AU2013202566C1 - Proteolytically cleavable fusion protein comprising a blood coagulation factor - Google Patents

Abstract

The invention relates to therapeutic fusion proteins in which a coagulation factor is fused to a half-life enhancing polypeptide, and both are connected by a linker peptide that is proteolytically cleavable. The cleavage of such linkers liberates the coagulation factor from any activity-compromising steric hindrance caused by the half-life enhancing polypeptide and, thereby, allows the generation of fusion proteins with high molar specific activity when tested in coagulation-related assays. Furthermore, the fact that the linker is cleavable can enhance the rates of inactivation and/or elimination after proteolytic cleavage of the peptide linker compared to the corresponding therapeutic fusion protein linked by the non-cleavable linker having the amino acid sequence GGGGGGV.

Description

74) Agent / Attorney

Watermark Intellectual Property Pty Ltd, L 1 109 Burwood Rd, Hawthorn, VIC, 3122, AU (56) Related Art

US 20050027109A1 US 20050266533 A1 EP 1444986 A1

2013202566 05 Apr 2013

ABSTRACT

The invention relates to therapeutic fusion proteins in which a coagulation factor is fused to a half-life enhancing polypeptide, and both are connected by a linker peptide that is proteolytically cleavable. The cleavage of such linkers liberates the coagulation factor from any activity-compromising steric hindrance caused by the half-life enhancing polypeptide and, thereby, allows the generation of fusion proteins with high molar specific activity when tested in coagulation-related assays. Furthermore, the fact that the linker is cleavable can enhance the rates of inactivation and/or elimination after proteolytic cleavage of the peptide linker compared to the corresponding therapeutic fusion protein linked by the non-cleavable linker having the amino acid sequence GGGGGGV.

2013202566 05 Apr 2013

2013202566 05 Apr 2013 λ~

PROTEOLYTICALIY CLEAVABLE FUSION PROTEIN COMPRISING A BLOOD COAGULATION FACTOR

Introduction

The present invention relates to the field of modified therapeutic fusion proteins with increased half-life compared to their non-modified parent therapeutic polypeptides. The invention specifically relates to coagulation factors fused to half-life enhancing polypeptides (HLEPs), which are connected by linker peptides that are proteolyticaliy cleavable. The cleavage of such linkers liberates the therapeutic polypeptide from any activity-compromising steric hindrance caused by the HLEP and thereby allows the generation of fusion proteins, which retain a high molar specific activity of the coagulation factor. In case the therapeutic fusion proteins are zymogens, those linkers are especially preferred that liberate the therapeutic polypeptide essentially simultaneous with its activation in vivo upon exposure to the corresponding protease(s). Another aspect of the present invention Is a faster inactivation rate of a given coagulation factor once the coagulation factor is activated and the peptide linker is proteolyticaliy cleaved in a coagulation-related mode and/or a faster elimination rate of a given coagulation factor once the coagulation factor is activated and the peptide linker Is proteolyticaliy cleaved in a coagulation-related mode compared to the corresponding fusion protein without cleavable linker.

The idea of the invention is demonstrated in particular by human vitamin Kdependent polypeptides Factor IX, Factor VII, and Factor Vila but the concept also may be applied to other coagulation factors. Any half-life enhancing polypeptide (HLEP) may be connected to the therapeutic polypeptide by a cleavable linker peptide, but albumin or immunoglobulins or fragments derived thereof like the Fc

2013202566 05 Apr 2013 fragment without an antigen binding domain are preferred HLEPs. The invention also relates to cDNA sequences coding for the therapeutic polypeptides and derivatives thereof genetically fused to a cDMA coding for HLEPs, such as human serum albumin linked by oligonucleotides that code for cieavable, intervening peptide linkers. Such encoded derivatives exhibit improved half-life and molar specific activities that are increased in comparison to their non-cleavable counterparts. The invention also relates to recombinant expression vectors containing such cDNA sequences, host ceils transformed with such recombinant expression vectors, recombinant polypeptides and derivatives which do have biological activities comparable to the unmodified wild type therapeutic polypeptide but having improved half-lifes. The invention also relates to processes for the manufacture of such recombinant proteins and their derivatives. The invention also covers a transfer vector for use in human gene therapy, which comprises such modified DMA sequences useful to increase half-life in vivo.

Background of the invention

Several recombinant, therapeutic polypeptides are commercially available for therapeutic and prophylactic use in humans. The patients in general benefit from the specific mode of action of the recombinant active ingredients but a disadvantage often is their limited availability due to their expensive and complex manufacturing processes. A reduction of the necessary dose or the frequency of administration of such products could improve this situation. A reduced frequency of administration could improve the convenience for the patient and, therefore, also the acceptance of the therapy. Several solutions have been described to achieve the goal of an increased in vivo half-life after administration. Solutions proposed recently include the formation of fusion proteins, especially in the case of polypeptides with a short in vivo half-life that can be increased significantly by fusion to a HLEP.

Ballance et al. (WO 01/79271) described fusion polypeptides of a multitude of different therapeutic polypeptides which, when fused to human serum albumin, are

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-3predicted to have an increased functional half-life in vivo and extended shelf-life. Long lists of potential fusion partners are described without showing by experimental data for almost any of these polypeptides that the respective albumin fusion proteins actually retain biological activity and have improved properties.

Among the list of therapeutic polypeptides mentioned as Examples are Factor IX and FVII/FVIIa.. Also described are fusions of FIX and FVII/FVIIa in which there is a peptide linker between albumin and FIX or FVII/FVIIa. However, the use of cleavabie linker peptides is not suggested.

Sheffield et al. (Sheffield W.P. et ai. (2004), Br, J. Haematol. 126: 565-573) expressed a murine Factor IX albumin fusion protein composed of murine FIX, a linker of 8 amino acids (GPG4TM), murine albumin and a peptide tag of 22 amino acids, and also a human Factor IX albumin fusion protein composed of human Factor IX, a linker of 7 amino acids (GsV) and human albumin. Using a one-stage,

FIX dependent clotting assay, the moiar specific activities of the murine FIXalbumin fusion protein (MFUST) and the human FiX-albumin fusion protein (HFUS) were at least two- to three-fold lower than that of their unfused counterparts, an effect attributed at least partially to a slower proteolytic activation process by FXIa. Sheffield did not use or suggest using a cleavabie linker between FIX and albumin.

Several patent applications describe the fusion of therapeutic polypeptides to immunoglobulin constant regions to extend the therapeutic polypeptide's in vivo half-life. WO 2002/04598, WO 2003/059935, WO 2004/081053, WO 2004/101740 and WO 2005/001025 include FIX as examples for the therapeutic polypeptide moiety. The latter two patent applications also describe FVII/FVIIa fused to immunoglobulin constant regions and find that fusion protein homodimers have inferior clotting activity compared to fusion proteins consisting of a monomer/dimer. Again, the use of cleavabie linker peptides is not suggested.

2013202566 05 Apr 2013 •4 In WO 91/09125 fusion proteins are disclosed that are joined by linkers which are cleavable by proteases of the blood coagulation cascade, but the fusion proteins are limited to those comprising fibrinolytic or antithrombotic proteins.

In WO 03/068934 chimeric molecules are described that are composed of at least one first component molecule, at least one linker and at least one second molecule, wherein the linker comprises an enzyme cleavage site to produce a non-naturaliy occurring linkage and cleavage site between the first and the second component molecule and wherein, upon cleavage of the chimeric molecule at the cleavage site, at least one of the component molecules is functionally active. The cleaving proteases may be coagulation factors like thrombin. Component molecules described among many others are FIX and FVIIa. However the therapeutic fusion proteins of the present invention are not disclosed, nor are improved properties of the therapeutic fusion proteins of the present invention disclosed such as increased molar specific activity, increased inactivation and/or elimination rates as compared to the therapeutic protein without cleavable Sinkers.

Description of the invention

There is a great medical need for coagulation factors which have a long half-life. In the prior art fusions of coagulation factors to half-life enhancing polypeptides have been suggested to achieve this goal. However, once a coagulation factor is activated during coagulation either by proteolytic cleavage of the zymogen (like FIX) or by contact of an already proteolyticaily pre'^activated factor to a second polypeptide (like FVIIa binding to Tissue Factor), it is no longer desirable to maintain the long half-life of the now activated coagulation factor as this might lead to thrombotic complications, as is already the case for a wild type coagulation factor as FVIIa (Aiedort L.M., J Thromb Haemost 2(10): 1700-1708 (2004)) and should be even more relevant if the activated factor would have an increased half-life. It is therefore one objective of the present invention to provide long-lived coagulation factors, which after activation or after availability of a cofactor have a half-life comparable to that of an unmodified coagulation factor.

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Fusions of the coagulation factors to half-life enhancing polypeptides as described in the prior art and as also shown in example 6 and 7 suffer in general from a reduced molar specific activity of the fused coagulation factor. Another aspect of the present invention is to provide coagulation factors with enhanced half-life, that show increased molar specific activity compared to the corresponding therapeutic fusion protein without a cleavable linker.

The invention is therefore about therapeutic fusion proteins comprising

a) a coagulation factor, its variants or derivatives,

b) a half-life enhancing polypeptide selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof and immunoglobulins without antigen binding domain including variants and derivatives thereof and

c) a peptide linker which joins the coagulation factor and the half-life enhancing polypeptide;

wherein the peptide linker is cleavable by proteases involved in coagulation or activated by coagulation enzymes and in that the therapeutic fusion protein has in comparison to the respective therapeutic fusion protein linked by a non-cleavable linker having the amino acid sequence GGGGGGV

i) an increased molar specific activity in at least one coagulation-related assay and/or ii) an increased inactivation rate of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode and/or iii) an increased elimination rate of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode.

Comprises/comprising and grammatical variations thereof when used in this 30 specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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As a consequence of the cleavable linker, after cleavage of the peptide linker in a coagulation-related mode, the coagulation factor more closely resembles the behaviour of the native, non-fused factor and does not show an increased half-life of the active factor with potentially prothrombotic effect.

Proteolytic cleavage in a coagulation-related mode in the sense of the invention, is any proteolytic cleavage that occurs as a consequence of the activation of at least one coagulation factor or coagulation cofactor.

The term activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode in the sense of the invention means that the coagulation factor is either activated almost in parallel to the proteolytic cleavage of the linker peptide, or that the coagulation factor was already activated before the proteolytic cleavage of the linker peptide. Activation may occur, for example by proteolytic cleavage of the coagulation factor or by binding to a cofactor.

A further aspect of the present invention is to provide therapeutic fusion proteins comprising

a) a coagulation factor, its variants or derivatives,

b) a half-life enhancing polypeptide selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof and immunoglobulins without antigen binding domain including variants and derivatives thereof and

c) a peptide linker which joins the coagulation factor and the half-life enhancing polypeptide;

wherein the peptide linker is cleavable by proteases involved in coagulation or activated by coagulation enzymes and in that the therapeutic fusion protein has in comparison to the respective therapeutic fusion protein linked by a non-cleavable linker having the amino acid sequence GGGGGGV

i) an increased molar specific activity in at least one coagulationrelated assay and/or

2013202566 05 Apr 2016 it) an increased inactivation rate of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulationrelated mode and/or iii) an increased elimination rate of the activated coagulation factor 5 after the peptide linker is proteolytically cleaved in a coagulationrelated mode and which have an enhanced in vivo recovery as compared to the in vivo recovery of the unmodified coagulation factor.

Preferred are therapeutic fusion proteins which have an enhanced in vivo recovery compared to the unmodified coagulation factor by at least 10%, more preferred by at least 25% and most preferred by 40% or more.

Preferred coagulation factors are vitamin-K dependent coagulation factors and fragments and variants thereof. Even more preferred are FIX, FVIII, FX, von Willebrand Factor (vWF) and fragments and variants thereof.

Preferred HLEPs are albumin and fragments or variants thereof and immunoglobulins including fragments and variants thereof.

The linker region in a preferred embodiment comprises a sequence of the therapeutic polypeptide to be administered or a variant thereof, which should result in a decreased risk of neoantigenic properties (formation of a novel potentially immunogenic epitope due to the occurrence of a peptide within the therapeutic antigen which does not exist in human proteins) of the expressed fusion protein. Also in case the therapeutic protein is a zymogen (e.g. needs to be proteolytically activated) the kinetics of the peptide linker cleavage will more closely reflect the coagulation-related activation kinetics of the zymogen. Thus, in such preferred embodiments a zymogen and a corresponding linker are activated and respectively cleaved, with comparable kinetics. For this reason, the present invention also particularly relates to fusion proteins of a zymogen and a HLEP, where the kinetics of the linker cleavage by relevant proteases is not delayed by

7a

2013202566 05 Apr 2016 more than a factor of 3, and most preferably not by more than a factor of 2 compared to the kinetics of the zymogen activation.

In a further embodiment, the linker peptide comprises cleavage sites for more 5 than one protease. This can be achieved either by a Sinker peptide that can be cleaved at

2013202566 05 Apr 2013 ~ δ * the same position by different proteases or by a Sinker peptide that provides two or more different cleavage sites. This may be advantageous circumstances where the therapeutic fusion protein must be activated by proteolytic cleavage to achieve enzymatic activity and where different proteases may contribute to this activation step. Tills is the case, for example, upon activation of FIX, which can either be achieved by FXta or by FVlia/Tissue Factor (TF),

Preferred embodiments of the invention are therapeutic fusion proteins wherein the linker is cleavable by the protease, that activates the coagulation factor, thereby ensuring that the cleavage of the linker is linked to the activation of the coagulation factor at a site at which coagulation occurs.

Other preferred therapeutic fusion proteins according to the invention are those, wherein the linker is cleavable by the coagulation factor which is part of the therapeutic fusion protein once it is activated, thus also ensuring that cleavage of the fusion protein is connected with a coagulatory event.

Other preferred therapeutic fusion proteins according to the invention are those, wherein the linker is cleavable by a protease, which itself is activated directly or indirectly by the activity of the coagulation factor which is part of the therapeutic fusion protein, thus also ensuring that cleavage of the fusion protein is connected with a coagulatory event.

One class of most preferred therapeutic fusion proteins are those wherein the linker is cleavable by FXIa and/or by FVIIa/TF and the coagulation factor is FIX

The gist of the invention is demonstrated in particular by the vitamin K-dependent polypeptide Factor IX, cleavable tinkers and albumin as the HLEP as well as its corresponding cDNA sequences. The invention also relates to cDNA sequences coding for any other coagulation factors which can be proteolyticaily activated or that are involved in the activation of other zymogens or polypeptides. These cDNAs

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-9are genetically fused to cDNA sequences coding for human serum albumin or other HLEPs, and are linked by oligonucleotides that code for intervening, cleavable peptide linkers. The expressed therapeutic fusion proteins exhibit molar specific activities which are increased in comparison to their non-cieavable counterparts.

The invention also relates to recombinant expression vectors containing such fused cDNA sequences, host cells transformed with such recombinant expression vectors, recombinant therapeutic fusion proteins and derivatives that have biological activities almost comparable to the unmodified wild type therapeutic polypeptides but having improved in vivo half-life. The invention also relates to processes for the manufacture of such recombinant polypeptides and their derivatives. The invention also covers a transfer vector for use in human gene therapy, which comprises such modified DNA sequences useful to increase product levels in vivo.

Preferred therapeutic fusion proteins according to the invention are those that have a molar specific activity, in particular a molar specific activity in at least one coagulation-related assay that is at least 25% increased compared to that of the therapeutic fusion protein without a cieavabie linker. More preferred are therapeutic fusion proteins in which the molar specific activity is increased by at least 50%, even more preferred those in which the molar specific activity is increased by at least 100%, in at least one of the different coagulation-related assays available.

Additional preferred embodiments of the present invention are therapeutic fusion proteins, wherein the inactivation rate of the activated coagulation factor after cleavage of the peptide linker which links the coagulation factor to the half-life enhancing polypeptide is increased by at least 10% as compared to the inactivation rate of the activated coagulation factor in a corresponding therapeutic fusion protein without a cieavabie linker. More preferred are therapeutic fusion proteins in which the inactivation rate is increased by at least 25%, even more preferred those in which the inactivation rate is increased by at least 50%.

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-IBAdditional preferred embodiments of the present invention are therapeutic fusion proteins, wherein the elimination rate of the coagulation factor after cleavage of the peptide linker that links the coagulation factor to the half-life enhancing polypeptide is increased by at least 10% as compared to the elimination rate of the coagulation factor in a corresponding therapeutic fusion protein without a cieavable linker. More preferred are therapeutic fusion proteins in which the elimination rate is increased by at least 25%, even more preferred those in which the elimination rate is increased by at least 50%.

Detailed description of the invention

Vitamin K-dependeml polypeptides

Vitamin K-dependent polypeptides as one group of the therapeutic polypeptides are polypeptides that are γ-carboxylated enzymatically in the liver using vitamin K as a cofactor. Such vitamin K-dependent polypeptides e.g. are Factors li, VII, IX, X, Protein G, Protein S, GASS, and Protein Z.

Human FIX

Human FIX, one member of the group of vitamin K-dependent polypeptides, is a single-chain glycoprotein with a molecular weight of 57 kDa, which is secreted by liver cells into the blood stream as an inactive zymogen of 415 amino acids. It contains 12 γ-earboxy-giutamic acid residues localized in the N-terminal Giadomain of the polypeptide. The Gia residues require vitamin K for their biosynthesis. Following the Gia domain there are two epidermal growth factor domains, an activation peptide, and a trypsin-type serine protease domain. Further posttransiationai modifications of FIX encompass hydroxyiation (Asp 64), N(Asn157 and Asn167) as well as O-type glyoosylation (Ser53, Ser61, Thr150, Thr169, and Thr172), sulfation (Tyr155), and phosphorylation (Ser158).

FIX is converted to its active form, Factor IXa, by proteoiysis of the activation peptide at Arg145-Aia146 and Arg180-Val181 leading to the formation of two polypeptide chains, an N-terminai light chain (18 kDa) and a C-terminal heavy chain

2013202566 05 Apr 2013 (28 kDa), which are held together by one disulfide bridge. Activation cleavage of Factor IX can be achieved in vitro e.g. by Factor Xla or Factor Vlla/TF. Factor IX is present in human plasma in a concentration of 5-10 pg/ml. Terminal plasma half-iife of Factor IX in humans was found to be about 15 to 18 hours (White GC et al. 1997,

Recombinant factor IX, Thromb Haemost 78: 261-285; Ewenstein BM et al. 2002. Pharmacokinetic analysis of plasma-derived and recombinant F IX concentrates in previously treated patients with moderate or severe hemophilia B, Transfusion 42:190-197).

Hemophilia B is caused by non-functional or missing Factor IX and is treated with

Factor IX concentrates from plasma or a recombinant form of Factor IX. As haemophilia B patients often receive at least biweekly prophylactic administrations of Factor IX to avoid spontaneous bleedings, it is desirable to increase the intervals of between administration by increasing the half-life of the Factor IX product applied. An improvement in plasma half-life would bring significant benefit to the patient. Up to now no pharmaceutical preparation of a Factor IX with improved plasma half-life is commercially available nor have any data been published showing F IX variants with prolonged in vivo half-life and almost unchanged molar specific activity in coagulation-related assays. Therefore, a great medical need still exists to develop forms of Factor IX which have a longer functional half-life in vivo.

Factor VII and Factor Vila

FVH is a single-chain glycoprotein with a molecular weight of 50 kDa, which is secreted by liver cells into the blood stream as an inactive zymogen of 408 amino acids. FVII is converted to Its active form Factor Vila, by proteolysis of the single peptide bond at Arg152-lle153 leading to the formation of two polypeptide chains, a N-terminal light chain (24 kDa) and a C-terminal heavy chain (28 kDa), which are held together by one disulfide bridge, in contrast to other vitamin K-dependent coagulation factors, no activation peptide is cleaved off during activation. Activation cleavage of Factor VIi can be achieved in vitro, for example, by Factor Xa, Factor IXa, Factor Vila, Factor Xlla, Factor Seven Activating Protease (FSAP), and

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-12thrombin. Mollerup et ai. (Biotechnol. Bioeng. (1995) 48: 501-505) reported that some cleavage also occurs in the heavy chain at Arg29G and/or Arg315,

Factor VII is present in plasma in a concentration of 500 ng/ml. About 1% or 5 ng/ml of Factor VII is present as activated Factor Vila. The terminal plasma half-life of

Factor VII was found to be about 4 hours and that of Factor Vila about 2 hours.

By administering supraphysiologieal concentrations of Factor Vila hemostasis can be achieved bypassing the need for Factor Villa and Factor IXa. The cloning of the cDNA for Factor VII (US 4,784,950) made it possible to develop activated Factor VII as a pharmaceutical. Factor Vila was successfully administered for the first time in 1988. Ever since the number of Indications of Factor Vila has grown steadily showing a potential to become an universal hemostatic agent to stop bleeding (Erhardtsen, 2002). However, the short terminal half-life of Factor Vila of approximately 2 hours and reduced in vivo recovery is limiting its application.

Therefore, a great medical need still exists to develop forms of Factor Vila which have an improved half-life but otherwise almost uncompromised molar specific activity, inactivation kinetics, and/or elimination kinetics after start of coagulation.

Therapeutic fusion proteins “Therapeutic fusion proteins” in the sense of this invention are coagulation factors fused to a half-life enhancing polypeptide that upon administration to a human or animal can produce a prophylactic or therapeutic effect. These therapeutic fusion proteins may be administered to a human or an animal via intravenous, intramuscular, oral, topical, parenteral or other routes. Specific classes of therapeutic fusion proteins covered, i.e. by the examples in this invention, are coagulation factors like e.g, vitamin K-dependent polypeptides linked to half-life enhancing polypeptides like e.g. albumin and immunoglobulins and their fragments or derivatives. The expression “therapeutic fusion protein is used interchangeable with fusion protein”.

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Half-life enhancing polypeptide (HLEP)

Albumin, albumin family members and immunoglobulines and their fragments or derivatives have been described above as examples of haif-Sife enhancing polypeptides (HLEPs). The terms “human serum albumin” (HSA) and human albumin” (HA) are used interchangeably in this application. The terms “albumin and “serum albumin” are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).

As used herein, “albumin” refers collectively to albumin polypeptide or amino acid sequence, or an albumin fragment or variant having one or more functional activities (e.g., biological activities) of albumin. In particular, “albumin” refers to human albumin or fragments thereof, especially the mature form of human albumin as shown in SEQ ID No:1 herein or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.

The albumin portion of the albumin fusion proteins may comprise the full length of the HA sequence as described above, or may Include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of specific domains of HA.

The albumin portion of the albumin fusion proteins of the invention may be a variant of normal HA, either natural or artificial. The therapeutic polypeptide portion of the fusion proteins of the invention may also be variants of the corresponding therapeutic polypeptides as described herein. The term “variants includes insertions, deletions, and substitutions, either conservative or non-conservative, either natural or artificial, where such changes do not substantially alter the active site, or active domain that confers the therapeutic activities of the therapeutic polypeptides.

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-14ln particular, the albumin fusion proteins of th© invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin. The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin-linked polypeptide may be from a different animal than the therapeutic polypeptide portion.

Generally speaking, an albumin fragment or variant will be at least 10, preferably at least 40, most preferably more than 70 amino acids long. The albumin variant may preferentially consist of or alternatively comprise at least one whole domain of albumin or fragments of said domains, for example domains 1 (amino acids 1-194 of SEQ ID NO:1), 2 (amino acids 195-387 of SEQ ID NO: 1), 3 (amino acids 388-585 of SEQ ID NO: 1), 1 + 2 (1-387 of SEQ ID NO: 1), 2 + 3 (195-585 of SEQ ID NO: 1) or 1 + 3 (amino acids 1-194 of SEQ ID NO: 1 + amino acids 388-585 of

SEQ ID NO: 1). Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain Sinker regions comprising residues Lys106 to Glu119, GSu292 to Val315, and Glu492 to Ala511.

The albumin portion of an albumin fusion protein of the invention may comprise at least one subdomain or domain of HA or conservative modifications thereof.

All fragments and variants of albumin are encompassed by the invention as fusion partners of a coagulation factor as long as they lead to a half-life extension of the therapeutic fusion protein in plasma of at least 25% as compared to the non-fused coagulation factor.

Besides albumin, alpha-fetoprotein, another member of the albumin family, has been claimed to enhance the half-life of an attached therapeutic polypeptide in vivo (WQ 2005/024044). The albumin family of proteins, evolutionarily related serum transport proteins, consists of albumin, alpha-fetoprotein (AFP; Beattie & Dugalczyk

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1982. Gene 20:415-422), afamin (AFM; Llchenstein et al. 1994. J. Biol. Chem, 269:18149-18154) and vitamin D binding protein (DBP; Cooke & David 1985. J. Clin. Invest. 78:2420-2424), Their genes represent a multigene cluster with structural and functional similarities mapping to the same chromosomal region in humans, mice and rat. The structural similarity of the albumin family members suggest their usability as HLEPs. It is therefore another object of the invention to use such albumin family members, fragments and variants thereof as HLEPs. The term “variants includes insertions, deletions and substitutions, either conservative or non-conservative as long as the desired function is still present.

Albumin family members may comprise the full length of the respective protein AFP, AFM and DBP, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids of the respective protein sequence or may include part or all of specific domains of the respective protein, as long as the HLEP fragments provide a half-iife extension of at least 25% as compared to the nonfused coagulation factor. Albumin family members of the therapeutic fusion proteins of the invention may include naturally occurring polymorphic variants of AFP, AFM and DBP.

IgG and IgG-fragments may also be used as HLEPs, as long as the HLEP fragments provide a half-life extension of at least 25% as compared to the nonfused coagulation factor. The therapeutic polypeptide portion is connected to the

IgG or the IgG fragments via a cleavable linker that allows high molar specific activities of the fusion protein. Examples for factor Vll/Vila and factor IX IgG fusion molecules are found, e.g., in WO 2005/001025 which is incorporated herein by reference in its entirety. It discloses i.e. a homodimer comprised of two factor Vli (factor Vila) molecules and two Fc molecules and a monomer/dimer hybrid comprised of one FVII (FVIIa) molecule and two Fc molecules, the monomer/dimer showing an about four times greater clotting activity than the homodimer. A linker

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-16sequence of the present invention liberating the FVII (FVIIa) molecules upon cleavage by a protease of the coagulation cascade like, e.g., FXIa, FXa, or FiXa could be able to elevate the clotting activity of the constructs and especially that of the homodimer to an activity level comparable to the monomer/dimer or even higher. A FIX»Fc fusion protein with cleavable linker is exempiarily shown in SEQ ID No 93, Cleavable linkers such as those shown in table 3a and 3b may be applied in this case.

The invention specifically relates to fusion proteins comprising linking a coagulation factor or fragment or variant thereof to the N- or C-terminus of a HLEP or fragment or variant thereof such that an intervening cieavable peptide linker is introduced between the therapeutic polypeptide and the HLEP such that the fusion protein formed has an increased in vivo half-life compared to the coagulation factor which has not been linked to a HLEP and that the fusion protein has an at least 25% higher molar specific activity compared to the corresponding fusion protein with non-cleavable Sinker in at least one of the different coagulation-related assays available.

“Coagulation factor” as used in this application include, but is not limited to, polypeptides consisting of Factor IX, Factor VII, Factor VIII, von Willebrand Factor, Factor V, Factor X, Factor XI, Factor XII, Factor XIII, Factor I, Factor II (Prothrombin), Protein C, Protein S, GASS, or Protein 2 as well as their activated forms. Furthermore, useful therapeutic polypeptides may be wild-type polypeptides or may contain mutations. Degree and location of glycosylation or other post25 translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. When referring to specific amino acid sequences, posttransiational modifications of such sequences ere encompassed in this application.

Coagulation factor within the above definition includes polypeptides that have the natural amino add sequence including any natural polymorphisms. It also indudes

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-17 2013202566 05 Apr polypeptides with a slightly modified amino acid sequence, for instance, a modified N-terminal or C~terminal end including terminal amino acid deletions or additions, as iong as those polypeptides substantially retain the activity of the respective therapeutic polypeptide. Variants included differ in one or more amino acid residues from the wild type sequence. Examples of such differences may include truncation of the N- and/or C-terminus by one or more amino acid residues (e.g. preferably 1 to 30 amino acid residues), or addition of one or more extra residues at the Nand/or C-terminus, as weli as conservative amino acid substitutions, i.e. substitutions performed within groups of amino acids with similar characteristics,

e.g. (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, and (6) aromatic amino acids. Examples of such conservative substitutions are shown in the following table.

Table 1

(1)	Alanine	Glycine
(2)	Aspartic acid	Glutamic acid
(3a)	Asparagine	Glutamine
(3b)	Serine	Threonine
(4)	Arginine	Histidine	Lysine
(5)	Isoleucine	Leucine	Methionine Valine
(6)	Phenylalanine	Tyrosine	Tryptophane

The in vivo half-life of the fusion proteins of the invention, in general determined as terminal half-life or β-haif-life, is usually at least about 25%, preferable at least about 50%, and more preferably more than 100% higher than the in vivo half-life of the non-fused polypeptide.

ο

CM

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The fusion proteins of the present invention have at least a 25%, preferably at least a 50%, more preferably an at least 100% increased molar specific activity compared to the corresponding fusion proteins without cleavable linkers.

considered here in particular) in this regard is defined as the activity expressed per mole (or e.g. nmole) of the therapeutic polypeptide or therapeutic fusion protein of interest. Calculation of the molar specific activity allows a direct comparison of the activity of the different constructs which is not affected by the different molecular weights or optical densities of the polypeptides studied. The molar specific activity may be calculated as exemplified in table 2 below for FIX and a FIX-HSA fusion

Table 2: Calculation of molar specific activity as shown for a purified FiX-HSA 15 fusion protein

Product	ODpKjnm, It)	MW	Act i wi Ey /Vo I/O Djgo (SU/L/OD288)	Molar optical density (OD^o) st 1 riioi/U	Calculation of molar specific activity (SU/mol)
Fix	13.3 53	57 GOO	determined for product	75810 (- MW x ODgso, n/IO)	=(A c i i v it y / Vo 1/0 D 2so) x (ODjug at 1 mol/L)
HSA	5.7 23	56 300		37791 (= MW x 00(280. 1%/fB)
FIX-HSA			determined for product	113601 (= sum of molar optical density of FIX and HSA)	(Actmty/Vol/ΟΟίββ) x (□Ojas at 1 mol/L)

P,g,g, Di Scipio et si., Biochem. 16: 698-706 (1377)

SX Gtotot et al, J. Exp. Med, 197(3): 315-322 (2003)

In order to determine a molar specific coagulation-related activity, any assay may be used that determines enzymatic or cofactor activities that are relevant to the coagulation process.

2013202566 05 Apr 2013

W~

Therefore “coagulation-related assays in the sens© of the invention is any assay which determines enzymatic or cofactor activities that are of relevance in the coagulation process or that is able to determine that either the intrinsic or the extrinsic coagulation cascade has been activated. The “coagulation-related” assay thus may be direct coagulation assays like aPTT, PT, or the thrombin generation assays. However, other assays like, e.g., chromogenic assays applied for specific coagulation factors are also included. Examples for such assays or corresponding reagents are Pathromtin® SL (aPTT assay, Dade Behring) or Thromborel® S (Prothrombin time assay, Dade Behring) with corresponding coagulation factor deficient plasma (Dade Behring), Thrombin generation assay kits {Technoclone, Thrombinoscope) using e.g, coagulation factor deficient plasma, chromogenic assays like Biophen Factor IX (Hyphen BioMed), Staclot® FVIIa-rTF (Roche Diagnostics GmbH), Coatest® Factor VIII:C/4 (Chromogenlx), or others.

For purposes of this invention, an increase in any one of the above assays or an equivalent coagulation-related assay is considered to show an increase in molar specific activity. For example, a 25% increase refers to a 25% increase in any of the above or an equivalent assay.

To determine whether therapeutic fusion proteins fall within the scope of the present invention, the standard against which the molar specific activity of these proteins is compared is a construct in which the respective coagulation factor and the respective HLEP are linked by a non-cleavable linker having the amino acid sequence GGGGGGV.

In the case of FIX, aPTT assays are often used for determination of coagulation activity. Such a coagulation assay (aPTT assay) is described in example 5 in more detail. However, other coagulation-related assays or assay principles may be applied to determine molar specific activity for FIX.

2013202566 05 Apr 2013

Recombinant therapeutic polypeptide drugs are usually expensive and not ail countries can afford costly therapies based on such drugs. Increasing the in vivo recovery of such drugs could make the use of these products cheaper and subsequently more patients would benefit from them. In the case of the fusion proteins of the present invention an increased in vivo recovery would also be a desirable advantage, “in vivo recovery” in the sense of the invention means the amount of product found in blood or plasma shortly after administration of the product. Therefore, for detection of the in vivo recovery in general the plasma content is determined a few minutes (e.g. 5 or 15 min) after administration of the product

Although it is desirable to have a high in vivo recovery and a long half-life for a nonactivated coagulation factor, it is advantageous to limit the half-life of a coagulation factor after its activation or the activation of its co-factor in order to avoid a prothrombotic risk. Therefore, after the coagulation process has been initiated, the half-life of the active coagulation factor should again be reduced. This can either be achieved by enhancing inactivation in a coagulation-related mode or by elimination of the coagulation factor.

Inactivation according to the present invention means the decrease of activity of the therapeutic polypeptide which can be caused, for example, by a complex formation of a coagulation factor and an inhibitor of the corresponding coagulation factor or by further proteolytic cleavage as known, e.g., in the case of FVill and FV.

The inactivation rate of an activated therapeutic fusion protein is defined as the rate the activity is declining, e.g,, by reaction with inhibitors or by proteolytic inactivation. The inactivation rate may be measured by following the molar specific activity of the activated coagulation factor over time in the presence of physiologic amounts of inhibitors of this coagulation factor. Alternatively, the inactivation rate may be determined after administration of the activated product to an animal followed by ο

CM at2013202566 05 Apr testing of plasma samples at an appropriate time frame using activity and antigen assays.

When for therapeutic fusion proteins a determination is needed whether these proteins fail within the scope of the present invention, the standard against which the inactivation rate of these therapeutic proteins is compared to, is a construct in which the respective coagulation factor and the respective HLEP are joined by a non-cleavable linker having the amino acid sequence GGGGGGV,

The elimination rate of an activated therapeutic fusion protein is defined as the rate 10 the polypeptide is eliminated from the circulation of humans or animate. The elimination rate may be determined by measuring the pharmacokinetics of the activated, therapeutic fusion protein after intravenous administration. Using an antigen assay, the elimination by direct removal from the circulation can be determined. Using an activity assay in addition, a specific removal and inactivation rate may be determined.

When for therapeutic fusion proteins a determination is needed whether these proteins fall within the scope of the present invention, the standard against which the elimination rate of these proteins is compared to, is a construct in which the respective coagulation factor and the respective HLEP are joined by the noncleavable linker having the amino acid sequence GGGGGGV,

According to this invention, the therapeutic polypeptide moiety is coupled to the HLEP moiety by a cleavable peptide linker. The linker should be non-immunogenic and should be flexible enough to allow cleavage by proteases. The cleavage of the linker should proceed comparably fast as the activation of the therapeutic polypeptide within the fusion protein, if the fusion protein is a zymogen.

The cleavable linker preferably comprises a sequence derived from

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2013202566 05 Apr

a) the therapeutic polypeptide to be administered itself if it contains proteolytic cleavage sites that are proteolytically cleaved during activation of the therapeutic polypeptide,

b) a substrate polypeptide of this therapeutic polypeptide, or

e) a substrate polypeptide cleaved by a protease which is activated or formed by the direct or indirect involvement of the therapeutic polypeptide.

The linker region in a more preferred embodiment comprises a sequence of the therapeutic polypeptide to be applied, which should result in a decreased risk of neoantigenlc properties of the expressed fusion protein. Also in case the therapeutic protein is a zymogen (e.g. needs to be proteolytically activated) the kinetics of the peptide linker cleavage will more closely reflect the coagulationrelated activation kinetics of the zymogen.

in a preferred embodiment, the therapeutic polypeptide is FIX zymogen and the HLEP is albumin. In this case the linker sequence is either derived from the sequences of the activation regions of FIX, from the cleavage region of any substrate of FIX like FX or FVII or from the cleavage region of any substrate polypeptide that is cleaved by a protease in whose activation FIXa is involved.

In a highly preferred embodiment the linker peptide is derived from FIX itself, in another preferred embodiment the linker peptide is derived from FX or FVII. In another preferred embodiment the linker sequence comprises two cleavage sequences that can be cleaved by FXia or FVlia/TF, two physiologically relevant activators of FIX.

Exemplary combinations of therapeutic polypeptide, cleavable Sinker and HLEP include the constructs listed in tables 3a and 3b but are not limited to these:

2013202566 05 Apr 2013

Table 3a: Examples of possible constructs

2013202566 05 Apr 2013

FIX		Γ.....hsa	FX
FIX	’gRGOWi.TRJTOGQE	i ksa ! fx	S1 .
FIX	PERGDNNLTniveGO	1 HSA	FX	no ·
FIX	ONiULTR W3SG	| HSA		in :
FIX	SVSQISraJR ΑΕϊνΤΡΓΓΥΟ	S Fc	Fix	52 ;
fix'	QSFNOFTBWSeeON	i re	FiX	53 ;
Fix 0-4123	SVSQTSkLTR AETWPO’vO	ί Fe	FiX	&
; FIX	A5KPQGR FvGG	1 HSAdeiDAH	FVO	112 ?
; FIX	KRNASKFG3R WGGXV	i HSA	FVii	&5 \
; FIX	PEEPGLR MKNNeEAED	!... HSA......	FVIII	£6 j
3 FIX	DNSPSFtQIR SWKKHPKT	I HSA	FVIII	07 j
3 FIX	LSKfiNAlEPR SFSGNSR HPS	1 fits	FMil	68
F8X	DED EMQSPR SFQKWTRHVFIA	I HSA	FVlil	69
FIX	SPHVLRN RAOSG SWG	HSA	FVHI	70
FMIorfMla	PEEPQLRMKNNeEAEOYDODLTDS	HSA	FMii	71
FMI or ΠΛβ	DDONSFSFiQiR GVAKKHPKTWVHYA^eEG	HSA	FMil	72
FMIorFMb	ISKN&AJEPR SFSQNSRHPSTRG&GFI'iA	HSA	FMil	H'™73 j
FMI orFMSs	DEDENGSPR SFQKXTRHYFSAA	HSA	FVW	14
JMii	0 YGMSSSPHVLR^ fi^SGS^QFKKVvf Q EFT Derived from dcsvsss sftg» <fiFMII.FEX, of Fibrlrogen Osrwed item cleave ββ shea «fVWF. FV9I. sr FIX	HSA | Η3&Γ™ ( HSA	fmss FtfSLFSXoiFqn FIX.iM8.SW	75 j
WVF	DiYD EDENQSPR SFQKKTRH VF!A	| HSA	FMil	’«......1
WF	DIN SPS FOR SWX Η P	HSA	FMls
VAF	lsknnajepr sfsqnsr hps	! HSA	FMi!	........J0.......ί

In the case of linkers derived from the N-terminal region of the FIX activation peptide, according to the natural polymorphism T148-A148 the sequences may also contain A instead of T at this position.

Table 3b: Examples of possible constructs with two or more cleavage

Variants and fragments of the described linkers are also encompassed in the present invention as long as the linker can still be cleaved by the protease or the proteases, that cleave the linkers of tables 3a and 3b or by the type of proteases

2013202566 05 Apr 2013

-25defined above. The term “variants” includes insertions, deletions and substitutions, either conservative or non-conservative.

Other combinations of the cleavage sequences described above and their variants 5 shall be included in the present invention.

In another embodiment, amino acid substitutions are included that change the posttranslational modification pattern of the peptide linker. These can be, for example, substitutions of amino acids that are glycosylated, sulphated, or phosphoryiated.

In another embodiment of the invention the peptide linker between the therapeutic polypeptide and the HLEP moiety contains consensus sites for the addition of posttransiational modifications. Preferably such modifications consist of glycosylation sites. More preferably, such modifications consist of at least one N15 glycosylation site of the structure Asn - X - Ser/Thr, wherein X denotes any amino acid except proline. Even more preferably such N-glycosylation sites are inserted close to the amino and/or carboxy terminus of the peptide linker such that they are capable of shielding potential neoepitopes which might develop at the sequences where the therapeutic polypeptide moiety is transitioning into the peptide linker or where the peptide linker is transitioning into the albumin moiety sequence.

2013202566 05 Apr 2013 >•«5

Brief Description of the Figures

Figure 1: in vitro activation of FiX-albumin fusion proteins by FXIa at 37®C at a molar ratio of FXIa to fusion protein of about 1:500. One fusion protein with non5 cieavable linker (1478/797) and two fusion proteins with cteavabie linker (1088/797 and 1089/797) were used. Samples were analyzed by SDS-PAGE under reducing conditions followed by Coomassie biue staining

Figure 2: Pharmakokinetics of activated rec FIX and FiX-albumin fusion proteins with and without cteavabie linker in comparison to non-activated fusion proteins.

Figure 3: Inactivation of activated rec FIX or FIX-albumin fusion protein by AT. Residual FIX activity was determined after 120 min using a non-activated partial thromboplastin time assay.

2013202566 05 Apr 2013

Examples:

Example 1: Generation of cDNAs encoding FIX and FIX-albumin fusion proteins

Factor IX coding sequence was amplified by PCR from a human liver cDNA library (ProQuest, Invstrogen) using primers We1403 and We1404 (SEQ ID NO 5 and 8). After a second round of PCR using primers We1405 and We1406 (SEQ ID NO 7 and 8) the resulting fragment was cloned into pCR4TOPO (Invitrogen). From there the FIX cDNA was transferred as an EcoRI Fragment into the EcoRI site of ptRESpuroS (BD Biosciences) wherein an internal Xhol site had been deleted previously. The resulting plasmid was designated pFIX-498 and was the expression vector for factor IX wild-type.

For the generation of albumin fusion constructs the FIX cDNA was reamplified by PCR under standard conditions using primers We2610 and We2611 (SEQ ID NO 9 and 10) deleting the stop codon and introducing an Xhol site instead. The resulting FIX fragment was digested with restriction endonucleases EcoRI and Xhol and ligated into an EcoRI / BamH1 digested pIRESpuroS together with one Xhol /

BamH1 digested linker fragment as described below.

Two different glycine / serine linker fragments without internal cleavage sites were generated: Oligonucleotides We2148 and We2150 (SEQ ID NO 11 and 12) were annealed in equimolar concentrations (10 pmol) under standard PCR conditions, filled up and amplified using a PCR protocol of a 2 min. initial denaturatfon at 94°C followed by 7 cycles of 15 sec. of denaturation at 94^CC, 15 sec. of annealing at 55®C and 15 sec. of elongation at 72^eC, and finalized by an extension step of 5 min at 72°C. The same procedure was performed using oligonucleotides We2156 and We2157 (SEQ ID NO 13 and 14). The resulting linker fragments were digested with restriction endonucleases Xhol and BamH1 and used separately in the above ο

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2013202566 05 Apr described ligation reaction. The resulting plasmids therefore contained the coding sequence for FIX and a C-terminai extension of a glycine / serine linker.

Two different cleavabie linker fragments derived from the activation sites of FIX were generated: Oligonucleotides We2335 and We2336 (SEQ ID NO 15 and 18), containing the activation cieavage site of the FIX light chain / activation peptide border region, ware annealed, filled, and amplified as described above. The resulting linker fragment was digested with restriction endonucleases Xhol and BamFH and used in the above described ligation reaction. The resulting plasmid therefore contained the coding sequence for FIX and a C-terminal extension of a cleavabie FIX sequence (amino acids 136 to 154 of SEQ ID NO 2). In a subsequent site directed mutagenesis reaction with a commercially available mutagenesis kit (QulckGhange XL Site Directed Mutagenesis Kit, Stratagene) using oligonucleotides We2636 and We2637 (SEQ ID NO 17 and 18) the Xhoi site was deleted.

For generation of the second cleavable linker fragment derived from FIX, the same procedure was performed using oligonucleotides We2337 and We2338 (SEQ ID NO 19 and 20) for linker construction. The resulting linker fragment was digested with restriction endonucleases Xhol and BamH1 and used in the above described ligation reaction. The resulting piasmid now contained the coding sequence for FIX and a C-terminal extension of a cleavabie FIX sequence derived from the activation cleavage site of the FIX activation peptide / heavy chain border region (amino acids 173 to 188 of SEQ ID NO 2). Oligonucleotides We2638 and We 2839 (SEQ ID NO

21 and 22) were used for deletion of the Xhol site as described above.

In the next cloning step the above generated plasmids were digested with BamH1 and a BamH1 fragment containing the cDNA of mature human albumin was inserted. This fragment had been generated by PGR on an albumin cDNA sequence using primers We1882 and We1902 (SEQ id no 23 and 24) under standard conditions.

2013202566 05 Apr 2013

The final plasmids with non-cleavable glyclne/serine linkers were designated pFIX980 (SEQ ID NO 30) and pFIX-986 (SEQ ID NO 31), respectively. The final plasmids with cleavable linkers derived from FIX sequences were designated pFIX1088 (SEQ ID NO 40) and pFIX-1089 (SEQ ID NO 49), respectively. Their linker sequences and the C-terminal FIX and N-terminal albumin sequences are outlined below. Proteolytic cleavage sites within the linkers are indicated with arrows, the FIX derived linker sequences are underlined.

EfTx 1 pdburffiml ^w™*^r^^r**«™************U Bssssscsssssssscccissssssso ¹⁰ .........

pFIX-980 j...KEKTKLTj SSfGGSfr GS pFIX-986 j...KEKTKLTl SSNGS(GGS)₃ NGS(GGS)₃ GGNGS jDAHKSRV-l

4pFIX-1088 ΓΚΕΚΤΚΪΙΙSVSQTSKLTR AETVFPDVP GSjjpAHKSEv2) pFIX-1089 ΓκΕΚΤΚί.ΐ1 QSFNDFTR WGGED GSjPAHt^EvTS

For expression in CHO celis the coding sequences for the FIX albumin fusion protein were transferred info vectors pIRESneoS (BD Bioseiences) or pcDNA3.1 (Invitrogen), respectively.

For efficient processing of the propeptide in cells expressing FIX in high amounts coexpression of Turin is required (Wasiey LC et aL 1993. PACE/Furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway. J. Biol. Chem. 268:8458-8485). Furin was amplified from a liver cDNA library (Ambion) using primers We1791 and We1792 (SEQ ID NO 25 and 26), A second round of PGR using primers We1808 and We18Q9 (SEQ ID NO 27 and 28) yielded a furin fragment where the carboxyterminal transmembrane domain (TM) was deleted and a stop codon introduced; this fragment was cloned into pCR4TOPO (Invitrogen). From there the furinATM cDNA was transferred as an EcoRI/Notl o

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2013202566 05 Apr

Fragment Into the EcoRI/Notl sites of plRESpuro3 (BD Biosciences) wherein an internal Xhol site had been deleted previously. The resulting plasmid was designated pFu-797. This plasmid was cotransfected with all FIX constructs in a 1:5 (pFu-797 : pFIX-xxx) molar ratio. The amino acid sequence of the secreted furin encoded by pFu-797 is given as SEQ-ID NO 29.

Example 2: Transfection and expression of FIX and FIX-albumin fusion proteins

Plasmids were grown up in E.coli TOPfO (Invitrogen) and purified using standard protocols (Qiagen). HEK-293 cells were transfected using the Lipofectamine 2000 reagent (Invitrogen) and grown up in serum-free medium (Invitrogen 293 Express) in the presence of 50 ng/ml Vitamin K and 4 pg/ml Puromycin. Transfected cell populations were spread through T-flasks info roller hotties or small-scale fermenters from which supernatants were harvested for purification,

Alternatively, CHO K1 or DG44 cells (Invitrogen) were transfected using the Lipofectamine 2000 reagent (Invitrogen) and grown up in serum-free medium (Invitrogen CD-CHO) in the presence of 50 ng/ml Vitamin K and 500-750 ng/ml Geneticin. High expressing clones were selected and spread through T-flasks into roller bottles or small-scale fermenters from which supernatants were harvested for purification.

Example 3: Purification of FIX and FIX - albumin fusion proteins

Ceil culture harvest containing FIX or FIX albumin fusion protein was applied on a

G-Sepharase FF column previously equilibrated with 50 mM TrisxHCI / 100 mM

NaCI buffer pH 8.0. Subsequently, the column was washed with equilibration buffer containing 200 mM NaCI. Elution of the bound FIX or FIX fusion protein was achieved by a salt gradient using 50 mM TrisxHCI / 200 mM NaCI buffer pH 8.0 as a basis. The eluate was further purified by column chromatography on a hydroxylapatit© resin. For this purpose, the eluate of the G-Sepharose FF column was loaded on a hydroxylapatite chromatography column equilibrated with 50 mM TrisxHCI / 100 mM NaCI buffer pH 7.2. The column was washed with the same

2013202566 05 Apr 2013 buffer and FIX or FIX-HSA were eluted using a potassium phosphate gradient at pH 7.2. The eluate was dialyzed to reduce the salt concentration and used for biochemical analysis as well as for determination of the pharmacokinetic parameters. FIX antigen and activity were determined as described in example 5.

Example 4: Alternative purification scheme of FIX and FIX - albumin fusion proteins

As described in example 3, cell culture harvest containing FIX or FIX albumin fusion protein was purified by chromatography on Q-Sepharose FF. The Q-Sepharase eluate was further purified by chromatography on a Heparirs-Fractogei column. For this purpose, the Heparin-Fractogel column was equilibrated using 50 mM Tris x HCI, 50 mM NaCI pH 8.0 buffer (EP), the Q-Sepharose FF eluate was applied and the column was washed with equilibration buffer containing 75 mM NaCI. FIX or FIX albumin fusion protein, respectively, was eluted using EP adjusted to 300 mM NaCI.

The Heparin-Fractogel eluate was further purified by chromatography on a hydroxylapatite chromatography column as described in example 3. The purified FIX resp. FIX albumin fusion protein concentrate was subjected to FIX activity and antigen determination according to example 5 and characterized by further in vitro and in vivo investigations.

Example 5: Determination of FIX activity and antigen

FIX activity was determined as clotting or coagulation activity (FIX:C) using commercially available aPTT reagents (Pathromtin SL and FIX depleted plasma, Dade Behring). An internal substandard calibrated against the WHO International

FIX concentrate Standard (96/854) was used as a reference.

FIX antigen (FIX:Ag) was determined by an ELISA acc. to standard protocols known to those skilled In the art. Briefly, microtiter piates were incubated with 100 pL per weli of the capture antibody (Paired antibodies for FIX ELISA 1:200, Cedarlane, but other sources of appropriate antibodies may also be applied) overnight at ambient temperature. After washing plates three times with washing buffer B (Sigma P3583), each well was incubated with 200 pL blocking buffer C

2013202566 05 Apr 2013

-32(Sigma F3888) for one hour at ambient temperature. After another three wash steps with buffer B, serial dilutions of the test sample in buffer B as well as serial dilutions of a substandard (SHP) in buffer B (volumes per well: 100 pL) were incubated for two hours at ambient temperature. After three wash steps with buffer

B, 100 pL of a 1:200 dilution of the detection antibody (Paired antibodies for FIX ELISA, peroxidase labelled, Cedarlane) in buffer B were added to each well and incubated for another two hours at ambient temperature. After three wash steps with buffer B, 100 pL of substrate solution (TMB, Dade Behring, OUVF) were added per well and incubated for 30 minutes at ambient temperature in the dark. Addition of 100 pL undiluted stop solution (Dade Behring, OSFA) prepared the samples for reading in a suitable microplate reader at 450 nm wavelength. Concentrations of test samples were then calculated using the standard curve with standard human plasma as reference.

Example 8: Comparison of FIX-activity/FIX-antigen ratio of different FIXalbumin fusion proteins in cell culture supernatant

Cell culture supernatants of HEK cells transfected with DNA constructs coding for FIX-albumin fusion proteins that contained different linker peptides were subjected to FIX activity and antigen testing as described above (see example 5). The ratio of

FIX:C to FlX:Ag was calculated representing a measure directiy proportional to molar specific activity of the different constructs.

The results shown in table 4 indicate that there is an increase in activity/antigen ratio upon introduction of cieavabie linkers info the FIX-HSA molecule. It also shows that the cieavabie linker peptide should have a length of more than two amino acids in order to provide cieariy increased activity/antigen ratios.

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Table 4: FJX:C/FIX:Ag ratios of FiX-albumin fusion proteins containing different linker peptides

FIX-HSA construct

1182/797

1366/797

1478/863

980/797

986/797

1483/863

1088/797

1365/797

1482/863'

1087/797

1089/797

1091/797

Linker

None

Ri

GGGGGGV (Sheffield et al.)

SS(GGS)₇GS

SSNGS(GGS)3NGS (GGS)3GGNGS

SVSQTSKLTR AETVFPDVD GSGGS

FiX:C/FiX:Ag < 0.031 < 0.068 4~~~

0.041

0.070

0.076

0.688

SVSQTSKLTR AETVFPDVD GS

SVSQTSKLTR AETVFPDVD

SVSQTSKLTR AETVFP

SVSQTSKLTR AETVFPDVD GS (FIX deltaKLT)

QSFNDFTR WGGED GS

PERGDNNLTR IVGGQE GS

0.832

0.680

0.482

0.472

0.532

ΠΓαΐΐΤ

Fold increase compared to fusion protein 980/797 with noncleavabie linker (GGGGGGV)

1.7

1.9

16.8

20.3

15.4

11.8

11.5

13.0

2.7

2013202566 05 Apr 2013

Example 7: Comparison of FIX and RX - albumin fusion proteins in respect to molar specific activity, terminal in vivo half-life and in vivo recovery in rats or rabbits

Purified recombinant wild type FIX (rFiX 496/797) and FIX-aibumin fusion proteins 5 (rFIX 980/797, rFIX 986/797, rFIX~ 1088/797 and rFIX 1089/797) were tested for

FIX activity in a clotting assay as described above. In parallel, the difference of the optical density at 280 and 320 nm was determined as a measure for protein concentration (OD280-320). The ratios of activity per GD280-320 were calculated and based on the molar optical densities the molar specific activities were calculated, in the following table 5 the results are summarized,

Table 5: Molar specific activities of wt FIX compared to FIX-albumin fusions

	Linker	Optical density	FIX clotting activity	Aesviiy/VeSOD	tifleiar specific activity*
		(00280-320)	(ILHnL)	(iU/mL/CD)	{(Ej/nmai)
rFK.wi (496/79?)	. . :	03798	,21,2	55.8	, 4,23
rF IX-KSA (non-deevabie, 1478/863 rFK-KSA (nen-ciesvabie, 880/797)	GGGGGGV <She«rclQ et ai)__ SS (GGS), G5	?91ίθ 1.1122	________5.8	2.0 . 3.0	_β,23 8,36
rFIX-KSA (rort-clesvabie, 9S6/797)	SS MGS (GGSB NGS (GGS)3 GGM G	0.8187	3.2	4.0	8,45
rFIX-KSA (deawbfe. 1888/797) rF IX-H5A ictesvab'e. 1 G83i7S7)	____PXEa ciesvgbig ____ _ rXEgeteavgbte ____ _	.., ... __0.4512	_______ii.s_______ ___1-,3___	........34.8____ _.. 25.0. .	__33? .. ___2^4.

* Molar specific activity based on activity, optical density and the following molar optical densities:

Molar optica! density of FIX: OD{280nm, 1 mol/L) = 75 810 Molar optica! density of albumin: OD{280rtm, 1 mol/L) = 37 791

Molar optica! density of FIX-aibumin fusion protein: OD(280nm, 1 mol/L) = 113 601

Taking the results summarized in Table 5 into account, it is surprising that two constructs that were generated according to the present invention show highly increased molar specific activities compared to the fusion proteins with non2013202566 05 Apr 2013

-3δcleavable linkers. In addition, the molar specific activity of these constructs was only moderately decreased compared to wild type rFIX.

in vitro investigations of the proteolytic cleavage reactions by Factor XIa (FXIa) 5 confirmed that FIX-albumin fusion proteins containing a cieavable linker like e.g. construct no. 1088/797 or 1089/797 are activated and in parallel the linker is cleaved resulting in release of the albumin moiety (Figure 1). The fusion protein with non-cieavable linker did not show a corresponding release of the albumin moiety.

In the case of FVlia as cleaving protease in the presence of tissue factor, the FIXaibumin fusion proteins 1088/797 or 1089/797 containing a cieavable linker also showed release of the albumin moiety in parallel to release of the FIX activation peptide (Data not shown).

In addition to determination of molar specific coagulation activity, the polypeptides no. 498/797, 980/797, 986/797, 1088/797 and 1089/797 described above were administered Intravenously to narcotized CD / Lewis rats (8 rats per substance) and/or rabbits (4 rabbits per substance) with a dose of 50 iU/kg body weight. Blood samples were drawn prior to test substance administration and at appropriate Intervals starting at 5 minutes after administration of the test substances. FIX antigen content was subsequently quantified by an ELISA assay specific for human Factor IX (see above). The mean values of the respective groups were used to caieuiate in vivo recovery after 5 min. Half-iives for each protein were calculated using the time points of the beta phase of elimination (terminal half-life) according to the formula La “ In2 / k, whereas k is the slope of the regression line obtained upon plotting FIX;Ag levels in logarithmic scale and time in linear scale.

Calculated in vivo half-lives are summarized in table 6. In rats as well as in rabbits the in vivo half-lives of the FIX-albumin fusion proteins were found to be significantly increased in comparison to non-fused wild-type recombinant FIX ο

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2013202566 05 Apr prepared inhouse or in comparison to the commercially available recombinant FIX product BeneFIX®. The in vivo half-lives of the albumin fusion proteins compared to BeneFIX® were increased to about 200-400%, depending on the animal species or construct used (Table 6).

To evaluate the in vivo recovery, the FIX antigen levels measured per mL of plasma at their maximum concentrations after intravenous administration (t ~ 5 min) were related to the amount of product applied per kg. Alternatively, a percentage was calculated by relating the determined antigen level (lU/mL) 5 min post infusion to the theoretical product level expected at 100 % recovery (product applied per kg divided by an assumed plasma volume of 40 mL per kg). The in vivo recoveries (IVR) of the FIX-albumin fusion proteins were significantly higher than the in vitro recoveries of rFIX (496/797) or BeneFIX® (Table 7).

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Table 6:

Terminal in vivo half-lives of FIX preparations derived from recombinant expression (BeneFIX®, rFIX 496/797) and FIX albumin fusion proteins (rFIX 980/797, rFIX 986/797, rFIX 1088/797, and rFIX 1089/797) after intravenous administration of 50 lU/kg into rats and/or 50 lU/kg into rabbits, respectively.

	Rat experiments PSR18-05, PSKG6435, PSR02-05	Rabbit experiment PSK11-05
Tesminai halflife (h)	relative to BeneFIX [%]	Terminal halflife (h)	relative to BeneFIX [%J
rFiX 498/797	4,5*	91	n.t	rt.t.
rFIX 380/73?	11,8*	234	36,9° 29,3^2^.5	410 326
rFIX 986/797	10,5*	212	n.t.	n.t.
rFIX 1088/797	8,3*	168	30,3°	337
rFiX 1089/797	10,5*	212	n.t	n.t.
BeneFIX	4,95* (mean of 5,3 and 4,6)	100	3,0°	100

* Determined between 120 and 1440 min ° Determined between 4 and 98 h

2013202566 05 Apr 2013 > 36 «

In vivo recoveries (amount of substance 5 minutes post administration) of recombinant FIX preparations (BeneFIX, rFIX 496/797) and FIX albumin fusion proteins (rFiX 1088/797, rFiX 1089/797) after intravenous administration of 50 iU /kg into rats. The percentage of in vivo recovery was calculated based on an assumed plasma volume of 40 mL/kg.

	rat experiment
	in vivo recovery iU/dL per lU/kg / [%]*	relative to BeneFIX (%]
rFIX 496/797	0.462/18,5	74.6
rFIX 1088/797	1,034/41.4	166.5
rFIX 1089/797	1.083/42.5	171.2
BeneFIX	0.621 / 24.8	100

• Caiciilaisc! based on a plasma volume of 40 mUkg

2013202566 05 Apr 2013

-39Example 8: In vitro activation of FIX albumin fusion proteins with/without cieavabte linker (1088/797 and 980/797) and determination of pharmacokinetics in rats

FIX-albumin fusion proteins and rec FIX were activated in vitro using commercially 5 available Factor Xla (Kordia). Briefly, identical molar amounts of FIX or FIX-albumin fusion protein (3.0 x 10'⁸ mol/L) were activated at 37°C in solution in the presence of FXIa (1,9 x 10⁸ mol/L) and CaCb (1,5 mmol/L) buffered at pH 8.8, After complete activation as shown by SDS-PAGE the reaction was stopped by addition of a 5x molar excess of C1-Inhibitor (Berinert P) based on the amount of FXIa. The samples were stored frozen below ~70“C until start of pharmacokinetic investigation.

A pharmacokinetic investigation of the activated FIX and the FIX-albumin fusion proteins was performed in rats as described in example 7 and the results were compared to a pharmacokinetic results covering ποη-activated fusion proteins.

It turned out that the activated fusion proteins demonstrated significantly reduced half-lifes as well as AUC’s compared to the non-activated molecules (Figure 2). Upon activation the FIX-fusion protein with cleavable linker (1088/797) showed a pharmacokinetic behaviour very similar to activated rec FIX (BeneFIX) whereas the activated fusion protein with non-cleavable linker (980/797) resuited in a higher initial as well as terminal half-life compared to activated fusion protein 1088/797 with cleavable linker. Therefore, the results clearly demonstrate that the cleavable linker results in increased elimination of the coagulation factor after activation and, therefore, avoids accumulation of potentially thrombogenic, activated fusion proteins with extended half-lives.

WO 2007/144173

PCT/EP2007/005246

2013202566 05 Apr 2013 «40*;

Example 9: Comparison of FIX«albumin fusion proteins wtth/wifbout cteavable linker in respect to inactivation rate of the activated coagulation factors by antithrombin 01 (AT)

FIX fusion proteins with (1088/797) and without (980/797) cleavabte linker were 5 activated by incubation with FXia as described in example 8. The activated factors were incubated with AT for 120 min and residual FIXa activity was determined using a manual FIX clotting assay method without activation (naPTT, see below) acc, to Schnitger and Gross. As control samples the activated FIX-albumin fusion proteins were used in presence of the same amount of AT but without incubation.

The FIX activity was determined with the aid of a non-activated partial thromboplastin time assay (naPTT) using FIX deficient plasma from Dade Behring. The samples were prediluted in a buffer of pH 6.8 containing His, Giy, Sucrose, and Tween 80. The whole determination was performed using coagulometers acc. to

Schnitger & Gross. A mixture of 0.1 mi F IX deficient plasma, 0.1 ml sample, and 0,1 ml of 0,1 % Phospholipids (Rhone-Pouienc-Nattermann, 1:3 prediluted in imidazole buffer supplemented with 1 % HSA) was incubated for 2 minutes at 37 °C. The coagulation reaction was initiated by adding 0,1 mi 0.025 mol/l CaClj solution and the clotting time was determined.

Figure 3 shows the results of a corresponding inactivation experiment. In the case of the fusion protein with cleavable linker (1088/797) an increase in clotting time from 210 to 540 sec (factor of 2.57x) demonstrated an accelerated inactivation process of FIXa activity by AT compared to a fusion protein with non-cleavable linker (980/797) that only showed an increase from 196 to 411 sec (factor of 2.10 x). Most probably, the albumin residue sterically affects the AT dependent inactivation process in the case of the fusion protein with non-cleavable linker whereas in the case of the fusion protein with cleavable linker the albumin residue is cleaved off resulting in an accelerated inactivation by AT.

-412013202566 26 Feb 2018

Example 10: Design of FIX-HSA Fusion Proteins with Reduced Immunogenicity

As there is with any fusion between two proteins, a slight risk is associated with a 5 neoepitope being created around the fusion point. It was therefore investigated whether the linker region as described in table 3a and 3b could be modified in order to decrease this risk.

In the course of this investigation all proposed linker sequences and the adjacent regions of FIX and HSA were analyzed for potential T-cell epitopes by way of prediction of binding capability to multiple MHC-II alleles. One of these approaches involved the PreDeFT analysis offered by the company EpiVax (146 Clifford St., Providence, R.l. 02903, USA) in which the input sequences were parsed into overlapping 9-mer frames where each frame overlaps the last by 8 amino acids. Each frame was then assessed for its ability to bind with a set of common HLA. These detailed findings were then summarized producing regional and overall assessments of immunogenic potential. Finally, any epitope clusters identified were screened against the non-redundant protein database at GenBank and EpiVax's own database of known MHC ligands and T-cell epitopes.

As a result of these in silico predictions the following FIX fusion proteins were cloned, expressed and purified

FIX- PVSQTSKLTRAETVFPDV-HSA SEQ ID NO: 113

FIX-PSVSQTSKLTRAETVFPDV-HSA SEQ ID NO: 114

Example 11 Neoantigenicity Test

FIX-HSA fusion proteins comprising linkers SEQ ID NO:113 or 114 can be shown to display a reduced immunogenicity compared to fusion proteins comprising different linkers or to display a comparable immunogenicity as compared to wild type factor FIX by the following neoantigenicity test.

-422013202566 26 Feb 2018

Products to be compared are administered subcutaneously into rabbits with or in the absence of Freund's adjuvant. The endpoint assay is a native Western blot.

A suitable dose of either the FIX-HSA fusion with a linker with reduced immunogenicity or a FIX-HSA fusion with a linker of enhanced immunogenicity or

FIX wild type can be administered as a slow bolus.

A sample can be taken about 30 to 40 days after the start of the immunization and be assayed by a Western blot method to ensure that the rabbits developed antibodies against each of the respective immunogens.

Antibodies against the test sample for which a potentially increased immunogenicity is to be measured are blocked by an excess of a control sample (e.g. wild type FIX) By doing so all of the antibodies present which formed against native epitopes are unable to react when that antibody is used as a probe in a

Western blot. The test and the control sample are run on the Western blot membrane and the blocked antibody is used as a probe.

If there were epitopes on the test samples (here FIX-HSA fusion protein with a linker) but not in the control sample (wild type FIX) that caused antibody formation in the rabbits, these antibodies would be detected after blocking as residual antibodies which would react with the test sample, but not the control sample. Likewise FIX-HSA with a linker having predicted reduced immunogenicity can be used as a control versus a FIX-HSA with a linker having predicted increased immunogenicity as a test sample.

Preferably the same Western blot is assayed with non-blocked antibodies raised against the test sample as a positive control. Here it is expected that test sample as well as control sample are detected in the Western blot assay.

If with blocked antibodies raised against the test sample both test and control sample are not detected it can be concluded that the test sample has no neoepitopes as compared to the control sample.

-432013202566 26 Feb 2018

If with blocked antibodies raised against the test sample only the test sample but not the control sample is detected it can be concluded that the test sample has neoepitopes as compared to the control sample.

For doing the analysis the IgG fraction of the hyper immune pooled serum can be purified using a protein A column from Pierce or other suppliers with a bed volume for example greater than 1 ml according to the instructions of the supplier. Preferably the rabbit sera are dilapidated for example with trichlorotrifluoroethane. Blocking is done for example by mixing 0.1-1.00 mg of the purified antiserum with

0.1 to 100 mg of the control sample in for example a 0.5 to 5 ml centrifuge cup, bringing the volume up to 0.5 to 3 ml. The tubes are then rotated slowly at room temperature of a minimum of 2 hours.

Nonblocked control antibodies to be used as a positive control can be prepared in the same way except that no control sample is used for blocking.

All blocked and nonblocked antibodies can be added to 3 to 4.5 ml of 5% dry milk solution in TBS+0.1% Tween-20 before incubation with their respective membrane.

Preferably Western blots are performed as native Western blots.

2013202566 26 Feb 2018

Claims (28)

1. CLAIMS:

   1. Therapeutic fusion protein comprising

   a) a coagulation factor, wherein the coagulation factor is FIX,

   b) a half-life enhancing polypeptide selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof, and immunoglobulins without antigen binding domain including variants and derivatives thereof, and

   c) a peptide linker which joins the coagulation factor and the half-life enhancing polypeptide;

   wherein the peptide linker is cleavable by proteases involved in coagulation or activated by coagulation enzymes and in that the therapeutic fusion protein has, in comparison to the respective therapeutic fusion protein linked by a non cleavable linker having the amino acid sequence GGGGGGV, an increased molar specific activity in at least one coagulation-related assay.
2. 2. Therapeutic fusion protein comprising

   a) a coagulation factor, wherein the coagulation factor is FIX,

   b) a half-life enhancing polypeptide selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof, and immunoglobulins without antigen binding domain including variants and derivatives thereof, and

   c) a peptide linker which joins the coagulation factor to the N- or Cterminus of the half-life enhancing polypeptide such that the intervening cleavable peptide linker is introduced between the coagulation factor and the half-life enhancing polypeptide;

   wherein the peptide linker is cleavable by proteases involved in coagulation or activated by coagulation enzymes and in that the therapeutic fusion protein has, in comparison to the respective therapeutic fusion protein linked by a non cleavable linker having the amino acid

   2013202566 26 Feb 2018 sequence GGGGGGV, an increased inactivation rate of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode.
3. 3. Therapeutic fusion protein comprising

   a) a coagulation factor, wherein the coagulation factor is FIX,

   b) a half-life enhancing polypeptide selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof, and immunoglobulins without antigen binding domain including variants and derivatives thereof, and

   c) a peptide linker which joins the coagulation factor to the N- or Cterminus of the half-life enhancing polypeptide such that the intervening cleavable peptide linker is introduced between the coagulation factor and the half-life enhancing polypeptide;

   wherein the peptide linker is cleavable by proteases involved in coagulation or activated by coagulation enzymes and in that the therapeutic fusion protein has, in comparison to the respective therapeutic fusion protein linked by a non cleavable linker having the amino acid sequence GGGGGGV, an increased elimination rate of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode.
4. 4. Therapeutic fusion protein according to any one of claims 1 to 3, wherein said fusion protein has a higher in vivo recovery compared to the in vivo recovery of the respective coagulation factor which is not fused to a halflife enhancing polypeptide.
5. 5. Therapeutic fusion protein according to any one of claims 1 to 4, wherein said fusion protein has an increased half-life in plasma compared to the half-life in plasma of the respective coagulation factor which is not fused to a half-life enhancing polypeptide.

   2013202566 26 Feb 2018
6. 6. Therapeutic fusion protein according to any one of claims 1 to 5, wherein the coagulation factor is a vitamin-K dependent coagulation factor.
7. 7. Therapeutic fusion protein according to any one of claims 1 to 6, wherein the half-life enhancing polypeptide is an immunoglobulin without an antigen binding domain, or a fragment or derivative thereof.
8. 8. Therapeutic fusion protein according to any one of claims 1 to 7 wherein the half life enhancing polypeptide is an immunoglobulin Fc region.
9. 9. Therapeutic fusion protein according to any one of claims 1 to 8, wherein the peptide linker is cleavabie by FXIa and/or FVIIa/TF.
10. 10. Therapeutic fusion protein according to any one of claims 1 and 4 to 9, wherein the molar specific coagulation-related activity of the therapeutic fusion protein is increased at least 25% compared to that of the therapeutic fusion protein linked by a non cleavabie linker having the amino acid sequence GGGGGGV in at least one of the different coagulation-related assays available.
11. 11. Therapeutic fusion protein according to any one of any one of claims 2 and 4 to 10, wherein the inactivation rate of the coagulation factor after cleavage of the peptide linker which links the coagulation factor to the halflife enhancing polypeptide is increased by at least 10% as compared to the inactivation rate of the coagulation factor in a corresponding therapeutic fusion linked by a non-cleavable linker having the amino acid sequence GGGGGGV.
12. 12. Therapeutic fusion protein according to any one of claims 3 to 11, wherein the elimination rate of the coagulation factor after cleavage of the peptide linker which links the coagulation factor to the half-life enhancing polypeptide is increased by at least 10% as compared to the elimination rate of the coagulation factor in a corresponding therapeutic fusion protein

    2013202566 26 Feb 2018 linked by a non-cleavable linker having the amino acid sequence GGGGGGV.
13. 13. Therapeutic fusion protein according to any one of claims 1 to 12, wherein the linker is cleavable by the protease or proteases, which activate the coagulation factor.
14. 14. Therapeutic fusion protein according to claim 13, wherein the kinetics of the peptide linker cleavage by the protease or proteases is not delayed by more than a factor of 3 compared to the kinetics of the activation of said coagulation factor.
15. 15. Therapeutic fusion protein according to any one claims 1 to 14, wherein the peptide linker comprises a sequence selected from the group consisting of linkers with one cleavage site and linkers with two cleavage sites and as defined herein in tables 3a and 3b.
16. 16. Therapeutic fusion protein according to any one of claims 1 to 15, wherein the peptide linker comprises SEQ ID NO: 113.
17. 17. Therapeutic fusion protein according to any one of claims 1 to 16 when used as a pharmaceutical substance.
18. 18. A polynucleotide encoding a therapeutic fusion protein according to any one of claims 1 to 17.
19. 19. A host cell comprising a polynucleotide according to claim 18.
20. 20. A method of producing a therapeutic fusion protein according to any one of claims 1 to 16, comprising culturing host cells according to claim 19 under conditions such that the therapeutic fusion protein is expressed.

    2013202566 26 Feb 2018
21. 21. A pharmaceutical composition comprising a therapeutic fusion protein according to any one of claims 1 to 16 or a polynucleotide according to claim 18.
22. 22. A pharmaceutical composition according to claim 21 formulated for intravenous or subcutaneous administration.
23. 23. The use of a therapeutic fusion protein according to any one of claims 1 to 16, of a polynucleotide according to claim 18, or of a host cell according to claim 19 for the manufacture of a medicament for the treatment or prevention of a blood coagulation disorder.
24. 24. The use according to claim 23, wherein the blood coagulation disorder is haemophilia B.
25. 25. The use according to claim 23 or 24, wherein the treatment comprises human gene therapy.
26. 26. A method of treatment or prevention of a blood coagulation disorder, comprising administering to a person in need of such treatment an effective amount of a therapeutic fusion protein according to any one of claims 1 to 16.
27. 27. The method of claim 26, wherein the blood coagulation disorder is haemophilia B.
28. 28. The method according to claim 26 or 27, wherein the person is administered the therapeutic protein intravenously or subcutaneously.

    CSL BEHRING GMBH

    WATERMARK PATENT AND TRADE MARK ATTORNEYS

    P31188AU01

    2013202566 05 Apr 2013

    FiX-HSA

    HC*KSA

    HSA

    FJX

    HC tc t«e 2 S 10 30 08 52ΰ240 430™π

    U78/883

    0 5 5 to 30 «0 t2Q2454£0min

    1088/797

    2 S 10 2C «0120 2i04iSmto

    1089/797

    2013202566 05 Apr 2013

    2/3

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    3/3

    2013202566 05 Apr 2013

    2OQ6_MQO4._All5_.PCT.ST2 5 SEQUENCE LISTING <110> CSL Behring GmbH csl Behring GmbH <12Q> Proteolytically cieavabie fusion proteins with high molar specific activity <130> 2OO6J4QO4_A115 <150> EP06012262.9 <151> 2006-06-14 <160> 112

    <170> ! Patentin version 3.3 <2io> : 1 <211> 585 <212> ! PRT <213> 1 Homo sapiens <400> : 1 ASD Ala Hi s Lys Ser Glu val Ala Hi s Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu val Leu lie Ala Phe Ala Gin Tvr Leu Gin 20 25 30 Gin Cys Pro Phe Glu Asp His val Lys Leu Val Asn Glu val Thr Glu 35 40 45 Phe Ala Lys Thr Cys val Ala Asp Glu Ser Ala Gl u Asn cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu cys Thr val Ala Thr Leu 65 70 75 so Arg Glu Thr Tyr Gly 85 Glu Met Ala Asp Cys 90 cys Ala Lys Gin Glu 95 Pro Glu Arg Asn GlU cys phe Leu Gin His Lys Asp Asp Asn Pro Asn Leu 100 105 110 pro Arg Leu val Arg Pro Gl u val ASp val Met cys Thr Ala Phe Hi s 115 120 125 Asp ASH Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu lie Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala pro Gl u Leu Leu Phe Phe Al a Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala Asp Lys Ala Al a 165 170 175

    Page 1

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    2OO6_MOO4_A115_PCT.ST2 5

    Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gin Arg Leu Lys cys Al a Ser Leu Gin Lys Phe Gly Glu 195 200 205 Arg Al a Phe Lys Ala Trp Ala val Ala Arg Leu ser Gl n Arg Phe Pro 210 215 220 Lys Ala Gl u Phe Ala Gl ll Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val Hi s Thr Glu Cys cys Hi s Gly ASp Leu Leu Glu cys Ala ASp ASp 245 250 255 Arg Al a Asp Leu Ala Lys Tyr lie Cys Glu Asn Gin Asp Ser He ser 260 265 270 Ser Lys Leu Lys Glu Cys cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 cys He Ala Glu val Glu Asn ASp Glu Met Pro Ala ASp Leu Pro ser 290 295 300 Leu Ala Ala Asp Phe Val Glu ser Lys Asp val cys Lys Asn Tyr Ala 305 310 315 320 Gl u Ala Lys Asp Val Phe Leu dy Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg Hi s pro Asp Tyr Ser Val val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys cys cys Ala Ala Ala ASp Pro Hi s Gl U 355 360 365 cys Tyr Ala Lys val Phe Asp Glu Phe Lys pro Leu Val Glu Gl u Pro 370 375 380 Gin Asn Leu lie Lys Gin Asn cys Glu Leu Phe Glu Gin Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gin Asrt Ala Leu Leu Val Arg Tyr Thr Lys Lys val pro 405 410 415 Gin val ser Thr Pro Thr Leu Val Gl u Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys cys Cys Lys Hi s Pro Glu Ala Lys Arg Met Pro cys

    435 440 445

    Page 2

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    2006_M004_All5._PCr.5T2 5

    Ala Glu ASp 450 Tyr Leu Ser val 455 val Leu Asn Gin Leu cys 460 val Leu Hi s Glu Lys Thr Pro Val ser ASp Arg Val Thr Lys Cys cys Thr Glu Ser 465 470 475 480 Leu val Asn Arg Arg Pro cys phe Ser Ala Leu Glu Val Asp Gl u Thr 485 490 495 Tyr val pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 lie cys Thr Leu ser Glu Lys Glu Arg Gin Xie Lys Lys Gin Thr Ala 515 520 525 Leu val Glu Leu val cys His Lys Pro Lys Ala Thr lvs Glu Gin Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe val Glu Lys Cys cys Lys 545 550 555 560 Ala ASp Asp Lys Glu Thr cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Glπ Ala Ala Leu Gly Leu 580 585 <210> 2 <211> 415 <212> PRT <213> Homo sapiens <400> 2 Tyr Asn Ser ely Lvs Leu Glu Glu Phe val Gin Gly Asn Leu Glu Arg 1 5 10 15 Gl u Cys Met Glu Glu Lys cys Ser Pne Glu Glu Ala Arg Glu val Phe 20 25 30 Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gin Tyr Val Asp Gly 35 40 45 ASp Gin cys Glu ser Asn pro cys Leu Asn Gly Gly ser Cys Lys ASp 50 55 60 Asp lie Asn Ser Tyr Gl U cys Trp Cys Pro Phe Gly Phe Glu Gly Lys 65 70 75 80 Asn cys Glu Leu Asp val Thr cys Asn lie Lys Asn Gly Arg Cys Gl u

    85 90 95

    Gin Phe cys Lys Asn ser Ala Asp Asn Lys val val cys ser Cys Thr Page 3

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    2OO6_MOO4_A115_PCT. ST25 100 105 110

    Glu Gly Tyr Arg Leu Ala Glu Asn Gin Lys 120 Ser Cys Glu 125 Pro Ala val 115 Pro Phe Pro cys Gly Arg val Ser val Ser Gin Thr Ser Lys Leu Thr 130 135 140 Arg Ala Glu Thr Val Phe Pro Asp val Asp Tyr val Asn Ser Thr Glu 145 150 155 160 Ala Gl U Thr lie Leu ASp Asn lie Thr Gl n ser Thr Gin ser Phe Asn 165 170 175 Asp Phe Thr Arg Val Val Gly Gly Glu ASp Ala Lys Pro Gly Gl n Phe 180 185 190 Pro Trp Gl n Val Val Leu Asn Gl y Lys Val Asp Al a Phe cys Gly sly 195 200 205 Ser lie val Asn Glu Lys ”rp lie Val Thr Al a Ala Hi s Cys Val Gl u 210 21.5 220 Thr Gly val Lys lie Thr val Val Ala Gly Gl U His Asn lie Glu Glu 225 230 235 240 Thr Gl u Hi s Thr Glu Gl n Lys Arg Asn val He Arg He He pro His 245 250 255 Hi s Asn Tyr Asn Ala Ala He Asn Lys Tyr Asn Hi s ASp lie Al a Leu 260 265 270 Leu Glu Leu Asp Gl u Pro Leu Val Leu Asn Ser Tyr Val Thr Pro lie 275 2.80 285 cys lie Ala Asp Lys Glu Tyr Thr Asn lie Phe Leu Lys Phe Gly Ser 290 295 300 Gly Tyr val Ser Gly Trp Gly Arg val Phe His Lys Gly Arg Ser Ala 305 310 315 320 Leu Val Leu Gin Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys 325 330 335 Leu Arg Ser Thr Lys Phe Thr lie Tyr Asn Asn Met Phe cys Ala Gly 340 345 350 Phe Hi s GlU Gly Gly Arg Asp Ser cys Gin Gly Asp Ser Gly Gly Pro 355 360 365 His Val Thr Glu val Gl Li Gl y Thr ser Phe Leu Thr Gly lie lie ser

    Page 4

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    2006_M004_All5_PCT.ST25 370 375 380

    Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly lie Tyr Thr Lys 385 390 395 400 val ser Arg Tyr val Asn Trp lie Lys Glu Lys Thr Lys Leu Thr 405 410 415 <210> 3 <211> 1386 <212> DHA <213> Homo sapiens <400> 3

    atgcagcgcg tgaaeatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt 120 ctgaatcggc oaaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180 gagagagaat gtatggaaga aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac 240 actgaaagaa caactgaatt ttggaagcag tatgttgatg gagatcagtg tgagtccaat 300 ccatgtttaa atggcggcag ttgcaaggat gacattaatt cctatgaatg ttggtgtccc 360 tttggatttg aaggaaagaa ctgtgaatta gatgtaacat gtaacattaa gaatggcaga 420 tgcgagcagt tttgtaaaaa tagtgctgat aacaaggtgg tttgctcctg tactgaggga 480 tatcgacttg cagaaaacca gaagtcctgt gaaccagcag tgccatttcc atgtggaaga 540 gtttctgttt cacaaacttc taagctcacc cgtgctgaga ctgtttttce tgatgtggac 600 tatgtaaatt ctactgaagc tgaaaeeatt ttggataaca tcactcaaag cacccaatca 660 tttaatgact tcactcgggt tgttggtgga gaagatgeca aaccaggtca attccettgg 720 caggttgttt tgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780 tggattgtaa ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt 840 gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgat tcgaattatt 900 cctcaccaca actaeaatgc agctattaat aagtacaacc atgacattgc ccttctggaa 960 ctggacgaac ccttagtgct aaacagctac gttacaccta tttgcattgc tgacaaggaa 1020 tacacgaaca tcttcctcaa atttggatct ggctatgtaa gtggctgggg aagagtcttc 1080 cacaaaggga gatcagcttt agttcttcag taccttagag ttctacttgt tgaccgagcc 1140 acatgtcttc gatctacaaa gttcaccatc tataacaaca tgttctgtgc tggcttccat 1200 gaaggaggta gagattcatg tcaaggagat agtgggggac cccatgttac tgaagtggaa 1260 gggaccagtt tcttaactgg aattattagc tggggtgaag agtgtgcaat gaaaggcaaa 1320 tatggaatat ataccaaggt atcccggtat gtcaactgga ttaaggaaaa aacaaagctc 1380

    acttaa 1386 <210> 4 <211> 1830

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    <212> DNA <213> Home ( sapiens 2006. _MOO4_A115„ PCT.ST25 <400> 4 atgaagtggg taacctttat tteccttctt tttetcttta geteggetta ttccaggggt 60 gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120 gaaaatttca aagccttggt gttgattgee tttgctcagt atcttcagca gtgtccattt 180 gaagatcatg taaaattagt gaatgaagta aetgaatttg caaaaacatg tgttgctgat 240 gagteagetg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300 gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360 gagagaaatg aatgettett gcaacacaaa gatgaeaacc caaacctccc cegattggtg 420 agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480 aaataettat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540 tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600 tgcctgttgc caaagctcga tgaaettegg gatgaaggga aggettegte tgccaaacag 660 agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720 gctegcctga gecagagatt tcccaaagct gagtttgcag aagcttccaa gttagtgaca 780 gatcttacca aagtccacac ggaatgctgc catggagate tgettgaatg tgctgatgac 840 agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900 gaatgctgtg aaaaacctct gttggaaaaa tcccaetgea ttgeegaagt ggaaaatgat 960 gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020 aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgeaaga 1080 aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140 ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200 tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga getttttgag 1260 cagcrtggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320 caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380 tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggte 1440 ctgaaccagt tatgtgtgtt geatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500 tgcacagaat ccttggtgaa caggcgacca tgettttcag ctctggaagt cgatgaaaca 1560 tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgeagatat atgeacaett 1620 tetgagaagg agagacaaat caagaaacaa actgcacttg ttgagctcgt gaaacacaag 1680 eeeaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740 aagtgctgca aggetgaega taaggagacc tgctttgccg aggagggtaa aaaaettgtt 1800 gctgcaagtc aagctgcctt aggettataa 1830

    <210> 5 page 6

    2013202566 05 Apr 2013

    2006....M004...A115...PCT, ST25 <211» 21 <212» DNA <213» Artificial Sequence <220» <223» Artificial <400» 5 ccactttcac aatctgctag c ' IX <210> 6 <211> 23 <212» DNA <213» Artificial sequence <220» <223» Artificial <400» 6 caattccaat gaattaacct tgg 23 <210» 7 <211» 21 <212» DNA <213> Artificial Sequence <220» <223» Artificial <400» 7 .

    atgcagcgcg tgaacatgat c IX <210» 8 <211> 25 <212» DNA «213» Artificial sequence <220» <223» Artificial <400» 8 tcattaagtg agctttgttt tttcc 23 <210» 9 <211» 21.

    <212» DNA <213» Artificial Sequence <220» <223» Artificial <400» 9 gattcgaatt cgcccttatg c 21

    <210» 10 <211» 32 <212> DNA <213» Artificial sequence <220» <223> Artifici al <400» 10

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    2 006J4004_All5__PCT. ST2 5 cgctcgaggt gagctttgtt ttttccttaa tc 32 <210> 11 <211> 52 <212> DNA <213> Artificial sequence <22Q>

    <223> Artificial <400> 11 ctcgagcggg ggatctggcg ggtctggagg ctctggaggg tcgggaggct ct <210> 12 <211> 46 <212> DNA <213> Artificial Sequence <22Q>

    <223> Artificial <400> 12 ggatccagat cccccagagc ctxcagagcc tcccgaccct ccagag 46' <210> 13 <211> 56 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 13 ctcgagcaat ggatctggcg ggtctggagg ctctggaggg tcgaatggct ctggag 56 <210> 14 <211> 64 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 14 ggatccgttc cctccagacc cgccagatcc cccagagcct ccagagccat tcgaccctcc 60 agag 64 <210> 15 <211> 46 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 15 cctcgagctc tgtgagccag acctccaagc tcaccagggc cgagac 46 <210> 16 <2.11> 44 <212> DNA

    Page 8

    2013202566 05 Apr 2013

    2006„M004„^115_mPCT.ST25 <213> Artificial Sequence <220>

    <223> Artificial <400> 16 gggatccgfc cacatcaggg aagacagtct cggccctggt gage <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <22O>

    <223> Artificial <400> 17 ggaaaaaaca aagctcactt ctgtgagcca gac

    <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <22O> <223> Artificial <4OO> 18

    gtctggctca cagaagtgag ctttgttttt tcc <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <223> Artificial <400> 19 cctcgagcag agcttcaatg acttcacccg ggtggtgg 38 <210> 20 <211> 41 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 20 gggatccatc ctccccgccc accacccggg tgaagtcatt g 41 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 21 ggaaaaaaca aagctcacte agagcttcaa tgac '34

    Pace 9

    2013202566 05 Apr 2013 <21Q>

    <211>

    <212>

    <213>

    <220>

    <223>

    2OQ6_MOQ4_A115_PCT.ST2 5

    DNA

    Artificial Sequence

    Artificial <400> 22 gtcattgaag ctctgagtga getttgtttt ttcc <210> 23 <211> 31 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 23 gtgggatccg atgcacacaa gagtgaggtt g M <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 24 cacggatccc tataagccta aggcagcttg acttg <210> 25 <211> IS <212> DNA <213> Artificial Sequence <220>

    <223> Artificial <400> 25 ., caaggagacg ggcgctcc IS <210> 26 <211> 19 <212> DNA <213> Artificial sequence <220>

    <223> Artificial <400> 26 gcccaaggag gggattggc IS <210> 2?

    <211> 30 <212> DNA <213> Artificial Sequence <22O>

    <223> Artificial

    Page 10

    2006_M004_A115„PCT.ST25

    2013202566 05 Apr 2013 <4OO> 27 .

    gtggaattca tggagctgag gccctggttg 30 <210> 28 <2U> 38 <212> DMA <213> Artificial sequence <22O>

    <22 3> Artificial <400> 28 .

    cacgcggccg ctcactacag ccgttgcccc gcctccac 38 <210> 29 <211> 704 <212> PRT

    <213> Homo Sap· iens <400> 29 Met Glu Leu Arg pro Trp Leu Leu Trp val Val Ala Ala Thr Gly Thr 1 5 10 15 Leu Val Leu Leu Ala Ala Asp Ala Gin Gly Gin Lys Val Phe Thr Asn 20 25 30 Thr Trp Ala val Arg lie Pro Gly Gly Pro Ala val Ala Asn Ser Val 35 40 45 Ala Arg Lys His Gly Phe Leu Asn Leu sly Gin lie Phe Gly ASp Tyr 50 55 60 Tyr His phe Trp His Arg Gly Val Thr Lys Arg Ser Leu ser Pro His 65 70 75 80 Arg Pro Arg His Ser Arg Leu Gin Arg Glu Pro Gl n Val Gin Trp Leu 85 90 95 Gl u Gin Gin val Ala Lys Arg Arg Thr Lys Arg Asp val Tyr Gin Gl u 100 105 110 Pro Thr Asp Pro Lys Phe Pro Gin Gin Trp Tyr Leu Ser Gly val Thr 115 120 125 Gin Arg Asp Leu Asn Val Lys Ala Ala Trp Ala Gin Gly Tyr Thr G1 y 130 135 140 Hi s Gly lie Val val Ser lie Leu ASp ASp Gly lie Glu Lys Asn His 145 150 155 160 pro ASp Leu Ala Gly Asn Tyr Asp Pro Gly Ala ser Phe Asp val Asn

    165 170 175

    Page 11

    2013202566 05 Apr 2013

    Asp Gin Asp Pro 180 Asp Pro Gin 2OO6_MOO4_A115_ PCT.ST25 Pro Arg 185 Tyr Thr Gin Met Asn 190 ASp Asn Arg Hi s Gly Thr Arg Cys Al a Gl y Gl U val Ala Al a val Al a Asn Asn 195 200 205 Gly val Cys Gly Val Gly val Ala Tyr Asn Ala Arg lie Gly Gly val 210 215 220 Arg Met Leu ASp Gly Glu Val Thr ASp Al a Val Glu Ala Arg Ser Leu 225 230 235 240 Gly Leu Asn pro Asn His lie Hi s lie Tyr Ser Al a Ser Trp dy Pro 245 250 255 Glu Asp ASp Gl y Lys Thr val ASp Gly Pro Ala Arg Leu Ala Glu Glu 260 265 270 Ala Phe Phe Arg dy Val Ser Gin Gly Arg Gly Gly Leu Gly Ser lie 275 280 285 Phe val Trp Ala Ser Gly Asn Gly Gly Arg Glu Hi s Asp Ser cys Asn 290 295 300 Cys Asp dy Tyr Thr Asn ser lie Tyr Thr Leu Ser lie Ser Ser Ala 305 310 315 320 Thr Gin Phe Gly Asn Val Pro Trp Tyr ser Glu Ala cys Ser Ser Thr 325 330 335 Leu Ala Thr Thr Tyr ser Ser Gly Asn Glrs Asn Glu Lys Gin lie val 340 345 350 Thr Thr Asp Leu Arg Gin Lys cys Thr Glu Ser His Thr Gly Thr Ser 355 360 365 Al a ser Ala pro Leu Ala Ala Gly ll e lie Ala Leu Thr Leu Glu Ala 370 375 380 Asn Lys Asn Leu Thr Trp Arg ASp Met Gin Hi s Leu val val Gin Thr 385 390 395 400 Ser Lys pro Ala His Leu Asn Ala Asn Asp Trp Al a Thr Asn Gl y val 405 410 415 Gly Arg Lys val Ser His Ser Tyr Gly Tyr Gly Leu Leu Asp Ala Gly 420 425 430 Al a Met val Ala Leu Ala Gin Asn Trp Thr Thr val Ala Pro Gin Arg 435 440 445

    page 12

    2013202566 05 Apr 2013

    Lys cys lie lie Asp 450 lie 2006„M004_All5_PCT.ST25 Leu 455 Thr Glu Pro Lys ASp 460 lie Gly Lys Arg Leu Gl u val Arg Lys Thr Val Thr Ala cys Leu Gly Gl u pro Asn Hi s 465 470 475 480 lie Thr Arg Leu Gl u His Ala Gin Ala Aro Leu Thr Leu Ser Tyr Asn 485 490 495 Arg Arg Gly Asp Leu Ala lie His Leu val Ser pro Het Gl y Thr Arg 500 505 510 ser Thr Leu Leu a! a Ala Arg Pro His Asp Tyr Ser Ala Asp Gly Phe 515 520 525 Ash Asp Trp Ala Phe Thr Thr Thr Hi s ser Trp Asp Glu ASp pro Ser 530 535 540 Gly Glu Trp val Leu Gl u He Glu Asn Thr Ser Glu Al a Asn Asn Tyr 545 550 555 560 Gly Thr Leu Thr Lys Phe Thr Leu val Leu Tyr Gly Thr Ala pro Glu 565 570 575 Gly Leu pro val Pro Pro Gl u ser ser Gly cys Lys Thr Leu Thr Ser 580 585 590 Ser Gin Ala Cys Val val cys Glu Gl U Gly Phe Ser Leu Hi s Gin Lys 595 600 605 Ser Cys val Gin His Cys Pro pro Gly Phe Ala Pro Gin Val Leu ASp 610 615 620 Thr His Tyr Ser Thr Glu ASH ASp Val Glu Thr II e Arg Ala Ser val 625 630 635 640 Cys Ala Pro Cys His Ala Ser Cys Ala Thr Cys Gin Gly Pro Ala Leu 645 650 655 Thr ASp cys Leu Ser Cys Pro ser His Ala Ser Leu Asp Pro val Glu 660 665 670 Gin Thr cys Ser Arg Gin Ser Gin Ser Ser Arg Glu ser Pro pro Gin 675 680 685 Gl n Gin Pro Pro Arg Leu Pro Pro Gl u Val Glu Ala Gly Gl Π Arg Leu 690 695 700

    <210> 30 <211> 25 <212> PRT <213> Artificial

    Page 13

    2013202566 05 Apr 2013

    2006„M004^All5„PCT,ST2 S <220>

    <223» Linker <400> 30 ser ser Gly Gly ser Gly Gly ser Gly Glv Ser Gly Gly Ser Gly Gly 1 5 10' 15

    Ser Glv Gly Ser Gly Gly Ser Gly Ser ' 20 25 <210> 31 <211> 31 <212> PRT <213» Artificial <220>

    <223> Linker <400> 31 ser ser Asrt Glv ser Gly Gly Ser Glv Gly Ser Gly Gly Ser Asn Gly 1 ' 5 10 15

    Ser Gly Gly ser Gly Gly Ser Gly Gly Ser Gly Gly Asn Gly Ser 20 25 30 <210> 32 <211> 17 <212> PRT <213> Artificial <220» <223> Linker <400> 32

    Arg Ala Glu Thr val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu

    1 5 Al 3l

    <210» 33 <211> 14 <212> PRT <213> Artificial <220> <223» Li nker <400> 33

    Arg Ala Glu Thr val Phe Pro Asp Val Asp Tyr val Asn Ser

    1 5 <210» 34 <211> 17 <212> PRT <213» Artificial

    Page 14

    2013202566 05 Apr 2013

    2OO6_MOO4_A115„PCT.ST25 <220» <223» Linker <400» 34

    Arg Ala Glu Ala val Phe Pro Asp Val Asp Tyr val Asn ser Thr Glu

    1 5 Ala

    <210» 35 <211> 19 <212» PRT <213» Artificial <220» <223> Li nker <400» 35

    Arg Ala Glu Ala Val Phe Pro Asp val Asp Tyr val Asn Ser Thr Git! 1 5 10 15

    Ala Gly ser <210» 36 <211» 19 <212» PR.T <213» Artificial <220>

    <223» Linker <400» 36

    Ser Val ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr val Phe Pro 1 5 10 15

    Asp val Asp

    <210» 37 <211» 21 <212> PRT <213> Artificial <220» <223» Linker <400» 37 Ser val Ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr val Phe Pro 1 5 10 15

    Asp val Asp Gly Ser 20

    Page 15

    2013202566 05 Apr 2013

    2006_M004_All5_PCT. ST25 <210> 38 <211> 19 <212> PRT <213> Artificial <220>

    <223> Linker <400> 38

    Ser Val ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 15 10 15

    Asp val Asp <210> 39 <211> 21 <212> PRT <213> Artificial <220>

    <223> Linker <400> 39

    Ser Val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 15 10 15

    Asp val Asp Gly Ser 20 <210> 40 <211> 22 <212> PRT <213> Artificial <22O>

    <223> Linker <400> 40

    Ser Val Ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr val Phe Pro 15 10 15

    Asp val Asp Asn Gly Ser 20

    <210> 41 <211> 19 <212> PRT <213> Artifici al <220> <223> Li nker <400> 41 Ser Val Ser Gin Thr ser Lys Leu Thr Arg Gly Glu Thr val Phe Pro 1 5 10 15

    Page 16

    2013202566 05 Apr 2013

    Asp Val Asp

    2006_M004_All5_PCT.ST25 <210> 42 <211> 19 <212> PRT <213> Artificial <220>

    <223> Linker <400> 42 ser val ser Gin Thr Ser Lys Leu Thr Arg Thr Glu Tbr val Phe Pro 15 10 15

    Asp Val Asp

    <210> 43 <211> 19 <212> PRT <213> Artificial <220> <223> Linker <400> 43 Ser Val ser Gin Thr Ser Lys Leu Thr Arg ser Glu Thr val Phe Pro 1 5 10 15

    Asp val Asp <210> 44 <211> 19 <212> PRT <213> Artificial <220>

    <223> Linker <400> 44

    Ser val Ser Gin Thr Ser Lys Leu Thr Arg Leu Glu Thr Val Phe Pro 15 10 15

    Asp val Asp

    <210> <211> <212> <213> 45 19 PRT Artificial <22O> <223> Li nker <400> 45

    Page 17

    2013202566 05 Apr 2013

    2006„MQ04_A115„PCT.ST2 5

    Ser Val Ser Gin Thr Ser Lys Leu Thr Arg Thr Glu Ala val Phe Pro 1 5 10' 15

    Asp Val Asp <210> 46 <211> 19 <212> PRT <213> Artificial <220>

    <223> Linker <400> 46

    Ser val ser Gin Thr Ser Lys Leu Thr Arg Gly Glu Ala Val Phe Pro 15 10 15

    Asp Val Asp <210> 47 <211> 14 <212> PRT <213> Artificial <22Q>

    <223> Linker <400> 47

    Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp val Asp 1 5 10 <210> 48 <211> 14 <212> PRT <213> Artificial <220>

    <223> Linker <400> 48

    Gin ser Phe Asn Asp Phe Thr Arq Val val Gly Gly Glu Asp 1 5 ~ 10 <210> 49 <211> 16 <212> PRT <213> Artificial <220>

    <223> Linker <400> 49

    Gin Ser Phe Asn Asp Phe Thr Arg val val Gly Gly Glu Asp Gly ser 1. 5 10 15

    Page 18

    2OQ6„MQO4_A115„PCT. ST25

    2013202566 05 Apr 2013 <210» 50 <211> 14 <212» PRT <213» Artificial <220» <223» Linker <400» 50

    Gin Ser Phe Asn Asp Phe Thr Arg Thr val Gly Gly Glu Asp 1 5 10 ' <210> 51 <211> 14 <212» PRT <213> Artificial <220» <223» Linker <400> 51

    Gin ser Phe Asn Asp Phe Thr Arg Leu val Gly Gly Glu Asp 1 5 10 <210» 52 <211» 14 <212» PRT <213» Artificial <220» <223» Linker <400» 52

    Gin Ser Phe Asn Asp Phe Thr Arg Gly Val Gly Gly Glu Asp 1 5 10 <210» 53 <211» 17 <212» PRT <213» Artificial <220» <223» Linker <400» 53

    Gin Ser Phe Asn Asp Phe Thr Arg val val Gly Gly Glu Asp Asn Gly 15 10 15

    Ser <210» 54 <211» 15 <212» PRT <213» Artificial <220» <223» Linker

    Page 19

    2013202566 05 Apr 2013

    2QQ6_M0Q4__All5__PCT. ST2 5 <4OO> 54

    Gin Ser Phe Asn Asp Phe Thr Arg Val val Gly Gly Glu Asp Asn

    1 5 <21O> 55 <211> 7 <212> PRT <213> Arti fi ci al <22O> <223> Li nker <400> 55

    Arg lie val Gly Gly Gin Glu 1 5 <21Q> 56 <211> 7 <212> PRT <213> Artificial <220>

    <223> Linker <400> 56

    Arg Leu Val Gly Gly Gin Glu 1 5 <21O> 57 <211> 7 <212> PRT <213> Artificial <220>

    <223> Linker <400> 57

    Arg Thr Val Gly Gly Gin Glu 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial <22O>

    <223> Linker <400> 58

    Arg val Val Gly Glv Gin Glu 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial <220>

    Page 20

    2013202566 05 Apr 2013 <223> Linker <400> 59

    Arg Ala Val Gly Gly Gin Glu 1 5

    2006_M004_A115„PCT.ST25 <210> 60 <211> 7 <212> PRT <213> Artificial <220>

    <223> Linker <400> 60

    Arg Gly Val Gly Gly Gin Glu 1 5 <210> 61 <211> 16 <212> PRT <213> Artificial <22O>

    <223> Linker <400> 61 pro Glu Arg Gly Asp Asn Asn Leu Thr Arg lie val Gly Gly Gin Glu

    1 5 <210> <211> <212> <213> 62 19 PRT Artificial <220> <223> Linker <400> 62

    Ser Val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 15 10 15

    Asp Val Asp <210> 63 <211> 15 <212> PRT <213> Artificial <220>

    <223> Linker <400> 63

    Gin Ser Phe Asn Asp Phe Thr Arg val Val Glv Gly Glu Asp Asn 1 5 10 15

    Page 21

    2013202566 05 Apr 2013

    2O06J4OO4_A115_.PCT.ST25 <210> 64 <2U> 19 <212> PRT <213> Artificial <22Q>

    <22 3> Linker <400> 64 ser val ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr val Pbe Pro 15 10 15

    Asp val Asp <210> 65 <211> 16 <212> PRT <213> Artificial <220>

    <223> Linker <400> 65

    Lys Arg Asrt Ala Ser Lys Pro Gin Gly Arg lie val Gly Gly Lys val 1 ' 5 10* '15 <210> 66 <211> 16 <212> PRT <213> Artificial <220>

    <223> Linker <400> 66

    Pro Glu Glu Pro Gin Leu Arg Met Lys Asrt Asrt Glu Glu Ala Glu Asp 15 10 15 <210> 67 <211> 19 <212> PRT .

    <213> Artificial <220>

    <223> Linker <400> 67

    Asp Asn Ser pro Ser Phe Xie Gin lie Arg ser val Ala Lys Lys His 15 10 15 pro Lys Thr <210> 68 <211> 20 <212> PRT <213> Artificial

    Page 22

    ST25

    2013202566 05 Apr 2013 <220» <223» Linker <400» 68

    Leu Ser Lys Asn Asn Ala lie Glu Pro Arg ser Phe Ser Gin Asn Ser 1 5 10 15

    Arg His Pro Ser 20 <210> 69 <211» 21 <212» PRT <213> Artificial <220» <223» Linker <400> 69

    Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 1 5 10 15

    His Tvr Phe lie Ala ' 20 <210» 70 <211» 16 <212» PRT <213» Artificial <220» <223» Linker <400» 70

    Ser Pro His val Leu Arg Asn Are Ala Gin Ser Gly Ser Val Pro Gin 1 5 10 15 <210» 71 <211» 24 <212» PRT <213» Artificial <220» <223» Linker <400» 71

    Pro Glu Glu Pro Gin Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp 15 10 15

    Tyr Asp Asp Asp Leu Thr Asp ser

    20 <210» 72 <211» 31 <212» PRT <213» Arti ficial

    Page 23

    2013202566 05 Apr 2013

    2OO6_MOO4_A115_PCT.ST25 <220» <223> Linker <400» 72

    Asp Asp Asp Asn Ser Pro ser Phe

    Lys His Pro Lys Thr Trp val His 20

    <210> <211> <212» <213> 73 28 PRT Artificial <220» <223» Li nker <400» 73 Leu Ser 1 Lys Asn Asn 5 Ala lie Glu Arg Hi s pro Ser Thr 20 Arg Gin Lys <210» <211» <212» <213» 74 22 PRT Artifi cial <220» <223» Linker <400» 74 Asp Glu 1 ί Asp Glu Asn 5 Gl n ser Pro His Tyr ¹ Phe lie Ala 20 Ala <210» <211» <212> <213> 75 32 PRT Artificial <220» <223> Linker <400» 75 Asp Tyr 1 ¹ Gly Met Ser 5 Ser Ser pro Ser Gly ’ ser val Pro 20 Girt Phe Lys

    ile Girt lie Arg Ser Val Ala Lys 10 15

    Tyr Ala Ala Glu Glu Glu Asp 25 30

    Pro Arg ser Phe ser Gin Asn ser 10 15

    Gin Phe Asn Ala 25

    Arg Ser Phe Gin Lys Lys Thr Arg 10 15

    His val Leu Arg Asn Arg Ala Gin 10 15

    Lys Val Val Phe Gin Glu Phe Thr 25 30

    Page 24

    2013202566 05 Apr 2013

    2OQ6_MO04„AI15„PCT,ST25 <210» 76 <211» 24 <212» PRT <213» Artificial <220» <223» Linker <400» 76

    Asp lie Tyr Asp Glu Asp Glu Asn Gin Ser Pro Arg ser Phe Gin Lys 15 10 15

    Lys Thr Arg His Tvr Phe lie Ala 20 <210» 77 <211> 17 <212> PRT <213> Artificial <220» <223» Linker <400» 77

    Asp Asn Ser Pro Ser Phe He Gin lie Arg ser val Ala Lys Lys Kis 15 10 15

    Pro <210» 78 <211» 20 <212» PRT <213» Artificial <220» <223» Linker <400» 78

    Leu Ser Lys Asn Asn Ala lie Glu Pro Arg ser Phe Ser Gin Asn ser 15 10 15

    Arq His pro ser 20

    <210» 79 <211» 36 <212» PRT <213» Arti fi ci al <220» <223» Linker <400» 79 ser val Ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15

    Page 25

    2013202566 05 Apr 2013

    2006_M004^All5_PCT. ST2 5

    Asp val Thr Gin Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg lie val 20 25 30

    Gly Gly Gin Glu 35 <210> 80 <211> 23 <212> PRT <213> Artificial <220>

    <223> Linker <400> 80

    Ser Lys Leu Thr Arg Ala Glu Thr val Phe Pro Asp Asn Asn L.eu Thr 15 10 15

    Arg lie Val Gly Glv Gin Glu 20 <210> 81 <211> 27 <212> PRT <213> Artificial <220>

    <223> Linker <400> 81

    Arc Ala Glu Thr Val Phe Pro Asp val Thr Gin Pro Glu Arg Gly Asp 1 ” 5 10 15

    Asn asp Leu Thr Arg lie val Gly Gly Gin Glu 20 25 <210> 82 <211> 22 <212> PRT <213> Artificial <22O>

    <223> Linker <400> 82

    Arg Ala Glu Thr Val Phe Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg 1 5 10 15

    He Val Gly Gly Gin Glu 20

    <210> 83 <211> 32 <212> PRT <213> Artificial

    Page 26 <22O>

    2013202566 05 Apr 2013

    2OO6_MOO4_A115„PCT.ST25 <223> Linker <4OO> 83 ser Val ser Gin Thr ser lvs Leu Thr Arg Ala Glu Thr val Phe Pro 15 10 15

    Asp val Asp Tyr val Asn Asn Leu Thr Arg lie val Gly Gly Gin Glu 20 25 30 <210> 84 <211> 30 <212> PRT <213> Artificial <220>

    <22 3> Linker <400> 84

    Ser Val ser Gin Thr ser lvs Leu Thr Arg Ala Glu Thr val Rhe Pro

    1 5 ' 10 15

    Asp Val Asp Asn Asn Leu Thr Arg lie val Gly Gly Gin Glu 20 25 30 <210> 85 <211> 30 <212> PRT <213> Artificial <220>

    <223> tinker <400> 85

    Ser val Ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 IS

    Asp val Asp Asn Asn Leu Thr Arg lie val Gly Gly Gin Glu 20 25 30 <210> 86 <211> 52 <212> PRT <213> Artificial <220>

    <223> Linker <400> 86

    Ser 1 Val ser Gin Thr 5 Ser Lys Leu Thr Arg Ala Glu 10 Thr val Phe 15 Pro Asp val ASp Tyr Val Asn Ser Thr Glu Ala Glu Thr lie Leu ASp Asn 20 25 30 lie Thr Gin Ser Thr Gin Ser Phe Asn Asp Phe Thr Arg val val Gly

    35 40 45

    Page 27

    2013202566 05 Apr 2013

    2OO6,..mOO4...A115...PCT, ST25

    Gly Glu Asp Ala <210> 87 <211> 32 <212> PRT <213> Artificial <220>

    <223> Linker <400> 87

    Ser Val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15

    Asp Val Gin Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp 20 25 30 <210> 88 <211> 32 <212> PRT <213> Artificial <220>

    <223> Linker <400> 88 ser Val Ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10' 15

    Asp Val Asp Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp 20 25 30 <210> 89 <211> 32 <212> PRT <213> Artificial <220>

    <223> Linker <400> 89

    Ser Val Ser Gin Thr ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 15 10 15

    Asp Val Asn Ala Ser Lys Pro Gin Gly Arg lie Val Gly Gly Lys Val

    20 <210> 90 <211> 32 <212> PRT <213> Artificial <220> <223> Linker

    Page 28

    2006...M004...A.115...PC.T, ST25

    2013202566 05 Apr 2013 <400> 90

    Ser val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr val Phe Pro 1 5 10 15

    Asp Val Asn Ala Ser Lys Pro Gin Gly Arg Leu Val Gly Gly Lys Val 20 25 30 <210» 91 <213> 32 <212» PRT <213» Artificial <220» <223» Linker <400» 91

    Ser val ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 15 10 15

    Asp val Asn Ala ser Lvs Pro Gin Gly Arg Thr Val Gly Gly Lys val 20 25 30 <210» 92 <211» 19 <212» PRT <213» Artificial <220» <223» Linker <400» 92

    Ser val ser Girt Thr Ser Lys Leu Thr Arg Ala Glu Thr val Phe Pro 15 10 15

    Asp val Asp <210» 93 <211» 666 <212» PRT <213» Homo Sapiens <400» 93

    Tyr Asn Ser Gly Lys Leu Gl u Gl u Phe val G i n Gly Asn Leu Glu Arg 1 5 10 15 Glu cys Met Glu Glu Lys Cys Ser Phe Glu Gl u Ala Arg Glu Val Phe 20 25 30 Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gin Tyr Val ASp cly 35 40 45 Asp Gin cys Glu Ser Asn pro Cys Leu Asn Gly Gly Ser cys Lys ASP 50 55 60

    Page 29

    2013202566 05 Apr 2013

    2006_M004_A115_PCT.ST25

    Asp 65 lie Asn Ser Tyr Glu 70 Cys Trp cys Pro Phe 75 Gly Phe Glu Gly Lys 80 Asn Cys Glu ΰ,ΘΙί Asp val Thr cys Asn lie Lys Asn Gly Arg Cys Gl u 85 90 95 Gin Phe cys lys Asn Ser Ala ASp Asn lvs val val cys ser cys Thr 100 105 no Glu dy Tyr Arg Leu Ala Gl u Asn Gin Lys ser cys Gl u Pro Ala val 115 120 125 Pro Phe Pro Cys Gly Arg val ser Val Ser Glr, Thr ser Lys Leu Thr 130 135 140 Arg Al a G S U Thr val Phe Pro Asp val ASp Tyr val Asn ser Thr Glu 145 150 155 160 Ala Glu Thr lie Leu Asp Asn He Thr Gin Ser Thr Gin Ser Phe Asn 165 170 175 Asp Phe Thr Arg val Val Gly Gly Glu Asp Ala Lys Pro Gly Gin Phe ISO 185 190 Pro Trp Gin Val Val Leu Asn Gl v Lys Val Asp Ala Phe cys Gly Gly 195 200 205 Ser lie Val Asn Glu Lys Trp ll e val Th r Ala Ala Hi s cys val Glu 210 215 220 Thr Gly val Lys lie Thr val val Ala Gly Glu Hi s Asn il e Gl u Glu 225 230 235 240 Thr Gl u His Thr Gl u Gin Lys Arg Asn val lie Arg lie lie Pro His 245 250 255 Hi s Asn Tyr Asn Ala Ala lie Asn Lys Tyr Asn His ASp lie Ala Leu 260 265 270 Leu Gl u Leu Asp Glu Pro Leu Val Leu Asn ser Tyr Val Thr Pro lie 275 280 285 Cys lie Al a Asp Lys Glu Tyr Thr Asn lie Phe Leu Lys Phe Gly Ser 290 295 300 Gly Tyr Val Ser Gly Trp dy Arg val phe Hi s Lys Gly Arg ser Ala 305 310 315 320 Leu Val Leu Gin Tyr Leu Arg val Pro Leu val ASp Arg Ala Thr Cys

    325 330 335

    Pace 30

    2013202566 05 Apr 2013

    2006_M004_A11S_PCT.ST25

    Leu Arg ser Thr 340 Lys Phe Thr I i e Tyr 345 Asn Asn Met Phe Cys 350 Ala Gly Phe His Glu Gly Gly Arg Asp Cys Gin Gly ASP Ser Gly Gly Pro 355 360 365 His Val Thr Glu val Glu Glv Thr Ser Phe Leu Thr Gl y He He Ser 370 375 380 Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly lie #» t y e Thr Lys 385 390 335 400 val Ser Arg Tyr val Asn Trp lie Lys Glu Lys Thr Lys Leu Thr Ser 405 410 415 val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro ASp 420 425 430 val Asp Glu Pro Lys ser Cys Asp Lys Thr His Thr Cys Pro pro Cys 435 440 445 Pro Ala Pro Glu Leu Leu Gly Gl y Pro ser val Phe Leu Phe pro Pro 450 455 460 Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Gl u Val Thr cys 465 470 475 480 val Val val Asp val Ser His Gl U Asp pro Glu val Lys Phe Asn Trp 485 490 495 Tyr Val Asp Gly Val G s ϋ val His Asn Ala Lys Thr Lys Pro Arg Glu 500 505 510 Glu Gl n Tyr Asn Ser Thr Tyr Arg Val val ser val Leu Thr Val Leu 515 520 525 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys cys Lys Val Ser Asn S30 535 540 Lys Ala Leu pro Ala Pro lie Glu Lys Thr lie ser cys Ala Lys Gly 545 550 555 560 dn Pro Arg Glu Pro Gin val Tyr Thr Leu Pro Pro ser Arg Glu Glu 565 570 575 Met Thr Lys Asn Gin val Ser Leu Thr cys Leu val Lys Gly Phe Tyr 580 585 590 Pro ser Asp lie Ala Val Gl u Trp Gl u Ser Asn dy Gin Pro Gl u Asn

    595 600 605

    Page 31

    2013202566 05 Apr 2013

    2006„M004~All5_,PCT. ST2 5

    Asrt Tyr Lys Thr Thr Pro Pro val Leu Asp Ser Asp Gly Ser Phe Phe 610 615 620 Leu Tyr Ser Lys Leu Thr Val ASP Lys Ser Arq Trp Gin Gin Gly Asn 625 630 635 640 Val Phe Ser CVS Ser V<3 3 Met Hi s Gl u Ala Leu Hi s Asn Hi s Tyr Thr 645 650 655 Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

    660 665 <210» 94 <211> 7 <212» PRT <213» Artificial . <22O>

    <223» Linker <400» 94

    Gly Gly Gly Gly Gly Gly val <210» 95 <211> 24 <212» PRT <213» Artificial <220>

    <223» Linker <400» 95 ser Val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 15 10 15

    Asp val Asp Gly Ser Gly Gly Ser 20 <210> 96 <211» 18 <212» PRT <213» Artificial <220» <223» Linker <400» 96 ser Val Ser Gin Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10’ 15

    Asp val <210» 97