WO2019201868A1

WO2019201868A1 - Coagulation factor based fusion protein with half-life extending polypeptide

Info

Publication number: WO2019201868A1
Application number: PCT/EP2019/059695
Authority: WO
Inventors: Tobias Cornvik; Joakim Nilsson; Erik Nordling; Stefan SVENSSON GELIUS
Original assignee: Swedish Orphan Biovitrum Ab (Publ)
Priority date: 2018-04-16
Filing date: 2019-04-15
Publication date: 2019-10-24

Abstract

A fusion protein is provided, comprising i) a coagulation factor protein selected from coagulation factor X (FX), coagulation factor IX (FIX) and variants thereof; and ii) a half-life extending polypeptide moiety comprising 2-80 units independently selected the amino acid sequences according to SEQ ID NO: 1: in which, independently: X1 is P or absent; X2 is V or absent; X3 is P or T; X4 is P or T; X5 is T or V; X6 is D, G or T; X8 is A, Q or S; X9 is E, G or K; X10 is A, E P or T; and X11 is A, P or T. The half-life extending polypeptide moiety has a generally unfolded conformation and provides a fusion protein with a large hydrodynamic radius that may avoid renal clearance. As a result, the biological half-life of the fusion protein is increased and the biological effect of the FX or FIX may thus be prolonged.

Description

COAGULATION FACTOR BASED FUSION PROTEIN WITH HALF-LIFE

EXTENDING POLYPEPTIDE

Field of the invention

The present invention relates to Factor X and Factor IX based fusion proteins comprising half-life extending polypeptides, and to uses of such fusion proteins.

Background

Vitamin K dependent proteases are crucial components of the coagulation cascade. Among them are Factor IX (FIX) and Factor X (FX) responsible for the bleeding disorders Haemophilia B (FIX deficiency) and FX deficiency, respectively. The overall domain structure of the two vitamin K dependent proteases are similar with an N-terminal c-carboxyglutamic acid (GLA) domain followed by two epidermal growth factor (EGF) domains and a c-terminal serine protease domain. The N- terminal GLA domain with it’s posttranslationally modified c-carboxyglutamic acids that is required for binding to phospholipid membranes are the common denominator for this family as the posttranslational modification requires vitamin K.

Coagulation Factor X (FX), also known as Stuart-Prower factor, is a vitamin K dependent serine protease (EC: 3.4.21.6, Uniprot accession code: P00742, Gene name: F10) of the coagulation cascade. In plasma, FX circulates as two chains joined by a disulfide bond, the light chain of 17 kDa and the heavy chain of 45 kDa. The light chain contains three domains, a c-carboxyglutamic acid (GLA) domain and two epidermal growth factor (EGF) domains. The GLA domain is necessary for a calcium ion dependent conformational change associated with phospholipid binding. The heavy chain contains the catalytic serine protease domain. It is positioned as the first enzyme in the common part of the extrinsic and the intrinsic pathway. FX is activated upon tissue factor exposure to plasma in the extrinsic pathway, in the intrinsic pathway the activation occurs through interaction with factor IXa, Factor Villa, calcium ions and acidic phospholipids. The activation occurs as the activation peptide is cleaved from the heavy chain and activated FX heavy chain is formed, also written as FXa heavy chain. Activated FX (FXa) is formed by the light chain and the FXa heavy chain that is held together by a disulfide bond. FX deficiency is a rare bleeding disorder that affects 1 :1 000 000 up to 1 :500 000 depending on sources. The disease affects both males and females as the gene is located on chromosome 13. The severity of the disease varies in the affected individuals, from severe cases which are evident during childhood to mild cases which are detected later during the life span of the affected individual. The symptoms range from easy bruising, nose bleeds, bleeding under the skin, blood in the urine, bleeding gums, bleeding after trauma or surgery, bleeding into the joints, and intracranial, pulmonary or gastrointestinal bleeds. As the deficiency also affect females they may also experience further complications in connection with menstruation and during pregnancy with increased risk for miscarriage and excessive bleeding at child birth.

Acquired FX deficiency may also present, often in patients with amyloidosis, where FX is trapped in amyloid fibrils and thus are cleared from the circulation. There a number of other indications such as cancer, myeloma, infection where an acquired FX deficiency has been found.

Up until recently only plasma concentrates or fresh frozen plasma were available for treating the patients that suffer from FX deficiency, now however there is a plasma derived highly purified FX product (pdFX) on the market. The half-life of the highly purified pdFX is in the range of 30 hours. For other pdFX the half-life has been reported to be in the range of 20 - 40 hours. Coagulation Factor IX (FIX), also known as the Christmas factor, is a vitamin K dependent serine protease (EC: 3.4.21.22, Uniprot accession code: P00740, Gene name: F9). It is the part of the intrinsic pathway of blood coagulation and it converts FX to its activated form FXa in the presence of calcium ions, phospholipids and activated FVIII. FIX deficiency, also known as Haemophilia B, is a bleeding disorder with the prevalence of 1 in 30,000 males, as the gene is carried on the X

chromosome. Several products to treat Haemophilia B are on the market today, both plasma derived and recombinant forms of the native FIX as well as forms with extended half-life. The half-life of recombinant FIX is reported to be on average 22 hours.

In plasma, FIX circulates as two chains, the light chain and the heavy chain, joined by a disulfide bond. The light chain contains three domains, a c-carboxyglutamic acid (GLA) domain and two epidermal growth factor (EGF) domains. The GLA domain is necessary for a calcium ion dependent conformational change associated with phospholipid binding. The heavy chain contains the catalytic serine protease domain. FIX is activated by tissue factor or by factor Xla. The activation occurs as the activation peptide is cleaved from the heavy chain and activated FIX heavy chain is formed (FIXa heavy chain). Activated FIX (FIXa) is formed by the light chain and the FIXa heavy chain that is held together by a disulfide bond.

In general, the effect of biologies is determined by their half-life in vivo. One of the major clearance mechanisms is filtration by the kidneys. As biologies most often are administrated by either intravenous (i.v., iv) or subcutaneous (s.c., sc) injection, the time span between each dose is of great importance. Meanwhile, these routes of administration, in particular intravenous injection, typically require the assistance of healthcare professionals and may also be uncomfortable, even painful, to the patient, and thus more frequent dosing increases patient discomfort and

inconvenience, and demands healthcare resources. This is in great contrast to dosing of a small molecule drug, which can often be administrated by less invasive routes, such as orally, intranasally or topically, as often as required, with much less effort and inconvenience.

One of the earliest attempts to address the problem of rapid clearance of biologies or biopharmaceuticals from circulation was to chemically attach a polyethylene glycol (PEG) polymer chain to a protein or peptide to increase the hydrodynamic radius of the drug, which translates to an increased apparent size in solution, such that it reaches a size that is not readily cleared by the kidneys. This technology, termed PEGylation, has shown to be successful, and is currently used in approved pharmaceutical products. However, the step of chemical attachment adds another process step to the manufacturing, resulting in an increased cost of the manufactured drug. Furthermore, attachment of a PEG moiety can occur at various sites of a protein or peptide, resulting in a product of greatly increased

inhomogeneity in which the location of the PEG chain varies among individual molecules. The nature of the PEG polymer itself also adds a degree of

inhomogeneity as the polymer is not monodisperse, but rather a collection of PEG polymers of similar, but not equal, length.

Contrary to the original belief that it was non-immunogenic and even capable of reducing immunogenicity also towards molecules to which it was linked, PEG has later been found to be immunogenic. In one example this led to a significantly increased clearance of the drug to which it was linked (PEG-uricase; Ganson NJ et al„ 2005).

With the aim to remove the additional manufacturing step and create a

monodisperse product, companies like Amunix Inc and XL-Protein GmbH have developed half-life extending technologies based on randomly non-repetitive protein sequences that can be used as fusion partners to prolong the biological half-life of therapeutic proteins and peptides (Podust et al. 2016 J Control Release, Schlapschy et al. 2013 Protein Eng Des Sel).

However, despite the advancements described above, there remains a need in the art for new means of prolonging the half-life of FX and FIX and/or increase the bioavailability in order to create a biopharmaceutical that has the potential to be used for prophylactic treatment.

Summary of the invention

It is an object of the present invention to at least partly reduce or avoid the problems of the prior art, and to provide a FX and FIX with extended biological half-life and/or increased bioavailability and facilitate prophylactic treatment.

These and other objects, which will be apparent to a skilled person from the present disclosure, are achieved by the different aspects of the invention as defined in the appended claims and as generally disclosed herein. In one aspect, the invention relates to a fusion protein comprising

i) a coagulation factor selected from coagulation factor X (FX),

coagulation factor IX (FIX) and variants thereof; and

ii) a half-life extending polypeptide moiety comprising 2-80 units, each unit being independently selected from the group consisting of all amino acid sequences according to SEQ ID NO: 1 :

X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 (SEQ ID NO: 1 ) in which, independently,

XI is P or absent;

X2 is V or absent;

X3 is P or T;

X4 is P or T;

X5 is T or V;

X6 is D, G or T;

X8 is A, Q or S;

X9 is E, G or K;

X10 is A, E P or T;

XI I is A, P or T.

The 2-80 units may be the same or different, within the definition of SEQ ID NO:1 set out above. Stated differently, the half-life extending polypeptide moiety comprises from 2 to 80 units, wherein each unit is an amino acid sequence independently selected from the group consisting of the individual sequences falling within the definition of SEQ ID NO:1. Preferably, each unit may be an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 2-1 1. The present inventors surprisingly found that a polypeptide moiety as defined above, which is based on or derived from the C-terminal domain of human bile salt- stimulated lipase (BSSL), can provide an excellent half-life extending moiety when fused to a coagulation factor selected from coagulation factor X (FX), coagulation factor IX (FIX) and variants thereof, to be used as a therapeutic. The half-life extending polypeptide moiety has a generally unfolded conformation under physiological conditions, and provides a fusion protein with a large hydrodynamic radius, and thus avoids, or at least reduces the rate of, renal clearance of the FX or FIX. Thus, the fusion protein including the half-life extending polypeptide moiety may have a biological half-life which is extended as compared to the biological half-life of the FX or FIX alone.

As used herein, the expressions“fused” and“fusion” refer to the artificial joining of two or more portions of chemical entities of the same kind, such as peptides, polypeptides, proteins, or nucleic acid sequences. A fusion protein as referred to herein typically comprises at least two polypeptide portions, which may be of different origin; for instance, a half-life extending polypeptide moiety, which may be derived from BSSL, and FX, FIX or a variant thereof. The fusion protein of the present invention is typically a non-naturally occurring entity.

In the context of the present invention, the amino acid sequences of the fusion partners of the fusion protein are referred to using the terms“polypeptide” and “polypeptide moiety”. Notably, these terms are intended to include amino acid sequences as short as 18 amino acids, which effectively represents the smallest version of the half-life extending polypeptide moiety (2 units each of 9 amino acids). An amino acid sequence of up to about 50 amino acids may sometimes be referred to as“peptide”; however, for the sake of simplicity, in the present specification, the amino acid sequences of the fusion protein will be referred to as“polypeptide” or “polypeptide moiety” throughout. The coagulation factor of the fusion protein is selected from coagulation factor X (FX), coagulation factor IX (FIX) and variants thereof. This means that the coagulation factor is selected from coagulation factor X (FX), variants of coagulation factor X (FX), coagulation factor IX (FIX) and variants of coagulation factor IX (FIX).

In embodiments, the coagulation factor is coagulation factor X or a variant thereof. Factor X and variants thereof is herein denoted“FX”. As an example, the FX may comprise mammalian FX, for example wild- type FX or FX variants such as conservative variants of FX. A conservative variant refers to a variant of Factor X having at least one amino acid substituted by another amino acid or an amino acid analogue that has at least one property similar to that of the original Factor X.

Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen- bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative substitution can be assessed by a variety of factors, such as, e.g., the physical properties of the amino acid being substituted or how the original amino acid would tolerate a substitution. The selections of which amino acid can be substituted for another amino acid in a peptide disclosed herein are known to a person of ordinary skill in the art. A conservative variant of Factor X may function in substantially the same manner as the original Factor X, and may thus be a variant of Factor X in any aspect of the present specification.

FX may for example be produced recombinantly, such as in in mammalian cells, such as in human cells.

In embodiments, said coagulation factor X (FX) or variant thereof is capable of being hydrolyzed into factor Xa, or a variant thereof, in vivo. Thus, the FX variant may have the same function as native factor X and may be activated into factor Xa, or a variant of factor Xa, in vivo.

In embodiments, the coagulation factor X (FX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 38 or a sequence that differs from SEQ ID NO: 38 by at most five deletions, insertions or substitutions.

In embodiments, the coagulation factor X (FX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 48, or a sequence that differs from SEQ ID NO: 48 by at most five deletions, insertions or substitutions. SEQ ID NO: 48 corresponds to positions 10 - 457 of SEQ ID NO: 38.

Furthermore, the coagulation factor X (FX) or variant thereof may comprise a light chain having the amino acid sequence according to SEQ ID NO: 58, or comprise a light chain having a sequence that differs from SEQ ID NO: 58 by at most five deletions, insertions or substitutions. Moreover, the coagulation factor X (FX) or variant thereof may comprise a heavy chain having the amino acid sequence according to SEQ ID NO: 59, or comprise a heavy chain having a sequence that differs from SEQ ID NO: 59 by at most five deletions, insertions or substitutions. Consequently, the coagulation factor X (FX) or variant thereof may comprise a light chain having the amino acid sequence according to SEQ ID NO: 58 and a heavy chain having the amino acid sequence according to SEQ ID NO: 59.

Factor X or variants thereof (FX) may also comprise active forms of factor X, such as factor Xa (FXa). Activated FX (FXa) is formed by the light chain and the FXa heavy chain held together by a disulfide bond. FXa may comprise the amino acid sequence according to SEQ ID NO: 60 or a sequence that differs from SEQ ID NO: 60 by at most five deletions, insertions or substitutions

However, in embodiments, the coagulation factor X (FX) is an inactive form of factor X, i.e. a form that may be activated by factor IXa and factor Vila, e.g. in vivo. Thus, in embodiments, the coagulation factor X or variant thereof may not comprise factor Xa or any variant thereof.

In embodiments, the coagulation factor is factor IX or a variant thereof. Factor IX and variants thereof is herein denoted“FIX”. As an example, the FIX may comprise mammalian FIX, for example wild-type FIX or FIX variants, such as conservative variants of FIX. A conservative variant refers to a variant of Factor IX having at least one amino acid substituted by another amino acid or an amino acid analogue that has at least one property similar to that of the original Factor IX. Examples of properties include, without limitation, similar size, topography, charge,

hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen- bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative substitution can be assessed by a variety of factors, such as, e.g., the physical properties of the amino acid being substituted or how the original amino acid would tolerate a substitution. The selections of which amino acid can be substituted for another amino acid in a peptide disclosed herein are known to a person of ordinary skill in the art. A conservative variant of Factor IX may function in substantially the same manner as the original Factor IX, and may thus be a variant of Factor IX in any aspect of the present specification.

FIX may for example be produced recombinantly, such as in in mammalian cells, such as in human cells.

In embodiments, said coagulation factor IX (FIX) or variant thereof is capable of being hydrolyzed into factor IXa, or a variant thereof, in vivo. Thus, the FIX variant may have the same function as native factor IX and may be activated into factor IXa, or a variant of factor IXa, in vivo.

In embodiments, the coagulation factor IX (FIX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 61 or a sequence that differs from SEQ ID NO: 61 by at most five deletions, insertions or substitutions.

In embodiments, the coagulation factor IX (FIX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 62, or a sequence that differs from SEQ ID NO: 62 by at most five deletions, insertions or substitutions. SEQ ID NO: 62 corresponds to positions 19 - 433 of SEQ ID NO: 61.

Furthermore, the coagulation factor IX (FIX) or variant thereof may comprise a light chain having the amino acid sequence according to SEQ ID NO: 63, or comprise a light chain having a sequence that differs from SEQ ID NO: 63 by at most five deletions, insertions or substitutions.

Moreover, the coagulation factor IX (FIX) or variant thereof may comprise a heavy chain having the amino acid sequence according to SEQ ID NO: 64, or comprise a heavy chain having a sequence that differs from SEQ ID NO: 64 by at most five deletions, insertions or substitutions. Consequently, the coagulation factor IX (FIX) or variant thereof may comprise a light chain having the amino acid sequence according to SEQ ID NO: 63 and a heavy chain having the amino acid sequence according to SEQ ID NO: 64. Factor IX or variants thereof (FIX) may also comprise active forms of factor IX, such as factor IXa (FIXa). Activated FIX (FIXa) is formed by the light chain and the FIXa heavy chain held together by a disulfide bond. FIXa may comprise the amino acid sequence according to SEQ ID NO: 65 or a sequence that differs from SEQ ID NO: 65 by at most five deletions, insertions or substitutions.

However, in embodiments, the coagulation factor IX (FIX) is an inactive form of factor IX, i.e. a form that may be activated by factor Xla and factor Vila, e.g. in vivo. Thus, in embodiments, the coagulation factor IX or variant thereof may not comprise factor IXa or any variant thereof. The expression“biological half-life” refers to the time it takes for the concentration of the substance in question in blood, serum or plasma to decrease to half of the initial concentration. The biological half-life may be

determined according to conventional methods known to persons of skill in the art. For instance, the biological half-life can be determined based on the concentration in serum, plasma or whole blood.

Preferably, the half-life extending polypeptide moiety extends the biological half-life of the FX or FIX by a factor of at least 1.5 in at least one species, typically humans.

In other words, the fusion protein preferably has a biological half-life that is at least 1.5 times that of FX or FIX alone. For example, the fusion protein may extend the biological half-life of FX or FIX by a factor of at least 2, at least 2.5, at least 3, at least 5, at least 10, at least 15, or at least 20. As a result of the increased biological half- life, the effect of FX or FIX may be prolonged in vivo.

From a dosing perspective, using the half-life extending polypeptide moiety as disclosed herein allows less frequent administration, which is beneficial for the patient, as well as from an economic perspective. For instance, instead of administration twice a week of a drug, the same or a similar biological or therapeutic effect may be attained by only one administration per week. Such a difference means a great improvement for patients, especially those who are required to come to a hospital or clinic to receive treatment, and/or where administration is physically uncomfortable or even painful. Additionally, by fewer doses and/or a longer time period between doses, adverse reactions caused by the mode of administration may be avoided; for instance, for subcutaneous injection, injection site reactions such as pain, eczema and rashes can be reduced or avoided, and for intravenous administration, infusions reactions involving e.g. fever or nausea can be reduced or avoided.

Another benefit of the half-life extending polypeptides used in the present invention resides in the increased hydrophilicity of the fusion protein due to the high number of hydrophilic residues in the half-life extending polypeptide. The increased

hydrophilicity may improve bioavailability of the fusion protein (relative to the bioavailability of FX or FIX as such) and increase systemic concentration, potentially allowing smaller and/or less frequent doses. As used herein,“bioavailability” refers to the dose fraction of a substance that reaches systemic circulation following administration via a different route than intravenous administration.

Another practical implication of the increased hydrophilicity is that subcutaneous administration may be a realistic option instead of intravenous administration. Where possible, subcutaneous administration is often preferred over intravenous infusion as subcutaneous injections in general are faster, less uncomfortable and require less medical training to perform compared to intravenous administration.

Additionally, the increased hydrophilicity of the fusion protein according to the invention may also be an advantage during the purification of a crude expression product. It was found that fusion proteins according to embodiments of the invention eluted earlier than FX or FIX as such using hydrophobic interaction chromatography (HIC) using gradient elution. This is considered a potentially very useful effect that could be the solution to problems relating to undesirable host cells proteins eluting simultaneously with FX or FIX. Hence, it may be possible to reduce the number of chromatography unit operations required to obtain a fusion protein of high purity.

In embodiments, the half-life extending polypeptide moiety comprises 6-70 units, such as 10-51 units, e.g. 7-18 units. In embodiments, the half-life extending peptide moiety comprises 10-68 of the units according to SEQ ID NO: 1. As an example, the half-life extending peptide moiety may comprise 17-51 of said units.

The half-life extending peptide moiety may form a contiguous sequence of 2-80, such as 10-80, such as 10-68, such as 17-51 , units of one or more sequence(s) as defined in SEQ ID NO: 1.

In embodiments, the fusion protein may comprise multiple half-life extending polypeptide moieties, each polypeptide moiety comprising 2-80 units as defined above. Such multiple half-life extending polypeptides may be of the same length (having the same number of units), or may be of different lengths. As an example, the total number of unit of all half-life extending polypeptide moieties fused to FX or FIX may be above 10, such as between 10-68, such as between 17-51.

Alternatively, the fusion protein may comprise one half-life extending polypeptide only, typically having 2-80, such as 10-80, such as 10-68, such as 17-51 , units as defined above.

In embodiments, the half-life extending polypeptide moiety may be positioned at the amino terminal (N-terminal) or at the carboxy terminal (C-terminal) of said FX or FIX. Preferably, the half-life extending polypeptide moiety is positioned at the carboxy terminal (C-terminal) of said FX or FIX.

In the case of multiple half-life extending polypeptides, at least one of said half-life extending polypeptides moieties may be positioned N-terminally or C-terminally of said FX or FIX. Preferably, at least one of the multiple half-life extending

polypeptides moieties is positioned at the carboxy terminal (C-terminal) of said FX or FIX.

Alternatively, or additionally, a half-life extending polypeptide moiety may constitute an insertion into, or replacement of a part of, the amino acid sequence of the FX or FIX. In the case of multiple half-life extending polypeptides, at least one of said half- life extending polypeptides moiety may optionally be positioned as an insertion into, or replacement of a part of, the amino acid sequence of the FX or FIX. An insertion or replacement may be made in a surface exposed loop of the tertiary structure of the FX or FIX, such that the half-life extending polypeptide moiety that constitutes an insertion into, or replacement of a part of, the amino acid sequence of the FX or FIX is exposed on the surface of the fusion protein.

In embodiments of the invention, at least one of the residues X3 and X4 of SEQ ID NO: 1 may be P. In some embodiments, at least one of X4 and X5 of SEQ ID NO: 1 may be T. In some embodiments, at least one of X10 and X1 1 of SEQ ID NO: 1 may be A or P. In some embodiments, X1 is P and X2 is V.

In embodiments of the invention, the half-life extending polypeptide moiety may comprise 2-80 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs: 2-11. These sequences represent human variants of SEQ ID NO: 1.

In some embodiments, the half-life extending polypeptide moiety may have SEQ ID NO: 2 in its N-terminal end, as is typically the case of naturally occurring sequences of human origin. For instance, the half-life extending polypeptide moiety may comprise at least 4 contiguous units in the following order: [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5], optionally preceded by SEQ ID NO: 2.

In embodiments of the invention, the half-life extending polypeptide moiety may comprise at least one sequence selected from SEQ ID NOs: 12-34. For example, the half-life extending polypeptide moiety may be selected from the group of amino acid sequences consisting of SEQ ID NO: 12-34. Alternatively, the half-life extending polypeptide moiety may comprise multiple copies, e.g. 2, or 3, optionally contiguous, copies of a sequence selected from the group consisting of SEQ ID NO: 12-34.

In embodiments of the invention, the half-life extending polypeptide moiety may comprise at least 4, at least 6, at least 8, at least 10, or at least 17 units of one or more amino acid sequence(s) according to SEQ ID NO: 1. Furthermore, in embodiments of the invention, the half-life extending polypeptide moiety may comprise up to 8, up to 10, up to 18, up to 34, up to 51 , up to 68 or up to 70 units of one or more amino acid sequence(s) according to SEQ ID NO: 1. Thus for example, the half-life extending polypeptide moiety may comprise from 7 to 18 units of one or more amino acid sequence(s) according to SEQ ID NO: 1 , such as 7 to 18 units independently selected from the group consisting of SEQ ID NO: 2-1 1.

Typically, the half-life extending polypeptide, or, in the case where the fusion protein comprises a plurality of half-life extending polypeptides, at least one of the half-life extending polypeptides, comprises at least two different amino acid sequences according to SEQ ID NO:1.

In embodiments of the invention, the half-life extending polypeptide may be fused to FX which alone has an apparent size in solution of at least 170 kDa. In

embodiments, the apparent size in solution of the fusion protein is larger than the apparent size in solution of FX alone, by a factor of at least 2. In embodiments, the hydrodynamic radius of the fusion protein may be at least 1.4 times as large, for instance 1.6 as large, as the hydrodynamic radius of FX alone. In embodiments the apparent size in solution of the fusion protein is larger than the apparent size in solution of FX alone, by a factor of at least 2, and up to a factor of 300.

In embodiments of the invention, the half-life extending polypeptide may be fused to FIX. In embodiments, the apparent size in solution of the fusion protein is larger than the apparent size in solution of FIX alone, by a factor of at least 2. In embodiments, the hydrodynamic radius of the fusion protein may be at least 1.4 times as large, for instance 1.6 as large, as the hydrodynamic radius of FIX alone. In embodiments the apparent size in solution of the fusion protein is larger than the apparent size in solution of FIX alone, by a factor of at least 2, and up to a factor of 300.

The apparent size increase provided by the half-life extending polypeptide may be at least partly explained by the unstructured or unfolded conformation of the half-life extending polypeptide. For instance, the half-life extending polypeptide may lack secondary structure elements such as ohelices and b-sheets, and thus the half-life extending polypeptide may be characterized as not contributing to the a-helix and/or b-sheet content of the fusion protein.

In embodiments of the invention, an amino acid sequence according to SEQ ID NO:1 may be of human origin. For example, the half-life extending polypeptide moiety may correspond to a naturally occurring human amino acid sequence. The use of a sequence of human origin may be advantageous as it is expected to contribute to a lower immunogenicity in human subjects. Nevertheless, sequences comprising or corresponding to naturally occurring repeating units of other species are also contemplated for use in a half-life extending polypeptide, alone or in combination with repeating units of human origin. Such other species particularly include non- human primates, e.g. gorilla, chimpanzee, orangutan, bonobo, and macaque.

In embodiments of the invention, each repeating unit according to SEQ ID NO:1 has one, or at most one, potential O-glycosylation site. Moreover, when the half-life extending polypeptide moiety has been produced in a mammalian expression system, each unit may comprise at most one O-glycosylation, and typically a majority, but not all, of said units comprises one O-glycosylation each. For instance, a certain number or share of said units may lack glycosylation. While some glycosylation may be beneficial as it may further contribute to the size increase, unspecific or an unknown glycosylation pattern may present practical problems during protein characterization. Hence, the limited and relatively well-defined glycosylation pattern of the half-life extending polypeptide moiety according to embodiments of the present invention is advantageous in this respect. Thus, in embodiments, each unit of the half-life extending polypeptide moiety comprises at most one O-glycosylation. In some embodiments however, in particular where the fusion protein is produced in non-mammalian cells, the half-life extending

polypeptide moiety may completely lack glycosylation. In some embodiments, the half-life extending polypeptide moiety may be designed to avoid O-glycosylation independently of the expression system used, i.e. also in cases where a mammalian expression system is used. In such embodiments, the half-life extending polypeptide moiety may comprise, or consist of, 2-80 units selected from the group consisting of SEQ ID NOs: 74-82. The fusion protein may have a biological half-life which is extended by a factor of at least 1.5 relative to the biological half-life of the FX or FIX alone. In embodiments, the fusion protein is further comprising a cleavage site between the coagulation factor X (FX) or a variant thereof and the half-life extending polypeptide moiety. The cleavage site may be a cleavage site for a protease.

In embodiments, the fusion protein is further comprising a cleavage site between the coagulation factor IX (FIX) or a variant thereof and the half-life extending polypeptide moiety. The cleavage site may be a cleavage site for a protease.

The cleavage site may be a cleavage site for thrombin and/or Factor IXa. Such cleavage sites may comprise or consist of a sequence selected from LTRIVGG (SEQ ID NO: 36) and LVPRGS (SEQ ID NO: 35). Another feasible route is cleavage by Factor Xla or FVIIa that cleaves the sequence VSQTSKLTRAETVFPDV (SEQ ID NO: 37) at the centrally positioned Arginine residue.

In embodiments, the fusion protein comprises a sequence selected from any one of sequences according to SEQ ID NO: 39-47. In embodiments, the fusion protein comprises a sequence that differs from any one of sequences SEQ ID NO: 39-47 by at most five deletions, insertions or substitutions.

In embodiments, the fusion protein comprises a sequence selected from any one of sequences according to SEQ ID NO: 49-57 or a sequence that differs from any one of sequences SEQ ID NO: 49-57 by at most five deletions, insertions or substitutions. Sequences according to SEQ ID NO: 49-57 correspond to the sequences according to SEQ ID NO: 39-47, but starting from position 10. In embodiments, the fusion protein comprises a sequence according to SEQ ID NO: 49, or a sequence that differs from sequence SEQ ID NO: 49 by at most five deletions, insertions or substitutions. In embodiments, the fusion protein comprises a sequence selected from any one of sequences according to SEQ ID NO: 66-72. In embodiments, the fusion protein comprises a sequence that differs from any one of sequences SEQ ID NO: 66-72 by at most five deletions, insertions or substitutions.

In embodiments, the fusion protein comprises a sequence according to SEQ ID NO: 67. In embodiments, the fusion protein comprises a sequence that differs from sequence SEQ ID NO: 67 by at most five deletions, insertions or substitutions.

In embodiments, the fusion protein has a hydrodynamic radius of at least 4.8 nm. Further, in embodiments, the fusion protein has an apparent size in solution of at least 170 kDa as determined by size exclusion chromatography.

In embodiments, the fusion protein is comprising a plurality of coagulation factor X (FX) or variants thereof or a plurality of coagulation factor IX (FIX) or variants thereof.

In another aspect, the invention provides a method of prolonging the biological half- life of FX or FIX, or a method of producing a fusion protein according to the above- mentioned first aspect of the invention, comprising the steps of:

a) providing a polynucleotide, typically a DNA construct, encoding a fusion protein as described above, comprising FX or FIX and a half-life extending polypeptide moiety;

b) introducing said polynucleotide into a cell;

c) maintaining said cell under conditions allowing expression of said fusion protein; and

d) isolating said fusion protein.

In some embodiments, the cell is a mammalian cell. Expression in mammalian expression systems may be beneficial as it may provide glycosylation of the fusion protein. In other embodiments, the cell may be a non-mammalian eukaryotic cell, such as a yeast cell, a plant cell or a non-mammalian animal cell. In other aspects, the invention provides a polynucleotide encoding a fusion protein as described herein, an expression vector comprising such a polynucleotide, and a cell, which may be a mammalian cell or a non-mammalian cell, comprising such an expression vector.

In another aspect, the invention provides a pharmaceutical composition comprising the fusion protein as described herein and a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition may be formulated for subcutaneous administration, and/or for intravenous administration.

In yet another aspect, the invention provides a fusion protein for use as a

medicament, and in particular for use as a medicament intended to be administered subcutaneously to a subject.

As an example, the fusion protein may be for use as a medicament for treating factor X deficiency. This may be when the coagulation factor protein is factor X (FX) or variants thereof. The factor X deficiency may be a FX deficiency located on chromosome 13 and/or an acquired FX deficiency e.g. in patients with amyloidosis, cancer, myeloma or infection.

As a further example, the fusion protein may be for use as a medicament for treating haemophilia B (factor IX deficiency). This may be when the coagulation factor protein is factor IX (FIX) or variants thereof.

Accordingly, in other aspects, the invention provides a method of treatment of a coagulation factor deficiency, comprising a step of administering, to a subject in need thereof, a fusion protein as described herein. When the coagulation factor deficiency is factor X deficiency, the fusion protein comprises FX as described herein. When the coagulation factor deficiency is Haemophilia B (factor IX deficiency), the fusion protein comprises FIX as described herein. Administration may occur intravenously or subcutaneously. In embodiments, the treatment may be a prophylactic treatment. In further aspects, the invention relates to the use of a half-life-extending polypeptide as defined herein for increasing the biological half-life of FX or FIX, as well as to the use of a half-life-extending polypeptide as defined herein for increasing the bioavailability of FX or FIX. As mentioned above, a distinct benefit of the half-life extending polypeptide moiety described herein is the increased hydrophilicity of the resulting fusion protein due to the high number of hydrophilic residues in the half-life extending polypeptide. The increased hydrophilicity may improve bioavailability and increase systemic concentration (e.g., serum concentration), potentially allowing smaller or less frequent doses. Another practical implication of an increased hydrophilicity is that for FX and FIX, subcutaneous administration may be a realistic option instead of intravenous administration.

It is noted that the invention relates to all possible combinations of the features recited in the claims.

Brief description of the drawings

Figure 1 is a schematic representation of a gene encoding FX or FIX (white) and one or more gene(s) encoding a half-life extending polypeptide moiety (shaded) according to embodiments of the invention.

Figure 2 is a computer generated representation of a fusion protein in which the coagulation factor is FX, according to embodiments of the invention. The model is of SEQ ID NO: 39; the structure of Factor X is modelled based on structures in pdb entries 1 p0S for the GLA domain and, the remaining domains are based on 3ens. The half-life extending polypeptide is shown in a low energy conformation. The Factor X portion is shown in ribbon representation whereas the half-life extending polypeptide is shown in surface representation

Figure 3 is a plot of size in solution and number of repeating units for FX and FX fusion proteins. Size determined by Size Exclusion Chromatography. Y-axis shows corresponding size for a globular protein in kDa. X-axis shows number of repeats in construct. Figure 4 is shows the results from Example 5. Thrombin cleavage of SEQ ID NO: 40. Lane: A, F and K SeeBlue plus 2 size marker; B Thrombin 1 :500, 0 h; C Thrombin 1 :500, 0.5 h; D Thrombin 1 :500, 2 h; E Thrombin 1 :500, 4 h; G Thrombin 1 :50, 0 h; H Thrombin 1 :50, 0.5 h; I Thrombin 1 :50, 2 h; J Thrombin 1 :50, 4 h; L Medium alone, 0 h, M Medium alone, 0.5 h; N Medium alone 2 h, O Medium alone, 4h

Detailed description

The human lactating mammary gland and pancreas produce a lipolytic enzyme, bile salt-stimulated lipase (BSSL), also referred to as bile salt-activated lipase (BAL) or carboxylic ester lipase (CEL). The protein is arranged in two domains, a large globular amino-terminal domain and a smaller but extended carboxy-terminal (C- terminal) domain (for a review, see e.g. Wang & Hartsuck (1993) Biochim. Biophys Acta 1166: 1-19). The present inventors surprisingly found that repetitive sequences based on or derived from the C-terminal domain of human BSSL can be successfully fused to FX or FIX and confer increased biological half-life of the fusion partner, thereby extending its biological or therapeutic effect in vivo, as demonstrated in the Examples below.

The C-terminal domain of human BSSL consists of repeating units of, or similar to, the formula“PVPPTGDSGAP”(SEQ ID NO: 5). Table 2 in Example 1 below lists the repeating units from human BSSL variants. The most common form of the C-terminal domain contains 18 repeating units (UniProt entry P19835). However, there are variations in the human population, both with regard to the number of repeating units, and the amino acid sequence of the individual repeating units. Furthermore, each repeating unit has one site that may be O-glycosylated, increasing the hydrophilicity and size of the region (Stromqvist et al. Arch. Biochem. Biophys.

1997). The C-terminal end of the domain is however hydrophobic, and has been shown to bind into the active site of BSSL and cause auto-inhibition of the enzyme. The most frequent human sequence of this hydrophobic portion is“QMPAVIRF” (SEQ ID NO: 83) (Chen et al. Biochemistry 1998).

It has previously been speculated that the C-terminal domain may be responsible for the stability of BSSL in vivo, for example its resistance to denaturation by acid and aggregation under physiological conditions (Loomes et al., Eur. J. Biochem. 1999, 266, 105-11 1 ). In contrast, another study of the cholesterol esterase structure showed that the C-terminal domain, which is enriched with Pro, Asp, Glu, Ser and Thr residues, is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that the protein may have a short half-life in vivo due to the repetitive sequences in the C-terminal domain (Kissel et al., Biochimica et Biophysica Acta 1989, 1006).

In the present invention, the extended biological half-life of a fusion protein comprising a half-life extending polypeptide moiety as defined herein, based on or derived from the C-terminal domain of human BSSL, is believed to be due mainly to the increased hydrodynamic radius of the protein. However, it is also envisaged that other mechanisms may contribute to the increased biological half-life.

As used herein, the expressions“fused” and“fusion” refer to the joining of two or more portions of chemical entities of the same kind, such as peptides, polypeptides, proteins, or nucleic acid sequences. A fusion protein as referred to herein typically comprises at least two polypeptide portions, which may be of different origin; for instance, FX or FIX and a half-life extending polypeptide moiety, which may be derived from BSSL. Generally, a fusion may contain the fused portions in any order and at any position; however, a fusion of genes is typically made in-frame (in-line), such that the open reading frames (ORFs) of the fused genes are maintained, as appreciated by persons of skill in the art.

Figure 1 schematically illustrates a nucleic acid construct encoding a fusion protein according to embodiments of the present invention, comprising a gene encoding FX or FIX (white bar), and a gene encoding a half-life extending polypeptide moiety (dashed bar). For simplicity other elements such as promoter or enhancer sequences and the like are not marked, although a person of skill in the art will appreciate that such elements may be included as necessary. For instance, the gene encoding FX or FIX may be preceded by a signalling peptide for expression in mammalian cells. As shown in Fig. 1 , the gene encoding the half-life extending polypeptide moiety may be located C-terminally (Fig. 1 b), N-terminally (Fig. 1 c) or both N- and C-terminally (Fig. 1d) to the gene encoding FX or FIX. Alternatively, a sequence encoding a half- life extending polypeptide moiety may be positioned within the boundaries of the gene encoding FX or FIX (in-line positioning). In such embodiments, sequences encoding half-life extending polypeptide moieties may optionally be present at multiple sites, e.g. at two sites as shown in Fig. 1 e, or more sites as desired, as long as the insertion does not disrupt the tertiary or folding structure of FX or FIX or the processing of FX into FXa or FIX into FIXa. In-line positioning of one or more half-life extending moieties may be combined with N- and/or C-terminal fusion(s).

The FX(s) or FIX(s) constituting the fusion partner(s) of the half-life extending polypeptide moiety may be any FX or FIX, or combination of FXs or FIXs, that may be suitable for use in treatment or prevention of any condition or disorder, where the biological function requires a certain systemic concentration of FX or FIX.

FX and FIX are a naturally occurring polypeptides, however, fused to the half-life extending polypeptide moiety, the resulting fusion protein will always be a non- naturally occurring entity. The fusion protein comprising a naturally occurring polypeptide may be recombinantly produced as described in the examples below.

Figure 2 illustrates a fusion protein according to embodiments of the present invention (PSI0727 of the Examples below, fusion protein represented by SEQ ID NO: 39), where FX is located N-terminally in the fusion protein and the half-life extending polypeptide moiety forming a tail at the C-terminal of FX, the half-life extending polypeptide of this example being represented by 17 repeating units according to SEQ ID NO: 20. FX is linked at its C-terminal portion to the half-life extending polypeptide via a peptide linker, here [G₄S]₂, linking the C-terminal end of FX to the N-terminal of the half-life extending polypeptide and thus forms a proximal part of the tail.

However, as explained above with reference to Figure 1 , the half-life extending polypeptide moiety is not necessarily located at the C-terminal of FX or FIX. In embodiments of the invention, the half-life extending polypeptide moiety may be located at the N-terminal of FX or FIX (Fig. 1c), or half-life extending moieties may be located each at the N-terminal and C-terminal, respectively (Fig. 1d). In other embodiments, one or more half-life extending polypeptides may be inserted at a position within FX or FIX (Fig. 1 e), for example in a position located in a surface- exposed loop of FX or FIX.

In some embodiments, the half-life extending polypeptide moiety may replace a specific sequence segment of FX or FIX. For instance, when positioned as an insert, the half-life extending polypeptide moiety may replace a part of a surface-exposed loop on FX or FIX.

In yet other embodiments, an in-line inserted half-life extending polypeptide moiety may be combined with either an N-terminal moiety, a C-terminal moiety, or both N- terminal and C-terminal half-life extending polypeptide moieties (Fig. 1f). Notably, in embodiments of the invention comprising multiple half-life extending moieties, located at different positions, each such half-life extending moiety may be

independently defined as described herein. Otherwise stated, each such half-life extending moiety may comprise from 2 to 80 units of an amino acid sequence according to SEQ ID NO: 1.

According to the invention, the half-life extending polypeptide moiety used for fusion with a FX or FIX comprises an amino acid sequence comprising 2-80 repeating units, each unit being independently selected from the group of amino acid sequences defined by SEQ ID NO: 1 :

X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 (SEQ ID NO: 1 ) in which, independently,

X1 is P or absent;

X2 is V or absent;

X3 is P or T;

X4 is P or T; X5 is T or V;

X6 is D, G or T;

X8 is A, Q or S;

X9 is E, G or K;

X10 is A, E P or T;

X11 is A, P or T.

As used herein, a“unit” refers to an occurrence of an amino acid sequence of the general formula according to SEQ ID NO: 1 as defined above, including for instance any of the sequences according to SEQ ID NOs: 2-1 1. The half-life extending polypeptide comprises from 2 to 80 such units, which may be the same or different, within the definition set out above. The units of the half-life extending polypeptide may also be referred to as“repeating units” although there is some variation in the amino acid sequence between individual units, and hence“repeating units” is not to be understood exclusively as the repetition of one and the same sequence. Stated differently, the half-life extending polypeptide moiety comprises from 2 to 80 units, wherein each unit is an amino acid sequence independently selected from the group consisting of the individual sequences falling within the definition of SEQ ID NO:1. The half-life extending polypeptide moiety may comprise a contiguous sequence of at least 18 amino acids (corresponding to 10 units that are both 9-meric versions of SEQ ID NO: 1 ), and typically up to 880 amino acids (corresponding to 80 units which are all 1 1-mer versions of SEQ ID NO: 1 ). The repeating units may be contiguous with one another, although it is also possible that the repeating units are separated by short spacing sequences. For instance, two repeating units may be separated by up to 10 amino acid residues that do not correspond to SEQ ID NO: 1 ; for instance, the short spacing sequence may be a peptide linker of the formula (G₄S)₂. In some embodiments, a spacing sequence may be up to 5 amino acid residues. In some embodiments one or more amino acid residue(s) may be positioned between two repeating units, e.g. to impart a desired functionality such as an N-glycosylation site, or to provide a site for another type of modification, for instance employing a single Cys residue. In some embodiments, a linker, such as one or more G₄S linkers, may be used as spacing sequences between adjacent repeating units. Hence, in view of this possibility, the contiguous sequence comprising up to 80 repeating units may be longer than 880 amino acids, for instance up to 900 amino acids or up to 1000 amino acids.

The repeating units of the half-life extending polypeptide moiety are defined by SEQ ID NO: 1 , which is based on the repeating units of human variants of the BSSL C- terminal domain, and which allows some variation of amino acid residues in positions X3, X4, X5, X6, X8, X9, X10 and X11. In contrast, the residues at positions X1 , X2 and 7 are fixed, although positions X1 and X2, may be absent. Typically, both X1 and X2 are absent, and in such embodiments, a repeating unit consists of 9 amino acids only.

A half-life extending polypeptide moiety comprising 10 to 80 units (repeating units) typically comprises several variants of the amino acid sequence motif generally defined by SEQ ID NO: 1 , such as at least two different variants according to SEQ ID NO: 1. For instance, in embodiments of the invention where the half-life extending polypeptide moiety comprises at least 4 units, it may comprise at least one unit of each of SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO: 5. In embodiments of the invention where the half-life extending polypeptide moiety comprises at least 2 units, these may be independently selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO: 5. Advantageously, the half-life extending polypeptide moiety may comprise SEQ ID NOs: 3-5 in this order, optionally preceded by SEQ ID NO: 2. A unit according to SEQ ID NO: 2 may especially be located at the N-terminal end of the half-life extending polypeptide moiety, representing the first unit of the half-life extending polypeptide moiety. While other specific variations of the repeating units (e.g. the units according to SEQ ID NOs: 3-11 ) may appear repeatedly, SEQ ID NO: 2, if present, typically only appears once, as the first repeating unit of the half-life extending polypeptide moiety.

The conformation of the half-life extending polypeptide moiety is generally unstructured. For instance, in embodiments of the invention, the half-life extending polypeptide does not contribute to the a-helix and/or b-sheet content of the fusion protein as determined by circular dichroism or FTIR (Fourier Transform Infrared Spectroscopy).

In embodiments of the invention, a repeating unit defined by SEQ ID NO: 1 is of human origin, and preferably all of the repeating units of the half-life extending polypeptide moiety correspond(s) to naturally occurring repeating units of a variant of the C-terminal domain of human BSSL. Such repeating units are represented by SEQ ID NOs: 2-1 1 (See also Table 2 in the Examples). In embodiments of the invention, all repeating units of the half-life extending polypeptide moiety are selected from the group consisting of SEQ ID NOs: 2-11 , e.g. SEQ ID NOs: 3-1 1. That is, the half-life extending polypeptide moiety may comprise 2-80 units, each independently selected from the group consisting of SEQ ID NO: 2-1 1 , e.g. SEQ ID NOs: 3-1 1. The use of a sequence of human origin may be advantageous as it is expected to contribute to a lower immunogenicity in human subjects compared to half-life extending moieties with repeating units of non-human or partly human origin, whether polypeptide based or other as used in the prior art.

Furthermore, in embodiments of the invention, the half-life extending polypeptide moiety comprises, or consists of, a sequence of repeating units that corresponds to a naturally occurring human sequence of repeating units. Examples of such natural human sequences of repeating units are presented in SEQ ID NO: 12-34. Typically, such sequences comprise, as the first five repeating units, in this order: [SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5], or, alternatively, as the first four repeating units, in this order: [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5]

Thus, in embodiments of the invention, the half-life extending polypeptide moiety comprises an amino acid sequence according to any one of in SEQ ID NO: 12-34. In some embodiments the half-life extending polypeptide moiety consists of a multiple of any one of SEQ ID NO: 12-34. For instance, the half-life extending polypeptide moiety may consist of three contiguous multiples, or copies, of an amino acid sequence according to any one of SEQ ID NOs: 12-34. For instance, SEQ ID NO: 20 comprises 17 units of an amino acid sequence according to SEQ ID NO:1 , and thus a three-copy multiple of SEQ ID NO: 20 comprises at least 51 units. However, it should be noted that the repeating units of the half-life extending polypeptide moiety can be independently selected from all units according to SEQ ID NO:1 and the invention is thus not limited to certain sequences of units being repeated.

Accordingly, for instance a 51 -unit half-life extending polypeptide moiety is not necessarily formed of three copies of a 17-unit sequence, but may be formed of any combination of units according to SEQ ID NO:1 , and in particular of any combination of repeating units selected from SEQ ID NOs: 2-11.

It was found that each repeating unit as defined above carries one potential O- glycosylation site. That is, upon expression in a mammalian environment allowing glycosylation, each repeating unit may be glycosylated at most one predetermined position, typically at a threonine (T, Thr) residue. For the repeating units of SEQ ID Nos: 2-1 1 , the potential sites of O-glycosylation are indicated in Table 2 (see Example 1 ). There may be an upper limit to the number of glycans, which is lower than the total number of units. That is, typically, less than all of the units of the half- life extending polypeptide moiety are glycosylated. For instance, out of a sequence of 17 units (such as SEQ ID NO: 13 or SEQ ID NO: 17) typically only 10 units are glycosylated. Hence, in embodiments, a majority of the units may be glycosylated, whereas a minority of the units may be non-glycosylated. Furthermore, the degree of glycosylation (e.g. the ratio of glycosylated units to non-glycoslyated units, or the like) may be possible to adjust according to known measures, e.g. by appropriately selecting the expression system and/or controlling the cultivation or expression conditions of the producer cells.

As mentioned above, the fusion protein comprising the half-life extending

polypeptide moiety according to the invention benefits from an increased biological half-life compared to that of FX or FIX alone. The increased biological half-life is mainly due to the increased size of the fusion protein vis-a-vis FX or FIX alone. The size of the fusion protein according to the invention is large enough to decrease clearance from circulation by the kidneys (renal clearance). The radius of the majority of the pores of the glomerular membrane are 4.5-5 nm. The membrane is negatively charged and thus are proteins that are negatively charged less prone to be cleared by the kidneys. For instance, negatively charged molecules may be significantly protected from renal clearance already at a hydrodynamic radius of 2.5 nm, while neutral molecules need a size of 3.5 nm to get a similar protection of renal clearance (Haraldsson et al Physiological

Reviews 88 (2) 451-487). For an uncharged globular protein, the size limit for renal clearance (below which a protein is secreted) is a molecular weight of about 60 kDa.

The actual molecular weight of a protein, as determined for instance by Multi Angle Light Scattering (MALS), corresponds to the theoretical molecular weight based on the amino acid composition, and any glycans bound. In contrast, the apparent size (or apparent molecular weight) in solution of a protein can be determined by Size Exclusion Chromatography (SEC), e.g. as described in Example 3 below, and yields an apparent molecular weight, or apparent size, of a protein that corresponds to the actual molecular weight of a globular protein. For proteins and peptides that do not have a globular conformation, the actual molecular weight may differ from the apparent molecular weight, or apparent size, in solution.

Typically, a non-globular protein or polypeptide may exhibit an apparent size in solution that is larger than its actual molecular weight. In the case of the present half- life extending polypeptides moieties, which typically have an unstructured, unfolded conformation, the inventors found that each repeating unit represented a size increase of at least 9 kDa, as determined by SEC (Figure 3, described in more detail below), even though the actual molecular weight was only about 1 kDa. This increase included the effect of increased glycosylation as well. Hence, the apparent size in solution of the fusion protein can be increased by at least 9 kDa for each unit contained in the fusion protein according to embodiments of the invention.

In total, the fusion protein may have an apparent size in solution, as determined by SEC, larger than the size of the FX or FIX alone by a factor of at least, at least 2, at least 2.5, at least 3, at least 5, at least 10, at least 20, or at least 50, and for example up to 20, up to 40, up to 60, up to 80, up to 100, up to 200, up to 250, or even up to a factor of 300.

The size of the half-life extending polypeptide moiety and of the fusion protein, respectively, may also be defined by the hydrodynamic radius, also referred to as the Stokes radius, measured in nanometers (nm). Both the apparent size in solution and the hydrodynamic radius are determined by Size Exclusion Chromatography (SEC), e.g. as described in Example 3 below.

In accordance with what has been said above with regard to apparent size in solution, the hydrodynamic radius of the fusion protein is typically large enough to avoid renal clearance. For comparison, human serum albumin, which has a size above the limit of renal clearance, has a hydrodynamic radius of 3.8 nm. The fusion protein may have a hydrodynamic radius that is at least 1.25 times as large, or at least 1.4 times as large, as the hydrodynamic radius of FX or FIX alone. For instance, the hydrodynamic radius of the fusion protein may represent an increase at least by a factor of 1.6, 2, 3, 5, 10, 20 or 50 of the hydrodynamic radius of the FX or FIX alone.

In addition to the number of repeating units in the half-life extending polypeptide moiety, also the location of the polypeptide moiety within the fusion protein may affect the size increase. For example, N-terminal or C-terminal location of a half-life extending polypeptide moiety is expected to provide a larger hydrodynamic radius compared to a half-life extending moiety located as an insert within the amino acid sequence of the FX or FIX (e.g. forming a surface loop).

Furthermore, the unfolded structure of the half-life extending moiety not only as such provides a large hydrodynamic radius, but it may contribute to the size increase because of the hydrophilic character of many of the amino acids of the repeating units, by binding of water molecules to the half-life extending polypeptide moiety, to further increase the hydrodynamic radius. Finally, glycosylation of some of the repeating units may further contribute to a larger size, as demonstrated in Example 3 below. It was found that a half-life extending polypeptide moiety of 17 repeating units increased the weight as measured by MALS by 22 kDa in addition to the weight added by the amino acid composition of the repeating units. This increase can be attributed to glycosylation of the repeating units.

Example 3 illustrates the relationship between the number of repeating units and the apparent size in solution according to various embodiments of the invention. In these embodiments, described in the Examples hereinbelow, a FX was fused to half-life extending polypeptide moieties of various lengths (different number of units: 10, 17, 34, and 51 , respectively). The inventors have found that the correlation between the size in solution and number of repeating units is linear in the investigated area. It was also found that the size in solution of one unit corresponds to a globular protein with molecular weight of at least 9 kDa. Hence, the size increase achieved by addition of a given number of units can be predicted. For instance, a polypeptide moiety having 80 repeating units would have an apparent size in solution

corresponding a globular protein of molecular weight of approximately 720 kDa.

As indicated above with reference to Figure 2, the fusion protein may comprise a linker, typically a peptide linker, linking FX or FIX to one or more half-life extending polypeptide moieties as described herein. Hence, in embodiments of the invention the fusion protein further comprises a peptide linker positioned between an amino acid sequence of the FX or FIX and an amino acid sequence of the half-life extending polypeptide moiety. For example, the peptide linker may be selected from -GS- and -(G₄S)_n, wherein n is an integer from 1 to 5, typically from 1 to 3, or from 2 to 3. The use of a linker may be advantageous in that it may reduce the occurrence of, or, in the case of n being at least 2, prevent the formation of neo epitopes and subsequent binding of such neo epitopes by antigen-presenting cells of the immune system. The linker may be a cleavable linker that comprises a cleavage site for a protein in vivo. The cleavage site may be a cleavage site susceptible for cleavage by a protease in the coagulation cascade.

The cleavage site may be a cleavage site for thrombin and/or Factor IXa. Such cleavage sites may comprise or consist of a sequence selected from LTRIVGG (SEQ ID NO: 36) and LVPRGS (SEQ ID NO: 35). This may allow for the half-life time to be extended in vivo until FX or FIX is activated. Then, the half-life extending polypeptide moiety may be cleaved off from the fusion protein to release an active form of FX or FIX without half-life time extending moieties as defined herein. It may be

advantageous to be capable of decreasing the half-life time of an activated form of FX or FIX (such as FXa or FIXa), i.e. upon initiation of the coagulation cascade.

Another suitable cleavage site may be VSQTSKLTRAETVFPDV (SEQ ID NO: 37), that may be cleaved at the centrally positioned Arginine residue.

The fusion proteins described herein can be produced by recombinant techniques using eukaryotic, such as mammalian, expression systems, using conventional methods known to persons of skill in the art. Example 2 and 6 below describes cloning and production of fusion proteins in which half-life extending polypeptide moieties are fused to FXs and FIXs, respectively. It should be noted that the invention is by no means limited to use of those cell types of Example 2 and 6; in contrast, suitable cell lines for production of fusion proteins are known to persons of skill in the art, and examples include, Pichia pastoris, Saccharomyces cerevisiae, algae, moss cells, plant cells such as carrot cells, and mammalian cells such as CHO, HEK-293, and HT1080.

The fusion protein may in some embodiments comprise further, or additional, C- terminal or N-terminal amino acid residues. For example, one or more of said one or more half-life extending polypeptide moieties may comprise further C- or N-terminal amino acid residues. Each additional amino acid residue may individually or collectively be added in order to, for example, improve production, purification, stabilization in vivo or in vitro, coupling, or detection of the polypeptide. Another example is additional amino acid residues that provide a“tag” for, inter alia, purification or detection of the fusion protein. Alternatively, a“tag” may be provided by replacement of terminal amino acid residues within e.g. one of said one or more half-life extending polypeptide moieties. Thus, in one embodiment, at least one of said one or more half-life extending polypeptides comprises a C-terminal unit comprising a tag, wherein said tag optionally is a peptide tag. This means that at least one of said one or more half-life extending polypeptide moieties, wherein each moiety comprises 2 to 80 units, comprises a C-terminal unit including a tag. Such a tag may, as set out above, for example improve purification or detection of the fusion protein. Further, said tag may constitute a replacement of a terminal part of said C- terminal unit, or may constitute a C-terminal addition to said C-terminal unit. When said tag constitutes a replacement, it may consist of 2-4 amino acid residues.

In particular, said C-terminal unit may have an amino acid sequence

PVPPTDDSKEPEA (SEQ ID NO: 73)

For market approved therapeutic products, accurate characterization is a necessary regulatory requirement, and for a glycosylated protein the exact position of any glycans must be known. The fact that each unit of the present half-life extending polypeptide moiety carries at most one O-glycosylation site may facilitate

characterization of a fusion protein expressed in mammalian systems.

A suitable protease for characterization of the half-life extending polypeptides according to the invention is pepsin, which cleaves after the acidic residues: glutamic acid (Glu, E) and aspartic acid (Asp, D). However, as pepsin typically will not cleave proximal to a glycosylated residue due to steric interference of the glycan with the protease, the repeating units that carry an O-glycosylation will have different cleavage patterns compared to non-glycosylated units. Based on this knowledge and in view of the limited and relatively predictable glycosylation pattern, characterization of the present fusion proteins using established methods, such as chromatographic methods and mass spectrometry, is greatly simplified compared to half-life extended moieties that are potentially glycosylated to a massive or unknown extent, making industrial expression of the present fusion proteins in mammalian systems more practically feasible. Another potential advantage of glycosylation of the half-life extending polypeptide moiety is that glycosylation may provide a means of increasing immune tolerance towards the fusion protein. O-glycans ending with a a2,6- linked terminal sialic acid can bind to CD22 or to Siglec-10, which are two inhibitory receptors of the sialic acid binding immunoglobulin-like lectin (Siglec) family. These receptors act by damping the signal from the B-cell receptor (BCR), which may lead to development of B-cell tolerance towards the fusion protein. Glycans of human proteins possess both a2,6- and a2,3-linked terminal sialic acid. In order to increase the sialic acid content with a2,6- linked terminal sialic acid in fusion proteins expressed in cells of human origin, the fusion protein of interest may be co-expressed with a2,6-sialyltransferase. Fusion proteins produced in Chinese hamster ovary (CHO) cells only have a2, 3-linkage due to the absence of a2,6-sialyltransferase expression. In order to introduce a2,6- linked terminal sialic acid in the O-glycans of fusion proteins produced in CHO cells, the fusion protein of interest may be co-expressed with a2,6-sialyltransferase.

In addition to the potentially improved immune tolerance, an increased sialic acid content may also have a beneficial effect on the half-life of the fusion protein, as the sialic acid may serve to shield any potential epitopes for other glycan receptors present among the O-glycans, thereby reducing or abolishing binding of the fusion protein to endocytic receptors.

In embodiments of the invention, each repeating unit according to SEQ ID NO: 1 has one, or at most one, potential O-glycosylation site. Moreover, when the half-life extending polypeptide moiety as disclosed herein has been produced in a mammalian expression system, each unit may comprise at most one O- glycosylation, and typically a majority, but not all, of said units comprises one O- glycosylation each. For instance, a certain number or share of said units may lack glycosylation. While some glycosylation may be beneficial as it may further contribute to the size increase, unspecific or an unknown glycosylation pattern may present practical problems during protein characterization. Hence, the limited and relatively well-defined glycosylation pattern of the half-life extending polypeptide moiety according to embodiments of the present invention is advantageous in this respect. In some embodiments however, in particular where the fusion protein is produced in non-mammalian cells, said one or more half-life extending polypeptide moieties may completely lack glycosylation. In such embodiments, one or more half- life extending polypeptide moieties of the fusion protein may each comprise 2 to 80 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs: 74-80. The final repeat is in some instances modified to incorporate a purification tag. In the same manner, a modified version of those can be designed to be O-glycan free and is assigned SEQ ID NO: 81

(PVPPVDDAKEPEA). These units (SEQ ID NO: 81 ) can be assembled into 16 repeats ended by a single C-terminal to form a half-life extending polypeptide moiety (SEQ ID NO: 82) that is almost identical in length as the most abundant natural form provided in SEQ ID NO: 20.

The fusion protein according to the invention has an increased hydrodynamic radius and apparent size in solution compared to the size of FX or FIX alone. As a consequence, at least in part of reduced renal clearance due to the size increase, the pharmacokinetic properties of the fusion protein are altered.

In other words, the fusion protein preferably has a biological half-life that is at least 1.5 times that of the FX or FIX alone. For example, the fusion protein may extend the biological half-life of the FX or FIX by a factor of at least 2, at least 3, at least 5, at least 10, at least 15, or at least 20. From a pharmacokinetic perspective, it may be desirable to extend the biological half-life as much as possible. However, as the half- life extending effect has been shown to be proportional to the size of the half-life extending moiety, and very large half-life extending polypeptide moieties may be undesirable for various reasons, such as feasibility of production or impediment of the biological activity of the FX or FIX, the half-life extension for a given FX or FIX may have to be balanced against other requirements, and the optimum half-life extension may thus be less than the theoretical maximum half-life extension achievable by the present invention. For instance, it may be desirable to use no more than three half-life extending polypeptide moieties of 80 units each (i.e. a total of 240 units distributed over three moieties), or no more than two half-life extending polypeptide moieties of 80 units each (i.e., a total of 160 units distributed over two moieties). An alternative conceivable upper limit to the half-life extending polypeptide moiety may be two moieties (e.g. one at the N-terminal and one at the C-terminal) of 68 units each. As an example, if FX has a biological half-life of around 30 hours, the half-life extending polypeptide moiety may extend the half-life by a factor up to 15. Further, if FIX has a biological half-life of around 22 hours, the half-life extending polypeptide moiety may extend the half-life by a factor up to 15.

Furthermore, the half-life extending polypeptide moiety may provide increased solubility to the fusion protein. In particular, the hydrophilic nature of the half-life extending polypeptide moiety, may be beneficial in that it may increase the bioavailability of a fusion protein that is administered subcutaneously, relative to the bioavailability of the FX or FIX alone. In such cases, the increased solubility of the fusion protein may promote transfer to the blood stream rather than remaining in the tissue extracellular matrix after injection. This could mean that for FXs or FIXs that otherwise require intravenous administration due to limited bioavailability, subcutaneous administration may be a realistic option if FX or FIX are fused to a half-life extending polypeptide moiety as described herein.

Thus, the half-life extending polypeptide moiety used in the present invention may be used as a means of extending the biological-half-life of a FX or FIX and possibly of adapting other pharmacokinetic properties thereof.

The fusion protein of the invention may be formulated as a pharmaceutical composition, for use in therapy and/or prevention of a condition, disorder or disease. The term "composition" as used herein should be understood as encompassing solid and liquid forms. A composition may preferably be a pharmaceutical composition, suitable for administration to a patient (e.g. a mammal) for example by injection or orally. The pharmaceutical composition typically includes the fusion protein according to the invention and at least one pharmaceutically acceptable carrier or substituent. The pharmaceutical composition may for instance comprise any one of a salt, a pH regulator, an oil, a preservative, an osmotically active agent, and any combination thereof.

The pharmaceutical composition may be formulated for any route of administration, including intravenous, subcutaneous, nasal, oral, and topical administration. For example, the composition may be formulated for intravenous or subcutaneous administration. The invention will be further described in the following examples.

Examples

Example 1: Identification of repeating units of human origin

A blast search was performed with the catalytic domain of Bile salt-stimulated lipase (BSSL) versus the non-redundant protein sequence database at the National Institute of Health (NIH), USA and identified 10 reported protein sequences for the protein of human origin that contained the whole or part of the C-terminal repetitive unstructured domain.

Material and methods

Blast at NIH was used to search for proteins of human origin that match the catalytic domain of Bile salt stimulated lipase with UniProt ID P19835 (Accession number CEL_HUMAN).

Results

The BLAST search resulted in finding 10 entries that contained both a significant portion of the catalytic domain and the C-terminal repetitive unstructured domain. The number of the repeating units in the domains differed and some variability among the sequence of the repeating units was noted, see Table 1 for the different hits. Each repeating domain is initiated by a truncated sequence of 9 residues, while the most prevalent repeating units are 11 residues long. In the table below, the repeating units are separated by a "~" sign for clarity. In the enclosed sequence listing, the repetitive portions are represented by SEQ ID NOs: 12-19.

Table 1. Variants of human BSSL-CTD

Table 2 below lists the unique sequences of repeating units of human origin, with reference to the sequence identity number in the enclosed sequence listing. Absent residues of the first sequence are marked by a dash. Potential sites of O- glycosylation are underlined.

Table 2. Units corresponding to repeating units found in human BSSL-CTD. Potential glycosylation site is underlined.

Hence, there exists a variety of lengths of the C-terminal domain in the human population. Furthermore, the order of the repeating units can vary in the human population. This could imply that variations in the order of the repeating units and the length of the entire domain motifs are allowed. Each unit carries one site that may be O-glycosylated. The most prevalent human form is made up of the combination of the following sequence of repeating units:

[SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 6] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 7] - [SEQ ID NO: 5] - [SEQ ID NO: 8] - [SEQ ID NO: 9]

Expressed differently:

[SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5]x8 - [SEQ ID NO: 6] - [SEQ ID NO: 5]x2 - [SEQ ID NO: 7] - [SEQ ID NO: 5] - [SEQ ID NO: 8] - [SEQ ID NO: 9] Example 2: Cloning and production of FX based fusion proteins

This Example describes the general strategies for cloning and production of fusion proteins based on human FX sequences, which were used in the Examples below.

Materials and methods

DNA constructions: DNA sequences encoding a set of FXs with and without half-life extending polypeptides were codon optimized for expression in CHO cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. The proteins with introduced cleavage sites for FIXa and Thrombin had the sequences introduced prior to the half-life extending

polypeptide, the arrow indicate the cleavage position. The genes were cloned in expression vectors for subsequent expression in Expi293 cells. Cultivation and purification: Expression of recombinant FXs or fusion proteins was performed using the Expi293 expression systems (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol. Supernatants were harvested by centrifugation 6 days after transfection of expression vectors and stored at -70°C. Table 3 lists the encoded protein sequences. Supernatants from the Expi293 cultures were thawed and filtered (0.22 mm) before purification. Each supernatant, containing the recombinant FX or fusion proteins was purified using conventional chromatography methods Recombinant FX or fusion proteins for use in animal studies were also subjected to an endotoxin removal step. Purified FX or fusion proteins were buffer exchanged to PBS (25 mM NaP + 125 mM NaCI, pH7.0) or TBS (25 mM Tris + 125 mM NaCI, pH7.0) and, unless otherwise stated, TBS was also the formulation buffer used in subsequent experiments. The purity of the fusion proteins was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight of each protein was analyzed using mass spectrometry (LC/MS or MALDI-TOF/MS). Results

Purification resulted in protein preparations with high purity, which was analyzed by SDS-PAGE stained with Coomassie Blue. The correct identity and molecular mass of each fusion protein were confirmed by mass spectrometry analysis. Table 3: Encoded protein sequences

Conclusions

Fusion proteins containing FX and half-life extending polypeptides of various lengths can be produced by constructing synthetic genes followed by expression in mammalian systems and purified to high purity using conventional techniques. Example 3: Biophysical characterization of fusion proteins

This Example describes the characterization of FX based fusion proteins containing half-life extending polypeptides, using unfused proteins as references, with respect to biophysical characteristics such as apparent size and molecular weight in solution and determination of hydrodynamic radius in solution by size exclusion

chromatography (SEC) and column calibration and Multi Angle Light Scattering (MALS).

Material and methods

The size of the fusion proteins, unfused proteins and PEGylated proteins in solution, were assessed by analytical gel filtration on an AKTA Micro (GE Healthcare Life Sciences) using a calibrated column Superdex 200 Increase 10/300 GL (GE

Healthcare Life Sciences). The column was calibrated with Gel Filtration Calibration Kit LMW (code no. 28-4038-41 , GE Healthcare Life Sciences) and Calibration Kit HMW (code no. 28-4038-42, GE Healthcare Life Sciences), containing 8 globular proteins in the size range of 6.5 to 669 kDa and Blue Dextran 2000, using a running buffer of 0.14 M NaCI, 0.0027 M KCI and 0.010 M Phosphate buffer pH 7.4

(Medicago PBS tablet cat#09-8912-100) with a flow rate of 750 mI/min at a temperature of 25°C. The corresponding size and hydrodynamic radius in solution can be calculated from the elution volume of a protein on a calibrated column by the methods described in appendix 10 of Handbook of Size Exclusion Chromatography Principles and Methods (order no 18-1022-18, GE Healtcare Life Sciences). The molecular weight of the proteins was determined by a connected MALS-RI system: Static light scattering detector miniDawn Tristar and Differential refractometer Optilab rEX , and the Astra V software (Wyatt Technology Europe, Germany).

The proteins of interest were analyzed under the same conditions as during the calibration.

Results

Table 4 presents the results for selected FX based molecules and reference molecules. Table 4. FX based molecules

Conclusions

All proteins displayed a higher MW by MALS measurements, an indication of that the proteins are glycosylated. Further, a positive correlation of length of the half-life extension polypeptide fusion and size in solution was observed.

Example 4: FX activity in a chromogenic assay

This Example describes the influence upon activity by the addition of the half-life extending polypeptides on FX, comparing FX and FX based fusion proteins produced in the same expression system.

Material and methods

The activity of FX and fusion proteins were assessed with Biophen Factor X chromogenic assay (Hyphen BioMed, ref. 221705, lot F1700281 P3) for measuring Factor X in plasma, essentially as described by the producer. The proteins were measured at a concentration 10 mg/ml.

Results

The relative activity compared to FX expressed in the same expression system is tabulated in Table 5.

Table 5. Activity in chromogenic assay, the activity of FX was set to 1

Conclusions

The fusion of FX to the half-life extending polypeptide has no detrimental influence on activity in the chromogenic assay.

Example 5: Cleavage and release of half-life extending polypeptide from fusion protein

This example describes that incorporation of a sequence susceptible for cleavage by a protease in the coagulation cascade may be used to release the half-life extending polypeptide upon initiation of coagulation.

Material and methods

Two different volume ratios of protease to protein medium was used; 1 :500 and 1 :50. The proteases used were Thrombin (Sigma-Aldrich, Product number: T9326) with a concentration of 0.32 mg/mL and FIXa (Thermo-fisher scientific, Product number: RP-43109) with a concentration of 2.8 mg/mL. As a control, protein medium alone was also included in the analyses. All samples were incubated at 37°C at 350 rpm and aliquots of a 100 mL were extracted at the time points 0, 0.5, 2 and 4 h, the aliquots were immediately freezed until analysis. All samples were analysed by SDS- PAGE.

Results

All time points for the samples of SEQ ID NO: 50 incubated with 1 :50 volume ratio of thrombin showed complete cleavage, for 1 :500 volume ratio the 0.5 h time point showed some un-cleaved fusion protein (see Fig. 3). No cleavage could be detected after incubation with FIXa.

Conclusion

The thrombin site is operational in the context of the fusion protein and could thus be utilized to release the half-life extending polypeptide as the coagulation cascade is activated.

Example 6: Cloning and production of FIX based fusion proteins

This Example describes the general strategies for cloning and production of fusion proteins based on human FIX sequences. Materials and methods

DNA constructions: DNA sequences encoding a set of FIXs with and without half-life extending polypeptides were codon optimized for expression in CHO cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. The proteins with introduced cleavage sites for Thrombin had the sequence

introduced prior to the half-life extending polypeptide, the arrow indicate the cleavage position. The genes were cloned in expression vectors for subsequent expression in Expi293 cells.

Cultivation and purification: Expression of recombinant FIX or fusion proteins was performed using the Expi293 expression systems (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol. Supernatants were harvested by centrifugation 6 days after transfection of expression vectors and stored at -70°C. Table 6 lists the encoded protein sequences.

Supernatants from the Expi293 cultures were thawed and filtered (0.22 mm) before purification. Each supernatant, containing the recombinant FIX or fusion proteins was purified using conventional chromatography methods. Recombinant FIX or fusion proteins for use in animal studies were also subjected to an endotoxin removal step. Purified FIX or fusion proteins were buffer exchanged to TBS (25 mM Tris + 125 mM NaCI, pH7.0) and, unless otherwise stated, TBS was also the formulation buffer used in subsequent experiments. The purity of the fusion proteins was analyzed by SDS-PAGE stained with Coomassie Blue and the molecular weight of each protein was analyzed using mass spectrometry (LC/MS or MALDI-TOF/MS).

Results

Purification resulted in protein preparations with high purity. The correct identity and molecular mass of each fusion protein were confirmed by mass spectrometry analysis. Table 6: Encoded protein sequences

*) In this protein,“17” denotes a 17 repeat, wherein the C-terminal unit is modified to represent the amino acid sequence PVPPTDDSKEPEA (SEQ ID NO: 73), which constitutes a two amino acid replacement, where the replacement (“EP” instead of“SK”) represents a tag.

Conclusions

Fusion proteins containing FIX and half-life extending polypeptides of various lengths can be produced by constructing synthetic genes followed by expression in mammalian systems and purified to high purity using

conventional techniques. Example 7; Biophysical characterization of FIX-fusion proteins

This Example describes the characterization of a FIX based fusion protein containing half-life extending polypeptides, using FIX as reference, with respect to biophysical characteristics such as apparent size and molecular weight in solution and determination of hydrodynamic radius in solution by size exclusion chromatography (SEC) and column calibration and Multi Angle Light Scattering (MALS).

Material and methods

The experiments were performed as described in Example 3. Results

Table 4 presents the results for selected FIX based molecules and reference molecules. Table 7. FIX based molecules

Example 8: Enhanced production of FX based fusion proteins and

char a cterization

This Example describes the general strategies for production of fusion proteins based on human FX sequences, which were used in the Examples below.

Materials and methods

DNA constructions: DNA sequences encoding a set of FXs with and without half-life extending polypeptides were codon optimized for expression in CHO cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. In order to increase the amount of gamma-carbylation present in the GLA domain the signal and propeptide of WT FX were exchanged with the corresponding ones from Thrombin. The genes were cloned in expression vectors for subsequent expression in Expi293 cells.

Cultivation and purification: Expression of recombinant FXs or fusion proteins was performed using the Expi293 expression systems (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol. In addition, the cells were co- transfected with genes for VKOR and Furin to enhance the production of fully mature FX. Harvest, purification and characterization were performed as in example 2.

Activity of the proteins were analysed as in Example 4. Results

Purification resulted in protein preparations with high purity, which was analyzed by SDS-PAGE stained with Coomassie Blue. The correct identity and molecular mass of each fusion protein were confirmed by mass spectrometry analysis. The activity of the material produced were comparable with plasma derived material.

Conclusions

Fusion proteins containing FX and half-life extending polypeptides of various lengths can be produced by constructing synthetic genes followed by expression in mammalian systems and purified to high purity using conventional techniques. The enhanced production methods allow for production of material with the same activity as plasma derived FX.

Example 9: Comparative study of pharmacokinetic properties of FX and FX based fusion protein

In this example, the intravenous pharmacokinetic properties (PK) of FX (SEQ ID NO:48) and FX-17 (SEQ ID NO:49), were investigated

Materials and methods

The study followed the design of a single intravenous (IV) dose in male Sprague- Dawley rats (N=3 per administration route and protein).

The dose and timepoints were as follows: 1 mg/kg for FX and 1.5 mg/kg for FX-17, 5 and 20 min and 1 , 4, 8, 24, 48, 72, 96 and 120 hours.

The serum concentrations were determined by a sandwich assay on the Meso Scale Discovery platform (Meso Scale Diagnostics). Individual concentration versus time profiles were compiled from the actual serum concentration measurements and nominal time points. Other exposure- and pharmacokinetic parameter estimates were determined by Non-Compartmental Analysis (using Phoenix WinNonlin 8.0); i.e. AUC (area under the plasma serum concentration-time curve from time zero to infinity), CL (clearance), V_ss (apparent volume of distribution at steady-state), MRT (mean residence time) and t_1/2z (terminal half-life). Results

The results are summarized in Table 8. Table 8. Median PK parameter estimates following an intravenous single dose

Conclusions

The longer mean residence time of the fusion protein may translate to a drug with longer dosing intervals.

Example 10: Design of repeating units that do not contain any O-glycosylation sites.

This example describes sequences of repeating units without O-glycan sites.

Materials and methods

By utilizing the variable positions in SEQ ID NO: 1 the following sequences were designed that lacked serine or threonine that could be O-glycosylated during cultivation in eukaryotic expression systems such as CHO, HEK or yeast. The sequences are listed in Table 9 below.

Table 9. Units corresponding to the general formula in SEQ ID NO: 1 without serine or threonine present.

The final repeat is in some instances modified to incorporate a purification tag. In the same manner, a modified version of those can be designed to be O-glycan free and is assigned SEQ ID NO: 81 (PVPPVDDAKEPEA). These units (SEQ ID NO: 74-80) can be assembled into 16 repeats ended by a single C-terminal modified repeat containing a purification tag (PVPPVDDAKEPEA, SEQ ID NO: 81 ) to form a half-life extending fusion partner (SEQ ID NO: 82) that is almost identical in length as the most abundant natural form provided in SEQ ID NO: 20.

Claims

1. A fusion protein comprising

i) a coagulation factor protein selected from coagulation factor X (FX), coagulation factor IX (FIX) and variants thereof; and

X1 -X2-X3-X4-X5-X6-D-X8-X9-X10-X11 (SEQ ID NO: 1 ) in which, independently,

X1 is P or absent;

X2 is V or absent;

X3 is P or T;

X4 is P or T;

X5 is T or V;

X6 is D, G or T; X8 is A, Q or S;

X9 is E, G or K;

X10 is A, E P or T;

X11 is A, P or T.

2. A fusion protein according to claim 1 , wherein said half-life extending peptide moiety comprises 10-68 of said units.

3. A fusion protein according to claim 1 or 2, wherein said half-life extending peptide moiety comprises 17-51 of said units.

4. A fusion protein according to any one of the preceding claims, wherein said half-life extending polypeptide moiety comprises 6-70 units, such as 10-51 units, e.g. 7-18 units.

5. A fusion protein according to any one of the preceding claims, wherein said half-life extending peptide moiety form a contiguous sequence of 2-80 units, such as 10-68 units, such as 17-51 units.

6. Afusion protein according to any one of the preceding claims, comprising multiple half-life extending polypeptide moieties, each polypeptide moiety comprising 2-80 units.

7. Afusion protein according to any one of the preceding claims, wherein said half- life extending polypeptide moiety, or at least one of said multiple half-life extending moieties, is positioned N-terminally or C-terminally of said coagulation factor protein.

8. A fusion protein according to any one of the preceding claims, wherein said half-life extending polypeptide moiety, or at least one of said multiple half-life extending moieties, is positioned C-terminally of said coagulation factor protein.

9. Afusion protein according to any one of the preceding claims, wherein said half- life extending polypeptide moiety, or at least one of said multiple half-life extending polypeptide moieties, constitutes an insertion into, or replacement of a part of, the amino acid sequence of the coagulation factor protein.

10. Afusion protein according to any one of the preceding claims, wherein said half- life extending polypeptide moiety comprises 2-80 units of one or more amino acid sequence(s) selected from the group consisting of SEQ ID NOs: 2-1 1 and 74-80.

1 1. A fusion protein according to any one of the preceding claims, wherein said half-life extending polypeptide moiety comprises at least one sequence selected from the group consisting of SEQ ID NOs: 12-34 and 82.

12. A fusion protein according to any one of the preceding claims, wherein said half-life extending polypeptide moiety is selected from the group consisting of SEQ ID NOs: 12-34 and 82.

13. A fusion protein according to any one of the preceding claims, having a hydrodynamic radius of at least 4.8 nm.

14. A fusion protein according to any one of the preceding claims, having an apparent size in solution of at least 170 kDa as determined by size exclusion chromatography.

15. Afusion protein according to any one of the preceding claims, wherein the amino acid sequence according to SEQ ID NO:1 is of human origin.

16. A fusion protein according to claim 15, wherein the half-life extending polypeptide moiety corresponds to a naturally occurring human amino acid sequence.

17. A fusion protein according to any one of the preceding claims, wherein each unit comprises at most one O-glycosylation.

18. Afusion protein according to any one of the preceding claims, further comprising a cleavage site between the coagulation factor protein and the half-life extending polypeptide moiety.

19. A fusion protein according to claim 18, wherein the cleavage site comprises a sequence selected from LTRIVGG (SEQ ID NO: 36), LVPRGS (SEQ ID NO: 35) and VSQTSKLTRAETVFPDV (SEQ ID NO: 37).

20. A fusion protein according to any one of the preceding claims, wherein the coagulation factor protein is coagulation factor X (FX) or a variant thereof.

21. A fusion protein according to claim 20, wherein said coagulation factor X (FX) or variant thereof is capable of being hydrolyzed into factor Xa or a variant thereof in vivo.

22. A fusion protein according to claim 20 or 21 , wherein said coagulation factor X (FX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 38 or a sequence that differs from SEQ ID NO: 38 by at most five deletions, insertions or substitutions.

23. A fusion protein according to any one of the preceding claims, wherein said coagulation factor X (FX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 48 or a sequence that differs from SEQ ID NO: 48 by at most five deletions, insertions or substitutions.

24. A fusion protein according to any one of the claims 20-22, wherein the fusion protein comprises a sequence selected from any one of sequences according to SEQ ID NO: 39-47 or 49-57 or a sequence that differs from any one of sequences according to SEQ ID NO: 39-47 or 49-57 by at most five deletions, insertions or substitutions.

25. A fusion protein according to any one of the claims 20-22, wherein the fusion protein comprises a sequence selected from any one of the sequences according to SEQ ID NOs: 49-57 or a sequence that differs from any one of sequences according to SEQ ID NO: 49-57 by at most five deletions, insertions or substitutions.

26. A fusion protein according to claim 25, wherein the fusion protein comprises SEQ ID NO: 49 or a sequence that differs from SEQ ID NO: 49 by at most five deletions, insertions or substitutions.

27. A fusion protein according to any one of the preceding claims, comprising a plurality of coagulation factor X (FX) or variants thereof.

28. Afusion protein according to any one of the claims 1-19, wherein the coagulation factor protein is coagulation factor IX (FIX) or a variant thereof.

29. A fusion protein according to claim 28, wherein said coagulation factor IX (FIX) or variant thereof is capable of being hydrolyzed into factor IXa or a variant thereof in vivo.

30. A fusion protein according to claim 28 or 29, wherein said coagulation factor IX (FIX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 61 or a sequence that differs from SEQ ID NO: 61 by at most five deletions, insertions or substitutions.

31. A fusion protein according to any one of claims 28 and 29, wherein said coagulation factor IX (FIX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 62 or a sequence that differs from SEQ ID NO: 62 by at most five deletions, insertions or substitutions.

32. A fusion protein according to any one of the claims 28 and 29, wherein said coagulation factor IX (FIX) or variant thereof comprises an amino acid sequence according to any one of SEQ ID NOs: 66-72 or a sequence that differs from any one of SEQ ID NO: 66-72 by at most five deletions, insertions or substitutions.

33. A fusion protein according to claim 32, wherein said coagulation factor IX (FIX) or variant thereof comprises the amino acid sequence according to SEQ ID NO: 67 or a sequence that differs from SEQ ID NO: 67 by at most five deletions, insertions or substitutions.

34. Afusion protein according to any one of claims 28-33, comprising a plurality of coagulation factor IX (FIX) or variants thereof.

35. A method of prolonging the biological half-life of a coagulation factor protein selected from factor X (FX), coagulation factor IX (FIX) and variants thereof, comprising the steps of:

a) providing a polynucleotide encoding a fusion protein according to any one of the claims 1 to 34;

b) introducing said polynucleotide into a cell;

d) isolating said fusion protein.

36. A polynucleotide encoding a fusion protein according to any one of the claims 1 to 34.

37. An expression vector comprising a polynucleotide according to claim 36.

38. A cell comprising an expression vector according to claim 37.

39. A pharmaceutical composition comprising the fusion protein according to any one of the claims 1 to 34 and a pharmaceutically acceptable carrier, optionally formulated for subcutaneous or intravenous administration.

40. A fusion protein according to any one of the claims 1 to 34 for use as a medicament, optionally to be administered subcutaneously or intravenously to a subject.

41. A fusion protein according to any one of the claims 20-27, for use as a medicament for treating factor X deficiency.

42. A fusion protein according to any one of the claims 28-34, for use as a medicament for treating Haemophilia B (factor IX deficiency).

43. A method of treatment of a coagulation factor deficiency, comprising a step of administering, to a subject in need thereof, a fusion protein according to any one of the claims 1-34.

44. The method according to claim 43, wherein the coagulation factor deficiency is factor X deficiency, and the method comprises a step of administering a fusion protein according to any one of the claims 20 to 27.

45. The method according to claim 43, wherein the coagulation factor deficiency is Haemophilia B (factor IX deficiency), and the method comprises a step of administering a fusion protein according to any one of the claims 28-34.

46. The method according to any one of the claims 43-45, wherein the fusion protein is administered intravenously.

47. The method according to any one of the claims 43-45, wherein the fusion protein is administered subcutaneously.

48. The method according to claim 44, wherein the treatment is a prophylactic treatment.

49. Use of a half-life-extending polypeptide as defined in any one of the claims 1 to 34, for increasing the bioavailability of a coagulation factor protein selected from factor X (FX), coagulation factor IX (FIX) and variants thereof.