccgggatgag ctgaccaaga accaggtcag cctgacctgc

	ctggtcaaag gcttctatcc cagcgacatc gccgtggagt

	gggagagcaa tgggcagccg gagaacaact acaagaccac

	gcctcccgtg ttggactccg acggctcctt cttcctctac

	agcaagctca ccgtggacaa gagcaggtgg cagcagggga

	acgtcttctc atgctccgtg atgcatgagg ctctgcacaa

	ccactacacg cagaagagcc tctccctgtc tccgggtaaa

	tga

FVII-011 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, Gla domain is italicized, linker region connecting FVII to Fc region is underlined, and linker connecting both Fcs sites is shown in bold


SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG

SDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV

DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA

KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

	FVII-011 DNA sequence
	atggtctccc aggccctcag gctcctctgc cttctgcttg

	ggcttcaggg ctgcctggct gcagtcttcg taacccagga

	ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac

	gcgttcctgg aggagctgcg gccgggctcc ctggagaggg

	agtgcaagga ggagcagtgc tccttcgagg aggcccggga

	gatcttcaag gacgcggaga ggacgaagct gttctggatt

	tcttacagtg atggggacca gtgtgcctca agtccatgcc

	agaatggggg ctcctgcaag gaccagctcc agtcctatat

	ctgcttctgc ctccctgcct tcgagggccg gaactgtgag

	acgcacaagg atgaccagct gatctgtgtg aacgagaacg

	gcggctgtga gcagtactgc agtgaccaca cgggcaccaa

	gcgctcctgt cggtgccacg aggggtactc tctgctggca

	gacggggtgt cctgcacacc cacagttgaa tatccatgtg

	gaaaaatacc tattctagaa aaaagaaatg ccagcaaacc

	ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg

	gagtgtccat ggcaggtcct gttgttggtg aatggagctc

	agttgtgtgg ggggaccctg atcaacacca tctgggtggt

	ctccgcggcc cactgtttcg acaaaatcaa gaactggagg

	aacctgatcg cggtgctggg cgagcacgac ctcagcgagc

	acgacgggga tgagcagagc cggcgggtgg cgcaggtcat

	catccccagc acgtacgtcc cgggcaccac caaccacgac

	atcgcgctgc tccgcctgca ccagcccgtg gtcctcactg

	accatgtggt gcccctctgc ctgcccgaac ggacgttctc

	tgagaggacg ctggccttcg tgcgcttctc attggtcagc

	ggctggggcc agctgctgga ccgtggcgcc acggccctgg

	agctcatggt cctcaacgtg ccccggctga tgacccagga

	ctgcctgcag cagtcacgga aggtgggaga ctccccaaat

	atcacggagt acatgttctg tgccggctac tcggatggca

	gcaaggactc ctgcaagggg gacagtggag gcccacatgc

	cacccactac cggggcacgt ggtacctgac gggcatcgtc

	agctggggcc agggctgcgc aaccgtgggc cactttgggg

	tgtacaccag ggtctcccag tacatcgagt ggctgcaaaa

	gctcatgcgc tcagagccac gcccaggagt cctcctgcga

	gccccatttc ccggtggcgg tggctccggc ggaggtgggt

	ccggtggcgg cggatcaggt gggggtggat caggcggtgg

	aggttccggt ggcgggggat ccgacaaaac tcacacatgc

	ccaccgtgcc cagctccgga actcctgggc ggaccgtcag

	tcttcctctt ccccccaaaa cccaaggaca ccctcatgat

	ctcccggacc cctgaggtca catgcgtggt ggtggacgtg

	agccacgaag accctgaggt caagttcaac tggtacgtgg

	acggcgtgga ggtgcataat gccaagacaa agccgcggga

	ggagcagtac aacagcacgt accgtgtggt cagcgtcctc

	accgtcctgc accaggactg gctgaatggc aaggagtaca

	agtgcaaggt ctccaacaaa gccctcccag cccccatcga

	gaaaaccatc tccaaagcca aagggcagcc ccgagaacca

	caggtgtaca ccctgccccc atcccgggat gagctgacca

	agaaccaggt cagcctgacc tgcctggtca aaggcttcta

	tcccagcgac atcgccgtgg agtgggagag caatgggcag

	ccggagaaca actacaagac cacgcctccc gtgttggact

	ccgacggctc cttcttcctc tacagcaagc tcaccgtgga

	caagagcagg tggcagcagg ggaacgtctt ctcatgctcc

	gtgatgcatg aggctctgca caaccactac acgcagaaga

	gcctctccct gtctccgggt aaaggtggcg gcggatcagg

	tgggggtgga tcaggcggtg gaggttccgg tggcggggga

	tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg

	aactcctggg aggaccgtca gtcttcctct tccccccaaa

	acccaaggac accctcatga tctcccggac ccctgaggtc

	acatgcgtgg tggtggacgt gagccacgaa gaccctgagg

	tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa

	tgccaagaca aagccgcggg aggagcagta caacagcacg

	taccgtgtgg tcagcgtcct caccgtcctg caccaggact

	ggctgaatgg caaggagtac aagtgcaagg tctccaacaa

	agccctccca gcccccatcg agaaaaccat ctccaaagcc

	aaagggcagc cccgagaacc acaggtgtac accctgcccc

	catcccgcga tgagctgacc aagaaccagg tcagcctgac

	ctgcctggtc aaaggcttct atcccagcga catcgccgtg

	gagtgggaga gcaatgggca gccggagaac aactacaaga

	ccacgcctcc cgtgttggac tccgacggct ccttcttcct

	ctacagcaag ctcaccgtgg acaagagcag gtggcagcag

	gggaacgtct tctcatgctc cgtgatgcat gaggctctgc

	acaaccacta cacgcagaag agcctctccc tgtctccggg

	taaatga

B domain deleted FVIII amino acid sequence: Signal peptide underlined; 14 amino acid linker (containing the remaining B domain) between the HC and LC sequence is double underlined, with the 5743/Q1638 fusion site indicated in bold.

1	MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL
	GELPVDARFP

51	PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL
	LGPTIQAEVY


101	DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR
	EKEDDKVFPG

151	GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG
	LIGALLVCRE

201	GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD
	AASARAWPKM

251	HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL
	EGHTFLVRNH

301	RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME
	AYVKVDSCPE

351	EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI
	RSVAKKHPKT

401	WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR
	KYKKVRFMAY

451	TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR
	PYNIYPHGIT

501	DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP
	TKSDPRCLTR

551	YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR
	NVILFSVFDE

601	NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV
	FDSLQLSVCL

651	HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF
	PFSGETVFMS

701	MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED
	SYEDISAYLL

751	SKNNAIEPRS F NPPVLKR HQR EITRTTL QSDQEEIDYD
	DTISVEMKKE


801	DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP
	HVLRNRAQSG

851	SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA
	EVEDNIMVTF

901	RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY
	FWKVQHHMAP

951	TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP
	AHGRQVTVQE


1001	FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN
	YRFHAINGYI

1051	MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV
	RKKEEYKMAL

1101	YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL
	VYSNKCQTPL

1151	GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK
	EPFSWIKVDL

1201	LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY
	RGNSTGTLMV

1251	FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME
	LMGCDLNSCS

1301	MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR
	SNAWRPQVNN

1351	PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS
	QDGHQWTLFF

1401	QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH
	QIALRMEVLG

1451	CEAQDLY

Full length FVIII amino acid sequence: Signal peptide underlined

1	MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL
	GELPVDARFP

51	PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL
	LGPTIQAEVY


101	DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR
	EKEDDKVFPG

151	GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG
	LIGALLVCRE

201	GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD
	AASARAWPKM

251	HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL
	EGHTFLVRNH

301	RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME
	AYVKVDSCPE

351	EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI
	RSVAKKHPKT

401	WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR
	KYKKVRFMAY

451	TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR
	PYNIYPHGIT

501	DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP
	TKSDPRCLTR

551	YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR
	NVILFSVFDE

601	NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV
	FDSLQLSVCL

651	HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF
	PFSGETVFMS

701	MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED
	SYEDISAYLL

751	SKNNAIEPRS FSQNSRHPST RQKQFNATTI PENDIEKTDP
	WFAHRTPMPK

801	IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPS
	PGAIDSNNSL

851	SEMTHFRPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL
	KKLDFKVSST

901	SNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL
	FGKKSSPLTE

951	SGGPLSLSEE NNDSKLLESG LMNSQESSWG KNVSSTESGR
	LFKGKRAHGP

1001	ALLTKDNALF KVSISLLKTN KTSNNSATNR KTHIDGPSLL
	IENSPSVWQN

1051	ILESDTEFKK VTPLIHDRML MDKNATALRL NHMSNKTTSS
	KNMEMVQQKK

1101	EGPIPPDAQN PDMSFFKMLF LPESARWIQR THGKNSLNSG
	QGPSPKQLVS

1151	LGPEKSVEGQ NFLSEKNKVV VGKGEFTKDV GLKEMVFPSS
	RNLFLTNLDN

1201	LHENNTHNQE KKIQEEIEKK ETLIQENVVL PQIHTVTGTK
	NFMKNLFLLS

1251	TRQNVEGSYD GAYAPVLQDF RSLNDSTNRT KKHTAHFSKK
	GEEENLEGLG

1301	NQTKQIVEKY ACTTRISPNT SQQNFVTQRS KRALKQFRLP
	LEETELEKRI

1351	IVDDTSTQWS KNMKHLTPST LTQIDYNEKE KGAITQSPLS
	DCLTRSHSIP

1401	QANRSPLPIA KVSSFPSIRP IYLTRVLFQD NSSHLPAASY
	RKKDSGVQES

1451	SHFLQGAKKN NLSLAILTLE MTGDQREVGS LGTSATNSVT
	YKKVENTVLP

1501	KPDLPKTSGK VELLPKVHIY QKDLFPTETS NGSPGHLDLV
	EGSLLQGTEG

1551	AIKWNEANRP GKVPFLRVAT ESSAKTPSKL LDPLAWDNHY
	GTQIPKEEWK

1601	SQEKSPEKTA FKKKDTILSL NACESNHAIA AINEGQNKPE
	IEVTWAKQGR

1651	TERLCSQNPP VLKRHQREIT RTTLQSDQEE IDYDDTISVE
	MKKEDFDIYD

1701	EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR
	AQSGSVPQFK

1751	KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI
	MVTFRNQASR

1801	PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH
	HMAPTKDEFD

1851	CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV
	TVQEFALFFT

1901	IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI
	NGYIMDTLPG

1951	LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY
	KMALYNLYPG

2001	VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC
	QTPLGMASGH

2051	IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI
	KVDLLAPMII

2101	HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG
	TLMVFFGNVD

2151	SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL
	NSCSMPLGME

2201	SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP
	QVNNPKEWLQ

2251	VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW
	TLFFQNGKVK

2301	VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM
	EVLGCEAQDL

2351	Y

FIX amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined


ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCT

FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR

VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW

QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII

PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF

HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE

GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL T

	FIX DNA sequence
	atgcagcgcg tgaacatgat catggcagaa tcaccaggcc

	tcatcaccat ctgcctttta ggatatctac tcagtgctga

	atgtacagtt tttcttgatc atgaaaacgc acaaaatt

	ctgaatcggc caaagaggta taattcaggt aaattggaag

	agtttgttca agggaatcta gagagagaat gtatggaaga

	aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac

	actgaaagaa caactgaatt ttggaagcag tatgttgatg

	gagatcagtg tgagtccaat ccatgtttaa atggcggcag

	ttgcaaggat gacattaatt cctatgaatg ttggtgtccc

	tttggatttg aaggaaagaa ctgtgaatta gatgtaacat

	gtaacattaa gaatggcaga tgcgagcagt tttgtaaaaa

	tagtgctgat aacaaggtgg tttgctcctg tactgaggga

	tatcgacttg cagaaaacca gaagtcctgt gaaccagcag

	tgccatttcc atgtggaaga gtttctgttt cacaaacttc

	taagctcacc cgtgctgaga ctgtttttcc tgatgtggac

	tatgtaaatt ctactgaagc tgaaaccatt ttggataaca

	tcactcaaag cacccaatca tttaatgact tcactcgggt

	tgttggtgga gaagatgcca aaccaggtca attcccttgg

	caggttgttt tgaatggtaa agttgatgca ttctgtggag

	gctctatcgt taatgaaaaa tggattgtaa ctgctgccca

	ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt

	gaacataata ttgaggagac agaacataca gagcaaaagc

	gaaatgtgat tcgaattatt cctcaccaca actacaatgc

	agctattaat aagtacaacc atgacattgc ccttctggaa

	ctggacgaac ccttagtgct aaacagctac gttacaccta

	tttgcattgc tgacaaggaa tacacgaaca tcttcctcaa

	atttggatct ggctatgtaa gtggctgggg aagagtcttc

	cacaaaggga gatcagcttt agttcttcag taccttagag

	ttccacttgt tgaccgagcc acatgtcttc gatctacaaa

	gttcaccatc tataacaaca tgttctgtgc tggcttccat

	gaaggaggta gagattcatg tcaaggagat agtgggggac

	cccatgttac tgaagtggaa gggaccagtt tcttaactgg

	aattattagc tggggtgaag agtgtgcaat gaaaggcaaa

	tatggaatat ataccaaggt atcccggtat gtcaactgga

	ttaaggaaaa aacaaagctc acttga

FX amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined


TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN

CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN GKACIPTGPY PCGKQTLERR

KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF NQTQPERGDN NLTRIVGGQE

CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ AKRFKVRVGD RNTEQEEGGE

AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP ACLPERDWAE STLMTQKTGI

VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ NMFCAGYDTK QEDACQGDSG

GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE

VITSSPLK

FX DNA sequence
atggggcgcc cactgcacct cgtcctgctc agtgcctccc tggctggcct cctgctgctc

ggggaaagtc tgttcatccg cagggagcag gccaacaaca tcctggcgag ggtcacgagg

gccaattcct ttcttgaaga gatgaagaaa ggacacctcg aaagagagtg catggaagag

acctgctcat acgaagaggc ccgcgaggtc tttgaggaca gcgacaagac gaatgaattc

tggaataaat acaaagatgg cgaccagtgt gagaccagtc cttgccagaa ccagggcaaa

tgtaaagacg gcctcgggga atacacctgc acctgtttag aaggattcga aggcaaaaac

tgtgaattat tcacacggaa gctctgcagc ctggacaacg gggactgtga ccagttctgc

cacgaggaac agaactctgt ggtgtgctcc tgcgcccgcg ggtacaccct ggctgacaac

ggcaaggcct gcattcccac agggccctac ccctgtggga aacagaccct ggaacgcagg

aagaggtcag tggcccaggc caccagcagc agcggggagg cccctgacag catcacatgg

aagccatatg atgcagccga cctggacccc accgagaacc ccttcgacct gcttgacttc

aaccagacgc agcctgagag gggcgacaac aacctcacca ggatcgtggg aggccaggaa

tgcaaggacg gggagtgtcc ctggcaggcc ctgctcatca atgaggaaaa cgagggtttc

tgtggtggaa ccattctgag cgagttctac atcctaacgg cagcccactg tctctaccaa

gccaagagat tcaaggtgag ggtaggggac cggaacacgg agcaggagga gggcggtgag

gcggtgcacg aggtggaggt ggtcatcaag cacaaccggt tcacaaagga gacctatgac

ttcgacatcg ccgtgctccg gctcaagacc cccatcacct tccgcatgaa cgtggcgcct

gcctgcctcc ccgagcgtga ctgggccgag tccacgctga tgacgcagaa gacggggatt

gtgagcggct tcgggcgcac ccacgagaag ggccggcagt ccaccaggct caagatgctg

gaggtgccct acgtggaccg caacagctgc aagctgtcca gcagcttcat catcacccag

aacatgttct gtgccggcta cgacaccaag caggaggatg cctgccaggg ggacagcggg

ggcccgcacg tcacccgctt caaggacacc tacttcgtga caggcatcgt cagctgggga

gagggctgtg cccgtaaggg gaagtacggg atctacacca aggtcaccgc cttcctcaag

tggatcgaca ggtccatgaa aaccaggggc ttgcccaagg ccaagagcca tgccccggag

gtcataacgt cctctccatt aaagtga

DNA sequence of FVII-066

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA ATTGTGGGGG GCAAGGTGTG CCCCAAAGGG

601	GAGTGTCCAT GGCAGGTCCT GTTGTTGGTG AATGGAGCTC AGTTGTGTGG GGGGACCCTG

661	ATCAACACCA TCTGGGTGGT CTCCGCGGCC CACTGTTTCG ACAAAATCAA GAACTGGAGG

721	AACCTGATCG CGGTGCTGGG CGAGCACGAC CTCAGCGAGC ACGACGGGGA TGAGCAGAGC

781	CGGCGGGTGG CGCAGGTCAT CATCCCCAGC ACGTACGTCC CGGGCACCAC CAACCACGAC

841	ATCGCGCTGC TCCGCCTGCA CCAGCCCGTG GTCCTCACTG ACCATGTGGT GCCCCTCTGC

901	CTGCCCGAAC GGACGTTCTC TGAGAGGACG CTGGCCTTCG TGCGCTTCTC ATTGGTCAGC

961	GGCTGGGGCC AGCTGCTGGA CCGTGGCGCC ACGGCCCTGG AGCTCATGGT CCTCAACGTG

1021	CCCCGGCTGA TGACCCAGGA CTGCCTGCAG CAGTCACGGA AGGTGGGAGA CTCCCCAAAT

1081	ATCACGGAGT ACATGTTCTG TGCCGGCTAC TCGGATGGCA GCAAGGACTC CTGCAAGGGG

1141	GACAGTGGAG GCCCACATGC CACCCACTAC CGGGGCACGT GGTACCTGAC GGGCATCGTC

1201	AGCTGGGGCC AGGGCTGCGC AACCGTGGGC CACTTTGGGG TGTACACCAG GGTCTCCCAG

1261	TACATCGAGT GGCTGCAAAA GCTCATGCGC TCAGAGCCAC GCCCAGGAGT CCTCCTGCGA

1321	GCCCCATTTC CCGGTGGCGG TGGCTCCGGC GGAGGTGGGT CCGGTGGCGG CGGATCAGGT

1381	GGGGGTGGAT CAGGCGGTGG AGGTTCCGGT GGCGGGGGAT CCGACAAAAC TCACACATGC

1441	CCACCGTGCC CAGCTCCGGA ACTCCTGGGA GGACCGTCAG TCTTCCTCTT CCCCCCAAAA

1501	CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG

1561	AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT

1621	GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC

1681	ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA

1741	GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA

1801	CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA AGAACCAGGT CAGCCTGACC

1861	TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG

1921	CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT CCGACGGCTC CTTCTTCCTC

1981	TACAGCAAGC TCACCGTCGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC

2041	GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT

2101	AAACGGCGCC GCCGGAGCGG TGGCGGCGGA TCAGGTGGGG GTGGATCAGG CGGTGGAGGT

2161	TCCGGTGGCG GGGGATCCGG CGGTGGAGGT TCCGGTGGGG GTGGATCAAG GAAGAGGAGG

2221	AAGAGGGCGC AGGTGCAGCT GCAGGAGTCT GGGGGAGGCT TGGTACAGCC TGGGGGGTCC

2281	CTGAGACTCT CCTGTGCAGC CTCTGGATTC ATGTTTAGCA GGTATGCCAT GAGCTGGGTC

2341	CGCCAGGCTC CAGGGAAGGG GCCAGAGTGG GTCTCAGGTA TTAGTGGTAG TGGTGGTAGT

2401	ACATACTACG CAGACTCCGT GAAGGGCCGG TTCACCGTCT CCAGAGACAA TTCCAAGAAC

2461	ACGCTGTATC TGCAAATGAA CAGCCTGAGA GCCGAGGACA CGGCTGTATA TTACTGCGCC

2521	CGGGGCGCCA CCTACACCAG CCGGAGCGAC GTGCCCGACC AGACCAGCTT CGACTACTGG

2581	GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA GGGAGTGCAT CCGCCCCAAA GCTTGAAGAA

2641	GGTGAATTTT CAGAAGCACG CGTATCTGAA CTGACTCAGG ACCCTGCTGT GTCTGTGGCC

2701	TTGGGACAGA CAGTCAGGAT CACATGCCAA GGAGACAGCC TCAGAAACTT TTATGCAAGC

2761	TGGTACCAGC AGAAGCCAGG ACAGGCCCCT ACTCTTGTCA TCTATGGTTT AAGTAAAAGG

2821	CCCTCAGGGA TCCCAGACCG ATTCTCTGCC TCCAGCTCAG GAAACACAGC TTCCTTGACC

2881	ATCACTGGGG CTCAGGCGGA AGATGAGGCT GACTATTACT GCCTGCTGTA CTACGGCGGC

2941	GGCCAGCAGG GCGTGTTCGG CGGCGGCACC AAGCTGACCG TCCTACGTCA GCCCAAGGCT

3001	GCCCCCTCGG TCACTCTGTT CCCGCCCTCT TCTGCGGCCG GTGGCGGTGG CTCCGGCGGA

3061	GGTGGGTCCG GTGGCGGCGG ATCAGGTGGG GGTGGATCAG GCGGTGGAGG TTCCGGTGGC

3121	GGGGGATCAG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCGGAACT CCTGGGCGGA

3181	CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT

3241	GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG

3301	TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC

3361	AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG

3421	GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC

3481	AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGATGAG

3541	CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC

3601	GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG

3661	TTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG

3721	CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG

3781	CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGA

FVII-066 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or SCE5 to Fc region is underlined, and linker with proprotein convertase processing sites is shown in bold

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

241	NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

301	LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

361	ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

421	YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC

481	PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN

541	AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

601	QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

661	YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KRRRRSGGGG SGGGGSGGGG

721	SGGGGSGGGG SGGGGSRKRR KRAQVQLQES GGGLVQPGGS LRLSCAASGF MFSRYAMSWV

781	RQAPGKGPEW VSGISGSGGS TYYADSVKGR FTVSRDNSKN TLYLQMNSLR AEDTAVYYCA

841	RGATYTSRSD VPDQTSFDYW GQGTLVTVSS GSASAPKLEE GEFSEARVSE LTQDPAVSVA

901	LGQTVRITCQ GDSLRNFYAS WYQQKPGQAP TLVIYGLSKR PSGIPDRFSA SSSGNTASLT

961	ITGAQAEDEA DYYCLLYYGG GQQGVFGGGT KLTVLRQPKA APSVTLFPPS SAAGGGGSGG

1021	GGSGGGGSGG GGSGGGGSGG GGSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP

1081	EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK

1141	EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI

1201	AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT

1261	QKSLSLSPGK *

DNA sequence for FVII-057

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCC

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGAGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAAGCG TGAGCCAGAC CAGCAAGCTG ACCCGGATTG
	TGGGGGGCAA GGTGTGCCCC

1501	AAAGGGGAGT GTCCATGGCA GGTCCTGTTG TTGGTGAATG
	GAGCTCAGTT GTGTGGGGGG

1561	ACCCTGATCA ACACCATCTG GGTGGTCTCC GCGGCCCACT
	GTTTCGACAA AATCAAGAAC

1621	TGGAGGAACC TGATCGCGGT GCTGGGCGAG CACGACCTCA
	GCGAGCACGA CGGGGATGAG

1681	CAGAGCCGGC GGGTGGCGCA GGTCATCATC CCCAGCACGT
	ACGTCCCGGG CACCACCAAC

1741	CACGACATCG CGCTGCTCCG CCTGCACCAG CCCGTGGTCC
	TCACTGACCA TGTGGTGCCC

1801	CTCTGCCTGC CCGAACGGAC GTTCTCTGAG AGGACGCTGG
	CCTTCGTGCG CTTCTCATTG

1861	GTCAGCGGCT GGGGCCAGCT GCTGGACCGT GGCGCCACGG
	CCCTGGAGCT CATGGTCCTC

1921	AACGTGCCCC GGCTGATGAC CCAGGACTGC CTGCAGCAGT
	CACGGAAGGT GGGAGACTCC

1981	CCAAATATCA CGGAGTACAT GTTCTGTGCC GGCTACTCGG
	ATGGCAGCAA GGACTCCTGC

2041	AAGGGGGACA GTGGAGGCCC ACATGCCACC CACTACCGGG
	GCACGTGGTA CCTGACGGGC

2101	ATCGTCAGCT GGGGCCAGGG CTGCGCAACC GTGGGCCACT
	TTGGGGTGTA CACCAGGGTC

2161	TCCCAGTACA TCGAGTGGCT GCAAAAGCTC ATGCGCTCAG
	AGCCACGCCC AGGAGTCCTC

2221	CTGCGAGCCC CATTTCCCGG TGGCGGTGGC TCCGGCGGAG
	GTGGGTCCGG TGGCGGCGGA

2281	TCAGGTGGGG GTGGATCAGG CGGTGGAGGT TCCGGTGGCG
	GGGGATCAGA CAAAACTCAC

2341	ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGAGGAC
	CGTCAGTCTT CCTCTTCCCC

2401	CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG
	AGGTCACATG CGTGGTGGTG

2461	GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT
	ACGTGGACGG CGTGGAGGTG

2521	CATAATGCCA AGACAAAGCC GCGGGAGGAG CAGTACAACA
	GCACGTACCG TGTGGTCAGC

2581	GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG
	AGTACAAGTG CAAGGTCTCC

2641	AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA
	AAGCCAAAGG GCAGCCCCGA

2701	GAACCACAGG TGTACACCCT GCCCCCATCC CGGGATGAGC
	TGACCAAGAA CCAGGTCAGC

2761	CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG
	CCGTGGAGTG GGAGAGCAAT

2821	GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGT
	TGGACTCCGA CGGCTCCTTC

2881	TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC
	AGCAGGGGAA CGTCTTCTCA

2941	TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC
	AGAAGAGCCT CTCCCTGTCT

3001	CCGGGTAAAT GA

FVII-057 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the Factor XIa cleavage site is shown in bold, and the Factor XIa cleavage site is shown in dashed underline

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481
541	WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN HDIALLRLHQ PVVLTDHVVP

601	LCLPERTFSE RTLAFVRFSL VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS

661	PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV

721	SQYIEWLQKL MRSEPRPGVL LRAPFPGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSDKTH

781	TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV

841	HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR

901	EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF

961	FLYSKLTVDK SRWOOGNVFS CSVMHEALHN HYTOKSLSLS PGK*

DNA sequence for FVII-058

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCC

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGAGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGACT TCCTGGCCGA GGGCGGCGGC GTGCGGATTG
	TGGGGGGCAA GGTGTGCCCC

1501	AAAGGGGAGT GTCCATGGCA GGTCCTGTTG TTGGTGAATG
	GAGCTCAGTT GTGTGGGGGG

1561	ACCCTGATCA ACACCATCTG GGTGGTCTCC GCGGCCCACT
	GTTTCGACAA AATCAAGAAC

1621	TGGAGGAACC TGATCGCGGT GCTGGGCGAG CACGACCTCA
	GCGAGCACGA CGGGGATGAG

1681	CAGAGCCGGC GGGTGGCGCA GGTCATCATC CCCAGCACGT
	ACGTCCCGGG CACCACCAAC

1741	CACGACATCG CGCTGCTCCG CCTGCACCAG CCCGTGGTCC
	TCACTGACCA TGTGGTGCCC

1801	CTCTGCCTGC CCGAACGGAC GTTCTCTGAG AGGACGCTGG
	CCTTCGTGCG CTTCTCATTG

1861	GTCAGCGGCT GGGGCCAGCT GCTGGACCGT GGCGCCACGG
	CCCTGGAGCT CATGGTCCTC

1921	AACGTGCCCC GGCTGATGAC CCAGGACTGC CTGCAGCAGT
	CACGGAAGGT GGGAGACTCC

1981	CCAAATATCA CGGAGTACAT GTTCTGTGCC GGCTACTCGG
	ATGGCAGCAA GGACTCCTGC

2041	AAGGGGGACA GTGGAGGCCC ACATGCCACC CACTACCGGG
	GCACGTGGTA CCTGACGGGC

2101	ATCGTCAGCT GGGGCCAGGG CTGCGCAACC GTGGGCCACT
	TTGGGGTGTA CACCAGGGTC

2161	TCCCAGTACA TCGAGTGGCT GCAAAAGCTC ATGCGCTCAG
	AGCCACGCCC AGGAGTCCTC

2221	CTGCGAGCCC CATTTCCCGG TGGCGGTGGC TCCGGCGGAG
	GTGGGTCCGG TGGCGGCGGA

2281	TCAGGTGGGG GTGGATCAGG CGGTGGAGGT TCCGGTGGCG
	GGGGATCAGA CAAAACTCAC

2341	ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGAGGAC
	CGTCAGTCTT CCTCTTCCCC

2401	CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG
	AGGTCACATG CGTGGTGGTG

2461	GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT
	ACGTGGACGG CGTGGAGGTG

2521	CATAATGCCA AGACAAAGCC GCGGGAGGAG CAGTACAACA
	GCACGTACCG TGTGGTCAGC

2581	GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG
	AGTACAAGTG CAAGGTCTCC

2641	AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA
	AAGCCAAAGG GCAGCCCCGA

2701	GAACCACAGG TGTACACCCT GCCCCCATCC CGGGATGAGC
	TGACCAAGAA CCAGGTCAGC

2761	CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG
	CCGTGGAGTG GGAGAGCAAT

2821	GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGT
	TGGACTCCGA CGGCTCCTTC

2881	TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC
	AGCAGGGGAA CGTCTTCTCA

2941	TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC
	AGAAGAGCCT CTCCCTGTCT

3001	CCGGGTAAAT GA

FVII-058 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, and the thrombin cleavage site is shown in dashed underline

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481
541	WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN HDIALLRLHQ PVVLTDHVVP

601	LCLPERTFSE RTLAFVRFSL VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS

661	PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV

721	SQYIEWLQKL MRSEPRPGVL LRAPFPGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSDKTH

781	TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV

841	HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR

901	EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF

961	FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK*

DNA sequence for FVII-059

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG GTTCCGGTGG CGGGGGATCC

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC TCCTGGGAGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG GGGGTGGATC AGGAGGAGGT

1441	GGTTCAACCA CCAAGATCAA GCCCCGGATT GTGGGGGGCA AGGTGTGCCC CAAAGGGGAG

1501	TGTCCATGGC AGGTCCTGTT GTTGGTGAAT GGAGCTCAGT TGTGTGGGGG GACCCTGATC

1561	AACACCATCT GGGTGGTCTC CGCGGCCCAC TGTTTCGACA AAATCAAGAA CTGGAGGAAC

1621	CTGATCGCGG TGCTGGGCGA GCACGACCTC AGCGAGCACG ACGGGGATGA GCAGAGCCGG

1681	CGGGTGGCGC AGGTCATCAT CCCCAGCACG TACGTCCCGG GCACCACCAA CCACGACATC

1741	GCGCTGCTCC GCCTGCACCA GCCCGTGGTC CTCACTGACC ATGTGGTGCC CCTCTGCCTG

1801	CCCGAACGGA CGTTCTCTGA GAGGACGCTG GCCTTCGTGC GCTTCTCATT GGTCAGCGGC

1861	TGGGGCCAGC TGCTGGACCG TGGCGCCACG GCCCTGGAGC TCATGGTCCT CAACGTGCCC

1921	CGGCTGATGA CCCAGGACTG CCTGCAGCAG TCACGGAAGG TGGGAGACTC CCCAAATATC

1981	ACGGAGTACA TGTTCTGTGC CGGCTACTCG GATGGCAGCA AGGACTCCTG CAAGGGGGAC

2041	AGTGGAGGCC CACATGCCAC CCACTACCGG GGCACGTGGT ACCTGACGGG CATCGTCAGC

2101	TGGGGCCAGG GCTGCGCAAC CGTGGGCCAC TTTGGGGTGT ACACCAGGGT CTCCCAGTAC

2161	ATCGAGTGGC TGCAAAAGCT CATGCGCTCA GAGCCACGCC CAGGAGTCCT CCTGCGAGCC

2221	CCATTTCCCG GTGGCGGTGG CTCCGGCGGA GGTGGGTCCG GTGGCGGCGG ATCAGGTGGG

2281	GGTGGATCAG GCGGTGGAGG TTCCGGTGGC GGGGGATCAG ACAAAACTCA CACATGCCCA

2341	CCGTGCCCAG CACCTGAACT CCTGGGAGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC

2401	AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC

2461	CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC

2521	AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC

2581	GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC

2641	CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG

2701	GTGTACACCC TGCCCCCATC CCGGGATGAG CTGACCAAGA ACCAGGTCAG CCTGACCTGC

2761	CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG

2821	GAGAACAACT ACAAGACCAC GCCTCCCGTG TTGGACTCCG ACGGCTCCTT CTTCCTCTAC

2881	AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG

2941	ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA

3001	TGA

FVII-059 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, and the thrombin cleavage site is shown in dashed underline

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481
541	LIAVLGEHDL SEHDGDEQSR RVAQVIIPST YVPGTTNHDI ALLRLHQPVV LTDHVVPLCL

601	PERTFSERTL AFVRFSLVSG WGQLLDRGAT ALELMVLNVP RLMTQDCLQQ SRKVGDSPNI

661	TEYMFCAGYS DGSKDSCKGD SGGPHATHYR GTWYLTGIVS WGQGCATVGH FGVYTRVSQY

721	IEWLQKLMRS EPRPGVLLRA PFPGGGGSGG GGSGGGGSGG GGSGGGGSGG GGSDKTHTCP

781	PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA

841	KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

901	VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

961	SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK *

DNA sequence for FVII-060

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCC

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGAGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGCCC TGCGGCCCCG GGTGGTGGGC GGCGCCGTGG
	TGGGGGGCAA GGTGTGCCCC

1501	AAAGGGGAGT GTCCATGGCA GGTCCTGTTG TTGGTGAATG
	GAGCTCAGTT GTGTGGGGGG

1561	ACCCTGATCA ACACCATCTG GGTGGTCTCC GCGGCCCACT
	GTTTCGACAA AATCAAGAAC

1621	TGGAGGAACC TGATCGCGGT GCTGGGCGAG CACGACCTCA
	GCGAGCACGA CGGGGATGAG

1681	CAGAGCCGGC GGGTGGCGCA GGTCATCATC CCCAGCACGT
	ACGTCCCGGG CACCACCAAC

1741	CACGACATCG CGCTGCTCCG CCTGCACCAG CCCGTGGTCC
	TCACTGACCA TGTGGTGCCC

1801	CTCTGCCTGC CCGAACGGAC GTTCTCTGAG AGGACGCTGG
	CCTTCGTGCG CTTCTCATTG

1861	GTCAGCGGCT GGGGCCAGCT GCTGGACCGT GGCGCCACGG
	CCCTGGAGCT CATGGTCCTC

1921	AACGTGCCCC GGCTGATGAC CCAGGACTGC CTGCAGCAGT
	CACGGAAGGT GGGAGACTCC

1981	CCAAATATCA CGGAGTACAT GTTCTGTGCC GGCTACTCGG
	ATGGCAGCAA GGACTCCTGC

2041	AAGGGGGACA GTGGAGGCCC ACATGCCACC CACTACCGGG
	GCACGTGGTA CCTGACGGGC

2101	ATCGTCAGCT GGGGCCAGGG CTGCGCAACC GTGGGCCACT
	TTGGGGTGTA CACCAGGGTC

2161	TCCCAGTACA TCGAGTGGCT GCAAAAGCTC ATGCGCTCAG
	AGCCACGCCC AGGAGTCCTC

2221	CTGCGAGCCC CATTTCCCGG TGGCGGTGGC TCCGGCGGAG
	GTGGGTCCGG TGGCGGCGGA

2281	TCAGGTGGGG GTGGATCAGG CGGTGGAGGT TCCGGTGGCG
	GGGGATCAGA CAAAACTCAC

2341	ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGAGGAC
	CGTCAGTCTT CCTCTTCCCC

2401	CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG
	AGGTCACATG CGTGGTGGTG

2461	GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT
	ACGTGGACGG CGTGGAGGTG

2521	CATAATGCCA AGACAAAGCC GCGGGAGGAG CAGTACAACA
	GCACGTACCG TGTGGTCAGC

2581	GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG
	AGTACAAGTG CAAGGTCTCC

2641	AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA
	AAGCCAAAGG GCAGCCCCGA

2701	GAACCACAGG TGTACACCCT GCCCCCATCC CGGGATGAGC
	TGACCAAGAA CCAGGTCAGC

2761	CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG
	CCGTGGAGTG GGAGAGCAAT

2821	GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGT
	TGGACTCCGA CGGCTCCTTC

2881	TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC
	AGCAGGGGAA CGTCTTCTCA

2941	TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC
	AGAAGAGCCT CTCCCTGTCT

3001	CCGGGTAAAT GA

FVII-060 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, and the thrombin cleavage site is shown in dashed underline

DNA sequence for FVII-061

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCC

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGAGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGCCC TGCGGCCCCG GGTGGTGGGC GGCGCCATTG
	TGGGGGGCAA GGTGTGCCCC

1501	AAAGGGGAGT GTCCATGGCA GGTCCTGTTG TTGGTGAATG
	GAGCTCAGTT GTGTGGGGGG

1561	ACCCTGATCA ACACCATCTG GGTGGTCTCC GCGGCCCACT
	GTTTCGACAA AATCAAGAAC

1621	TGGAGGAACC TGATCGCGGT GCTGGGCGAG CACGACCTCA
	GCGAGCACGA CGGGGATGAG

1681	CAGAGCCGGC GGGTGGCGCA GGTCATCATC CCCAGCACGT
	ACGTCCCGGG CACCACCAAC

1741	CACGACATCG CGCTGCTCCG CCTGCACCAG CCCGTGGTCC
	TCACTGACCA TGTGGTGCCC

1801	CTCTGCCTGC CCGAACGGAC GTTCTCTGAG AGGACGCTGG
	CCTTCGTGCG CTTCTCATTG

1861	GTCAGCGGCT GGGGCCAGCT GCTGGACCGT GGCGCCACGG
	CCCTGGAGCT CATGGTCCTC

1921	AACGTGCCCC GGCTGATGAC CCAGGACTGC CTGCAGCAGT
	CACGGAAGGT GGGAGACTCC

1981	CCAAATATCA CGGAGTACAT GTTCTGTGCC GGCTACTCGG
	ATGGCAGCAA GGACTCCTGC

2041	AAGGGGGACA GTGGAGGCCC ACATGCCACC CACTACCGGG
	GCACGTGGTA CCTGACGGGC

2101	ATCGTCAGCT GGGGCCAGGG CTGCGCAACC GTGGGCCACT
	TTGGGGTGTA CACCAGGGTC

2161	TCCCAGTACA TCGAGTGGCT GCAAAAGCTC ATGCGCTCAG
	AGCCACGCCC AGGAGTCCTC

2221	CTGCGAGCCC CATTTCCCGG TGGCGGTGGC TCCGGCGGAG
	GTGGGTCCGG TGGCGGCGGA

2281	TCAGGTGGGG GTGGATCAGG CGGTGGAGGT TCCGGTGGCG
	GGGGATCAGA CAAAACTCAC

2341	ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGAGGAC
	CGTCAGTCTT CCTCTTCCCC

2401	CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG
	AGGTCACATG CGTGGTGGTG

2461	GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT
	ACGTGGACGG CGTGGAGGTG

2521	CATAATGCCA AGACAAAGCC GCGGGAGGAG CAGTACAACA
	GCACGTACCG TGTGGTCAGC

2581	GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG
	AGTACAAGTG CAAGGTCTCC

2641	AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA
	AAGCCAAAGG GCAGCCCCGA

2701	GAACCACAGG TGTACACCCT GCCCCCATCC CGGGATGAGC
	TGACCAAGAA CCAGGTCAGC

2761	CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG
	CCGTGGAGTG GGAGAGCAAT

2821	GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGT
	TGGACTCCGA CGGCTCCTTC

2881	TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC
	AGCAGGGGAA CGTCTTCTCA

2941	TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC
	AGAAGAGCCT CTCCCTGTCT

3001	CCGGGTAAAT GA

FVII-061 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, and the thrombin cleavage site is shown in dashed underline

DNA sequence for FVII-062

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCA

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGCGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGGTG GTGGAGGATC CATTGTGGGG GGCAAGGTGT
	GCCCCAAAGG GGAGTGTCCA

1501	TGGCAGGTCC TGTTGTTGGT GAATGGAGCT CAGTTGTGTG
	GGGGGACCCT GATCAACACC

1561	ATCTGGGTGG TCTCCGCGGC CCACTGTTTC GACAAAATCA
	AGAACTGGAG GAACCTGATC

1621	GCGGTGCTGG GCGAGCACGA CCTCAGCGAG CACGACGGGG
	ATGAGCAGAG CCGGCGGGTG

1681	GCGCAGGTCA TCATCCCCAG CACGTACGTC CCGGGCACCA
	CCAACCACGA CATCGCGCTG

1741	CTCCGCCTGC ACCAGCCCGT GGTCCTCACT GACCATGTGG
	TGCCCCTCTG CCTGCCCGAA

1801	CGGACGTTCT CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT
	CATTGGTCAG CGGCTGGGGC

1861	CAGCTGCTGG ACCGTGGCGC CACGGCCCTG GAGCTCATGG
	TCCTCAACGT GCCCCGGCTG

1921	ATGACCCAGG ACTGCCTGCA GCAGTCACGG AAGGTGGGAG
	ACTCCCCAAA TATCACGGAG

1981	TACATGTTCT GTGCCGGCTA CTCGGATGGC AGCAAGGACT
	CCTGCAAGGG GGACAGTGGA

2041	GGCCCACATG CCACCCACTA CCGGGGCACG TGGTACCTGA
	CGGGCATCGT CAGCTGGGGC

2101	CAGGGCTGCG CAACCGTGGG CCACTTTGGG GTGTACACCA
	GGGTCTCCCA GTACATCGAG

2161	TGGCTGCAAA AGCTCATGCG CTCAGAGCCA CGCCCAGGAG
	TCCTCCTGCG AGCCCCATTT

2221	CCCGGTGGCG GTGGCTCCGG CGGAGGTGGG TCCGGTGGCG
	GCGGATCAGG TGGGGGTGGA

2281	TCAGGCGGTG GAGGTTCCGG TGGCGGGGGA TCAGACAAAA
	CTCACACATG CCCACCGTGC

2341	CCAGCACCTG AACTCCTGGG AGGACCGTCA GTCTTCCTCT
	TCCCCCCAAA ACCCAAGGAC

2401	ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
	TGGTGGACGT GAGCCACGAA

2461	GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG
	AGGTGCATAA TGCCAAGACA

2521	AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG
	TCAGCGTCCT CACCGTCCTG

2581	CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG
	TCTCCAACAA AGCCCTCCCA

2641	GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
	CCCGAGAACC ACAGGTGTAC

2701	ACCCTGCCCC CATCCCGGGA TGAGCTGACC AAGAACCAGG
	TCAGCCTGAC CTGCCTGGTC

2761	AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA
	GCAATGGGCA GCCGGAGAAC

2821	AACTACAAGA CCACGCCTCC CGTGTTGGAC TCCGACGGCT
	CCTTCTTCCT CTACAGCAAG

2881	CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT
	TCTCATGCTC CGTGATGCAT

2941	GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
	TGTCTCCGGG TAAATGA

FVII-062 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVIIa light chain or heavy chain to Fc region is underlined, and linker region connecting the Fc and the FVIIa heavy chain is shown in bold

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481	GSGGGGSIVG GKVCPKGECP WQVLLLVNGA QLCGGTLINT IWVVSAAHCF DKIKNWRNLI

541	AVLGEHDLSE HDGDEQSRRV AQVIIPSTYV PGTTNHDIAL LRLHQPVVLT DHVVPLCLPE

601	RTFSERTLAF VRFSLVSGWG QLLDRGATAL ELMVLNVPRL MTQDCLQQSR KVGDSPNITE

661	YMFCAGYSDG SKDSCKGDSG GPHATHYRGT WYLTGIVSWG QGCATVGHFG VYTRVSQYIE

721	WLQKLMRSEP RPGVLLRAPF PGGGGSGGGG SGGGGSGGGG SGGGGSGGGG SDKTHTCPPC

781	RAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT

841	KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY

901	TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

961	LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK*

DNA sequence for FVII-090

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCA

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGCGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGCCC TGCGGCCCCG GATTGTGGGG GGCAAGGTGT
	GCCCCAAAGG GGAGTGTCCA

1501	TGGCAGGTCC TGTTGTTGGT GAATGGAGCT CAGTTGTGTG
	GGGGGACCCT GATCAACACC

1561	ATCTGGGTGG TCTCCGCGGC CCACTGTTTC GACAAAATCA
	AGAACTGGAG GAACCTGATC

1621	GCGGTGCTGG GCGAGCACGA CCTCAGCGAG CACGACGGGG
	ATGAGCAGAG CCGGCGGGTG

1681	GCGCAGGTCA TCATCCCCAG CACGTACGTC CCGGGCACCA
	CCAACCACGA CATCGCGCTG

1741	CTCCGCCTGC ACCAGCCCGT GGTCCTCACT GACCATGTGG
	TGCCCCTCTG CCTGCCCGAA

1801	CGGACGTTCT CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT
	CATTGGTCAG CGGCTGGGGC

1861	CAGCTGCTGG ACCGTGGCGC CACGGCCCTG GAGCTCATGG
	TCCTCAACGT GCCCCGGCTG

1921	ATGACCCAGG ACTGCCTGCA GCAGTCACGG AAGGTGGGAG
	ACTCCCCAAA TATCACGGAG

1981	TACATGTTCT GTGCCGGCTA CTCGGATGGC AGCAAGGACT
	CCTGCAAGGG GGACAGTGGA

2041	GGCCCACATG CCACCCACTA CCGGGGCACG TGGTACCTGA
	CGGGCATCGT CAGCTGGGGC

2101	CAGGGCTGCG CAACCGTGGG CCACTTTGGG GTGTACACCA
	GGGTCTCCCA GTACATCGAG

2161	TGGCTGCAAA AGCTCATGCG CTCAGAGCCA CGCCCAGGAG
	TCCTCCTGCG AGCCCCATTT

2221	CCCGGTGGCG GTGGCTCCGG CGGAGGTGGG TCCGGTGGCG
	GCGGATCAGG TGGGGGTGGA

2281	TCAGGCGGTG GAGGTTCCGG TGGCGGGGGA TCAGACAAAA
	CTCACACATG CCCACCGTGC

2341	CCAGCACCTG AACTCCTGGG AGGACCGTCA GTCTTCCTCT
	TCCCCCCAAA ACCCAAGGAC

2401	ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
	TGGTGGACGT GAGCCACGAA

2461	GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG
	AGGTGCATAA TGCCAAGACA

2521	AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG
	TCAGCGTCCT CACCGTCCTG

2581	CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG
	TCTCCAACAA AGCCCTCCCA

2641	GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
	CCCGAGAACC ACAGGTGTAC

2701	ACCCTGCCCC CATCCCGGGA TGAGCTGACC AAGAACCAGG
	TCAGCCTGAC CTGCCTGGTC

2761	AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA
	GCAATGGGCA GCCGGAGAAC

2821	AACTACAAGA CCACGCCTCC CGTGTTGGAC TCCGACGGCT
	CCTTCTTCCT CTACAGCAAG

2881	CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT
	TCTCATGCTC CGTGATGCAT

2941	GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
	TGTCTCCGGG TAAATGA

FVII-090 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, and the thrombin cleavage site is shown in dashed underline

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481
541	AVLGEHDLSE HDGDEQSRRV AQVIIPSTYV PGTTNHDIAL LRLHQPVVLT DHVVPLCLPE

601	RTFSERTLAF VRFSLVSGWG QLLDRGATAL ELMVLNVPRL MTQDCLQQSR KVGDSPNITE

661	YMFCAGYSDG SKDSCKGDSG GPHATHYRGT WYLTGIVSWG QGCATVGHFG VYTRVSQYIE

721	WLQKLMRSEP RPGVLLRAPF PGGGGSGGGG SGGGGSGGGG SGGGGSGGGG SDKTHTCPPC

781	RAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT

841	KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY

901	TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

961	LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK*

DNA sequence for FVII-100

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCC

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGAGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGCCC TGCGGCCCCG GATTGTGGGG GGCAAGGTGT
	GCCCCAAAGG GGAGTGTCCA

1501	TGGCAGGTCC TGTTGTTGGT GAATGGAGCT CAGTTGTGTG
	GGGGGACCCT GATCAACACC

1561	ATCTGGGTGG TCTCCGCGGC CCACTGTTTC GACAAAATCA
	AGAACTGGAG GAACCTGATC

1621	GCGGTGCTGG GCGAGCACGA CCTCAGCGAG CACGACGGGG
	ATGAGCAGAG CCGGCGGGTG

1681	GCGCAGGTCA TCATCCCCAG CACGTACGTC CCGGGCACCA
	CCAACCACGA CATCGCGCTG

1741	CTCCGCCTGC ACCAGCCCGT GGTCCTCACT GACCATGTGG
	TGCCCCTCTG CCTGCCCGAA

1801	CGGACGTTCT CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT
	CATTGGTCAG CGGCTGGGGC

1861	CAGCTGCTGG ACCGTGGCGC CACGGCCCTG GAGCTCATGG
	TCCTCAACGT GCCCCGGCTG

1921	ATGACCCAGG ACTGCGAGGC CAGCTACCCC GGCAAGATCA
	CGGAGTACAT GTTCTGTGCC

1981	GGCTACTCGG ATGGCAGCAA GGACTCCTGC AAGGGGGACA
	GTGGAGGCCC ACATGCCACC

2041	CACTACCGGG GCACGTGGTA CCTGACGGGC ATCGTCAGCT
	GGGGCCAGGG CTGCGCAACC

2101	GTGGGCCACT TTGGGGTGTA CACCAGGGTC TCCCAGTACA
	TCGAGTGGCT GCAAAAGCTC

2161	ATGCGCTCAG AGCCACGCCC AGGAGTCCTC CTGCGAGCCC
	CATTTCCCGG TGGCGGTGGC

2221	TCCGGCGGAG GTGGGTCCGG TGGCGGCGGA TCAGGTGGGG
	GTGGATCAGG CGGTGGAGGT

2281	TCCGGTGGCG GGGGATCAGA CAAAACTCAC ACATGCCCAC
	CGTGCCCAGC ACCTGAACTC

2341	CTGGGAGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA
	AGGACACCCT CATGATCTCC

2401	CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC
	ACGAAGACCC TGAGGTCAAG

2461	TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA
	AGACAAAGCC GCGGGAGGAG

2521	CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG
	TCCTGCACCA GGACTGGCTG

2581	AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC
	TCCCAGCCCC CATCGAGAAA

2641	ACCATCTCCA AAGCCAAAGG GCAGCCCCGA GAACCACAGG
	TGTACACCCT GCCCCCATCC

2701	CGGGATGAGC TGACCAAGAA CCAGGTCAGC CTGACCTGCC
	TGGTCAAAGG CTTCTATCCC

2761	AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG
	AGAACAACTA CAAGACCACG

2821	CCTCCCGTGT TGGACTCCGA CGGCTCCTTC TTCCTCTACA
	GCAAGCTCAC CGTGGACAAG

2881	AGCAGGTGGC AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA
	TGCATGAGGC TCTGCACAAC

2941	CACTACACGC AGAAGAGCCT CTCCCTGTCT
	CCGGGTAAAT GA

FVII-100 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, the thrombin cleavage site is shown in dashed underline, and the trypsin 170 loop region is wave underlined

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481
541	AVLGEHDLSE HDGDEQSRRV AQVIIPSTYV PGTTNHDIAL LRLHQPVVLT DHVVPLCLPE

601
661	GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL

721	MRSEPRPGVL LRAPFPGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSDKTH TCPPCPAPEL

781	LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE

841	QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS

901	RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK

961	SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK*

DNA sequence for FVII-115

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGTGGCGGTG
	GCTCCGGCGG AGGTGGGTCC

601	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCA

661	GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC
	TCCTGGGCGG ACCGTCAGTC

721	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

781	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

841	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

901	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

961	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

1021	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGGGATGA GCTGACCAAG

1081	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

1141	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

1201	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTCGACA
	AGAGCAGGTG GCAGCAGGGG

1261	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

1321	CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG
	GGGGTGGATC AGGCGGTGGA

1381	GGTTCCGGTG GCGGGGGATC CGGCGGTGGA GGTTCCGGTG
	GGGGTGGATC AGGAGGAGGT

1441	GGTTCAGCCC TGCGGCCCCG GATTGTGGGG GGCAAGGACT
	GCCCCAAAGG GGAGTGTCCA

1501	TGGCAGGTCC TGTTGTTGGT GAATGGAGCT CAGTTGTGTG
	GGGGGACCCT GATCAACACC

1561	ATCTGGGTGG TCTCCGCGGC CCACTGTTTC GACAAAATCA
	AGAACTGGAG GAACCTGATC

1621	GCGGTGCTGG GCGAGCACGA CCTCAGCGAG CACGACGGGG
	ATGAGCAGAG CCGGCGGGTG

1681	GCGCAGGTCA TCATCCCCAG CACGTACGTC CCGGGCACCA
	CCAACCACGA CATCGCGCTG

1741	CTCCGCCTGC ACCAGCCCGT GGTCCTCACT GACCATGTGG
	TGCCCCTCTG CCTGCCCGAA

1801	CGGACGTTCT CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT
	CATTGGTCAG CGGCTGGGGC

1861	CAGCTGCTGG ACCGTGGCGC CACGGCCCTG GTACTCCAAG
	TCCTCAACGT GCCCCGGCTG

1921	ATGACCCAGG ACTGCCTGCA GCAGTCACGG AAGGTGGGAG
	ACTCCCCAAA TATCACGGAG

1981	TACATGTTCT GTGCCGGCTA CTCGGATGGC AGCAAGGACT
	CCTGCAAGGG GGACAGTGGA

2041	GGCCCACATG CCACCCACTA CCGGGGCACG TGGTACCTGA
	CGGGCATCGT CAGCTGGGGC

2101	CAGGGCTGCG CAACCGTGGG CCACTTTGGG GTGTACACCA
	GGGTCTCCCA GTACATCGAG

2161	TGGCTGCAAA AGCTCATGCG CTCAGAGCCA CGCCCAGGAG
	TCCTCCTGCG AGCCCCATTT

2221	CCCGGTGGCG GTGGCTCCGG CGGAGGTGGG TCCGGTGGCG
	GCGGATCAGG TGGGGGTGGA

2281	TCAGGCGGTG GAGGTTCCGG TGGCGGGGGA TCAGACAAAA
	CTCACACATG CCCACCGTGC

2341	CCAGCACCTG AACTCCTGGG AGGACCGTCA GTCTTCCTCT
	TCCCCCCAAA ACCCAAGGAC

2401	ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG
	TGGTGGACGT GAGCCACGAA

2461	GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG
	AGGTGCATAA TGCCAAGACA

2521	AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG
	TCAGCGTCCT CACCGTCCTG

2581	CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG
	TCTCCAACAA AGCCCTCCCA

2641	GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC
	CCCGAGAACC ACAGGTGTAC

2701	ACCCTGCCCC CATCCCGGGA TGAGCTGACC AAGAACCAGG
	TCAGCCTGAC CTGCCTGGTC

2761	AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA
	GCAATGGGCA GCCGGAGAAC

2821	AACTACAAGA CCACGCCTCC CGTGTTGGAC TCCGACGGCT
	CCTTCTTCCT CTACAGCAAG

2881	CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT
	TCTCATGCTC CGTGATGCAT

2941	GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC
	TGTCTCCGGG TAAATGA

FVII-115 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII light chain or heavy chain to Fc region is underlined, linker region connecting the Fc and the thrombin cleavage site is shown in bold, the thrombin cleavage site is shown in dashed underline, and the three point mutations in FVIIa (V158D, E296V and M298Q) are in bold and underlined

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV

241	FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

361	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG

421	NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

481
541	AVLGEHDLSE HDGDEQSRRV AQVIIPSTYV PGTTNHDIAL LRLHQPVVLT DHVVPLCLPE

601	RTFSERTLAF VRFSLVSGWG QLLDRGATAL V L Q VLNVPRL MTQDCLQQSR KVGDSPNITE

661	YMFCAGYSDG SKDSCKGDSG GPHATHYRGT WYLTGIVSWG QGCATVGHFG VYTRVSQYIE

721	WLQKLMRSEP RPGVLLRAPF PGGGGSGGGG SGGGGSGGGG SGGGGSGGGG SDKTHTCPPC

781	RAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT

841	KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY

901	TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

961	LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK*

DNA sequence for FVII-118

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCGCC CTGCGGCCCC
	GGATTGTGGG GGGCAAGGTG

601	TGCCCCAAAG GGGAGTGTCC ATGGCAGGTC CTGTTGTTGG
	TGAATGGAGC TCAGTTGTGT

661	GGGGGGACCC TGATCAACAC CATCTGGGTG GTCTCCGCGG
	CCCACTGTTT CGACAAAATC

721	AAGAACTGGA GGAACCTGAT CGCGGTGCTG GGCGAGCACG
	ACCTCAGCGA GCACGACGGG

781	GATGAGCAGA GCCGGCGGGT GGCGCAGGTC ATCATCCCCA
	GCACGTACGT CCCGGGCACC

841	ACCAACCACG ACATCGCGCT GCTCCGCCTG CACCAGCCCG
	TGGTCCTCAC TGACCATGTG

901	GTGCCCCTCT GCCTGCCCGA ACGGACGTTC TCTGAGAGGA
	CGCTGGCCTT CGTGCGCTTC

961	TCATTGGTCA GCGGCTGGGG CCAGCTGCTG GACCGTGGCG
	CCACGGCCCT GGAGCTCATG

1021	GTCCTCAACG TGCCCCGGCT GATGACCCAG GACTGCCTGC
	AGCAGTCACG GAAGGTGGGA

1081	GACTCCCCAA ATATCACGGA GTACATGTTC TGTGCCGGCT
	ACTCGGATGG CAGCAAGGAC

1141	TCCTGCAAGG GGGACAGTGG AGGCCCACAT GCCACCCACT
	ACCGGGGCAC GTGGTACCTG

1201	ACGGGCATCG TCAGCTGGGG CCAGGGCTGC GCAACCGTGG
	GCCACTTTGG GGTGTACACC

1261	AGGGTCTCCC AGTACATCGA GTGGCTGCAA AAGCTCATGC
	GCTCAGAGCC ACGCCCAGGA

1321	GTCCTCCTGC GAGCCCCATT TCCCGGTGGC GGTGGCTCCG
	GCGGAGGTGG GTCCGGTGGC

1381	GGCGGATCAG GTGGGGGTGG ATCAGGCGGT GGAGGTTCCG
	GTGGCGGGGG ATCCGACAAA

1441	ACTCACACAT GCCCACCGTG CCCAGCTCCG GAACTCCTGG
	GCGGACCGTC AGTCTTCCTC

1501	TTCCCCCCAA AACCCAAGGA CACCCTCATG ATCTCCCGGA
	CCCCTGAGGT CACATGCGTG

1561	GTGGTGGACG TGAGCCACGA AGACCCTGAG GTCAAGTTCA
	ACTGGTACGT GGACGGCGTG

1621	GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT
	ACAACAGCAC GTACCGTGTG

1681	GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG
	GCAAGGAGTA CAAGTGCAAG

1741	GTCTCCAACA AAGCCCTCCC AGCCCCCATC GAGAAAACCA
	TCTCCAAAGC CAAAGGGCAG

1801	CCCCGAGAAC CACAGGTGTA CACCCTGCCC CCATCCCGGG
	ATGAGCTGAC CAAGAACCAG

1861	GTCAGCCTGA CCTGCCTGGT CAAAGGCTTC TATCCCAGCG
	ACATCGCCGT GGAGTGGGAG

1921	AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC
	CCGTGTTGGA CTCCGACGGC

1981	TCCTTCTTCC TCTACAGCAA GCTCACCGTG GACAAGAGCA
	GGTGGCAGCA GGGGAACGTC

2041	TTCTCATGCT CCGTGATGCA TGAGGCTCTG CACAACCACT
	ACACGCAGAA GAGCCTCTCC

2101	CTGTCTCCGG GTAAAGGTGG CGGCGGATCA GGTGGGGGTG
	GATCAGGCGG TGGAGGTTCC

2161	GGTGGCGGGG GATCAGACAA AACTCACACA TGCCCACCGT
	GCCCAGCACC TGAACTCCTG

2221	GGAGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG
	ACACCCTCAT GATCTCCCGG

2281	ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG
	AAGACCCTGA GGTCAAGTTC

2341	AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA
	CAAAGCCGCG GGAGGAGCAG

2401	TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC
	TGCACCAGGA CTGGCTGAAT

2461	GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC
	CAGCCCCCAT CGAGAAAACC

2521	ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT
	ACACCCTGCC CCCATCCCGC

2581	GATGAGCTGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG
	TCAAAGGCTT CTATCCCAGC

2641	GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA
	ACAACTACAA GACCACGCCT

2701	CCCGTGTTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA
	AGCTCACCGT GGACAAGAGC

2761	AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC
	ATGAGGCTCT GCACAACCAC

2821	TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAATGA

FVII-118 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, the thrombin cleavage site is shown in dashed underline, the linker region connecting FVII heavy chain to Fc region is underlined, and the linker region connecting the Fc regions is shown in bold

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181
241	KNWRNLIAVL GEHDLSEHDG DEQSRRVAQV IIPSTYVPGT TNHDIALLRL HQPVVLTDHV

301	VPLCLPERTF SERTLAFVRF SLVSGWGQLL DRGATALELM VLNVPRLMTQ DCLQQSRKVG

361	DSPNITEYMF CAGYSDGSKD SCKGDSGGPH ATHYRGTWYL TGIVSWGQGC ATVGHFGVYT

421	RVSQYIEWLQ KLMRSEPRPG VLLRAPFPGG GGSGGGGSGG GGSGGGGSGG GGSGGGGSDK

481	THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV

541	EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ

601	PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG

661	SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKGGGGS GGGGSGGGGS

721	GGGGSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF

781	NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT

841	ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP

901	PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK*

DNA sequence for FVII-119

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCGGA GGAGGTGGTT
	CAGCCCTGCG GCCCCGGATT

601	GTGGGGGGCA AGGTGTGCCC CAAAGGGGAG TGTCCATGGC
	AGGTCCTGTT GTTGGTGAAT

661	GGAGCTCAGT TGTGTGGGGG GACCCTGATC AACACCATCT
	GGGTGGTCTC CGCGGCCCAC

721	TGTTTCGACA AAATCAAGAA CTGGAGGAAC CTGATCGCGG
	TGCTGGGCGA GCACGACCTC

781	AGCGAGCACG ACGGGGATGA GCAGAGCCGG CGGGTGGCGC
	AGGTCATCAT CCCCAGCACG

841	TACGTCCCGG GCACCACCAA CCACGACATC GCGCTGCTCC
	GCCTGCACCA GCCCGTGGTC

901	CTCACTGACC ATGTGGTGCC CCTCTGCCTG CCCGAACGGA
	CGTTCTCTGA GAGGACGCTG

961	GCCTTCGTGC GCTTCTCATT GGTCAGCGGC TGGGGCCAGC
	TGCTGGACCG TGGCGCCACG

1021	GCCCTGGAGC TCATGGTCCT CAACGTGCCC CGGCTGATGA
	CCCAGGACTG CCTGCAGCAG

1081	TCACGGAAGG TGGGAGACTC CCCAAATATC ACGGAGTACA
	TGTTCTGTGC CGGCTACTCG

1141	GATGGCAGCA AGGACTCCTG CAAGGGGGAC AGTGGAGGCC
	CACATGCCAC CCACTACCGG

1201	GGCACGTGGT ACCTGACGGG CATCGTCAGC TGGGGCCAGG
	GCTGCGCAAC CGTGGGCCAC

1261	TTTGGGGTGT ACACCAGGGT CTCCCAGTAC ATCGAGTGGC
	TGCAAAAGCT CATGCGCTCA

1321	GAGCCACGCC CAGGAGTCCT CCTGCGAGCC CCATTTCCCG
	GTGGCGGTGG CTCCGGCGGA

1381	GGTGGGTCCG GTGGCGGCGG ATCAGGTGGG GGTGGATCAG
	GCGGTGGAGG TTCCGGTGGC

1441	GGGGGATCCG ACAAAACTCA CACATGCCCA CCGTGCCCAG
	CTCCGGAACT CCTGGGCGGA

1501	CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC
	TCATGATCTC CCGGACCCCT

1561	GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC
	CTGAGGTCAA GTTCAACTGG

1621	TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC
	CGCGGGAGGA GCAGTACAAC

1681	AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC
	AGGACTGGCT GAATGGCAAG

1741	GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC
	CCATCGAGAA AACCATCTCC

1801	AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC
	TGCCCCCATC CCGGGATGAG

1861	CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG
	GCTTCTATCC CAGCGACATC

1921	GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT
	ACAAGACCAC GCCTCCCGTG

1981	TTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA
	CCGTGGACAA GAGCAGGTGG

2041	CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG
	CTCTGCACAA CCACTACACG

2101	CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA GGTGGCGGCG
	GATCAGGTGG GGGTGGATCA

2161	GGCGGTGGAG GTTCCGGTGG CGGGGGATCA GACAAAACTC
	ACACATGCCC ACCGTGCCCA

2221	GCACCTGAAC TCCTGGGAGG ACCGTCAGTC TTCCTCTTCC
	CCCCAAAACC CAAGGACACC

2281	CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG
	TGGACGTGAG CCACGAAGAC

2341	CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG
	TGCATAATGC CAAGACAAAG

2401	CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA
	GCGTCCTCAC CGTCCTGCAC

2461	CAGGACTGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT
	CCAACAAAGC CCTCCCAGCC

2521	CCCATCGAGA AAACCATCTC CAAAGCCAAA GGGCAGCCCC
	GAGAACCACA GGTGTACACC

2581	CTGCCCCCAT CCCGCGATGA GCTGACCAAG AACCAGGTCA
	GCCTGACCTG CCTGGTCAAA

2641	GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA
	ATGGGCAGCC GGAGAACAAC

2701	TACAAGACCA CGCCTCCCGT GTTGGACTCC GACGGCTCCT
	TCTTCCTCTA CAGCAAGCTC

2761	ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT
	CATGCTCCGT GATGCATGAG

2821	GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT
	CTCCGGGTAA ATGA

FVII-119 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, the thrombin cleavage site with GGGS linker is shown in dashed underline, the linker region connecting FVII heavy chain to Fc region is underlined, and the linker region connecting the Fc regions is shown in bold

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181
241	CFDKIKNWRN LIAVLGEHDL SEHDGDEQSR RVAQVIIPST YVPGTTNHDI ALLRLHQPVV

301	LTDHVVPLCL PERTFSERTL AFVRFSLVSG WGQLLDRGAT ALELMVLNVP RLMTQDCLQQ

361	SRKVGDSPNI TEYMFCAGYS DGSKDSCKGD SGGPHATHYR GTWYLTGIVS WGQGCATVGH

421	FGVYTRVSQY IEWLQKLMRS EPRPGVLLRA PFPGGGGSGG GGSGGGGSGG GGSGGGGSGG

481	GGSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW

541	YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS

601	KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

661	LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK GGGGSGGGGS

721	GGGGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED

781	PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA

841	PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN

901	YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

DNA sequence for FVII-127

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCGCC CTGCGGCCCC
	GGATTGTGGG GGGCAAGGTG

601	TGCCCCAAAG GGGAGTGTCC ATGGCAGGTC CTGTTGTTGG
	TGAATGGAGC TCAGTTGTGT

661	GGGGGGACCC TGATCAACAC CATCTGGGTG GTCTCCGCGG
	CCCACTGTTT CGACAAAATC

721	AAGAACTGGA GGAACCTGAT CGCGGTGCTG GGCGAGCACG
	ACCTCAGCGA GCACGACGGG

781	GATGAGCAGA GCCGGCGGGT GGCGCAGGTC ATCATCCCCA
	GCACGTACGT CCCGGGCACC

841	ACCAACCACG ACATCGCGCT GCTCCGCCTG CACCAGCCCG
	TGGTCCTCAC TGACCATGTG

901	GTGCCCCTCT GCCTGCCCGA ACGGACGTTC TCTGAGAGGA
	CGCTGGCCTT CGTGCGCTTC

961	TCATTGGTCA GCGGCTGGGG CCAGCTGCTG GACCGTGGCG
	CCACGGCCCT GGAGCTCATG

1021	GTCCTCAACG TGCCCCGGCT GATGACCCAG GACTGCGAGG
	CCAGCTACCC CGGCAAGATC

1081	ACGGAGTACA TGTTCTGTGC CGGCTACTCG GATGGCAGCA
	AGGACTCCTG CAAGGGGGAC

1141	AGTGGAGGCC CACATGCCAC CCACTACCGG GGCACGTGGT
	ACCTGACGGG CATCGTCAGC

1201	TGGGGCCAGG GCTGCGCAAC CGTGGGCCAC TTTGGGGTGT
	ACACCAGGGT CTCCCAGTAC

1261	ATCGAGTGGC TGCAAAAGCT CATGCGCTCA GAGCCACGCC
	CAGGAGTCCT CCTGCGAGCC

1321	CCATTTCCCG GTGGCGGTGG CTCCGGCGGA GGTGGGTCCG
	GTGGCGGCGG ATCAGGTGGG

1381	GGTGGATCAG GCGGTGGAGG TTCCGGTGGC GGGGGATCAG
	ACAAAACTCA CACATGCCCA

1441	CCGTGCCCAG CTCCGGAACT CCTGGGCGGA CCGTCAGTCT
	TCCTCTTCCC CCCAAAACCC

1501	AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT
	GCGTGGTGGT GGACGTGAGC

1561	CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG
	GCGTGGAGGT GCATAATGCC

1621	AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTACC
	GTGTGGTCAG CGTCCTCACC

1681	GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT
	GCAAGGTCTC CAACAAAGCC

1741	CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG
	GGCAGCCCCG AGAACCACAG

1801	GTGTACACCC TGCCCCCATC CCGGGATGAG CTGACCAAGA
	ACCAGGTCAG CCTGACCTGC

1861	CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT
	GGGAGAGCAA TGGGCAGCCG

1921	GAGAACAACT ACAAGACCAC GCCTCCCGTG TTGGACTCCG
	ACGGCTCCTT CTTCCTCTAC

1981	AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA
	ACGTCTTCTC ATGCTCCGTG

2041	ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC
	TCTCCCTGTC TCCGGGTAAA

2101	GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG
	GTTCCGGTGG CGGGGGATCA

2161	GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC
	TCCTGGGAGG ACCGTCAGTC

2221	TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT
	CCCGGACCCC TGAGGTCACA

2281	TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA
	AGTTCAACTG GTACGTGGAC

2341	GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
	AGCAGTACAA CAGCACGTAC

2401	CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC
	TGAATGGCAA GGAGTACAAG

2461	TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA
	AAACCATCTC CAAAGCCAAA

2521	GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT
	CCCGCGATGA GCTGACCAAG

2581	AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC
	CCAGCGACAT CGCCGTGGAG

2641	TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA
	CGCCTCCCGT GTTGGACTCC

2701	GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA
	AGAGCAGGTG GCAGCAGGGG

2761	AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
	ACCACTACAC GCAGAAGAGC

2821	CTCTCCCTGT CTCCGGGTAA ATGA

FVII-127 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, the thrombin cleavage site is shown in dashed underline, the trypsin 170 loop region is wave underlined, the linker region connecting FVII heavy chain to Fc region is underlined, and the linker region connecting the Fc regions is shown in bold

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181
241	KNWRNLIAVL GEHDLSEHDG DEQSRRVAQV IIPSTYVPGT TNHDIALLRL HQPVVLTDHV

301
361	TEYMFCAGYS DGSKDSCKGD SGGPHATHYR GTWYLTGIVS WGQGCATVGH FGVYTRVSQY

421	IEWLQKLMRS EPRPGVLLRA PFPGGGGSGG GGSGGGGSGG GGSGGGGSGG GGSDKTHTCP

481	PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA

541	KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

601	VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

661	SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK GGGGSGGGGS GGGGSGGGGS

721	DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

781	GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

841	GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

901	DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

DNA sequence for FVII-125

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA ATTGTGGGGG GCAAGGTGTG CCCCAAAGGG

601	GAGTGTCCAT GGCAGGTCCT GTTGTTGGTG AATGGAGCTC AGTTGTGTGG GGGGACCCTG

661	ATCAACACCA TCTGGGTGGT CTCCGCGGCC CACTGTTTCG ACAAAATCAA GAACTGGAGG

721	AACCTGATCG CGGTGCTGGG CGAGCACGAC CTCAGCGAGC ACGACGGGGA TGAGCAGAGC

781	CGGCGGGTGG CGCAGGTCAT CATCCCCAGC ACGTACGTCC CGGGCACCAC CAACCACGAC

841	ATCGCGCTGC TCCGCCTGCA CCAGCCCGTG GTCCTCACTG ACCATGTGGT GCCCCTCTGC

901	CTGCCCGAAC GGACGTTCTC TGAGAGGACG CTGGCCTTCG TGCGCTTCTC ATTGGTCAGC

961	GGCTGGGGCC AGCTGCTGGA CCGTGGCGCC ACGGCCCTGG AGCTCATGGT CCTCAACGTG

1021	CCCCGGCTGA TGACCCAGGA CTGCCTGCAG CAGTCACGGA AGGTGGGAGA CTCCCCAAAT

1081	ATCACGGAGT ACATGTTCTG TGCCGGCTAC TCGGATGGCA GCAAGGACTC CTGCAAGGGG

1141	GACAGTGGAG GCCCACATGC CACCCACTAC CGGGGCACGT GGTACCTGAC GGGCATCGTC

1201	AGCTGGGGCC AGGGCTGCGC AACCGTGGGC CACTTTGGGG TGTACACCAG GGTCTCCCAG

1261	TACATCGAGT GGCTGCAAAA GCTCATGCGC TCAGAGCCAC GCCCAGGAGT CCTCCTGCGA

1321	GCCCCATTTC CCGGTGGCGG TGGCTCCGGC GGAGGTGGGT CCGGTGGCGG CGGATCAGGT

1381	GGGGGTGGAT CAGGCGGTGG AGGTTCCGGT GGCGGGGGAT CCGACATCGT GATGACCCAG

1441	GCCGCCCCCA GCGTGCCCGT GACCCCCGGC GAGAGCGTGA GCATCAGCTG CCGGAGCAGC

1501	CGGAGCCTGC TGCACAGCAA CGGCAACACC TACCTGTGCT GGTTCCTGCA GCGGCCCGGC

1561	CAGAGCCCCC AGCTGCTGAT CTACCGGATG AGCAACCTGG CCAGCGGCGT GCCCGACCGG

1621	TTCAGCGGCA GCGGCAGCGG CACCGCCTTC ACCCTGCGGA TCAGCCGGGT GGAGGCCGAG

1681	GACGTGGGCG TGTACTACTG CATGCAGCAC CTGGAGTACC CCTTCACCTT CGGCAGCGGC

1741	ACCAAGCTGG AGATCAAGCG GGGCGGCGGC GGCAGCGGCG GCGGCGGCAG CGGCGGCGGC

1801	GGCAGCCAGG TGCAGCTGCA GCAGAGCGGC GCCGAGCTGG TGCGGCCCGG CACCAGCGTG

1861	AAGATCAGCT GCAAGGCCAG CGGCTACACC TTCACCAACT ACTGGCTGGG CTGGGTGAAG

1921	CAGCGGCCCG GCCACGGCCT GGAGTGGATC GGCGACATCT ACCCCGGCGG CGGCTACAAC

1981	AAGTACAACG AGAACTTCAA GGGCAAGGCC ACCCTGACCG CCGACACCAG CAGCAGCACC

2041	GCCTACATGC AGCTGAGCAG CCTGACCAGC GAGGACAGCG CCGTGTACTT CTGCGCCCGG

2101	GAGTACGGCA ACTACGACTA CGCCATGGAC AGCTGGGGCC AGGGCACCAG CGTGACCGTG

2161	AGCAGCTGA

FVII-125 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, and linker region connecting FVII to AP3 is bold, and AP3 scFv is italicized

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTGTVE YPCGKIPILE

181	KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

241	NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

301	LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

361	ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

421	YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGS DIVMTQ

461	AAPSVPVTPG ESVSISCRSS RSLLHSNGNT YLCWFLQRPG QSPQLLIYRM SNLASGVPDR

541	FSGSGSGTAF TLRISRVEAE DVGVYYCMQH LEYPFTFGSG TKLEIKR GGG GSGGGGSGGG

601	GSQVQLQQSG AELVRPGTSV KISCKASGYT FTNYWLGWVK QRPGHGLEWI GDIYPGGGYN

661	KYNENFKGKA TLTADTSSST AYMQLSSLTS EDSAVYFCAR EYGNYDYAMD SWGQGTSVTV

721	SS*

DNA sequence for FVII-067

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA ATTGTGGGGG
	GCAAGGTGTG CCCCAAAGGG

601	GAGTGTCCAT GGCAGGTCCT GTTGTTGGTG AATGGAGCTC
	AGTTGTGTGG GGGGACCCTG

661	ATCAACACCA TCTGGGTGGT CTCCGCGGCC CACTGTTTCG
	ACAAAATCAA GAACTGGAGG

721	AACCTGATCG CGGTGCTGGG CGAGCACGAC CTCAGCGAGC
	ACGACGGGGA TGAGCAGAGC

781	CGGCGGGTGG CGCAGGTCAT CATCCCCAGC ACGTACGTCC
	CGGGCACCAC CAACCACGAC

841	ATCGCGCTGC TCCGCCTGCA CCAGCCCGTG GTCCTCACTG
	ACCATGTGGT GCCCCTCTGC

901	CTGCCCGAAC GGACGTTCTC TGAGAGGACG CTGGCCTTCG
	TGCGCTTCTC ATTGGTCAGC

961	GGCTGGGGCC AGCTGCTGGA CCGTGGCGCC ACGGCCCTGG
	AGCTCATGGT CCTCAACGTG

1021	CCCCGGCTGA TGACCCAGGA CTGCCTGCAG CAGTCACGGA
	AGGTGGGAGA CTCCCCAAAT

1081	ATCACGGAGT ACATGTTCTG TGCCGGCTAC TCGGATGGCA
	GCAAGGACTC CTGCAAGGGG

1141	GACAGTGGAG GCCCACATGC CACCCACTAC CGGGGCACGT
	GGTACCTGAC GGGCATCGTC

1201	AGCTGGGGCC AGGGCTGCGC AACCGTGGGC CACTTTGGGG
	TGTACACCAG GGTCTCCCAG

1261	TACATCGAGT GGCTGCAAAA GCTCATGCGC TCAGAGCCAC
	GCCCAGGAGT CCTCCTGCGA

1321	GCCCCATTTC CCGGTGGCGG TGGCTCCGGC GGAGGTGGGT
	CCGGTGGCGG CGGATCAGGT

1381	GGGGGTGGAT CAGGCGGTGG AGGTTCCGGT GGCGGGGGAT
	CCGACAAAAC TCACACATGC

1441	CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG
	TCTTCCTCTT CCCCCCAAAA

1501	CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA
	CATGCGTGGT GGTGGACGTG

1561	AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG
	ACGGCGTGGA GGTGCATAAT

1621	GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT
	ACCGTGTGGT CAGCGTCCTC

1681	ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA
	AGTGCAAGGT CTCCAACAAA

1741	GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA
	AAGGGCAGCC CCGAGAACCA

1801	CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA
	AGAACCAGGT CAGCCTGACC

1861	TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG
	AGTGGGAGAG CAATGGGCAG

1921	CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT
	CCGACGGCTC CTTCTTCCTC

1981	TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG
	GGAACGTCTT CTCATGCTCC

2041	GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA
	GCCTCTCCCT GTCTCCGGGT

2101	AAAGGTGGCG GCGGATCAGG TGGGGGTGGA TCAGGCGGTG
	GAGGTTCCGG TGGCGGGGGA

2161	TCAGACAAAA CTCACACATG CCCACCGTGC CCAGCACCTG
	AACTCCTGGG AGGACCGTCA

2221	GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA
	TCTCCCGGAC CCCTGAGGTC

2281	ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG
	TCAAGTTCAA CTGGTACGTG

2341	GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
	AGGAGCAGTA CAACAGCACG

2401	TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
	GGCTGAATGG CAAGGAGTAC

2461	AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG
	AGAAAACCAT CTCCAAAGCC

2521	AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC
	CATCCCGCGA TGAGCTGACC

2581	AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT
	ATCCCAGCGA CATCGCCGTG

2641	GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA
	CCACGCCTCC CGTGTTGGAC

2701	TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTCG
	ACAAGAGCAG GTGGCAGCAG

2761	GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC
	ACAACCACTA CACGCAGAAG

2821	AGCCTCTCCC TGTCTCCGGG TAAAGGTGGC GGTGGCTCCG
	GCGGAGGTGG GTCCGGTGGC

2881	GGCGGATCAG GTGGGGGTGG ATCAGGCGGT GGAGGTTCCG
	GTGGCGGGGG ATCAGCGCAG

2941	GTGCAGCTGC AGGAGTCTGG GGGAGGCTTG GTACAGCCTG
	GGGGGTCCCT GAGACTCTCC

3001	TGTGCAGCCT CTGGATTCAT GTTTAGCAGG TATGCCATGA
	GCTGGGTCCG CCAGGCTCCA

3061	GGGAAGGGGC CAGAGTGGGT CTCAGGTATT AGTGGTAGTG
	GTGGTAGTAC ATACTACGCA

3121	GACTCCGTGA AGGGCCGGTT CACCGTCTCC AGAGACAATT
	CCAAGAACAC GCTGTATCTG

3181	CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTATATT
	ACTGCGCCCG GGGCGCCACC

3241	TACACCAGCC GGAGCGACGT GCCCGACCAG ACCAGCTTCG
	ACTACTGGGG CCAGGGAACC

3301	CTGGTCACCG TCTCCTCAGG GAGTGCATCC GCCCCAAAGC
	TTGAAGAAGG TGAATTTTCA

3361	GAAGCACGCG TATCTGAACT GACTCAGGAC CCTGCTGTGT
	CTGTGGCCTT GGGACAGACA

3421	GTCAGGATCA CATGCCAAGG AGACAGCCTC AGAAACTTTT
	ATGCAAGCTG GTACCAGCAG

3481	AAGCCAGGAC AGGCCCCTAC TCTTGTCATC TATGGTTTAA
	GTAAAAGGCC CTCAGGGATC

3541	CCAGACCGAT TCTCTGCCTC CAGCTCAGGA AACACAGCTT
	CCTTGACCAT CACTGGGGCT

3601	CAGGCGGAAG ATGAGGCTGA CTATTACTGC CTGCTGTACT
	ACGGCGGCGG CCAGCAGGGC

3661	GTGTTCGGCG GCGGCACCAA GCTGACCGTC CTACGTCAGC
	CCAAGGCTGC CCCCTCGGTC

3721	ACTCTGTTCC CGCCCTCTTC TGCGGCCTGA

FVII-067 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVIIa to Fc region is underlined, linker connecting both Fc regions is dashed underlined and linker connecting the Fc region to SCE5 is in bold


61 SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE
121 THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE
151 KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR
241 NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC
301 LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN
361 ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ
421 YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC
481 PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN
541 AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP
601 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL

781 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA
841 KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
901 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG
961 GGSGGGGSGG GGSGGGGSAQ VQLQESGGGL VQPGGSLRLS CAASGFMFSR YAMSWVRQAP
1021 GKGPEWVSGI SGSGGSTYYA DSVKGRFTVS RDNSKNTLYL QMNSLRAEDT AVYYCARGAT
1081 YTSRSDVPDQ TSFDYWGQGT LVTVSSGSAS APKLEEGEFS EARVSELTQD PAVSVALGQT
1141 VRITCQGDSL RNFYASWYQQ KPGQAPTLVI YGLSKRPSGI PDRFSASSSG NTASLTITGA
1201 QAEDEADYYC LLYYGGGQQG VFGGGTKLTV LRQPKAAPSV TLFPPSSAA*

DNA sequence for FVII-094

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT


61	GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC
	ACCGGCGCCG GCGCGCCAAC

121	GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG
	AGTGCAAGGA GGAGCAGTGC

181	TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA
	GGACGAAGCT GTTCTGGATT

241	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

301	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

361	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

421	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

481	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

541	AAAAGAAATG CCAGCAAACC CCAAGGCCGA ATTGTGGGGG
	GCAAGGTGTG CCCCAAAGGG

601	GAGTGTCCAT GGCAGGTCCT GTTGTTGGTG AATGGAGCTC
	AGTTGTGTGG GGGGACCCTG

661	ATCAACACCA TCTGGGTGGT CTCCGCGGCC CACTGTTTCG
	ACAAAATCAA GAACTGGAGG

721	AACCTGATCG CGGTGCTGGG CGAGCACGAC CTCAGCGAGC
	ACGACGGGGA TGAGCAGAGC

781	CGGCGGGTGG CGCAGGTCAT CATCCCCAGC ACGTACGTCC
	CGGGCACCAC CAACCACGAC

841	ATCGCGCTGC TCCGCCTGCA CCAGCCCGTG GTCCTCACTG
	ACCATGTGGT GCCCCTCTGC

901	CTGCCCGAAC GGACGTTCTC TGAGAGGACG CTGGCCTTCG
	TGCGCTTCTC ATTGGTCAGC

961	GGCTGGGGCC AGCTGCTGGA CCGTGGCGCC ACGGCCCTGG
	AGCTCATGGT CCTCAACGTG

1021	CCCCGGCTGA TGACCCAGGA CTGCCTGCAG CAGTCACGGA
	AGGTGGGAGA CTCCCCAAAT

1081	ATCACGGAGT ACATGTTCTG TGCCGGCTAC TCGGATGGCA
	GCAAGGACTC CTGCAAGGGG

1141	GACAGTGGAG GCCCACATGC CACCCACTAC CGGGGCACGT
	GGTACCTGAC GGGCATCGTC

1201	AGCTGGGGCC AGGGCTGCGC AACCGTGGGC CACTTTGGGG
	TGTACACCAG GGTCTCCCAG

1261	TACATCGAGT GGCTGCAAAA GCTCATGCGC TCAGAGCCAC
	GCCCAGGAGT CCTCCTGCGA

1321	GCCCCATTTC CCGATATCGG TGGCGGTGGC TCCGGCGGAG
	GTGGGTCCGG TGGCGGCGGA

1381	TCAGGTGGGG GTGGATCAGG CGGTGGAGGT TCCGGTGGCG
	GGGGATCAGC GCAGGTGCAG

1441	CTGCAGGAGT CTGGGGGAGG CTTGGTACAG CCTGGGGGGT
	CCCTGAGACT CTCCTGTGCA

1501	GCCTCTGGAT TCATGTTTAG CAGGTATGCC ATGAGCTGGG
	TCCGCCAGGC TCCAGGGAAG

1561	GGGCCAGAGT GGGTCTCAGG TATTAGTGGT AGTGGTGGTA
	GTACATACTA CGCAGACTCC

1621	GTGAAGGGCC GGTTCACCGT CTCCAGAGAC AATTCCAAGA
	ACACGCTGTA TCTGCAAATG

1681	AACAGCCTGA GAGCCGAGGA CACGGCTGTA TATTACTGCG
	CCCGGGGCGC CACCTACACC

1741	AGCCGGAGCG ACGTGCCCGA CCAGACCAGC TTCGACTACT
	GGGGCCAGGG AACCCTGGTC

1801	ACCGTCTCCT CAGGGAGTGC ATCCGCCCCA AAGCTTGAAG
	AAGGTGAATT TTCAGAAGCA

1861	CGCGTATCTG AACTGACTCA GGACCCTGCT GTGTCTGTGG
	CCTTGGGACA GACAGTCAGG

1921	ATCACATGCC AAGGAGACAG CCTCAGAAAC TTTTATGCAA
	GCTGGTACCA GCAGAAGCCA

1981	GGACAGGCCC CTACTCTTGT CATCTATGGT TTAAGTAAAA
	GGCCCTCAGG GATCCCAGAC

2041	CGATTCTCTG CCTCCAGCTC AGGAAACACA GCTTCCTTGA
	CCATCACTGG GGCTCAGGCG

2101	GAAGATGAGG CTGACTATTA CTGCCTGCTG TACTACGGCG
	GCGGCCAGCA GGGCGTGTTC

2161	GGCGGCGGCA CCAAGCTGAC CGTCCTACGT CAGCCCAAGG
	CTGCCCCCTC GGTCACTCTG

2221	TTCCCGCCCT CTTCTGCGGC CTGA

FVII-094 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, and linker region connecting FVII to SCE5 is underlined

1
61	SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE

121	THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

181	KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR

241	NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC

301	LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN

351	ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ

421	YIEWLQKLMR SEPRPGVLLR APFPDIGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSAQVQ

461	LQESGGGLVQ PGGSLRLSCA ASGFMFSRYA MSWVRQAPGK GPEWVSGISG SGGSTYYADS

541	VKGRFTVSRD NSKNTLYLQM NSLRAEDTAV YYCARGATYT SRSDVPDQTS FDYWGQGTLV

601	TVSSGSASAP KLEEGEFSEA RVSELTQDPA VSVALGQTVR ITCQGDSLRN FYASWYQQKP

661	GQAPTLVIYG LSKRPSGIPD RFSASSSGNT ASLTITGAQA EDEADYYCLL YYGGGQQGVF

721	GGGTKLTVLR QPKAAPSVTL FPPSSAA*

DNA sequence for FVII-028

1	ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG
	GGCTTCAGGG CTGCCTGGCT

61	GCGGAAGTGC AGCTGGTGCA GTCTGGAGCT GAGGTGAATA
	AGCCTGGGGC CTCAGTGAAG

121	GTCTCCTGCA AGGCTTCTGG ATACACCTTC ACCGGCTACT
	ATATGCACTG GGTGCGACAG

181	GCCCCTGGAC AAGGGCTTGA GTGGATGGGA TGGATCAACC
	CTAACAGTGG TGGCACAAAC

241	TATGCACAGA AGTTTCAGGG CTGGGTCACC ATGACCAGGG
	ACACGTCCAT CAGCACCGCC

301	TACATGGAGC TGAGCAGGCT GAGATCTGAC GACACGGCCG
	TGTATTACTG TGCGAGAGGC

361	CGTGCTTTGT ATAACCGGAA CGACCGGTCC CCCAACTGGT
	TCGACCCCTG GGGCCAGGGA

421	ACCCTGGTCA CCGTCTCCTC AGGGAGTGCA TCCGCCCCAA
	CCCTTAAACT TGAAGAAGGT

481	GAATTTTCAG AAGCACGCGT ACAGGCTGTG CTGACTCAGC
	CGCCCTCGGT GTCAGTGGCC

541	CCAGGACAGA CGGCCAGGAT TACCTGTGGG GGAAACAACA
	TTGGAAGTAA AAGTGTGCAG

601	TGGTACCAGC AGAAGCCAGG CCAGGCCCCT GTGCTGGTCG
	TCTATGATGA TAGCGACCGG

661	CCCTCAGGGA TCCCTGAGCG ATTCTCTGGC TCCAACTCTG
	GGAACATGGC CACCCTGACC

721	ATCAGCAGGG TCGAAGCCGG GGATGAGGCC GACTATTACT
	GTCAGGTGTG GGATAGTAGT

781	AGTGATCATG TGGTATTCGG CGGAGGGACC AAGCTGACCG
	TCCTAGGTCA GCCCAAGGCT

841	GCCCCCTCGG TCACTCTGTT CCCGCCGTCC GCGGCCGCTA
	GGACGAAGCT GTTCTGGATT

901	TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC
	AGAATGGGGG CTCCTGCAAG

961	GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT
	TCGAGGGCCG GAACTGTGAG

1021	ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG
	GCGGCTGTGA GCAGTACTGC

1081	AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG
	AGGGGTACTC TCTGCTGGCA

1141	GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG
	GAAAAATACC TATTCTAGAA

1201	AAAAGAAATG CCAGCAAACC CCAAGGCCGA ATTGTGGGGG
	GCAAGGTGTG CCCCAAAGGG

1261	GAGTGTCCAT GGCAGGTCCT GTTGTTGGTG AATGGAGCTC
	AGTTGTGTGG GGGGACCCTG

1321	ATCAACACCA TCTGGGTGGT CTCCGCGGCC CACTGTTTCG
	ACAAAATCAA GAACTGGAGG

1381	AACCTGATCG CGGTGCTGGG CGAGCACGAC CTCAGCGAGC
	ACGACGGGGA TGAGCAGAGC

1441	CGGCGGGTGG CGCAGGTCAT CATCCCCAGC ACGTACGTCC
	CGGGCACCAC CAACCACGAC

1501	ATCGCGCTGC TCCGCCTGCA CCAGCCCGTG GTCCTCACTG
	ACCATGTGGT GCCCCTCTGC

1561	CTGCCCGAAC GGACGTTCTC TGAGAGGACG CTGGCCTTCG
	TGCGCTTCTC ATTGGTCAGC

1621	GGCTGGGGCC AGCTGCTGGA CCGTGGCGCC ACGGCCCTGG
	AGCTCATGGT CCTCAACGTG

1681	CCCCGGCTGA TGACCCAGGA CTGCCTGCAG CAGTCACGGA
	AGGTGGGAGA CTCCCCAAAT

1741	ATCACGGAGT ACATGTTCTG TGCCGGCTAC TCGGATGGCA
	GCAAGGACTC CTGCAAGGGG

1801	GACAGTGGAG GCCCACATGC CACCCACTAC CGGGGCACGT
	GGTACCTGAC GGGCATCGTC

1861	AGCTGGGGCC AGGGCTGCGC AACCGTGGGC CACTTTGGGG
	TGTACACCAG GGTCTCCCAG

1921	TACATCGAGT GGCTGCAAAA GCTCATGCGC TCAGAGCCAC
	GCCCAGGAGT CCTCCTGCGA

1981	GCCCCATTTC CCGGTGGCGG TGGCTCCGGC GGAGGTGGGT
	CCGGTGGCGG CGGATCAGGT

2041	GGGGGTGGAT CAGGCGGTGG AGGTTCCGGT GGCGGGGGAT
	CCGACAAAAC TCACACATGC

2101	CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG
	TCTTCCTCTT CCCCCCAAAA

2161	CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA
	CATGCGTGGT GGTGGACGTG

2221	AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG
	ACGGCGTGGA GGTGCATAAT

2281	GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT
	ACCGTGTGGT CAGCGTCCTC

2341	ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA
	AGTGCAAGGT CTCCAACAAA

2401	GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA
	AAGGGCAGCC CCGAGAACCA

2461	CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA
	AGAACCAGGT CAGCCTGACC

2521	TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG
	AGTGGGAGAG CAATGGGCAG

2581	CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT
	CCGACGGCTC CTTCTTCCTC

2641	TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG
	GGAACGTCTT CTCATGCTCC

2701	GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA
	GCCTCTCCCT GTCTCCGGGT

2761	AAAGGTGGCG GCGGATCAGG TGGGGGTGGA TCAGGCGGTG
	GAGGTTCCGG TGGCGGGGGA

2821	TCCGACAAAA CTCACACATG CCCACCGTGC CCAGCACCTG
	AACTCCTGGG AGGACCGTCA

2881	GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA
	TCTCCCGGAC CCCTGAGGTC

2941	ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG
	TCAAGTTCAA CTGGTACGTG

3001	GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG
	AGGAGCAGTA CAACAGCACG

3061	TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT
	GGCTGAATGG CAAGGAGTAC

3121	AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG
	AGAAAACCAT CTCCAAAGCC

3181	AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC
	CATCCCGCGA TGAGCTGACC

3241	AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT
	ATCCCAGCGA CATCGCCGTG

3301	GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA
	CCACGCCTCC CGTGTTGGAC

3361	TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG
	ACAAGAGCAG GTGGCAGCAG

3421	GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC
	ACAACCACTA CACGCAGAAG

3481	AGCCTCTCCC TGTCTCCGGG TAAATGA

FVII-028 amino acid sequence. Signal sequence is shown in dotted underline, linker region connecting FVII to Fc region is underlined, linker connecting both Fcs sites is shown in bold, and MB9 is italicized

1
61	APGQGLEWMG WINPNSGGTN YAQKFQGWVT MTRDTSISTA YMELSRLRSD DTAVYYCARG

121	RALYNRNDRS PNWFDPWGQG TLVTVSSGSA SAPTLKLEEG EFSEARVQAV LTQPPSVSVA

181	PGQTARITCG GNNIGSRSVQ WYQQKPGQAP VLVVYDDSDR PSGIPERFSG SNSGNMATLT

241	ISRVEAGDEA DYYCQVWDSS SDHVVFGGGT KLTVLGQPRA APSVTLFPPS AAARTKLFWI

301	SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE THKDDQLICV NENGGCEQYC

351	SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE KRNASKPQGR IVGGKVCPKG

421	ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR NLIAVLGEHD LSEHDGDEQS

481	RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC LPERTFSERT LAFVRFSLVS

541	GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN ITEYMFCAGY SDGSKDSCKG

601	DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ YIEWLQKLMR SEPRPGVLLR

661	APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC PPCPAPELLG GPSVFLFPPK

721	PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL

781	TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT

841	CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS

901	VMHEALHNHY TQKSLSLSPG KGGGGSGGGG SGGGGSGGGG SDKTHTCPPC PAPELLGGPS

961	VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST

1021	YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT

1081	KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

1141	GNVFSCSVMH EALHNHYTQK SLSLSPGK*

DNA sequence FVII-039
1 ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG GGCTTCAGGG CTGCCTGGCT

61 GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC ACCGGCGCCG GCGCGCCAAC

121 GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG AGTGCAAGGA GGAGCAGTGC

181 TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA GGACGAAGCT GTTCTGGATT

241 TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC AGAATGGGGG CTCCTGCAAG

301 GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT TCGAGGGCCG GAACTGTGAG

361 ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG GCGGCTGTGA GCAGTACTGC

421 AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG AGGGGTACTC TCTGCTGGCA

481 GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG GAAAAATACC TATTCTAGAA

541 AAAAGAAATG CCAGCAAACC CCAAGGCCGA GGCGGAGGAG ACTTCACTCG GGTTGTGGGG

601 GGCAAGGTGT GCCCCAAAGG GGAGTGTCCA TGGCAGGTCC TGTTGTTGGT GAATGGAGCT

661 CAGTTGTGTG GGGGGACCCT GATCAACACC ATCTGGGTGG TCTCCGCGGC CCACTGTTTC

721 GACAAAATCA AGAACTGGAG GAACCTGATC GCGGTGCTGG GCGAGCACGA CCTCAGCGAG

781 CACGACGGGG ATGAGCAGAG CCGGCGGGTG GCGCAGGTCA TCATCCCCAG CACGTACGTC

841 CCGGGCACCA CCAACCACGA CATCGCGCTG CTCCGCCTGC ACCAGCCCGT GGTCCTCACT

901 GACCATGTGG TGCCCCTCTG CCTGCCCGAA CGGACGTTCT CTGAGAGGAC GCTGGCCTTC

961 GTGCGCTTCT CATTGGTCAG CGGCTGGGGC CAGCTGCTGG ACCGTGGCGC CACGGCCCTG

1021 GAGCTCATGG TCCTCAACGT GCCCCGGCTG ATGACCCAGG ACTGCCTGCA GCAGTCACGG

1081 AAGGTGGGAG ACTCCCCAAA TATCACGGAG TACATGTTCT GTGCCGGCTA CTCGGATGGC

1141 AGCAAGGACT CCTGCAAGGG GGACAGTGGA GGCCCACATG CCACCCACTA CCGGGGCACG

1201 TGGTACCTGA CGGGCATCGT CAGCTGGGGC CAGGGCTGCG CAACCGTGGG CCACTTTGGG

1261 GTGTACACCA GGGTCTCCCA GTACATCGAG TGGCTGCAAA AGCTCATGCG CTCAGAGCCA

1321 CGCCCAGGAG TCCTCCTGCG AGCCCCATTT CCCGGTGGCG GTGGCTCCGG CGGAGGTGGG

1381 TCCGGTGGCG GCGGATCAGG TGGGGGTGGA TCAGGCGGTG GAGGTTCCGG TGGCGGGGGA

1441 TCCGACAAAA CTCACACATG CCCACCGTGC CCAGCTCCGG AACTCCTGGG CGGACCGTCA

1501 GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC

1561 ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG

1621 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA CAACAGCACG

1681 TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGG CAAGGAGTAC

1741 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAGCC

1801 AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC CATCCCGGGA TGAGCTGACC

1861 AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG

1921 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC CGTGTTGGAC

1981 TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG ACAAGAGCAG GTGGCAGCAG

2041 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG

2101 AGCCTCTCCC TGTCTCCGGG TAAAGGTGGC GGCGGATCAG GTGGGGGTGG ATCAGGCGGT

2161 GGAGGTTCCG GTGGCGGGGG ATCAGACAAA ACTCACACAT GCCCACCGTG CCCAGCACCT

2221 GAACTCCTGG GAGGACCGTC AGTCTTCCTC TTCCCCCCAA AACCCAAGGA CACCCTCATG

2281 ATCTCCCGGA CCCCTGAGGT CACATGCGTG GTGGTGGACG TGAGCCACGA AGACCCTGAG

2341 GTCAAGTTCA ACTGGTACGT GGACGGCGTG GAGGTGCATA ATGCCAAGAC AAAGCCGCGG

2401 GAGGAGCAGT ACAACAGCAC GTACCGTGTG GTCAGCGTCC TCACCGTCCT GCACCAGGAC

2461 TGGCTGAATG GCAAGGAGTA CAAGTGCAAG GTCTCCAACA AAGCCCTCCC AGCCCCCATC

2521 GAGAAAACCA TCTCCAAAGC CAAAGGGCAG CCCCGAGAAC CACAGGTGTA CACCCTGCCC

2581 CCATCCCGCG ATGAGCTGAC CAAGAACCAG GTCAGCCTGA CCTGCCTGGT CAAAGGCTTC

2641 TATCCCAGCG ACATCGCCGT GGAGTGGGAG AGCAATGGGC AGCCGGAGAA CAACTACAAG

2701 ACCACGCCTC CCGTGTTGGA CTCCGACGGC TCCTTCTTCC TCTACAGCAA GCTCACCGTG

2761 GACAAGAGCA GGTGGCAGCA GGGGAACGTC TTCTCATGCT CCGTGATGCA TGAGGCTCTG

2821 CACAACCACT ACACGCAGAA GAGCCTCTCC CTGTCTCCGG GTAAATGA

FVII-039 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, the FXIa cleavage site is shown in dashed underline, the linker region connecting FVII heavy chain to Fc region is underlined, and the linker region connecting the Fc regions is shown in bold


61 SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE
121 THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

241 DKIKNWRNLI AVLGEHDLSE HDGDEQSRRV AQVIIPSTYV PGTTNHDIAL LRLHQPVVLT
301 DHVVPLCLPE RTFSERTLAF VRFSLVSGWG QLLDRGATAL ELMVLNVPRL MTQDCLQQSR
361 KVGDSPNITE YMFCAGYSDG SKDSCKGDSG GPHATHYRGT WYLTGIVSWG QGCATVGHFG
421 VYTRVSQYIE WLQKLMRSEP RPGVLLRAPF PGGGGSGGGG SGGGGSGGGG SGGGGSGGGG
481 SDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV
541 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA
601 KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
661 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG
721 GGSGGGGSDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
781 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI
841 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK
901 TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK*

DNA sequence for FVII-040
1 ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG GGCTTCAGGG CTGCCTGGCT

61 GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC ACCGGCGCCG GCGCGCCAAC

121 GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG AGTGCAAGGA GGAGCAGTGC

181 TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA GGACGAAGCT GTTCTGGATT

241 TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC AGAATGGGGG CTCCTGCAAG

301 GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT TCGAGGGCCG GAACTGTGAG

361 ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG GCGGCTGTGA GCAGTACTGC

421 AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG AGGGGTACTC TCTGCTGGCA

461 GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG GAAAAATACC TATTCTAGAA

541 AAAAGAAATG CCAGCAAACC CCAAGGCGGC GGAGGAGACT TCACTCGGGT TGTGGGGGGC

601 AAGGTGTGCC CCAAAGGGGA GTGTCCATGG CAGGTCCTGT TGTTGGTGAA TGGAGCTCAG

661 TTGTGTGGGG GGACCCTGAT CAACACCATC TGGGTGGTCT CCGCGGCCCA CTGTTTCGAC

721 AAAATCAAGA ACTGGAGGAA CCTGATCGCG GTGCTGGGCG AGCACGACCT CAGCGAGCAC

781 GACGGGGATG AGCAGAGCCG GCGGGTGGCG CAGGTCATCA TCCCCAGCAC GTACGTCCCG

841 GGCACCACCA ACCACGACAT CGCGCTGCTC CGCCTGCACC AGCCCGTGGT CCTCACTGAC

901 CATGTGGTGC CCCTCTGCCT GCCCGAACGG ACGTTCTCTG AGAGGACGCT GGCCTTCGTG

961 CGCTTCTCAT TGGTCAGCGG CTGGGGCCAG CTGCTGGACC GTGGCGCCAC GGCCCTGGAG

1021 CTCATGGTCC TCAACGTGCC CCGGCTGATG ACCCAGGACT GCCTGCAGCA GTCACGGAAG

1061 GTGGGAGACT CCCCAAATAT CACGGAGTAC ATGTTCTGTG CCGGCTACTC GGATGGCAGC

1141 AAGGACTCCT GCAAGGGGGA CAGTGGAGGC CCACATGCCA CCCACTACCG GGGCACGTGG

1201 TACCTGACGG GCATCGTCAG CTGGGGCCAG GGCTGCGCAA CCGTGGGCCA CTTTGGGGTG

1261 TACACCAGGG TCTCCCAGTA CATCGAGTGG CTGCAAAAGC TCATGCGCTC AGAGCCACGC

1321 CCAGGAGTCC TCCTGCGAGC CCCATTTCCC GGTGGCGGTG GCTCCGGCGG AGGTGGGTCC

1381 GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG GTTCCGGTGG CGGGGGATCC

1441 GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCGGAAC TCCTGGGCGG ACCGTCAGTC

1501 TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA

1561 TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTACGTGGAC

1621 GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC

1681 CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG

1741 TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA

1801 GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG

1861 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG

1921 TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT GTTGGACTCC

1981 GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG

2041 AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC

2101 CTCTCCCTGT CTCCGGGTAA AGGTGGCGGC GGATCAGGTG GGGGTGGATC AGGCGGTGGA

2161 GGTTCCGGTG GCGGGGGATC AGACAAAACT CACACATGCC CACCGTGCCC AGCACCTGAA

2221 CTCCTGGGAG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC

2281 TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC

2341 AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG

2401 GAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG

2461 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG

2521 AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCA

2581 TCCCGCGATG AGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAA AGGCTTCTAT

2641 CCCAGCGACA TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC

2701 ACGCCTCCCG TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC

2761 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC

2821 AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AATGA

FVII-040 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, the FXIa cleavage site is shown in dashed underline, the linker region connecting FVII heavy chain to Fc region is underlined, and the linker region connecting the Fc regions is shown in bold


61 SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE
121 THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE

241 KIKNWRNLIA VLGEHDLSEH DGDEQSRRVA QVIIPSTYVP GTTNHDIALL RLHQPVVLTD
301 HVVPLCLPER TFSERTLAFV RFSLVSGWGQ LLDRGATALE LMVLNVPRLM TQDCLQQSRK
361 VGDSPNITEY MFCAGYSDGS KDSCKGDSGG PHATHYRGTW YLTGIVSWGQ GCATVGHFGV
421 YTRVSQYIEW LQKLMRSEPR PGVLLRAPFP GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS
461 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
541 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
601 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
661 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG
721 GSGGGGSDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV
781 KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE
841 KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT
901 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

DNA sequence for FIX-042
1 ATGCAGCGCG TGAACATGAT CATGGCAGAA TCACCAGGCC TCATCACCAT CTGCCTTTTA

61 GGATATCTAC TCAGTGCTGA ATGTACAGGT TTGTTTCCTT TTTTAAAATA CATTGAGTAT

121 GCTTGCCTTT TAGATATAGA AATATCTGAT GCTGTCTTCT TCACTAAATT TTGATTACAT

181 GATTTGACAG CAATATTGAA GAGTCTAACA GCCAGCACGC AGGTTGGTAA GTACTGTGGG

241 AACATCACAG ATTTTGGCTC CATGCCCTAA AGAGAAATTG GCTTTCAGAT TATTTGGATT

301 AAAAACAAAG ACTTTCTTAA GAGATGTAAA ATTTTCATGA TGTTTTCTTT TTTGCTAAAA

361 CTAAAGAATT ATTCTTTTAC ATTTCAGTTT TTCTTGATCA TGAAAACGCC AACAAAATTC

461 TGAATCGGCC AAAGAGGTAT AATTCAGGTA AATTGGAAGA GTTTGTTCAA GGGAATCTAG

461 AGAGAGAATG TATGGAAGAA AAGTGTAGTT TTGAAGAAGC ACGAGAAGTT TTTGAAAACA

541 CTGAAAGAAC AACTGAATTT TGGAAGCAGT ATGTTGATGG AGATCAGTGT GAGTCCAATC

601 CATGTTTAAA TGGCGGCAGT TGCAAGGATG ACATTAATTC CTATGAATGT TGGTGTCCCT

661 TTGGATTTGA AGGAAAGAAC TGTGAATTAG ATGTAACATG TAACATTAAG AATGGCAGAT

721 GCGAGCAGTT TTGTAAAAAT AGTGCTGATA ACAAGGTGGT TTGCTCCTGT ACTGAGGGAT

181 ATCGACTTGC AGAAAACCAG AAGTCCTGTG AACCAGCAGT GCCATTTCCA TGTGGAAGAG

841 TTTCTGTTTC ACAAACTTCT AAGCTCACCC GTGCTGAGAC TGTTTTTCCT GATGTGGACT

901 ATGTAAATTC TACTGAAGCT GAAACCATTT TGGATAACAT CACTCAAAGC ACCCAATCAT

961 TTAATGACTT CACTCGGGTT GTTGGTGGAG AAGATGCCAA ACCAGGTCAA TTCCCTTGGC

1021 AGGTTGTTTT GAATGGTAAA GTTGATGCAT TCTGTGGAGG CTCTATCGTT AATGAAAAAT

1081 GGATTGTAAC TGCTGCCCAC TGTGTTGAAA CTGGTGTTAA AATTACAGTT GTCGCAGGTG

1141 AACATAATAT TGAGGAGACA GAACATACAG AGCAAAAGCG AAATGTGATT CGAATTATTC

1201 CTCACCACAA CTACAATGCA GCTATTAATA AGTACAACCA TGACATTGCC CTTCTGGAAC

1281 TGGACGAACC CTTAGTGCTA AACAGCTACG TTACACCTAT TTGCATTGCT GACAAGGAAT

1321 ACACGAACAT CTTCCTCAAA TTTGGATCTG GCTATGTAAG TGGCTGGGGA AGAGTCTTCC

1381 ACAAAGGGAG ATCAGCTTTA GTTCTTCAGT ACCTTAGAGT TCCACTTGTT GACCGAGCCA

1441 CATGTCTTCG ATCTACAAAG TTCACCATCT ATAACAACAT GTTCTGTGCT GGCTTCCATG

1501 AAGGAGGTAG AGATTCATGT CAAGGAGATA GTGGGGGACC CCATGTTACT GAAGTGGAAG

1561 GGACCAGTTT CTTAACTGGA ATTATTAGCT GGGGTGAAGA GTGTGCAATG AAAGGCAAAT

1621 ATGGAATATA TACCAAGGTG TCCCGGTATG TCAACTGGAT TAAGGAAAAA ACAAAGCTCA

1681 CTGACAAAAC TCACACATGC CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG

1741 TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA

1801 CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG

1861 ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT

1921 ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA

1981 AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA

2041 AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA

2101 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG

2161 AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT

2221 CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG

2281 GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA

2341 GCCTCTCCCT GTCTCCGGGT AAAGGTGGCG GCGGATCAGG TGGGGGTGGA TCAGGCGGTG

2401 GAGGTTCCGG TGGCGGGGGA TCAGACAAAA CTCACACATG CCCACCGTGC CCAGCACCTG

2461 AACTCCTGGG AGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA

2521 TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG

2581 TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG

2641 AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT

2701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG

2761 AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC

2821 CATCCCGCGA TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT

2881 ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA

2941 CCACGCCTCC CGTGTTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG

3001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC

3061 ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATGA

FIX-042 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, and linker region connecting the Fc regions is underlined


61 ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP
121 FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR
181 VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW
241 QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII
301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF
361 HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE
421 GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL TDKTHTCPPC PAPELLGGPS
481 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST
541 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT
601 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
661 GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG GGSGGGGSDK THTCPPCPAP
721 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR
781 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP
841 PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
901 DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK*

DNA sequence for FIX-068

1 ATGCAGCGCG TGAACATGAT CATGGCAGAA TCACCAGGCC TCATCACCAT CTGCCTTTTA

61 GGATATCTAC TCAGTGCTGA ATGTACAGGT TTGTTTCCTT TTTTAAAATA CATTGAGTAT

121 GCTTGCCTTT TAGATATAGA AATATCTGAT GCTGTCTTCT TCACTAAATT TTGATTACAT

181 GATTTGACAG CAATATTGAA GAGTCTAACA GCCAGCACGC AGGTTGGTAA GTACTGTGGG

241 AACATCACAG ATTTTGGCTC CATGCCCTAA AGAGAAATTG GCTTTCAGAT TATTTGGATT

301 AAAAACAAAG ACTTTCTTAA GAGATGTAAA ATTTTCATGA TGTTTTCTTT TTTGCTAAAA

361 CTAAAGAATT ATTCTTTTAC ATTTCAGTTT TTCTTGATCA TGAAAACGCC AACAAAATTC

421 TGAATCGGCC AAAGAGGTAT AATTCAGGTA AATTGGAAGA GTTTGTTCAA GGGAATCTAG

481 AGAGAGAATG TATGGAAGAA AAGTGTAGTT TTGAAGAAGC ACGAGAAGTT TTTGAAAACA

541 CTGAAAGAAC AACTGAATTT TGGAAGCAGT ATGTTGATGG AGATCAGTGT GAGTCCAATC

601 CATGTTTAAA TGGCGGCAGT TGCAAGGATG ACATTAATTC CTATGAATGT TGGTGTCCCT


661 TTGGATTTGA AGGAAAGAAC TGTGAATTAG ATGTAACATG TAACATTAAG AATGGCAGAT

721 GCGAGCAGTT TTGTAAAAAT AGTGCTGATA ACAAGGTGGT TTGCTCCTGT ACTGAGGGAT

781 ATCGACTTGC AGAAAACCAG AAGTCCTGTG AACCAGCAGT GCCATTTCCA TGTGGAAGAG

841 TTTCTGTTTC ACAAACTTCT AAGCTCACCC GTGCTGAGAC TGTTTTTCCT GATGTGGACT

901 ATGTAAATTC TACTGAAGCT GAAACCATTT TGGATAACAT CACTCAAAGC ACCCAATCAT

961 TTAATGACTT CACTCGGGTT GTTGGTGGAG AAGATGCCAA ACCAGGTCAA TTCCCTTGGC

1021 AGGTTGTTTT GAATGGTAAA GTTGATGCAT TCTGTGGAGG CTCTATCGTT AATGAAAAAT

1061 GGATTGTAAC TGCTGCCCAC TGTGTTGAAA CTGGTGTTAA AATTACAGTT GTCGCAGGTG

1141 AACATAATAT TGAGGAGACA GAACATACAG AGCAAAAGCG AAATGTGATT CGAATTATTC

1201 CTCACCACAA CTACAATGCA GCTATTAATA AGTACAACCA TGACATTGCC CTTCTGGAAC

1261 TGGACGAACC CTTAGTGCTA AACAGCTACG TTACACCTAT TTGCATTGCT GACAAGGAAT

1321 ACACGAACAT CTTCCTCAAA TTTGGATCTG GCTATGTAAG TGGCTGGGGA AGAGTCTTCC

1301 ACAAAGGGAG ATCAGCTTTA GTTCTTCAGT ACCTTAGAGT TCCACTTGTT GACCGAGCCA

1441 CATGTCTTCG ATCTACAAAG TTCACCATCT ATAACAACAT GTTCTGTGCT GGCTTCCATG

1501 AAGGAGGTAG AGATTCATGT CAAGGAGATA GTGGGGGACC CCATGTTACT GAAGTGGAAG

1561 GGACCAGTTT CTTAACTGGA ATTATTAGCT GGGGTGAAGA GTGTGCAATG AAAGGCAAAT

1621 ATGGAATATA TACCAAGGTG TCCCGGTATG TCAACTGGAT TAAGGAAAAA ACAAAGCTCA

1601 CTGACAAAAC TCACACATGC CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG

1741 TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA

1801 CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG

1061 ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT

1921 ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA

1901 AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA

2041 AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA

2101 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG

2161 AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT

2221 CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTCGA CAAGAGCAGG TGGCAGCAGG

2261 GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA

2341 GCCTCTCCCT GTCTCCGGGT AAACGGCGCC GCCGGAGCGG TGGCGGCGGA TCAGGTGGGG

2401 GTGGATCAGG CGGTGGAGGT TCCGGTGGCG GGGGATCCGG CGGTGGAGGT TCCGGTGGGG

2461 GTGGATCAAG GAAGAGGAGG AAGAGGGCGC AGGTGCAGCT GCAGGAGTCT GGGGGAGGCT

2521 TGGTACAGCC TGGGGGGTCC CTGAGACTCT CCTGTGCAGC CTCTGGATTC ATGTTTAGCA

2581 GGTATGCCAT GAGCTGGGTC CGCCAGGCTC CAGGGAAGGG GCCAGAGTGG GTCTCAGGTA

2641 TTAGTGGTAG TGGTGGTAGT ACATACTACG CAGACTCCGT GAAGGGCCGG TTCACCGTCT

2101 CCAGAGACAA TTCCAAGAAC ACGCTGTATC TGCAAATGAA CAGCCTGAGA GCCGAGGACA

2761 CGGCTGTATA TTACTGCGCC CGGGGCGCCA CCTACACCAG CCGGAGCGAC GTGCCCGACC

2821 AGACCAGCTT CGACTACTGG GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA GGGAGTGCAT

2861 CCGCCCCAAA GCTTGAAGAA GGTGAATTTT CAGAAGCACG CGTATCTGAA CTGACTCAGG

2941 ACCCTGCTGT GTCTGTGGCC TTGGGACAGA CAGTCAGGAT CACATGCCAA GGAGACAGCC

3001 TCAGAAACTT TTATGCAAGC TGGTACCAGC AGAAGCCAGG ACAGGCCCCT ACTCTTGTCA

3061 TCTATGGTTT AAGTAAAAGG CCCTCAGGGA TCCCAGACCG ATTCTCTGCC TCCAGCTCAG

3121 GAAACACAGC TTCCTTGACC ATCACTGGGG CTCAGGCGGA AGATGAGGCT GACTATTACT

3181 GCCTGCTGTA CTACGGCGGC GGCCAGCAGG GCGTGTTCGG CGGCGGCACC AAGCTGACCG

3241 TCCTACGTCA GCCCAAGGCT GCCCCCTCGG TCACTCTGTT CCCGCCCTCT TCTGCGGCCG

3301 GTGGCGGTGG CTCCGGCGGA GGTGGGTCCG GTGGCGGCGG ATCAGGTGGG GGTGGATCAG

3361 GCGGTGGAGG TTCCGGTGGC GGGGGATCAG ACAAAACTCA CACATGCCCA CCGTGCCCAG

3421 CACCGGAACT CCTGGGCGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC

3461 TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC

3541 CTGAGGTCAA GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC

3601 CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC

3661 AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC

3721 CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC

3781 TGCCCCCATC CCGCGATGAG CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG

3841 GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT

3901 ACAAGACCAC GCCTCCCGTG TTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA

3961 CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG

4021 CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGA

FIX-068 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting SCE5 to Fc region is underlined, and linker with proprotein convertase processing sites is shown in bold


61 ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP
121 FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR
181 VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW
241 QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII
301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF
361 HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE
421 GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL TDKTHTCPPC PAPELLGGPS
481 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST
541 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT
601 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
661 GNVFSCSVMH EALHNHYTQK SLSLSPGKRR RRSGGGGSGG GGSGGGGSGG GGSGGGGSGG
721 GGSRERRKRA QVQLQESGGG LVQPGGSLRL SCAASGFMFS RYAMSWVRQA PGKGPEWVSG
781 ISGSGGSTYY ADSVKGRFTV SRDNSKNTLY LQMNSLRAED TAVYYCARGA TYTSRSDVPD
841 QTSFDYWGQG TLVTVSSGSA SAPKLEEGEF SEARVSELTQ DPAVSVALGQ TVRITCQGDS
901 LRNFYASWYQ QKPGQAPTLV IYGLSKRPSG IPDRFSASSS GNTASLTITG AQAEDEADYY
961 CLLYYGGGQQ GVFGGGTKLT VLRQPKAAPS VTLFPPSSAA GGGGSGGGGS GGGGSGGGGS
1021 GGGGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED
1081 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
1141 PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN
1201 YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

DNA sequence for FIX-088
1 ATGCAGCGCG TGAACATGAT CATGGCAGAA TCACCAGGCC TCATCACCAT CTGCCTTTTA

61 GGATATCTAC TCAGTGCTGA ATGTACAGGT TTGTTTCCTT TTTTAAAATA CATTGAGTAT

121 GCTTGCCTTT TAGATATAGA AATATCTGAT GCTGTCTTCT TCACTAAATT TTGATTACAT

181 GATTTGACAG CAATATTGAA GAGTCTAACA GCCAGCACGC AGGTTGGTAA GTACTGTGGG

241 AACATCACAG ATTTTGGCTC CATGCCCTAA AGAGAAATTG GCTTTCAGAT TATTTGGATT

301 AAAAACAAAG ACTTTCTTAA GAGATGTAAA ATTTTCATGA TGTTTTCTTT TTTGCTAAAA

361 CTAAAGAATT ATTCTTTTAC ATTTCAGTTT TTCTTGATCA TGAAAACGCC AACAAAATTC

421 TGAATCGGCC AAAGAGGTAT AATTCAGGTA AATTGGAAGA GTTTGTTCAA GGGAATCTAG

481 AGAGAGAATG TATGGAAGAA AAGTGTAGTT TTGAAGAAGC ACGAGAAGTT TTTGAAAACA

541 CTGAAAGAAC AACTGAATTT TGGAAGCAGT ATGTTGATGG AGATCAGTGT GAGTCCAATC

601 CATGTTTAAA TGGCGGCAGT TGCAAGGATG ACATTAATTC CTATGAATGT TGGTGTCCCT

661 TTGGATTTGA AGGAAAGAAC TGTGAATTAG ATGTAACATG TAACATTAAG AATGGCAGAT

721 GCGAGCAGTT TTGTAAAAAT AGTGCTGATA ACAAGGTGGT TTGCTCCTGT ACTGAGGGAT

781 ATCGACTTGC AGAAAACCAG AAGTCCTGTG AACCAGCAGT GCCATTTCCA TGTGGAAGAG

841 TTTCTGTTTC ACAAACTTCT AAGCTCACCC GTGCTGAGAC TGTTTTTCCT GATGTGGACT

901 ATGTAAATTC TACTGAAGCT GAAACCATTT TGGATAACAT CACTCAAAGC ACCCAATCAT

961 TTAATGACTT CACTCGGGTT GTTGGTGGAG AAGATGCCAA ACCAGGTCAA TTCCCTTGGC

1021 AGGTTGTTTT GAATGGTAAA GTTGATGCAT TCTGTGGAGG CTCTATCGTT AATGAAAAAT

1081 GGATTGTAAC TGCTGCCCAC TGTGTTGAAA CTGGTGTTAA AATTACAGTT GTCGCAGGTG

1141 AACATAATAT TGAGGAGACA GAACATACAG AGCAAAAGCG AAATGTGATT CGAATTATTC

1201 CTCACCACAA CTACAATGCA GCTATTAATA AGTACAACCA TGACATTGCC CTTCTGGAAC

1261 TGGACGAACC CTTAGTGCTA AACAGCTACG TTACACCTAT TTGCATTGCT GACAAGGAAT

1321 ACACGAACAT CTTCCTCAAA TTTGGATCTG GCTATGTAAG TGGCTGGGGA AGAGTCTTCC

1381 ACAAAGGGAG ATCAGCTTTA GTTCTTCAGT ACCTTAGAGT TCCACTTGTT GACCGAGCCA

1441 CATGTCTTCG ATCTACAAAG TTCACCATCT ATAACAACAT GTTCTGTGCT GGCTTCCATG

1501 AAGGAGGTAG AGATTCATGT CAAGGAGATA GTGGGGGACC CCATGTTACT GAAGTGGAAG

1551 GGACCAGTTT CTTAACTGGA ATTATTAGCT GGGGTGAAGA GTGTGCAATG AAAGGCAAAT

1621 ATGGAATATA TACCAAGGTG TCCCGGTATG TCAACTGGAT TAAGGAAAAA ACAAAGCTCA

1681 CTGACAAAAC TCACACATGC CCACCGTGCC CAGCTCCGGA ACTCCTGGGC GGACCGTCAG

1741 TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA

1801 CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG

1861 ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT

1921 ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA

1981 AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA

2041 AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA

2101 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG

2151 AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT

2221 CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG

2281 GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA

2341 GCCTCTCCCT GTCTCCGGGT AAAGGTGGCG GCGGATCAGG TGGGGGTGGA TCAGGCGGTG

2401 GAGGTTCCGG TGGCGGGGGA TCAGACAAAA CTCACACATG CCCACCGTGC CCAGCACCTG

2461 AACTCCTGGG AGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA

2521 TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG

2581 TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG

2641 AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT

2101 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG

2751 AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC

2821 CATCCCGCGA TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT

2881 ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA

2941 CCACGCCTCC CGTGTTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTCG

3001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGC

3061 ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAAGGTGGC GGTGGCTCCG

3121 GCGGAGGTGG GTCCGGTGGC GGCGGATCAG GTGGGGGTGG ATCAGGCGGT GGAGGTTCCG

3181 GTGGCGGGGG ATCAGCGCAG GTGCAGCTGC AGGAGTCTGG GGGAGGCTTG GTACAGCCTG

3241 GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAT GTTTAGCAGG TATGCCATGA

3301 GCTGGGTCCG CCAGGCTCCA GGGAAGGGGC CAGAGTGGGT CTCAGGTATT AGTGGTAGTG

3361 GTGGTAGTAC ATACTACGCA GACTCCGTGA AGGGCCGGTT CACCGTCTCC AGAGACAATT

3421 CCAAGAACAC GCTGTATCTG CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTATATT

3481 ACTGCGCCCG GGGCGCCACC TACACCAGCC GGAGCGACGT GCCCGACCAG ACCAGCTTCG

3541 ACTACTGGGG CCAGGGAACC CTGGTCACCG TCTCCTCAGG GAGTGCATCC GCCCCAAAGC

3601 TTGAAGAAGG TGAATTTTCA GAAGCACGCG TATCTGAACT GACTCAGGAC CCTGCTGTGT

3661 CTGTGGCCTT GGGACAGACA GTCAGGATCA CATGCCAAGG AGACAGCCTC AGAAACTTTT

3721 ATGCAAGCTG GTACCAGCAG AAGCCAGGAC AGGCCCCTAC TCTTGTCATC TATGGTTTAA

3781 GTAAAAGGCC CTCAGGGATC CCAGACCGAT TCTCTGCCTC CAGCTCAGGA AACACAGCTT

3841 CCTTGACCAT CACTGGGGCT CAGGCGGAAG ATGAGGCTGA CTATTACTGC CTGCTGTACT

3901 ACGGCGGCGG CCAGCAGGGC GTGTTCGGCG GCGGCACCAA GCTGACCGTC CTACGTCAGC

3961 CCAAGGCTGC CCCCTCGGTC ACTCTGTTCC CGCCCTCTTC TGCGGCCTGA

FIX-088 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker connecting both Fc regions is underlined and linker connecting the Fc region to SCE5 is in bold


61 ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP
121 FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR
161 VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW
241 QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII
301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF
361 HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE
421 GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL TDKTHTCPPC PAPELLGGPS
481 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST
541 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT
601 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
661 GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG GGSGGGGSDK THTCPPCPAP
721 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR
781 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP
841 PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV
901 DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKGGGGS GGGGSGGGGS GGGGSGGGGS
961 GGGGSAQVQL QESGGGLVQP GGSLRLSCAA SGFMFSRYAM SWVRQAPGKG PEWVSGISGS
1021 GGSTYYADSV KGRFTVSRDN SKNTLYLQMN SLRAEDTAVY YCARGATYTS RSDVPDQTSF
1081 DYWGQGTLVT VSSGSASAPK LEEGEFSEAR VSELTQDPAV SVALGQTVRI TCQGDSLRNF
1141 YASWYQQKPG QAPTLVIYGL SKRPSGIPDR FSASSSGNTA SLTITGAQAE DEADYYCLLY
1201 YGGGQQGVFG GGTKLTVLRQ PKAAPSVTLF PPSSAA*

DNA sequence for FIX-089

1	ATGCAGCGCG TGAACATGAT CATGGCAGAA TCACCAGGCC TCATCACCAT CTGCCTTTTA

61	GGATATCTAC TCAGTGCTGA ATGTACAGGT TTGTTTCCTT TTTTAAAATA CATTGAGTAT

121	GCTTGCCTTT TAGATATAGA AATATCTGAT GCTGTCTTCT TCACTAAATT TTGATTACAT

181	GATTTGACAG CAATATTGAA GAGTCTAACA GCCAGCACGC AGGTTGGTAA GTACTGTGGG

241	AACATCACAG ATTTTGGCTC CATGCCCTAA AGAGAAATTG GCTTTCAGAT TATTTGGATT

301	AAAAACAAAG ACTTTCTTAA GAGATGTAAA ATTTTCATGA TGTTTTCTTT TTTGCTAAAA

361	CTAAAGAATT ATTCTTTTAC ATTTCAGTTT TTCTTGATCA TGAAAACGCC AACAAAATTC

421	TGAATCGGCC AAAGAGGTAT AATTCAGGTA AATTGGAAGA GTTTGTTCAA GGGAATCTAG

481	AGAGAGAATG TATGGAAGAA AAGTGTAGTT TTGAAGAAGC ACGAGAAGTT TTTGAAAACA

541	CTGAAAGAAC AACTGAATTT TGGAAGCAGT ATGTTGATGG AGATCAGTGT GAGTCCAATC

601	CATGTTTAAA TGGCGGCAGT TGCAAGGATG ACATTAATTC CTATGAATGT TGGTGTCCCT

661	TTGGATTTGA AGGAAAGAAC TGTGAATTAG ATGTAACATG TAACATTAAG AATGGCAGAT

721	GCGAGCAGTT TTGTAAAAAT AGTGCTGATA ACAAGGTGGT TTGCTCCTGT ACTGAGGGAT

781	ATCGACTTGC AGAAAACCAG AAGTCCTGTG AACCAGCAGT GCCATTTCCA TGTGGAAGAG

841	TTTCTGTTTC ACAAACTTCT AAGCTCACCC GTGCTGAGAC TGTTTTTCCT GATGTGGACT

901	ATGTAAATTC TACTGAAGCT GAAACCATTT TGGATAACAT CACTCAAAGC ACCCAATCAT

961	TTAATGACTT CACTCGGGTT GTTGGTGGAG AAGATGCCAA ACCAGGTCAA TTCCCTTGGC

1021	AGGTTGTTTT GAATGGTAAA GTTGATGCAT TCTGTGGAGG CTCTATCGTT AATGAAAAAT

1081	GGATTGTAAC TGCTGCCCAC TGTGTTGAAA CTGGTGTTAA AATTACAGTT GTCGCAGGTG

1141	AACATAATAT TGAGGAGACA GAACATACAG AGCAAAAGCG AAATGTGATT CGAATTATTC

1201	CTCACCACAA CTACAATGCA GCTATTAATA AGTACAACCA TGACATTGCC CTTCTGGAAC

1261	TGGACGAACC CTTAGTGCTA AACAGCTACG TTACACCTAT TTGCATTGCT GACAAGGAAT

1321	ACACGAACAT CTTCCTCAAA TTTGGATCTG GCTATGTAAG TGGCTGGGGA AGAGTCTTCC

1381	ACAAAGGGAG ATCAGCTTTA GTTCTTCAGT ACCTTAGAGT TCCACTTGTT GACCGAGCCA

1441	CATGTCTTCG ATCTACAAAG TTCACCATCT ATAACAACAT GTTCTGTGCT GGCTTCCATG

1501	AAGGAGGTAG AGATTCATGT CAAGGAGATA GTGGGGGACC CCATGTTACT GAAGTGGAAG

1561	GGACCAGTTT CTTAACTGGA ATTATTAGCT GGGGTGAAGA GTGTGCAATG AAAGGCAAAT

1621	ATGGAATATA TACCAAGGTG TCCCGGTATG TCAACTGGAT TAAGGAAAAA ACAAAGCTCA

1681	CTGACAAAAC TCACACATGC CCACCGTGCC CAGCTCCGGA ACTCCTGGGA GGACCGTCAG

1741	TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA

1801	CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG

1861	ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT

1921	ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA

1981	AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA

2041	AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA

2101	AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG

2161	AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT

2221	CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTCGA CAAGAGCAGG TGGCAGCAGG

2281	GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA

2341	GCCTCTCCCT GTCTCCGGGT AAAGGCGGTG GCGGTTCAGG TGGAGGAGGG TCAGGCGGTG

2401	GTGGATCCGG CGGGGGCGGA TCCGGTGGCG GAGGGTCAGG CGGTGGCGGA TCAGCCTGCA

2461	CCGAGCGGAT GGCCCTGCAC AACCTGTGCG GTGGCGGTGG CTCCGGCGGA GGTGGGTCCG

2521	GTGGCGGCGG ATCAGGTGGG GGTGGATCAG GCGGTGGAGG TTCCGGTGGC GGGGGATCCG

2581	ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCGGAACT CCTGGGCGGA CCGTCAGTCT

2641	TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT

2701	GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG

2761	GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTACC

2821	GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT

2881	GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG

2941	GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGATGAG CTGACCAAGA

3001	ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT

3061	GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG TTGGACTCCG

3121	ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA

3181	ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC

3241	TCTCCCTGTC TCCGGGTAAA TGA

FIX-089 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker regions connecting OS1 to Fc regions are underlined, and OS1 peptide is italicized


61 ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP
121 FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR
181 VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW
241 QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII
301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF
361 HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE
421 GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL TDKTHTCPPC PAPELLGGPS
481 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST
541 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT
601 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ
661 GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGSGGGGSGG GGSGGGGSGG GGSGGGGS AC
721 TERMALHNLC GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV
781 FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
841 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK
901 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
961 NVFSCSVMHE ALHNHYTQKS LSLSPGK*

DNA sequence for FIX-090
1 ATGCAGCGCG TGAACATGAT CATGGCAGAA TCACCAGGCC TCATCACCAT CTGCCTTTTA

61 GGATATCTAC TCAGTGCTGA ATGTACAGGT TTGTTTCCTT TTTTAAAATA CATTGAGTAT

121 GCTTGCCTTT TAGATATAGA AATATCTGAT GCTGTCTTCT TCACTAAATT TTGATTACAT

181 GATTTGACAG CAATATTGAA GAGTCTAACA GCCAGCACGC AGGTTGGTAA GTACTGTGGG

241 AACATCACAG ATTTTGGCTC CATGCCCTAA AGAGAAATTG GCTTTCAGAT TATTTGGATT

301 AAAAACAAAG ACTTTCTTAA GAGATGTAAA ATTTTCATGA TGTTTTCTTT TTTGCTAAAA

351 CTAAAGAATT ATTCTTTTAC ATTTCAGTTT TTCTTGATCA TGAAAACGCC AACAAAATTC

421 TGAATCGGCC AAAGAGGTAT AATTCAGGTA AATTGGAAGA GTTTGTTCAA GGGAATCTAG

481 AGAGAGAATG TATGGAAGAA AAGTGTAGTT TTGAAGAAGC ACGAGAAGTT TTTGAAAACA

541 CTGAAAGAAC AACTGAATTT TGGAAGCAGT ATGTTGATGG AGATCAGTGT GAGTCCAATC

601 CATGTTTAAA TGGCGGCAGT TGCAAGGATG ACATTAATTC CTATGAATGT TGGTGTCCCT

661 TTGGATTTGA AGGAAAGAAC TGTGAATTAG ATGTAACATG TAACATTAAG AATGGCAGAT

721 GCGAGCAGTT TTGTAAAAAT AGTGCTGATA ACAAGGTGGT TTGCTCCTGT ACTGAGGGAT

781 ATCGACTTGC AGAAAACCAG AAGTCCTGTG AACCAGCAGT GCCATTTCCA TGTGGAAGAG

841 TTTCTGTTTC ACAAACTTCT AAGCTCACCC GTGCTGAGAC TGTTTTTCCT GATGTGGACT

901 ATGTAAATTC TACTGAAGCT GAAACCATTT TGGATAACAT CACTCAAAGC ACCCAATCAT

961 TTAATGACTT CACTCGGGTT GTTGGTGGAG AAGATGCCAA ACCAGGTCAA TTCCCTTGGC

101 AGGTTGTTTT GAATGGTAAA GTTGATGCAT TCTGTGGAGG CTCTATCGTT AATGAAAAAT

1081 GGATTGTAAC TGCTGCCCAC TGTGTTGAAA CTGGTGTTAA AATTACAGTT GTCGCAGGTG

1141 AACATAATAT TGAGGAGACA GAACATACAG AGCAAAAGCG AAATGTGATT CGAATTATTC

1201 CTCACCACAA CTACAATGCA GCTATTAATA AGTACAACCA TGACATTGCC CTTCTGGAAC

1261 TGGACGAACC CTTAGTGCTA AACAGCTACG TTACACCTAT TTGCATTGCT GACAAGGAAT

1321 ACACGAACAT CTTCCTCAAA TTTGGATCTG GCTATGTAAG TGGCTGGGGA AGAGTCTTCC

1381 ACAAAGGGAG ATCAGCTTTA GTTCTTCAGT ACCTTAGAGT TCCACTTGTT GACCGAGCCA

1441 CATGTCTTCG ATCTACAAAG TTCACCATCT ATAACAACAT GTTCTGTGCT GGCTTCCATG

1501 AAGGAGGTAG AGATTCATGT CAAGGAGATA GTGGGGGACC CCATGTTACT GAAGTGGAAG

1561 GGACCAGTTT CTTAACTGGA ATTATTAGCT GGGGTGAAGA GTGTGCAATG AAAGGCAAAT

1621 ATGGAATATA TACCAAGGTG TCCCGGTATG TCAACTGGAT TAAGGAAAAA ACAAAGCTCA

1681 CTGGTGGCGG TGGCTCCGGC GGAGGTGGGT CCGGTGGCGG CGGATCAGGT GGGGGTGGAT

1741 CAGGCGGTGG AGGTTCCGGT GGCGGGGGAT CAGCGCAGGT GCAGCTGCAG GAGTCTGGGG

1801 GAGGCTTGGT ACAGCCTGGG GGGTCCCTGA GACTCTCCTG TGCAGCCTCT GGATTCATGT

1861 TTAGCAGGTA TGCCATGAGC TGGGTCCGCC AGGCTCCAGG GAAGGGGCCA GAGTGGGTCT

1921 CAGGTATTAG TGGTAGTGGT GGTAGTACAT ACTACGCAGA CTCCGTGAAG GGCCGGTTCA

1981 CCGTCTCCAG AGACAATTCC AAGAACACGC TGTATCTGCA AATGAACAGC CTGAGAGCCG

2041 AGGACACGGC TGTATATTAC TGCGCCCGGG GCGCCACCTA CACCAGCCGG AGCGACGTGC

2101 CCGACCAGAC CAGCTTCGAC TACTGGGGCC AGGGAACCCT GGTCACCGTC TCCTCAGGGA

2161 GTGCATCCGC CCCAAAGCTT GAAGAAGGTG AATTTTCAGA AGCACGCGTA TCTGAACTGA

2221 CTCAGGACCC TGCTGTGTCT GTGGCCTTGG GACAGACAGT CAGGATCACA TGCCAAGGAG

2251 ACAGCCTCAG AAACTTTTAT GCAAGCTGGT ACCAGCAGAA GCCAGGACAG GCCCCTACTC

2341 TTGTCATCTA TGGTTTAAGT AAAAGGCCCT CAGGGATCCC AGACCGATTC TCTGCCTCCA

2401 GCTCAGGAAA CACAGCTTCC TTGACCATCA CTGGGGCTCA GGCGGAAGAT GAGGCTGACT

2461 ATTACTGCCT GCTGTACTAC GGCGGCGGCC AGCAGGGCGT GTTCGGCGGC GGCACCAAGC

2521 TGACCGTCCT ACGTCAGCCC AAGGCTGCCC CCTCGGTCAC TCTGTTCCCG CCCTCTTCTG

2501 CGGCCTGA

FIX-90 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, and linker regions connecting FIX to SCE5 is underlined

1
61	ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP

121	FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR

181	VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW

241	QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII

301	PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF

351	HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE

421	GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL TGGGGSGGGG SGGGGSGGGG

461	SGGGGSGGGG SAQVQLQESG GGLVQPGGSL RLSCAASGEM FSRYAMSWVR QAPGKGPEWV

541	SGISGSGGST YYADSVKGRF TVSRDNSKNT LYLQMNSLRA EDTAVYYCAR GATYTSRSDV

601	PDQTSFDYWG QGTLVTVSSG SASAPKLEEG EFSEARVSEL TQDPAVSVAL GQTVRITCQG

661	DSLRNFYASW YQQKPGQAPT LVIYGLSKRP SGIPDRFSAS SSGNTASLTI TGAQAEDEAD

721	YYCLLYYGGG QQGVFGGGTK LTVLRQPKAA PSVTLFPPSS AA*

DNA sequence for FVII-088
1 ATGGTCTCCC AGGCCCTCAG GCTCCTCTGC CTTCTGCTTG GGCTTCAGGG CTGCCTGGCT

61 GCAGTCTTCG TAACCCAGGA GGAAGCCCAC GGCGTCCTGC ACCGGCGCCG GCGCGCCAAC

121 GCGTTCCTGG AGGAGCTGCG GCCGGGCTCC CTGGAGAGGG AGTGCAAGGA GGAGCAGTGC

181 TCCTTCGAGG AGGCCCGGGA GATCTTCAAG GACGCGGAGA GGACGAAGCT GTTCTGGATT

241 TCTTACAGTG ATGGGGACCA GTGTGCCTCA AGTCCATGCC AGAATGGGGG CTCCTGCAAG

301 GACCAGCTCC AGTCCTATAT CTGCTTCTGC CTCCCTGCCT TCGAGGGCCG GAACTGTGAG

361 ACGCACAAGG ATGACCAGCT GATCTGTGTG AACGAGAACG GCGGCTGTGA GCAGTACTGC

421 AGTGACCACA CGGGCACCAA GCGCTCCTGT CGGTGCCACG AGGGGTACTC TCTGCTGGCA

481 GACGGGGTGT CCTGCACACC CACAGTTGAA TATCCATGTG GAAAAATACC TATTCTAGAA

541 AAAAGAAATG CCAGCAAACC CCAAGGCCGA ATTGTGGGGG GCAAGGTGTG CCCCAAAGGG

601 GAGTGTCCAT GGCAGGTCCT GTTGTTGGTG AATGGAGCTC AGTTGTGTGG GGGGACCCTG

661 ATCAACACCA TCTGGGTGGT CTCCGCGGCC CACTGTTTCG ACAAAATCAA GAACTGGAGG

721 AACCTGATCG CGGTGCTGGG CGAGCACGAC CTCAGCGAGC ACGACGGGGA TGAGCAGAGC

181 CGGCGGGTGG CGCAGGTCAT CATCCCCAGC ACGTACGTCC CGGGCACCAC CAACCACGAC

841 ATCGCGCTGC TCCGCCTGCA CCAGCCCGTG GTCCTCACTG ACCATGTGGT GCCCCTCTGC

901 CTGCCCGAAC GGACGTTCTC TGAGAGGACG CTGGCCTTCG TGCGCTTCTC ATTGGTCAGC

961 GGCTGGGGCC AGCTGCTGGA CCGTGGCGCC ACGGCCCTGG AGCTCATGGT CCTCAACGTG

1021 CCCCGGCTGA TGACCCAGGA CTGCCTGCAG CAGTCACGGA AGGTGGGAGA CTCCCCAAAT

1081 ATCACGGAGT ACATGTTCTG TGCCGGCTAC TCGGATGGCA GCAAGGACTC CTGCAAGGGG

1141 GACAGTGGAG GCCCACATGC CACCCACTAC CGGGGCACGT GGTACCTGAC GGGCATCGTC

1201 AGCTGGGGCC AGGGCTGCGC AACCGTGGGC CACTTTGGGG TGTACACCAG GGTCTCCCAG

1261 TACATCGAGT GGCTGCAAAA GCTCATGCGC TCAGAGCCAC GCCCAGGAGT CCTCCTGCGA

1321 GCCCCATTTC CCGGTGGCGG TGGCTCCGGC GGAGGTGGGT CCGGTGGCGG CGGATCAGGT

1381 GGGGGTGGAT CAGGCGGTGG AGGTTCCGGT GGCGGGGGAT CCGACAAAAC TCACACATGC

1441 CCACCGTGCC CAGCTCCGGA ACTCCTGGGA GGACCGTCAG TCTTCCTCTT CCCCCCAAAA

1501 CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTG

1561 AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGG ACGGCGTGGA GGTGCATAAT

1621 GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC

1681 ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA

1741 GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCA

1801 CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA AGAACCAGGT CAGCCTGACC

1851 TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG

1921 CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGTTGGACT CCGACGGCTC CTTCTTCCTC

1961 TACAGCAAGC TCACCGTCGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC

2041 GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT

2101 AAACGGCGCC GCCGGAGCGG TGGCGGCGGA TCAGGTGGGG GTGGATCAGG CGGTGGAGGT

2161 TCCGGTGGCG GGGGATCCGG CGGTGGAGGT TCCGGTGGGG GTGGATCAAG GAAGAGGAGG

2221 AAGAGGGACA TCGTGATGAC CCAGGCCGCC CCCAGCGTGC CCGTGACCCC CGGCGAGAGC

2291 GTGAGCATCA GCTGCCGGAG CAGCCGGAGC CTGCTGCACA GCAACGGCAA CACCTACCTG

2341 TGCTGGTTCC TGCAGCGGCC CGGCCAGAGC CCCCAGCTGC TGATCTACCG GATGAGCAAC

2401 CTGGCCAGCG GCGTGCCCGA CCGGTTCAGC GGCAGCGGCA GCGGCACCGC CTTCACCCTG

2451 CGGATCAGCC GGGTGGAGGC CGAGGACGTG GGCGTGTACT ACTGCATGCA GCACCTGGAG

2521 TACCCCTTCA CCTTCGGCAG CGGCACCAAG CTGGAGATCA AGCGGGGCGG CGGCGGCAGC

2561 GGCGGCGGCG GCAGCGGCGG CGGCGGCAGC CAGGTGCAGC TGCAGCAGAG CGGCGCCGAG

2641 CTGGTGCGGC CCGGCACCAG CGTGAAGATC AGCTGCAAGG CCAGCGGCTA CACCTTCACC

2701 AACTACTGGC TGGGCTGGGT GAAGCAGCGG CCCGGCCACG GCCTGGAGTG GATCGGCGAC

2761 ATCTACCCCG GCGGCGGCTA CAACAAGTAC AACGAGAACT TCAAGGGCAA GGCCACCCTG

2821 ACCGCCGACA CCAGCAGCAG CACCGCCTAC ATGCAGCTGA GCAGCCTGAC CAGCGAGGAC

2881 AGCGCCGTGT ACTTCTGCGC CCGGGAGTAC GGCAACTACG ACTACGCCAT GGACAGCTGG

2941 GGCCAGGGCA CCAGCGTGAC CGTGAGCAGC GGTGGCGGTG GCTCCGGCGG AGGTGGGTCC

3001 GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG GTTCCGGTGG CGGGGGATCA

3051 GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCGGAAC TCCTGGGCGG ACCGTCAGTC

3121 TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA

3161 TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTACGTGGAC

3241 GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC

3301 CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG

3351 TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA

3421 GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG

3481 AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG

3541 TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT GTTGGACTCC

3601 GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG

3661 AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC

3721 CTCTCCCTGT CTCCGGGTAA ATGA

FVII-088 amino acid sequence. Signal sequence is shown in dotted underline, propeptide is double underlined, linker region connecting FVII or AP3 to Fc region is underlined, the AP3 scFv italicized, and linker with proprotein convertase processing sites is shown in bold


61 SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE
121 THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE
181 KRNASKPQGR IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR
241 NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS TYVPGTTNHD IALLRLHQPV VLTDHVVPLC
301 LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ QSRKVGDSPN
361 ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG HFGVYTRVSQ
421 YIEWLQKLMR SEPRPGVLLR APFPGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSDKTHTC
481 PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN
541 AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP
601 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL
661 YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG KRRRRSGGGG SGGGGSGGGG
721 SGGGGSGGGG SGGGGSRKRR KR DIVMTQAA PSVPVTPGES VSISCRSSRS LLHSNGNTYL
781 CWFLQRPGQS PQLLIYRMSN LASGVPDRFS GSGSGTAFTL RISRVEAEDV GVYYCMQHLE
841 YPFTFGSGTK LEIKRGGGGS GGGGSGGGGS QVQLQQSGAE LVRPGTSVKI SCRASGYTFT
901 NYWLGWVKQR PGHGLEWIGD TYPGGGYNKY NENFKGRATL TADTSSSTAY MQLSSLTSED
961 SAVYFCAREY GNYDYAMDSW GQGTSVTVSS GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS
1021 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
1081 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
1141 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
1201 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

DNA sequence for FVIII-041

1	ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG CTTTAGTGCC

61	ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG ACTATATGCA AAGTGATCTC

121	GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGC CAAAATCTTT TCCATTCAAC

181	ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC

241	GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301	GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT TCATGCTGTT

361	GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG ATGATCAGAC CAGTCAAAGG

421	GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GGAAGCCATA CATATGTCTG GCAGGTCCTG

481	AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT

541	GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601	GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT TTTTGCTGTA

661	TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT CCTTGATGCA GGATAGGGAT

721	GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCA ATGGTTATGT AAACAGGTCT

781	CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC

841	ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

901	CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC ACTCTTGATG

961	GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC ACCAACATGA TGGCATGGAA

1021	GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAAC TACGAATGAA AAATAATGAA

1081	GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT

1141	GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT

1201	TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT AGTCCTCGCC

1261	CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG GCCCTCAGCG GATTGGTAGG

1321	AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAA CCTTTAAGAC TCGTGAAGCT

1381	ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG

1441	TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT

1501	GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT GAAGGATTTT

1561	CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG TGACTGTAGA AGATGGGCCA

1621	ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTA GTTTCGTTAA TATGGAGAGA

1681	GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA

1741	AGAGGAAACC AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG

1801	AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC AGCTGGAGTG

1861	CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC ACAGCATCAA TGGCTATGTT

1921	TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGG CATACTGGTA CATTCTAAGC

1981	ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA

2041	ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG

2101	ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG GAACAGAGGC

2161	ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA CTGGTGATTA TTACGAGGAC

2221	AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAAACA ATGCCATTGA ACCAAGAAGC

2281	TTCTCTCAAA ACCCACCAGT CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT

2341	CAGTCAGATC AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA

2401	GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA AAAGAAAACA

2461	CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT ATGGGATGAG TAGCTCCCCA

2521	CATGTTCTAA GAAACAGGGC TCAGAGTGGC AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC

2581	CAGGAATTTA CTGATGGCTC CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT

2641	TTGGGACTCC TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC

2701	AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA TGAGGAAGAT

2761	CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC CTAATGAAAC CAAAACTTAC

2821	TTTTGGAAAG TGCAACATCA TATGGCACCC ACTAAAGATG AGTTTGACTG CAAAGCCTGG

2881	GCTTATTTCT CTGATGTTGA CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT

2941	CTGGTCTGCC ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA

3001	TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC TGAAAATATG

3061	GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG ATCCCACTTT TAAAGAGAAT

3121	TATCGCTTCC ATGCAATCAA TGGCTACATA ATGGATACAC TACCTGGCTT AGTAATGGCT

3181	CAGGATCAAA GGATTCGATG GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT

3241	ATTCATTTCA GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG

3301	TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA AGCTGGAATT

3361	TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG GGATGAGCAC ACTTTTTCTG

3421	GTGTACAGCA ATAAGTGTCA GACTCCCCTG GGAATGGCTT CTGGACACAT TAGAGATTTT

3481	CAGATTACAG CTTCAGGACA ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT

3541	TCCGGATCAA TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG

3601	TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA GTTCTCCAGC

3661	CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG GGAAGAAGTG GCAGACTTAT

3721	CGAGGAAATT CCACTGGAAC CTTAATGGTC TTCTTTGGCA ATGTGGATTC ATCTGGGATA

3781	AAACACAATA TTTTTAACCC TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT

3841	TATAGCATTC GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC

3901	ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC TTCATCCTAC

3961	TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC GACTTCACCT CCAAGGGAGG

4021	AGTAATGCCT GGAGACCTCA GGTGAATAAT CCAAAAGAGT GGCTGCAAGT GGACTTCCAG

4081	AAGACAATGA AAGTCACAGG AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG

4141	TATGTGAAGG AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT

4201	CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC TGTGGTGAAC

4261	TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC ACCCCCAGAG TTGGGTGCAC

4321	CAGATTGCCC TGAGGATGGA GGTTCTGGGC TGCGAGGCAC AGGACCTCTA CGACAAAACT

4381	CACACATGCC CACCGTGCCC AGCTCCAGAA CTCCTGGGCG GACCGTCAGT CTTCCTCTTC

4441	CCCCCAAAAC CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG

4501	GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA CGGCGTGGAG

4561	GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA ACAGCACGTA CCGTGTGGTC

4621	AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA AGGAGTACAA GTGCAAGGTC

4681	TCCAACAAAG CCCTCCCAGC CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC

4741	CGAGAACCAC AGGTGTACAC CCTGCCCCCA TCCCGGGATG AGCTGACCAA GAACCAGGTC

4801	AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGC

4861	AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG TGTTGGACTC CGACGGCTCC

4921	TTCTTCCTCT ACAGCAAGCT CACCGTGGAC AAGAGCAGGT GGCAGCAGGG GAACGTCTTC

4981	TCATGCTCCG TGATGCATGA GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG

5041	TCTCCGGGTA AAGGTGGCGG CGGATCAGGT GGGGGTGGAT CAGGCGGTGG AGGTTCCGGT

5101	GGCGGGGGAT CAGACAAAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGA

5161	GGACCGTCAG TCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC

5221	CCTGAGGTCA CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC

5281	TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC

5341	AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC

5401	AAGGAGTACA AGTGCAAGGT CTCCAACAAA GCCCTCCCAG CCCCCATCGA GAAAACCATC

5461	TCCAAAGCCA AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC TCCCGCGAT

5521	GAGCTGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC

5581	ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC

5641	GTGTTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTGGA CAAGAGCAGG

5701	TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC

5761	ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AAATGA

FVIII-041 amino acid sequence. Signal sequence is shown in dotted underline, and linker region connecting the Fc regions is underlined


61 TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY DTVVITLKNM ASHPVSLHAV
121 GVSYWKASEG AEYDDQTSQR EKEDDKVFPG GSHTYVWQVL KENGPMASDP LCLTYSYLSH
181 VDLVKDLNSG LIGALLVCRE GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD
241 AASARAWPKM HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH
301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE EPQLRMKNNE
361 EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT WVHYIAAEEE DWDYAPLVLA
421 PDDRSYKSQY LNNGPQRIGR KYKKVREMAY TDETFKTREA IQHESGILGP LLYGEVGDTL
481 LIIFKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP
541 TKSDPRCLTR YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE
601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL HEVAYWYILS
661 IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS MENPGLWILG CHNSDFRNRG
721 MTALLKVSSC DKNTGDYYED SYEDISAYLL SKNNAIEPRS FSQNPPVLKR HQREITRTTL
781 QSDQEEIDYD DTISVEMKKE DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP
841 HVLRNRAQSG SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF
901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP TKDEFDCKAW
961 AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE FALFFTIFDE TKSWYFTENM
1021 ERNCRAPCNI QMEDPTFKEN YRFHAINGYI MDTLPGLVMA QDQRIRWYLL SMGSNENIHS
1081 IHFSGHVFTV RKKEEYKMAL YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL
1141 VYSNKCQTPL GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL
1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV FFGNVDSSGI
1261 KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS MPLGMESKAI SDAQITASSY
1321 FTNMFATWSP SKARLHLQGR SNAWRPQVNN PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM
1381 YVKEFLISSS QDGHQWTLFF QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH
1441 QIALRMEVLG CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV
1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV
1561 SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES
1621 NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL
1681 SPGKGGGGSG GGGSGGGGSG GGGSDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT
1741 PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG
1801 KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD
1861 IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY
1921 TQKSLSLSPG K*

DNA sequence for FVIII-108

1	ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG CTTTAGTGCC

61	ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG ACTATATGCA AAGTGATCTC

121	GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGC CAAAATCTTT TCCATTCAAC

181	ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC

241	GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301	GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT TCATGCTGTT

361	GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG ATGATCAGAC CAGTCAAAGG

421	GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GGAAGCCATA CATATGTCTG GCAGGTCCTG

481	AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT

541	GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601	GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT TTTTGCTGTA

661	TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT CCTTGATGCA GGATAGGGAT

721	GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCA ATGGTTATGT AAACAGGTCT

781	CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC

841	ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

901	CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC ACTCTTGATG

961	GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC ACCAACATGA TGGCATGGAA

1021	GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAAC TACGAATGAA AAATAATGAA

1081	GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT

1141	GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT

1201	TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT AGTCCTCGCC

1261	CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG GCCCTCAGCG GATTGGTAGG

1321	AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAA CCTTTAAGAC TCGTGAAGCT

1381	ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG

1441	TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT

1501	GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT GAAGGATTTT

1561	CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG TGACTGTAGA AGATGGGCCA

1621	ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTA GTTTCGTTAA TATGGAGAGA

1681	GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA

1741	AGAGGAAACC AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG

1801	AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC AGCTGGAGTG

1861	CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC ACAGCATCAA TGGCTATGTT

1921	TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGG CATACTGGTA CATTCTAAGC

1981	ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA

2041	ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG

2101	ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG GAACAGAGGC

2161	ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA CTGGTGATTA TTACGAGGAC

2221	AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAAACA ATGCCATTGA ACCAAGAAGC

2281	TTCTCTCAAA ACCCACCAGT CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT

2341	CAGTCAGATC AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA

2401	GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA AAAGAAAACA

2461	CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT ATGGGATGAG TAGCTCCCCA

2521	CATGTTCTAA GAAACAGGGC TCAGAGTGGC AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC

2581	CAGGAATTTA CTGATGGCTC CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT

2641	TTGGGACTCC TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC

2701	AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA TGAGGAAGAT

2761	CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC CTAATGAAAC CAAAACTTAC

2821	TTTTGGAAAG TGCAACATCA TATGGCACCC ACTAAAGATG AGTTTGACTG CAAAGCCTGG

2881	GCTTATTTCT CTGATGTTGA CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT

2941	CTGGTCTGCC ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA

3001	TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC TGAAAATATG

3061	GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG ATCCCACTTT TAAAGAGAAT

3121	TATCGCTTCC ATGCAATCAA TGGCTACATA ATGGATACAC TACCTGGCTT AGTAATGGCT

3181	CAGGATCAAA GGATTCGATG GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT

3241	ATTCATTTCA GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG

3301	TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA AGCTGGAATT

3361	TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG GGATGAGCAC ACTTTTTCTG

3421	GTGTACAGCA ATAAGTGTCA GACTCCCCTG GGAATGGCTT CTGGACACAT TAGAGATTTT

3481	CAGATTACAG CTTCAGGACA ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT

3541	TCCGGATCAA TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG

3601	TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA GTTCTCCAGC

3661	CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG GGAAGAAGTG GCAGACTTAT

3721	CGAGGAAATT CCACTGGAAC CTTAATGGTC TTCTTTGGCA ATGTGGATTC ATCTGGGATA

3781	AAACACAATA TTTTTAACCC TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT

3841	TATAGCATTC GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC

3901	ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC TTCATCCTAC

3961	TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC GACTTCACCT CCAAGGGAGG

4021	AGTAATGCCT GGAGACCTCA GGTGAATAAT CCAAAAGAGT GGCTGCAAGT GGACTTCCAG

4081	AAGACAATGA AAGTCACAGG AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG

4141	TATGTGAAGG AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT

4201	CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC TGTGGTGAAC

4261	TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC ACCCCCAGAG TTGGGTGCAC

4321	CAGATTGCCC TGAGGATGGA GGTTCTGGGC TGCGAGGCAC AGGACCTCTA CGACAAAACT

4381	CACACATGCC CACCGTGCCC AGCACCTGAA CTCCTGGGAG GACCGTCAGT CTTCCTCTTC

4441	CCCCCAAAAC CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG

4501	GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA CGGCGTGGAG

4561	GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA ACAGCACGTA CCGTGTGGTC

4621	AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA AGGAGTACAA GTGCAAGGTC

4681	TCCAACAAAG CCCTCCCAGC CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC

4741	CGAGAACCAC AGGTGTACAC CCTGCCCCCA TCCCGCGATG AGCTGACCAA GAACCAGGTC

4801	AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGC

4861	AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG TGTTGGACTC CGACGGCTCC

4921	TTCTTCCTCT ACAGCAAGCT CACCGTCGAC AAGAGCAGGT GGCAGCAGGG GAACGTCTTC

4981	TCATGCTCCG TGATGCATGA GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG

5041	TCTCCGGGTA AACGGCGCCG CCGGAGCGGT GGCGGCGGAT CAGGTGGGGG TGGATCAGGC

5101	GGTGGAGGTT CCGGTGGCGG GGGATCCGGC GGTGGAGGTT CCGGTGGGGG TGGATCAAGG

5161	AAGAGGAGGA AGAGGGCGCA GGTGCAGCTG CAGGAGTCTG GGGGAGGCTT GGTACAGCCT

5221	GGGGGGTCCC TGAGACTCTC CTGTGCAGCC TCTGGATTCA TGTTTAGCAG GTATGCCATG

5281	AGCTGGGTCC GCCAGGCTCC AGGGAAGGGG CCAGAGTGGG TCTCAGGTAT TAGTGGTAGT

5341	GGTGGTAGTA CATACTACGC AGACTCCGTG AAGGGCCGGT TCACCGTCTC CAGAGACAAT

5401	TCCAAGAACA CGCTGTATCT GCAAATGAAC AGCCTGAGAG CCGAGGACAC GGCTGTATAT

5461	TACTGCGCCC GGGGCGCCAC CTACACCAGC CGGAGCGACG TGCCCGACCA GACCAGCTTC

5521	GACTACTGGG GCCAGGGAAC CCTGGTCACC GTCTCCTCAG GGAGTGCATC CGCCCCAAAG

5581	CTTGAAGAAG GTGAATTTTC AGAAGCACGC GTATCTGAAC TGACTCAGGA CCCTGCTGTG

5641	TCTGTGGCCT TGGGACAGAC AGTCAGGATC ACATGCCAAG GAGACAGCCT CAGAAACTTT

5701	TATGCAAGCT GGTACCAGCA GAAGCCAGGA CAGGCCCCTA CTCTTGTCAT CTATGGTTTA

5761	AGTAAAAGGC CCTCAGGGAT CCCAGACCGA TTCTCTGCCT CCAGCTCAGG AAACACAGCT

5821	TCCTTGACCA TCACTGGGGC TCAGGCGGAA GATGAGGCTG ACTATTACTG CCTGCTGTAC

5881	TACGGCGGCG GCCAGCAGGG CGTGTTCGGC GGCGGCACCA AGCTGACCGT CCTACGTCAG

5941	CCCAAGGCTG CCCCCTCGGT CACTCTGTTC CCGCCCTCTT CTGCGGCCGG TGGCGGTGGC

6001	TCCGGCGGAG GTGGGTCCGG TGGCGGCGGA TCAGGTGGGG GTGGATCAGG CGGTGGAGGT

6061	TCCGGTGGCG GGGGATCAGA CAAAACTCAC ACATGCCCAC CGTGCCCAGC ACCGGAACTC

6121	CTGGGCGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC

6181	CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG

6241	TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC GCGGGAGGAG

6301	CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG TCCTGCACCA GGACTGGCTG

6361	AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA

6421	ACCATCTCCA AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATCC

6481	CGGGATGAGC TGACCAAGAA CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTATCCC

6541	AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG AGAACAACTA CAAGACCACG

6601	CCTCCCGTGT TGGACTCCGA CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG

6661	AGCAGGTGGC AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC

6721	CACTACACGC AGAAGAGCCT CTCCCTGTCT CCGGGTAAAT GA

FVIII-108 amino acid sequence. Signal sequence is shown in dotted underline, linker region connecting SCE5 to Fc region is underlined, and linker with proprotein convertase processing sites is shown in bold

1
61	TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY DTVVITLKNM ASHPVSLHAV

121	GVSYWKASEG AEYDDQTSQR EKEDDKVFPG GSHTYVWQVL KENGPMASDP LCLTYSYLSH

181	VDLVKDLNSG LIGALLVCRE GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

241	AASARAWPKM HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH

301	RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE EPQLRMKNNE

351	EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT WVHYIAAEEE DWDYAPLVLA

421	PDDRSYKSQY LNNGPQRIGR KYKKVREMAY TDETFKTREA IQHESGILGP LLYGEVGDTL

481	LIIFKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

541	TKSDPRCLTR YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE

601	NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL HEVAYWYILS

661	IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS MENPGLWILG CHNSDFRNRG

721	MTALLKVSSC DKNTGDYYED SYEDISAYLL SKNNAIEPRS FSQNPPVLKR HQREITRTTL

781	QSDQEEIDYD DTISVEMKKE DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

841	HVLRNRAQSG SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF

901	RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP TKDEFDCKAW

951	AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE FALFFTIFDE TKSWYFTENM

1021	ERNCRAPCNI QMEDPTFKEN YRFHAINGYI MDTLPGLVMA QDQRIRWYLL SMGSNENIHS

1081	IHFSGHVFTV RKKEEYKMAL YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

1141	VYSNKCQTPL GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1201	LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV FFGNVDSSGI

1261	KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS MPLGMESKAI SDAQITASSY

1321	FTNMFATWSP SKARLHLQGR SNAWRPQVNN PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM

1301	YVKEFLISSS QDGHQWTLFF QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

1441	QIALRMEVLG CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1501	VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV

1551	SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES

1621	NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL

1681	SPGKRRRRSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGSR KRRKRAQVQL QESGGGLVQP

1741	GGSLRLSCAA SGEMFSRYAM SWVRQAPGKG PEWVSGISGS GGSTYYADSV KGRFTVSRDN

1801	SKNTLYLQMN SLRAEDTAVY YCARGATYTS RSDVPDQTSF DYWGQGTLVT VSSGSASAPK

1861	LEEGEFSEAR VSELTQDPAV SVALGQTVRI TCQGDSLRNF YASWYQQKPG QAPTLVIYGL

1921	SKRPSGIPDR FSASSSGNTA SLTITGAQAE DEADYYCLLY YGGGQQGVFG GGTKLTVLRQ

1901	PKAAPSVTLF PPSSAAGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSDKTH TCPPCPAPEL

2041	LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE

2101	QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS

2161	RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK

2221	SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK*

DNA sequence for pSYN-FVIII-049
1 ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG CTTTAGTGCC

61 ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG ACTATATGCA AAGTGATCTC

121 GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGC CAAAATCTTT TCCATTCAAC

181 ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC

241 GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT

301 GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT TCATGCTGTT

361 GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG ATGATCAGAC CAGTCAAAGG

421 GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GGAAGCCATA CATATGTCTG GCAGGTCCTG

481 AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT

541 GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA

601 GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT TTTTGCTGTA

661 TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT CCTTGATGCA GGATAGGGAT

721 GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCA ATGGTTATGT AAACAGGTCT

161 CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC

841 ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT

901 CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC ACTCTTGATG

961 GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC ACCAACATGA TGGCATGGAA

1021 GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAAC TACGAATGAA AAATAATGAA

1081 GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT

1141 GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT

1201 TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT AGTCCTCGCC

1261 CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG GCCCTCAGCG GATTGGTAGG

1321 AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAA CCTTTAAGAC TCGTGAAGCT

1381 ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG

1441 TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT

1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT GAAGGATTTT

1561 CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG TGACTGTAGA AGATGGGCCA

1621 ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTA GTTTCGTTAA TATGGAGAGA

1601 GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA

1141 AGAGGAAACC AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG

1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC AGCTGGAGTG

1851 CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC ACAGCATCAA TGGCTATGTT

1921 TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGG CATACTGGTA CATTCTAAGC

1961 ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA

2041 ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG

2101 ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG GAACAGAGGC

2161 ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA CTGGTGATTA TTACGAGGAC

2221 AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAAACA ATGCCATTGA ACCAAGAAGC

2201 TTCTCTCAAA ACCCACCAGT CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT

2341 CAGTCAGATC AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA

2401 GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA AAAGAAAACA

2451 CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT ATGGGATGAG TAGCTCCCCA

2521 CATGTTCTAA GAAACAGGGC TCAGAGTGGC AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC

2561 CAGGAATTTA CTGATGGCTC CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT

2641 TTGGGACTCC TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC

2101 AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA TGAGGAAGAT

2761 CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC CTAATGAAAC CAAAACTTAC

2821 TTTTGGAAAG TGCAACATCA TATGGCACCC ACTAAAGATG AGTTTGACTG CAAAGCCTGG

2881 GCTTATTTCT CTGATGTTGA CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT

2941 CTGGTCTGCC ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA

3001 TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC TGAAAATATG

3051 GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG ATCCCACTTT TAAAGAGAAT

31:1 TATCGCTTCC ATGCAATCAA TGGCTACATA ATGGATACAC TACCTGGCTT AGTAATGGCT

3161 CAGGATCAAA GGATTCGATG GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT

3241 ATTCATTTCA GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG

3301 TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA AGCTGGAATT

3361 TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG GGATGAGCAC ACTTTTTCTG

3421 GTGTACAGCA ATAAGTGTCA GACTCCCCTG GGAATGGCTT CTGGACACAT TAGAGATTTT

3481 CAGATTACAG CTTCAGGACA ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT

3541 TCCGGATCAA TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG

3601 TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA GTTCTCCAGC

3661 CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG GGAAGAAGTG GCAGACTTAT

3721 CGAGGAAATT CCACTGGAAC CTTAATGGTC TTCTTTGGCA ATGTGGATTC ATCTGGGATA

3181 AAACACAATA TTTTTAACCC TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT

3841 TATAGCATTC GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC

3901 ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC TTCATCCTAC

3951 TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC GACTTCACCT CCAAGGGAGG

4021 AGTAATGCCT GGAGACCTCA GGTGAATAAT CCAAAAGAGT GGCTGCAAGT GGACTTCCAG

4081 AAGACAATGA AAGTCACAGG AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG

4141 TATGTGAAGG AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT

4201 CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC TGTGGTGAAC

4261 TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC ACCCCCAGAG TTGGGTGCAC

4321 CAGATTGCCC TGAGGATGGA GGTTCTGGGC TGCGAGGCAC AGGACCTCTA CGACAAAACT

4381 CACACATGCC CACCGTGCCC AGCACCTGAA CTCCTGGGAG GACCGTCAGT CTTCCTCTTC

4441 CCCCCAAAAC CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG

4501 GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA CGGCGTGGAG

4551 GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA ACAGCACGTA CCGTGTGGTC

4621 AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA AGGAGTACAA GTGCAAGGTC

4681 TCCAACAAAG CCCTCCCAGC CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC

4141 CGAGAACCAC AGGTGTACAC CCTGCCCCCA TCCCGCGATG AGCTGACCAA GAACCAGGTC

4801 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGC

4861 AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG TGTTGGACTC CGACGGCTCC

4921 TTCTTCCTCT ACAGCAAGCT CACCGTCGAC AAGAGCAGGT GGCAGCAGGG GAACGTCTTC

4981 TCATGCTCCG TGATGCATGA GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG

5041 TCTCCGGGTA AACGGCGCCG CCGGAGCGGT GGCGGCGGAT CAGGTGGGGG TGGATCAGGC

5101 GGTGGAGGTT CCGGTGGCGG GGGATCCGGC GGTGGAGGTT CCGGTGGGGG TGGATCAAGG

5151 AAGAGGAGGA AGAGGGACAA AACTCACACA TGCCCACCGT GCCCAGCTCC AGAACTCCTG

5221 GGCGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG

5281 ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC

5341 AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG

5401 TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT

5461 GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC

5521 ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG

5581 GATGAGCTGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC

5641 GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT

5101 CCCGTGTTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT GGACAAGAGC

5761 AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC

5821 TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAATGA

FVIII-049 amino acid sequence. Signal sequence is shown in dotted underline, and linker with proprotein convertase processing sites is shown in bold


61 TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY DTVVITLKNM ASHPVSLHAV
121 GVSYWKASEG AEYDDQTSQR EKEDDKVFPG GSHTYVWQVL KENGPMASDP LCLTYSYLSH
181 VDLVKDLNSG LIGALLVCRE GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD
241 AASARAWPKM HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH
301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE EPQLRMKNNE
361 EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT WVHYIAAEEE DWDYAPLVLA
421 PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY TDETFKTREA IQHESGILGP LLYGEVGDTL
481 LIIFKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP
541 TKSDPRCLTR YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE
601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL HEVAYWYILS
661 IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS MENPGLWILG CHNSDFRNRG
721 MTALLKVSSC DKNTGDYYED SYEDISAYLL SKNNAIEPRS FSQNPPVLKR HQREITRTTL
781 QSDQEEIDYD DTISVEMKKE DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP
841 HVLRNRAQSG SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF
901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP TKDEFDCKAW
961 AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE FALFFTIFDE TKSWYFTENM
1021 ERNCRAPCNI QMEDPTFKEN YRFHAINGYI MDTLPGLVMA QDQRIRWYLL SMGSNENIHS
1081 IHFSGHVFTV RKKEEYKMAL YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL
1141 VYSNKCQTPL GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL
1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV FFGNVDSSGI
1261 KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS MPLGMESKAI SDAQITASSY
1321 FTNMFATWSP SKARLHLQGR SNAWRPQVNN PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM
1381 YVKEFLISSS QDGHQWTLFF QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH
1441 QIALRMEVLG CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV
1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV
1561 SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES
1621 NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL
1681 SPGKRRRRSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGSR KRRKRDKTHT CPPCPAPELL
1741 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
1801 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
1861 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS
1921 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK*

Claims

1. A chimeric clotting factor which comprises i) a clotting factor selected from the group consisting of FVII, FIX and FX and ii) a targeting moiety and, optionally, iii) a spacer moiety between the clotting factor and the targeting moiety.

2. The chimeric clotting factor of claim 1, which comprises a structure represented by the formula A B C, wherein A is the clotting factor; wherein B is a spacer moiety; and wherein C is at least one targeting moiety which binds to platelets.

3-4. (canceled)

5. The chimeric clotting factor of claim 1, further comprising a scaffold moiety and, optionally, a second spacer moiety.

6. (canceled)

7. The chimeric clotting factor of claim 1, further comprising a dimeric Fc region comprising a first Fc moiety, F1 and a second Fc moiety, F2.

8. The chimeric clotting factor of claim 7, wherein the chimeric clotting factor is expressed as a polypeptide comprising a cleavable single chain Fc (cscFc) linker interposed between the two Fc moieties, wherein the cscFc linker is adjacent to at least one enzymatic cleavage site which results in cleavage of the cscFc polypeptide linker.

9-19. (canceled)

20. The chimeric clotting factor of claim 7, wherein the targeting moiety is fused to at least one of F1 and F2.

21-23. (canceled)

24. The chimeric clotting factor of claim 1, wherein the targeting moiety is selected from the group consisting of: an antibody molecule, an antigen binding fragment of an antibody molecule, an scFv molecule, a receptor binding portion of a receptor, and a peptide.

25-31. (canceled)

32. The chimeric clotting factor of claim 1, wherein the clotting factor is Factor VII.

33. (canceled)

34. The chimeric clotting factor of claim 1, wherein the clotting factor is Factor IX.

35. (canceled)

36. The chimeric clotting factor of claim 1, wherein the clotting factor is Factor X.

37-39. (canceled)

40. The chimeric clotting factor of claim 1, wherein the chimeric clotting factor comprises a heterologous enzymatic cleavage site not naturally present in the clotting factor.

41-43. (canceled)

44. A polypeptide comprising FVII, which FVII comprises a heterologous enzymatic cleavage site activatable by a component of the clotting cascade.

45. The polypeptide of claim 44, wherein the polypeptide comprises a scaffold moiety and, optionally, a spacer moiety.

46. The chimeric clotting factor of claim 45, wherein the scaffold moiety is a dimeric Fc region comprising a first Fc moiety, F1 and a second Fc moiety, F2.

47-70. (canceled)

71. A nucleic acid molecule encoding a chimeric clotting factor of claim 1.

72. A vector comprising the nucleic acid molecule of claim 71.

73-74. (canceled)

75. A host cell comprising the vector of claim 72, wherein the host cell expresses an enzyme capable of intracellular processing.

76-77. (canceled)

78. A method for producing a chimeric clotting factor comprising culturing the host cell of claim 75 in culture and recovering the chimeric clotting factor from the medium.

79. A processed, heterodimeric polypeptide comprising two polypeptide chains, wherein said processed, heterodimeric polypeptide is made by expressing the vector of claim 78 in a cell cultured in cell culture medium and isolating the mature, heterodimeric polypeptide from the culture medium.

80. A pharmaceutical composition comprising the chimeric clotting factor claim 1 and a pharmaceutically acceptable carrier.

81. (canceled)

82. A method for improving hemostasis in a subject, comprising administering to a subject in need thereof an effective amount of the composition of claim 1.