Factor IX variant polypeptides for administration to soft tissue

Abstract

The present invention relates to Factor IX variant polypeptides for administration to soft tissue.

Description

[0001] Related application data

[0002] This application claims priority to European Patent Application No. EP22200505.0, entitled "Factor IX variant polypeptides for administration to soft tissue", filed on October 10, 2022, the entire content of which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application is filed together with a sequence listing in electronic form. The entire content of the sequence listing is incorporated herein by reference. Technical Field

[0005] The present invention relates to Factor IX (FIX) variant polypeptides for administration to soft tissues such as skin tissue (including subcutaneous administration) or mucosal tissue (such as gastrointestinal mucosal tissue), and their use in therapy. Specifically, described herein are such FIX variant polypeptides that have increased hemostatic efficacy when administered to soft tissues as compared to wild-type FIX.

[0006] Background

[0007] Human coagulation FIX plays a key role in the formation of blood clots. FIX has been used for the prevention and treatment of bleeding disorders such as hemophilia B. Maintaining an appropriate level of FIX activity in plasma is crucial for preventing bleeding in patients with hemophilia B. Without treatment, the lack of FIX activity can cause serious harm to patients and may even lead to death.

[0008] The general view in the art is that increasing the circulating time of FIX protein in plasma is important for maintaining an appropriate level of FIX activity and achieving hemostasis. Hemostasis is the mechanism that causes blood vessels to stop bleeding. Therefore, current treatments for hemophilia B include intravenous administration of FIX proteins with an extended half-life in plasma, including and

[0009] Gene therapies for the treatment of hemophilia B are currently being investigated. Current methods use adeno-associated virus (AAV) vectors to deliver the FIX transgene (Reference 1). This method has been used in clinical trials, and during the 5-year period after treatment with adeno-associated virus expressing a high-activity variant of FIX, the annual bleeding rate was significantly reduced (Reference 2). However, some patients may be more suitable for FIX protein replacement therapy rather than gene therapy.

[0010] Recently, the relevance of an extravascular FIX depot has emerged as a concept that may contribute to hemostasis. An extravascular FIX depot refers to non-circulating FIX that is bound outside of plasma, in the extravascular space (such as the vascular endothelium or the subendothelial extracellular matrix). Understanding the balance between extravascular FIX and circulating FIX has opened up new possibilities for identifying novel and improved strategies for treating hemophilia B. The present invention is based on the surprising realization that when specifically administered to soft tissues (including subcutaneous administration), the hemostatic efficacy of FIX can be improved by using certain FIX variant polypeptides that reduce binding to the extracellular matrix.

[0011] Disclosure of the invention

[0012] The present invention provides advantageous FIX variant polypeptides that have increased hemostatic efficacy when the FIX variant polypeptides are specifically administered to soft tissues (such as when administered subcutaneously). The present invention is particularly suitable for use with FIX variant polypeptides (such as the K5A variant) that have reduced binding to the extracellular matrix compared to wild-type FIX. For example, the K5A variant has been described previously (Reference 3), but it has not been demonstrated that it has an unexpectedly improved hemostatic efficacy when specifically administered to soft tissues. As now shown in the present disclosure, the inventors have advantageously identified that when these FIX variant polypeptides are administered to soft tissues, they are more readily taken up into the circulation. These examples demonstrate that this results in higher levels of bioavailable FIX in the plasma, leading to significantly improved hemostatic efficacy compared to administering wild-type FIX by the same route or administering the same variant but by the intravenous route, a finding that is particularly unexpected. Thus, these FIX variant polypeptides are particularly suitable for treating and preventing bleeding disorders, such as hemophilia B, when administered to soft tissues.

[0013] Accordingly, in one aspect, the present invention provides a factor IX (FIX) variant polypeptide for use in a method of treating or preventing a bleeding disorder, the method comprising administering the FIX variant polypeptide to a soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX. SEQ ID NO:1 is an example of the wild-type FIX polypeptide sequence referred to herein and below.

[0014] As an alternative to any aspect and embodiment described herein using a FIX variant polypeptide (“K5A”) comprising the amino acid alanine at a position corresponding to position 5 of wild-type factor IX, the FIX variant polypeptide may instead comprise the amino acid lysine at a position corresponding to position 10 of wild-type factor IX (“V10K”). Both variants show reduced binding to the extracellular matrix compared to wild-type FIX (Reference 4).

[0015] The present invention also provides a method for treating or preventing a bleeding disorder in a subject, comprising administering to the soft tissue of the subject a therapeutically or prophylactically effective amount of a FIX variant polypeptide, wherein the FIX variant polypeptide comprises alanine at the position corresponding to position 5 of wild-type factor IX.

[0016] The present invention also provides the use of a FIX variant polypeptide in the preparation of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX variant polypeptide is to be administered to the soft tissue of the subject and wherein the FIX variant polypeptide comprises alanine at the position corresponding to position 5 of wild-type factor IX.

[0017] The present invention also provides a FIX variant polypeptide for treating or preventing a bleeding disorder, wherein the FIX variant polypeptide is to be administered to the soft tissue of the subject and wherein the FIX variant polypeptide comprises alanine at the position corresponding to position 5 of wild-type factor IX.

[0018] In some embodiments, the bleeding disorder is hemophilia B (also known as congenital factor IX deficiency).

[0019] In some embodiments, the FIX variant polypeptide further comprises (i.e., in addition to alanine at the position corresponding to position 5 of wild-type factor IX) lysine at the position corresponding to position 10 of wild-type factor IX.

[0020] In some embodiments, the FIX variant polypeptide further comprises (i.e., in addition to alanine at the position corresponding to position 5 of wild-type factor IX and optionally lysine at the position corresponding to position 10 of wild-type factor IX) leucine at the position corresponding to position 338 of wild-type factor IX.

[0021] In other embodiments, the FIX variant polypeptide further comprises (i.e., in addition to alanine at the position corresponding to position 5 of wild-type factor IX and optionally lysine at the position corresponding to position 10 of wild-type factor IX) an amino acid other than arginine (e.g., an amino acid selected from the group consisting of valine, threonine, and tryptophan) at the position corresponding to position 338 of wild-type factor IX in combination with histidine at the position corresponding to position 410 of wild-type factor IX. In certain such embodiments, the FIX variant polypeptide comprises valine at the position corresponding to position 338 of wild-type factor IX and arginine at the position corresponding to position 410 of wild-type factor IX.

[0022] In some embodiments, the FIX variant polypeptide further comprises (i.e., in addition to the alanine amino acid at the position corresponding to position 5 of wild-type factor IX, and optionally the lysine amino acid at the position corresponding to position 10 of wild-type factor IX) a tyrosine amino acid at the position corresponding to position 318 of wild-type factor IX, a glutamate amino acid at the position corresponding to position 338 of wild-type factor IX, and an arginine amino acid at the position corresponding to position 343 of wild-type factor IX.

[0023] The factor IX variant polypeptide can have an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO:1 over the full length of SEQ ID NO:1.

[0024] In any of the embodiments described herein, the factor IX variant polypeptide can have the sequence of SEQ ID NO:1 with substitutions other than those specified herein (e.g., SEQ ID NO:1 with lysine at position 5 replaced with alanine, etc.).

[0025] In some embodiments, the FIX variant polypeptide comprises a half-life enhancing moiety, e.g., albumin including its variants and derivatives, a polypeptide of the albumin family including its variants and derivatives, an immunoglobulin without an antigen-binding domain (e.g., only the Fc portion), or polyethylene glycol.

[0026] In some embodiments, the FIX variant polypeptide further comprises a cleavable peptide linker between the FIX variant polypeptide and the half-life enhancing moiety.

[0027] In some embodiments, the soft tissue is skin tissue or gastrointestinal tissue (e.g., mucosal gastrointestinal tissue). In certain embodiments, the soft tissue is skin tissue, including subcutaneous tissue. In certain such embodiments, the FIX variant polypeptide is administered subcutaneously. For example, the FIX variant polypeptide is used in a method for treating or preventing a bleeding disorder, the method comprising subcutaneously administering the FIX variant polypeptide, wherein the FIX variant polypeptide comprises an alanine amino acid at the position corresponding to position 5 of wild-type factor IX.

[0028] In alternative embodiments, the FIX variant polypeptide is administered to the gastrointestinal tissue using an oral drug delivery device. For example, the FIX variant polypeptide is used in a method for treating or preventing a bleeding disorder, the method comprising administering the FIX variant polypeptide to the gastrointestinal tissue using an oral drug delivery device, wherein the FIX variant polypeptide comprises an alanine amino acid at the position corresponding to position 5 of wild-type factor IX.

[0029] Another aspect of the present invention provides a pharmaceutical composition comprising a FIX variant polypeptide for use in a method of treating or preventing a bleeding disorder, the method comprising administering the pharmaceutical composition to soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0030] The present invention also provides a method of treating or preventing a bleeding disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising a FIX variant polypeptide to soft tissue of the subject, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0031] The present invention also provides the use of a pharmaceutical composition comprising a FIX variant polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the pharmaceutical composition is to be administered to soft tissue of the subject and wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0032] The present invention also provides a pharmaceutical composition comprising a FIX variant polypeptide for treating or preventing a bleeding disorder, wherein the pharmaceutical composition is to be administered to soft tissue in the subject and wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0033] It is apparent that, as disclosed herein, FIX variant polypeptides comprising additional mutations (i.e., in addition to the amino acid alanine at the position corresponding to position 5 of wild-type factor IX) can be used in any of the above aspects and embodiments.

[0034] Definitions

[0035] Unless otherwise indicated, the practice of the present invention will employ conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacy within the skill of the art. Such techniques are well explained in the literature.

[0036] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It is understood that since the polypeptides of the present invention may be based on, for example, antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the "polypeptide" may occur as a single chain or as two or more associated chains.

[0037] The percent sequence identity between two amino acid sequences means the percentage of identical amino acids in the two sequences being compared when aligned. The percent sequence identity is calculated as the percentage of identical amino acids in the aligned sequences. A sequence "has" (or "have") x% sequence identity with another sequence means that the sequence is x% identical to that other sequence.

[0038] The term "wild-type Factor IX" refers to a Factor IX polypeptide sequence that occurs naturally and has the FIX activity of typical native FIX (e.g., as found in normal human plasma). The sequence of the polypeptide relative to the naturally occurring polypeptide sequence has not been artificially modified. This means that no amino acid in the naturally occurring polypeptide sequence has been replaced by a different amino acid. SEQ ID NO:1 is an example of a wild-type polypeptide sequence, but as exemplified below, the term also encompasses functional fragments, truncations, etc. For example, the term includes polypeptides having a modified N-terminus or C-terminus, including terminal amino acid deletions or additions, provided that these polypeptides substantially retain the activity of wild-type Factor IX. The term also includes any natural polymorphic variants of Factor IX. For example, a common natural polymorphic variant that occurs at a frequency of 33% is a Factor IX polypeptide that presents an alanine (A) at the position corresponding to position T148 of SEQ ID NO:1. This T148A polymorphic variant is shown as SEQ ID NO:20. Thus, all references herein to SEQ ID NO:1 may also refer to SEQ ID NO:20. Although these polymorphic variants occur naturally in the general population, at least some of them are associated with phenotypic effects, such as the T148A (reference 56) has been described in the literature.

[0039] The terms "FIX variant polypeptide", "FIX variant", "variant", "FIX polypeptide", etc. are used interchangeably herein and, unless otherwise expressly stated, all refer to FIX variant polypeptides. FIX variant polypeptides include full-length FIX protein or biologically active fragments of FIX protein, i.e., the polypeptide is capable of activating factor X (i.e., generating factor Xa). The factor IX variant polypeptides of the present invention are derived from the polypeptide sequence of wild-type factor IX (SEQ ID NO:1). The variant differs from wild-type factor IX at one or more amino acid positions corresponding to those in wild-type factor IX, i.e., the variant has one or more amino acid substitutions relative to the corresponding positions in wild-type factor IX. The numbering refers to the amino acid positions in wild-type factor IX as defined in SEQ ID NO:1. An exemplary polynucleotide coding sequence for the polypeptide of SEQ ID NO:1 is provided by SEQ ID NO:2.

[0040] To avoid any doubt, all FIX variant polypeptides described herein have FIX clotting activity. For example, they have the clotting activity of wild-type FIX, or they may even have a higher clotting activity than wild-type FIX; the clotting activity can be measured by standard assays known to those skilled in the art.

[0041] As shown in SEQ ID NO:3, the factor IX variant polypeptide can also be derived from wild-type factor IX including a signal and / or propeptide. SEQ ID NO:3 includes both a signal peptide (aa 1-28) and a propeptide (aa 29-46). The polypeptide of SEQ ID NO:3 is known in the art as the precursor of human factor IX, or prepropeptide factor IX. Factor IX with a propeptide but lacking a signal peptide is also referred to as propeptide factor IX. An exemplary polynucleotide coding sequence for the polypeptide of SEQ ID NO:3 is shown as SEQ ID NO:4.

[0042] The factor IX variant polypeptide can also be derived from one or more functional fragments of wild-type factor IX. For example, it can be derived from activated factor IX containing two fragments of factor IX (which lacks the middle "activation peptide" present in SEQ ID NO:1). SEQ ID NO:17 and 18 show the light and heavy chains of human activated factor IX, respectively, which are held together by disulfide bridges. Another example is isoform 2 of human factor IX, which lacks a 38-aa fragment at positions 47-84 of SEQ ID NO:1.

[0043] Alternatively, the factor IX variant polypeptide can be derived from a truncation or fusion of wild-type factor IX.

[0044] The term "polypeptide sequence derived from wild-type factor IX" (or similar phrasing) means that when two sequences are aligned, the factor IX variant polypeptide has a certain degree of sequence identity with the wild-type factor IX polypeptide. For example, as described above, the factor IX variant polypeptide can have a sequence identity of at least 70% or the like with SEQ ID NO:1. The factor IX variant polypeptide has biological activity, i.e., it is capable of activating factor X (i.e., generating factor Xa).

[0045] The factor IX variant polypeptide can be provided as an "isolated" or "purified" polypeptide. This term can refer to a polypeptide produced by expressing the isolated nucleic acid molecule of the present invention. Alternatively, this term can refer to a protein that has been sufficiently separated from other proteins with which it is naturally associated (e.g., present in a "substantially pure" form). "Isolated" does not mean excluding the presence of artificial or synthetic mixtures with other compounds or materials, or impurities that do not interfere with the basic activity, e.g., they may be present due to incomplete purification or addition of stabilizers.

[0046] Unless otherwise specified, "FIX protein" or "FIX polypeptide" herein refers to the weight of the FIX portion in the protein / polypeptide (e.g., as defined in SEQ ID NO:9), i.e., excluding the weight of any additional portions such as fusion partners (e.g., albumin).

[0047] The terms "administration" or "administering" are used interchangeably herein. Unless otherwise specifically stated, the term administration refers to administration into soft tissue.

[0048] The terms "treatment", "therapy" and "healing" are used interchangeably herein and refer to therapeutic measures that cure, alleviate, relieve the symptoms of a diagnosed pathological condition or disorder and / or stop the progression of a diagnosed pathological condition or disorder. Unless otherwise specified, the terms "treatment", "therapy" and "healing" can include prevention. The terms "treatment", "therapy" and "healing" also include on-demand treatment. If administering the factor IX variant polypeptide described herein to a subject (e.g., a person with factor IX deficiency such as hemophilia B) results in a therapeutic or preventive effect, the disorder is treated or prevented. This means that the plasma level of factor IX activity in the subject increases at least temporarily after treatment when measured using at least one factor IX assay. The factor IX activity can be determined using an in vitro aPTT-based one-stage clotting assay (references 5 and 6) or a tail clip model (e.g., as described in the examples). Such an increase may be clinically relevant, e.g., the frequency or intensity of bleeding events is reduced.

[0049] "Therapeutically effective amount" means that amount of the Factor IX variant polypeptide which, when administered to a subject as a single dose or as part of a series, is effective for treatment. "Prophylactically effective amount" means that amount of the Factor IX variant polypeptide which, when administered to a subject as a single dose or as part of a series, is effective for prophylaxis. Such methods are effective in treating or preventing disorders that require procoagulant activity (e.g., preventing, reducing or inhibiting bleeding), said disorders including but not limited to hemophilia, specifically hemophilia B.

[0050] The term "reduced binding" or "decreased binding" refers to a Factor IX variant polypeptide having a reduced binding of FIX to the extracellular matrix as compared to wild-type FIX, and includes FIX variants that exhibit no binding to the extracellular matrix. The binding of FIX to the extracellular matrix can be determined by various known bioassays, such as the competitor binding assay described in (Reference 4).

[0051] To avoid any doubt, the FIX variant polypeptides with reduced binding for use in the present invention retain FIX coagulant activity. For example, they have the coagulant activity of wild-type FIX, or they may even have a higher coagulant activity than wild-type FIX. Coagulant activity can be evaluated by assays known in the art. Any reference to a method of treatment that includes administering a FIX variant polypeptide to a subject also covers said FIX variant polypeptide for use in said method of treatment, and the use of said FIX variant polypeptide in said method of treatment, and the use of said FIX variant polypeptide in the preparation of a medicament for treating a disease.

[0052] The term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, rabbits, rodents, etc., which will be the recipient of a particular treatment. The subject is preferably a human. Generally, the terms "subject" and "patient" may be used interchangeably herein to refer to a human subject.

[0053] The term "pharmaceutically acceptable" means a substance that is approved or approvable by a regulatory agency of the Federal or a State government of the United States or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals (including humans).

[0054] The term "pharmaceutically acceptable excipient, carrier or adjuvant" or "acceptable pharmaceutical carrier" refers to an excipient, carrier or adjuvant that can be administered to a patient together with at least one agent of the present disclosure, and which, when administered in a dose sufficient to provide a therapeutic effect, does not destroy its pharmaceutical activity and is non-toxic. Generally, pharmaceutically acceptable excipients, carriers or adjuvants are considered by those skilled in the art and the U.S. FDA to be the inactive ingredients of any formulation.

[0055] The term "substantially pure" refers to a preparation comprising at least 75% by weight of the Factor IX variant polypeptide, specifically at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, or at least 96%, 97%, 98% or 99% by weight, e.g., 90-99% by weight or more of the Factor IX variant polypeptide. Purity can be measured by methods applicable to the compound of interest (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0056] The term "comprising" encompasses "including" as well as "consisting of", "consisting solely of" and / or "consisting essentially of", e.g., a composition "comprising" X can consist exclusively of X or can include additional substances, e.g., X + Y. It is also understood that wherever embodiments are described in the language of "consisting essentially of" herein, similar embodiments described in the language of "consisting of" are also provided.

[0057] The term "about" associated with a numerical value x is optional and means, e.g., x ± 10%.

[0058] The term "substantially" does not exclude "completely", e.g., a composition "substantially free of" Y can be completely free of Y. Where necessary, the term "substantially" can be omitted from the definitions of the present invention.

[0059] The term "and / or", e.g., "X and / or Y" should be understood to mean "X and Y" or "X or Y" and should be regarded as providing explicit support for both meanings or either meaning.

[0060] As used herein, the verb "comprising" and its inflected forms are used in its non-limiting sense, meaning including the item(s) following the word but not excluding items not specifically mentioned. In addition, where necessary, the verb "consisting of" can be replaced by "consisting essentially of", which means that the product as defined herein can contain additional components other than the specifically identified components, provided that such additional components do not alter the unique characteristics of the present invention.

[0061] Unless otherwise specified, a process or method comprising a number of steps can include additional steps at the start or end of the method, or can include additional intermediate steps. Also, where appropriate, steps can be combined, omitted or performed in an alternative order.

[0062] As used in the present disclosure and claims, unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" include the plural forms.

[0063] The entire contents of all patents and literature references cited in this specification are incorporated herein by reference.

[0064] This document describes various embodiments of the present invention. It is understood that the features specified in each embodiment can be combined with other specified features to provide further embodiments. Specifically, embodiments emphasized herein as suitable, exemplary, or preferred can be combined with each other (unless they are mutually exclusive).

[0065] Factor IX (FIX) variant polypeptide

[0066] The present invention relates to the use of a FIX variant polypeptide having reduced binding to the extracellular matrix relative to wild-type FIX for use in a treatment by administering the FIX variant polypeptide to soft tissue (e.g., subcutaneous tissue).

[0067] Extra-vascular FIX

[0068] The concept of extra-vascular FIX was first reported in 1983, when it was shown that FIX was able to bind to endothelial cells (reference 7). Subsequently in 1987, Stern et al. demonstrated that a large amount of FIX could be present in the extra-vascular space and that there was a rapid, reversible equilibrium between plasma and extra-vascular FIX (reference 8). Later studies demonstrated the direct binding of FIX to endothelial cells. In vitro experiments showed that the zymogen form of FIX reversibly binds to vascular endothelium (references 9, 10, and 11) and may bind to platelets (reference 12).

[0069] Experiments in hemophilia B (HB) mice have shown that FIX can occupy extravascular reservoirs and provide hemostatic protection for more than 7 days, while it is not detected in plasma (Reference 13). This study also estimated that these extravascular reservoirs contain significantly more FIX than in circulation. To characterize the phenomenon of extravascular FIX reservoirs, Cheung et al. mutated the vitamin K-dependent γ-carboxyglutamic acid (Gla) domain of FIX at residue 5 (lysine) or residue 10 (valine), which was reported to strongly affect their interaction with endothelial cells. Specifically, single-point mutations of lysine to alanine (FIXK5A) or to arginine (FIXK5R) at residue 5 of the FIX molecule resulted in altered endothelial cell binding affinity (Reference 3). The FIXK5R variant showed higher in vitro binding affinity to endothelial cells than wild-type FIX (FIXWT), while the FIXK5A variant failed to bind bovine endothelial cells but retained normal coagulation activity. In a subsequent study (Reference 4), Cheung et al. hypothesized that the extracellular matrix (specifically collagen IV, perhaps) is the FIX binding site on endothelial cells. Subsequent in vivo studies in HB mice found that in a saphenous vein bleeding model, infusion of FIXK5R into HB mice provided better hemostatic protection than infusion of wild-type FIX. In contrast, HB mice infused with FIXK5A showed reduced coagulation (Reference 14). On this basis, the authors proposed that FIX binding to collagen IV provides a more persistent extravascular reservoir of FIX and thus better hemostatic protection (see also References 15 and 16).

[0070] Thus, it is not obvious from these studies that FIX variant polypeptides with reduced binding to the extracellular matrix (such as the K5A variant) can be used to treat bleeding disorders, let alone that they can provide increased hemostatic efficacy when administered to soft tissues. The present inventors have realized that when FIX is specifically administered to soft tissues (e.g., subcutaneous administration), FIX variant polypeptides with reduced binding to the extracellular matrix actually provide enhanced hemostatic protection. For example, these examples show that FIX variant polypeptides with reduced binding to the extracellular matrix (e.g., the K5A variant) have higher hemostatic efficacy after subcutaneous administration compared to wild-type FIX. Without wishing to be bound by any particular theory, it is speculated that this higher hemostatic efficacy after subcutaneous administration is due to the fact that these FIX variant polypeptides can be more readily released into plasma circulation after subcutaneous administration because they have a lower intensity of interaction with the extracellular matrix (such as collagen IV) present at the administration site in the extravascular space. Unexpectedly, when these FIX variants (e.g., the K5A variant) are subcutaneously administered, the lack of a bound extravascular reservoir of FIX does not seem to have a negative impact on their hemostatic efficacy, which is contrary to the effects of those variants using other administration routes (e.g., intravenous) described previously.

[0071] Thus, the FIX variant polypeptides for use in the present invention have reduced binding to the extracellular matrix (such as collagen IV). Examples of FIX variant polypeptides with reduced binding for use in the present invention include: FIX variant polypeptides containing the amino acid alanine at the position corresponding to position 5 of wild-type factor IX, FIX variant polypeptides containing the amino acid lysine at the position corresponding to position 10 of wild-type factor IX, or more generally, FIX variant polypeptides containing an amino acid with any hydrophobic or uncharged side chain at the position corresponding to position 5 of wild-type factor IX, or FIX variant polypeptides containing an amino acid with a positively charged side chain at the position corresponding to position 10 of wild-type factor IX, provided that they retain FIX clotting activity, for example, they have the clotting activity of wild-type FIX, or they may even have a higher clotting activity than wild-type FIX; the clotting activity can be measured by standard assays known to those skilled in the art. Amino acids containing hydrophobic side chains (at pH 7) include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids containing uncharged side chains (at pH 7) include serine, threonine, asparagine, and glutamine. Amino acids containing positively charged side chains (at pH 7) include lysine, arginine, and histidine. In a preferred embodiment, the FIX variant polypeptide contains the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0072] In some embodiments, the FIX variant polypeptide contains the amino acid alanine at the position corresponding to position 5 of wild-type factor IX, but does not contain the amino acid lysine at the position corresponding to position 10 of wild-type factor IX (as an alternative, valine can be used at position 10).

[0073] The FIX variant polypeptides for use in the present invention can also contain two or more mutations (such as at positions 5 and 10), which reduce the binding of the polypeptide to the extracellular matrix. For example, in some embodiments, the FIX variant polypeptide contains the amino acid alanine at the position corresponding to position 5 of wild-type factor IX and the amino acid lysine at the position corresponding to position 10 of wild-type factor IX. In other embodiments, the FIX variant polypeptide contains an amino acid with a hydrophobic or uncharged side chain at the position corresponding to position 5 of wild-type factor IX and an amino acid with a positively charged side chain at the position corresponding to position 10 of wild-type factor IX.

[0074] Accordingly, in one aspect, the present invention provides a Factor IX (FIX) variant polypeptide for use in a method of treating or preventing a disease or disorder, the method comprising administering the FIX variant polypeptide to soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX.

[0075] The present invention also provides a method of treating or preventing a disease or disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of a FIX variant polypeptide to soft tissue in the subject, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX.

[0076] The present invention also provides the use of a FIX variant polypeptide in the preparation of a medicament for treating or preventing a disease or disorder in a subject, wherein the FIX variant polypeptide is to be administered to soft tissue in the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX.

[0077] The present invention also provides a FIX variant polypeptide for treating or preventing a disease or disorder, wherein the FIX variant polypeptide is to be administered to soft tissue in the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX.

[0078] Additional Factor IX mutations

[0079] In a further embodiment, the FIX variant polypeptide for use in the present invention may further comprise further mutations relative to wild-type Factor IX which are capable of increasing the coagulation activity (e.g., increasing the specific activity) relative to wild-type Factor IX. Such variant polypeptides are also referred to herein as "hyperactive" FIX polypeptides or hyperactive FIX variant polypeptides. Other terms in the art are used synonymously, e.g., "hyperactive" FIX variants. These variants have the biological function of Factor IX, i.e., the variant is capable of generating Factor Xa, optionally after the Factor IX variant polypeptide is converted to its active form (Factor IXa) by excision of the activation peptide. The variant is capable of generating a higher level of Factor Xa than wild-type FIX activity. Activation cleavage of Factor IX can be achieved in vitro, e.g., by Factor XIa or Factor VIIa / TF. Suitable in vitro assays for measuring Factor IX activity are known to those skilled in the art (e.g., one-stage clotting assays such as the aPTT assay, chromogenic assays, etc.).

[0080] Exemplary highly active Factor IX variant polypeptides contain leucine (L) at the position corresponding to position 338 of wild-type Factor IX, where wild-type Factor IX normally has arginine (R) at this position (“R338L”). One such exemplary polypeptide is the “Padua” mutant described in reference 17. See SEQ ID NO:10. The specific activity of the “Padua” mutant is generally at least about 5-8 times higher than that of wild-type Factor IX.

[0081] Thus, in some embodiments, the Factor IX (FIX) variant polypeptide for use in the present invention contains alanine at the position corresponding to position 5 of wild-type Factor IX and leucine at the position corresponding to position 338 of wild-type Factor IX.

[0082] Other exemplary highly active Factor IX variants are E410H, E410K, R338V, and R338L+E410K, and those described in reference 18, e.g., containing the amino acid H at the position corresponding to position 410 of wild-type Factor IX, and containing an amino acid other than R at the position corresponding to position 338 of wild-type Factor IX, e.g., containing an amino acid selected from the group consisting of V, T, and W at the position corresponding to position 338 of wild-type Factor IX, e.g., R338V+E410H, R338T+E410H, R338W+E410H, and R338L+E410H. Another useful variant is R318Y+R338E+T343R.

[0083] Thus, in some embodiments, the FIX variant polypeptide for use in the present invention contains alanine at the position corresponding to position 5 of wild-type Factor IX, an amino acid selected from valine, threonine, and tryptophan at the position corresponding to position 338 of wild-type Factor IX, and histidine at the position corresponding to position 410 of wild-type Factor IX. In a specific embodiment, the FIX variant polypeptide for use in the present invention contains alanine at the position corresponding to position 5 of wild-type Factor IX, valine at the position corresponding to position 338 of wild-type Factor IX, and histidine at the position corresponding to position 410 of wild-type Factor IX.

[0084] As described above, another highly active Factor IX variant for use in the present invention is the Dalcinonacog alpha variant (also known as CB 2679d), see SEQ ID NO:19. Dalcinonacog alpha has three amino acid substitutions in two loops within the FIX protein. Based on the mature FIX sequence numbering, (1) R318Y, located in the "150 loop", stabilizes activated FIX (FIXa), directly interacts with the substrate Factor X (FX) and provides antithrombin resistance; (2) R338E and (3) T343R, both located in the "170 loop", significantly enhance the affinity for the cofactor activated Factor VIII (FVIIIa) and increase the catalytic activity of FIXa. R318Y / R338E / T343R refers to R150Y / R170E / T175R in the classical chymotrypsin numbering (Reference 19), and R364Y / R384E / T389R in the Human Genome Variation Society (HGVS) nomenclature, which includes a 46 amino acid propeptide (Reference 20). Thus, in some embodiments, the FIX variant polypeptide for use in the present invention comprises the amino acid alanine at the position corresponding to position 5 of wild-type Factor IX, the amino acid tyrosine at the position corresponding to position 318 of wild-type Factor IX, the amino acid glutamate at the position corresponding to position 338 of wild-type Factor IX, and the amino acid arginine at the position corresponding to position 343 of wild-type Factor IX.

[0085] Further exemplary highly active Factor IX variant polypeptides include those listed in Table 1 below (Reference 21).

[0086] Table 1

[0087]

[0088] The numbers in Table 1 refer to positions in the mature FIX protein (SEQ ID NO:1) without the propeptide sequence. Activity was determined using a one-stage clotting assay.

[0089] By using methods known in the art to determine the specific (molar) activity of the Factor IX polypeptide and comparing this activity to wild-type Factor IX, those skilled in the art are able to identify and validate these and other highly active Factor IX variant polypeptides.

[0090] The Factor IX variant polypeptide can be derived from a Factor IX polypeptide sequence of any mammalian species. In a specific embodiment, the Factor IX variant polypeptide is derived from a Factor IX polypeptide sequence of human origin. The gene ID: 2158 (https: / / www.ncbi.nlm.nih.gov / gene / 2158), GenBank accession number NM_000133.3 (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000133.3), NP_000124.1 (https: / / www.ncbi.nlm.nih.gov / protein / NP_000124.1?report=genpept) and UniProt entry P00740 (https: / / www.uniprot.org / uniprot / P00740) provide examples of the amino acid and / or nucleotide sequences of wild-type human Factor IX.

[0091] The Factor IX variant polypeptide according to the present invention can be derived from mature (i.e., excluding the signal peptide and propeptide) wild-type Factor IX, such as of human origin, the amino acid sequence of which is shown in SEQ ID NO:1. This polypeptide sequence is "isoform 1" of human Factor IX.

[0092] Route of administration

[0093] These examples show that when administered into the subcutaneous tissue, the FIX variant polypeptide containing the amino acid alanine at the position corresponding to position 5 of wild-type Factor IX has a greater hemostatic effect than wild-type FIX. Without wishing to be bound by any specific theory, it is speculated that the reason these FIX variant polypeptides have a greater hemostatic effect after subcutaneous administration is because they have a lower binding strength to the extracellular matrix and thus are released into the circulation faster from the extracellular space. Therefore, based on these data, it seems reasonable that the disclosed FIX variant polypeptides have a greater hemostatic effect compared to wild-type FIX when administered more generally into soft tissues.

[0094] Accordingly, a Factor IX (FIX) variant polypeptide for use in the present invention is administered to soft tissue. Those skilled in the art can understand this term. For example, soft tissue administration is defined by the US FDA as administration into any soft tissue (https: / / www.fda.gov / drugs / data-standards-manual-monographs / route-administration). Soft tissue is any tissue in the body that has not been hardened by the process of ossification or calcification (such as bone and teeth). In one embodiment, the soft tissue does not include muscle tissue. In another embodiment, the soft tissue does not include liver tissue. In a preferred embodiment, the soft tissue to which the FIX variant polypeptide is administered is skin tissue (including subcutaneous tissue) or mucosal tissue (including gastrointestinal mucosal tissue).

[0095] The FIX variant polypeptide is for administration to a subject, such as an animal, typically a human subject.

[0096] The methods and uses described herein do not involve intravenous administration of the FIX variant polypeptide. For example, the present invention provides a Factor IX (FIX) variant polypeptide for use in a method of treating or preventing a disease (e.g., a bleeding disorder), wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type Factor IX, and wherein the FIX variant polypeptide is not administered intravenously.

[0097] In some embodiments, the methods and uses described herein do not involve intramuscular administration of the FIX variant polypeptide.

[0098] Administration into soft tissue immediately exposes the FIX variant polypeptide to components in the extracellular space (referred to as the interstitial space) between cells. For example, after the FIX variant polypeptide is administered into soft tissue, at least a portion of the FIX variant polypeptide is directly delivered to the extracellular space, while another portion of the FIX variant polypeptide may be delivered into cells or taken up by cells and then the FIX variant polypeptide is secreted out of the cells, into the extracellular space. Accordingly, the methods and uses described herein are different from gene-based (e.g., viral or non-viral vector) methods, wherein a nucleic acid sequence encoding FIX is administered (e.g., into muscle tissue) and the FIX polypeptide is produced intracellularly.

[0099] In a preferred embodiment, the soft tissue is skin tissue. For the purposes of the present disclosure, skin includes three main layers - the hypodermis (subcutaneous tissue) is the innermost layer of the skin; the dermis is the middle layer, and the epidermis is the outermost layer. Subcutaneous administration (e.g., subcutaneous injection) refers to the administration of a substance into the hypodermis. To avoid any doubt, in the context of the present disclosure, reference to "administered to the skin" or "administered to skin tissue" encompasses subcutaneous administration (administered into the hypodermis). Additionally, subcutaneous administration characterized by administration "under" or "beneath" or "below" the skin (or synonyms) is also encompassed by the present invention.

[0100] Thus, in some embodiments, the FIX variant polypeptide is administered to skin tissue. In some embodiments, the FIX variant polypeptide is administered into subcutaneous (hypodermal) tissue, dermal tissue, or epidermal tissue. Thus, the administration can be subcutaneous, intradermal, topical (e.g., epidermal), or transdermal (e.g., by transdermal injection or absorption). In a preferred embodiment, the FIX variant polypeptide is administered subcutaneously.

[0101] In a preferred embodiment, the factor IX (FIX) variant polypeptide for use in the present invention comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX and is administered subcutaneously. For example, the present invention provides a method for treating or preventing a disease in a subject, which comprises subcutaneously administering an effective amount of a FIX variant polypeptide that comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0102] The present invention also provides a factor IX (FIX) variant polypeptide for use in a method for treating or preventing a disease in a subject, the method comprising subcutaneously administering the FIX variant polypeptide, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0103] Also provided is the use of a factor IX variant polypeptide in the preparation of a medicament for treating or preventing a disease in a subject, wherein the FIX variant polypeptide is to be administered subcutaneously to the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0104] The present invention also provides the use of a FIX variant polypeptide for treating or preventing a disease in a subject, comprising subcutaneously administering the FIX variant polypeptide to the subject, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0105] In some embodiments, the FIX variant polypeptide is administered to mucosal tissue, such as gastrointestinal mucosal tissue. In some embodiments, the FIX variant polypeptide is administered enterally (through the human gastrointestinal tract). Examples of enteral administration include oral, sublingual, gastric, and rectal administration.

[0106] An orally administrable FIX variant polypeptide can be injected into the mucosal tissue of the gastrointestinal tract using a drug delivery device (also referred to as an applicator) that autonomously positions itself to engage with GI tissue and inject the drug into the GI tissue. Exemplary drug delivery devices are described in reference 30. Exemplary devices include the SOMA (self-orienting millimeter-scale applicator) (reference 31), BIONDD TM (reference 32), and RaniPill TM (references 33 and 34). In some embodiments, the FIX variant polypeptide is injected into the mucosal tissue of the stomach, for example, using the BIONDD TM device. The BIONDD TM is designed to insert biodegradable spikes carrying the drug into the stomach wall. It consists of a capsule that attaches to and delivers the drug to the stomach tissue.

[0107] Bleeding disorders

[0108] In a preferred embodiment, the factor IX variant polypeptides described herein are used for the treatment or prevention of bleeding disorders. The bleeding disorder can be any disease that requires a coagulant (e.g., to prevent, reduce, or inhibit bleeding). An exemplary bleeding disorder is hemophilia, specifically hemophilia B.

[0109] Accordingly, the present invention provides FIX variant polypeptides for use in a method of treating or preventing a bleeding disorder, the method comprising administering the FIX variant polypeptide to soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX.

[0110] The present invention also provides a method of treating or preventing a bleeding disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of a FIX variant polypeptide to the soft tissue of the subject, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX.

[0111] The present invention also provides the use of a FIX variant polypeptide in the preparation of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX variant polypeptide is to be administered to soft tissue of the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX.

[0112] The present invention also provides a FIX variant polypeptide for treating or preventing a bleeding disorder, wherein the FIX variant polypeptide is to be administered to soft tissue of the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX.

[0113] The treatment or prevention can include on-demand control of bleeding episodes, perioperative bleeding management, and / or routine prophylaxis to prevent or reduce the frequency of bleeding episodes. For example, the treatment can include on-demand control of bleeding episodes or perioperative bleeding management. The prophylaxis can include prevention of bleeding episodes or reduction of the frequency of bleeding episodes.

[0114] The subject is typically human. The subject can be an adult or a child. The baseline (untreated or unprophylaxed) plasma factor IX activity of the subject can be 40% or lower, 30% or lower, 20% or lower, 10% or lower, 5% or lower, 4% or lower, 3% or lower, 2% or lower, 1 to 5% or 1% or lower compared to the plasma factor IX activity of a healthy subject. In a specific embodiment, the subject is a pediatric subject (child), for example, 18 years of age or younger. In one embodiment, the subject is not a suitable candidate for FIX gene therapy.

[0115] In some embodiments, the FIX variant polypeptide is administered at a dose of 20 IU / kg to 350 IU / kg. In certain embodiments, the FIX variant polypeptide is administered at a dose of 30 IU / kg to 300 IU / kg, 30 IU / kg to 250 IU / kg, 50 IU / kg to 200 IU / kg, or 50 IU / kg to 150 IU / kg. In some embodiments, the FIX variant polypeptide is administered at a dose of about 25 IU / kg, 30 IU / kg, 50 IU / kg, 75 IU / kg, 100 IU / kg, 150 IU / kg, 200 IU / kg, 250 IU / kg, 300 IU / kg, or 350 IU / kg. In certain embodiments, the FIX variant polypeptide is administered at a dose of about 50 IU / kg, 100 IU / kg, or 150 IU / kg.

[0116] In one embodiment, the FIX polypeptide is administered in a composition that does not contain antithrombotic substances (such as heparin).

[0117] Bleeding disorders include hemophilia (hemophilia A, hemophilia B, hemophilia A and B patients with inhibitory antibodies; specifically hemophilia B), at least one coagulation factor deficiency (e.g., factor VII, IX, X, XI, V, XII, II, and / or von Willebrand factor; specifically factor IX), FV / FVIII combined deficiency, vitamin K epoxide reductase CI deficiency, γ-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule anticoagulants (i.e., FXa inhibitors); and platelet disorders such as giant platelet syndrome (Bernard Soulier syndrome), Glanzmann thrombasthenia, and storage pool deficiency.

[0118] In a preferred embodiment, the above method or use is for treating or preventing bleeding in a subject with hemophilia B, which is also known in the art as congenital factor IX deficiency.

[0119] One way to represent factor IX activity in plasma is as a percentage relative to normal human plasma. Another way to represent factor IX activity in plasma is in international units (IU) relative to the international standard of factor IX in plasma. 1 IU of factor IX activity in plasma is equivalent to the amount of factor IX in 1 mL of normal human plasma.

[0120] One way to check the prophylactic or therapeutic efficacy is to measure the plasma factor IX activity in the subject after prophylaxis or treatment and compare it with the plasma factor IX activity in the subject before prophylaxis or treatment. An increase in factor IX activity after prophylaxis or treatment (e.g., from <1% or 1%-5% or 5-40% of normal human plasma to, e.g., peak levels of 15%, 20%, >25%, >30%, >35%, >40%, >50%, or >60% of normal human plasma, e.g., from <5% to >5% (such as to 5-40%)) indicates a prophylactic or therapeutic effect. In clinical trials, a factor IX level of 5-10% of normal human serum is used as the target for achieving bleeding control in prophylactic treatment.

[0121] A prophylactic or therapeutic effect can also be achieved when the factor IX activity after prophylaxis or treatment is sufficient to prevent, reduce, or inhibit bleeding.

[0122] The factor IX activity after prophylaxis or treatment can result in a trough value of at least 15-40%, or can even exceed the pathological range (e.g., peak levels >40% of normal human serum).

[0123] Factor IX activity can be measured using any Factor IX activity assay known to those skilled in the art, such as using the aPTT assay (a decrease in the aPTT value indicates an increase in Factor IX activity). Thus, in a preferred embodiment, Factor IX activity is determined using an in vitro aPTT-based one-stage clotting assay (References 5 and 6).

[0124] When administered in vivo to a subject, the Factor IX variant polypeptides for use in the present invention can have a higher specific molar activity than the corresponding wild-type Factor IX polypeptide. Such highly active variants are as described above. For example, the % increase in plasma Factor IX activity (e.g., measured using an in vitro aPTT-based one-stage clotting assay) using the Factor IX variant polypeptides described herein can be higher compared to using the same molar amount of the corresponding wild-type Factor IX polypeptide. Another way of describing it is that the aPTT time in a serum sample is shorter after administration of the Factor IX variant polypeptides described herein compared to using the same molar amount of the corresponding wild-type Factor IX polypeptide.

[0125] Preparation of Factor IX variant polypeptides

[0126] The Factor IX variant polypeptides for use in the present invention can be prepared using standard techniques well known to those skilled in the art. For example, the cDNA sequence of wild-type Factor IX (e.g., SEQ ID NO:2) can be modified using standard mutagenesis techniques (e.g., site-directed mutagenesis) so that it encodes the desired Factor IX variant polypeptide, e.g., encoding the amino acid alanine at the position corresponding to position 5 of wild-type Factor IX (where lysine (K) is encoded at that position). For the purpose of recombinant protein production, an N-terminal leader peptide can be used, based on the native Factor IX leader peptide (as shown in SEQ ID NO:3) or alternatives known to those skilled in the art. The cDNA sequence can be inserted into a suitable expression plasmid to express the recombinant Factor IX variant polypeptide. This is typically carried out using mammalian cells (e.g., HEK for transient expression or CHO cell line for stable expression), but other types of cells that can produce glycosylated and correctly folded proteins can also be used. The recombinant Factor IX variant polypeptide can subsequently be purified, e.g., using anion exchange chromatography.

[0127] The Factor IX variant polypeptides can be combined with other agents and / or pharmaceutically acceptable carriers.

[0128] Fusions and conjugates

[0129] The Factor IX variant polypeptides for use in the present invention can also be provided as part of a fusion with another moiety (e.g., with albumin (e.g., linked via a cleavable linker)).

[0130] The Factor IX variant polypeptide can be provided fused to one or more additional moieties or conjugated to one or more additional moieties. The one or more additional moieties are generally different from Factor IX, i.e., they do not have the biological function of Factor IX as defined above (they do not have the ability to generate Factor Xa). This means that a fragment of Factor IX (e.g., a linker containing a polypeptide sequence fragment derived from Factor IX) that does not itself have the function of Factor IX can be such an "one or more additional moieties", i.e., they are not part of the Factor IX moiety, but they can be part of a molecule containing the Factor IX moiety.

[0131] Half-life enhancing moiety and linker

[0132] In an exemplary embodiment, the FIX variant polypeptide is linked to a half-life enhancing moiety. The half-life enhancing moiety can comprise one or more polypeptides (half-life enhancing polypeptides, HLEPs). In one embodiment, the HLEP is albumin, e.g., recombinant human albumin. In another embodiment, the HLEP is a fragment of an antibody (immunoglobulin), e.g., an Fc fragment, e.g., IgG Fc, such as IgG1 Fc. Alternatively, the HLEP can be the C-terminal peptide (CTP) of human chorionic gonadotropin. The HLEP can also be an unstructured recombinant polypeptide (e.g., XTEN). Such molecules are also referred to in the art as fusion polypeptides.

[0133] The FIX variant polypeptide can be linked to the HLEP via a cleavable linker (specifically, a cleavable peptide linker). Generally, the cleavable linker can be cleaved by a protease that is the same as that for activated Factor IX. Thus, such a cleavable linker provides a high molar specific activity of the fusion polypeptide.

[0134] The FIX variant polypeptide can also be pegylated, i.e., one or more polyethylene glycol moieties are conjugated to the FIX variant polypeptide using methods known in the art.

[0135] The FIX variant polypeptide for use in the present invention can comprise one half-life enhancing moiety or more than one half-life enhancing moiety. Thus, the term "half-life enhancing moiety" encompasses one or more half-life enhancing moieties. The half-life enhancing moieties can be of the same type. The half-life enhancing moieties can be of different types. For example, the FIX variant polypeptide can be linked to XTEN (e.g., XTEN72) and additionally to an Fc domain (e.g., human IgG1 Fc).

[0136] Preferably, compared to the unfused FIX variant polypeptide, the half-life enhancing moiety is capable of extending the in vivo (plasma) half-life of the FIX variant polypeptide by at least about 25%. Preferably, the half-life enhancing moiety is capable of extending the in vivo (plasma) half-life of the FIX variant polypeptide by at least about 50%, and more preferably by more than 100%. The in vivo half-life is typically determined as the terminal half-life or the β-half-life.

[0137] Albumin

[0138] As used herein, "albumin" generally refers to albumin polypeptides or amino acid sequences, or albumin fragments, variants or analogs having one or more functional activities (biological activities) of albumin. Specifically, "albumin" may refer to human albumin (HA) or fragments thereof, particularly the mature form of human albumin, as shown by SEQ ID NO:5 herein. The albumin may also be derived from other species, specifically other vertebrates. The albumin moiety of the fusion polypeptide may comprise the full length of the HA sequence as set forth in SEQ ID NO:5, or it may include one or more of its fragments, which are capable of stabilizing or extending the therapeutic activity of the factor IX variant polypeptide. Such fragments may be 10 or more amino acids in length, or may include about 15, 20, 25, 30, 50 or more contiguous amino acids from the HA sequence, or may include a portion or all of a specific domain of HA. These and other suitable albumin moieties (including variants) are described in reference 36.

[0139] Structurally related family members of the albumin family can also be used as HLEP. For example, alpha-fetoprotein (AFP, reference 35) is a member of the albumin family and can also be used to enhance the half-life of the factor IX variant polypeptide. Such half-life enhancing polypeptides are described in reference 36. Another option is afamin (AFM, reference 37) or vitamin D binding polypeptide (DBP, reference 38). Fragments of these polypeptides can also be used.

[0140] In embodiments using albumin HLEP, the albumin is typically provided as a genetic fusion with the factor IX moiety. This means that a single cDNA molecule encodes the factor IX moiety and the albumin moiety, optionally with an intervening sequence encoding a linker (e.g., a cleavable linker).

[0141] Immunoglobulin

[0142] Immunoglobulins (Igs) or fragments thereof can also be used as HLEP. Examples of suitable immunoglobulins are IgG or IgG fragments, such as the Fc region. The Fc region can be an Fc domain (e.g., two polypeptide chains, each polypeptide chain containing a hinge region (or a part of the hinge region), a CH2 region, and a CH3 region). Thus, the Factor IX variant polypeptide can be fused directly or via a linker to the Fc domain. In embodiments using a linker, the linker can be cleavable.

[0143] Monomers, dimers, and hybrids are all encompassed. For example, the Factor IX variant polypeptide can be a heterodimer comprising two polypeptide chains, wherein the first chain comprises a Factor IX portion linked to the hinge region (or a part of the hinge region), CH2 region, and CH3 region of an immunoglobulin (e.g., IgG1), and the second chain comprises the hinge region (or a part of the hinge region), CH2 region, and CH3 region of an immunoglobulin (e.g., IgG1).

[0144] In another embodiment, the Factor IX variant polypeptide is a homodimer comprising two polypeptide chains, wherein each chain comprises a Factor IX portion linked to the hinge region (or a part of the hinge region), CH2 region, and CH3 region of an immunoglobulin (e.g., IgG1).

[0145] In a further embodiment, the Factor IX variant polypeptide is a monomer, and the monomer comprises a Factor IX portion linked to the hinge region (or a part of the hinge region), CH2 region, and CH3 region of an immunoglobulin (e.g., IgG1).

[0146] For example, other examples of suitable Factor IX IgG Fc fusion molecule configurations are found in reference 39.

[0147] Exemplary Fc polypeptides (derived from the human IgG1 Fc domain) are shown in SEQ ID NO:6. Another exemplary Fc polypeptide (derived from the human IgG1 Fc domain) is shown in SEQ ID NO:7.

[0148] In any of these embodiments, the Factor IX portion can be linked directly or via a linker to the Fc portion. In embodiments using a linker, the linker can be cleavable or non - cleavable. In a specific embodiment, the linker is cleavable. An exemplary cleavable linker is shown in SEQ ID NO:8.

[0149] An exemplary Fc portion is the Fc portion of Eftrenonacogα See also references 40, 41, or 42.

[0150] The C - terminal peptide (CTP) of human chorionic gonadotropin

[0151] Another exemplary half-life enhancing moiety is the C-terminal peptide (CTP) of human chorionic gonadotropin. CTP is based on the native peptide of 31 amino acids in length, i.e., the C-terminal peptide of the β-chain of human chorionic gonadotropin (hCG).

[0152] One or more units of CTP may be fused to the Factor IX moiety. The one or more units of CTP may be fused to the N-terminus and / or C-terminus of Factor IX, preferably to the C-terminus.

[0153] In one embodiment, the Factor IX variant polypeptide is Factor IX modified with CTP, which comprises the Factor IX variant polypeptide as described herein linked to 3 to 5 CTPs, optionally wherein the CTPs are linked to the C-terminus of the Factor IX variant polypeptide. In a specific embodiment, three tandem units of CTP are linked to the Factor IX variant polypeptide, optionally linked to the C-terminus of the Factor IX variant polypeptide.

[0154] In any of these embodiments, at least one of the CTPs may be linked to the Factor IX moiety via a linker. The linker may be a peptide bond. The linker may be cleavable.

[0155] In an exemplary embodiment, the CTP sequence comprises SEQ ID NO:11. In another exemplary embodiment, the CTP sequence comprises SEQ ID NO:12. In another exemplary embodiment, the CTP sequence comprises SEQ ID NO:13.

[0156] Other suitable CTP sequences and related methods are known to those skilled in the art, for example, see references 43, 44 or 45.

[0157] Unstructured recombinant polypeptides

[0158] Another exemplary half-life enhancing moiety is an unstructured recombinant polypeptide. An example of such an unstructured recombinant polypeptide is XTEN, for example, see reference 46.

[0159] Thus, in one embodiment, the Factor IX variant polypeptide is a Factor IX variant polypeptide fused to at least one XTEN. XTEN can be fused to the Factor IX moiety by insertion into the Factor IX variant polypeptide sequence while maintaining the biological activity of Factor IX. For example, the XTEN can be inserted between two adjacent amino acids at a position in the activation peptide of Factor IX, and when the XTEN is inserted, the inserted position does not prevent cleavage of the activation peptide during blood coagulation. Alternatively, XTEN can be fused to the C-terminus and / or N-terminus of the Factor IX, preferably the C-terminus. XTEN can be fused to the C-terminus and / or N-terminus (preferably the C-terminus) of the Factor IX via a linker (e.g., a cleavable linker). The linker can be cleaved by thrombin.

[0160] A preferred XTEN is XTEN72. An exemplary XTEN72 sequence is shown as SEQ ID NO:14. Alternative XTEN sequences are shown as SEQ ID NO:15. Other suitable sequences and methods are disclosed in, for example, references 47, 48, or 49.

[0161] In a specific embodiment, the Factor IX variant polypeptide comprises XTEN72 linked to the activation peptide of Factor IX, and wherein the Factor IX moiety is further linked to a human IgG1 Fc domain at the C-terminus of the Factor IX moiety.

[0162] PEGylation

[0163] Another exemplary half-life enhancing moiety is polyethylene glycol (PEG). Glycosylated PEGylation is within the scope of the term "PEGylation" as used herein. For example, a PEG moiety of about 40 kDa can be covalently linked to the Factor IX variant polypeptide, e.g., via a specific N-linked glycan within the activation peptide.

[0164] An example of a glycosylated PEG moiety is nonacog β (see also reference 50), wherein the non-reducing end of an average of one glycan at N157 or N167 of Factor IX (numbered according to SEQ ID NO:1) is linked to neuraminic acid and conjugated to two PEG polymers (total average molecular weight of the polymers is about 42 kDa) via an amino group.

[0165] PEGylation of Factor IX polypeptides is also taught in, for example, references 51, 52, and 53.

[0166] Linker

[0167] Factor IX variant polypeptides comprising a half-life enhancing portion can use a cleavable linker, specifically a proteolytically cleavable linker. The linker is typically located between the Factor IX polypeptide portion and the half-life enhancing portion. The linker can release the Factor IX portion after the linker is cleaved by a protease of the blood coagulation cascade (e.g., a protease that is also capable of converting Factor IX to its activated form (e.g., FXIa or VIIa / tissue factor (TF))). When the HLEP is albumin, the cleavable linker is particularly useful.

[0168] While it is desirable to have an enhanced in vivo half-life of Factor IX, once the Factor IX is activated, it is desirable to limit its half-life to reduce the risk of prothrombotic effects, especially for highly active Factor IX variant polypeptides. Thus, in some embodiments, the cleavable linker couples the Factor IX variant polypeptide to the half-life enhancing portion, thereby providing the Factor IX variant polypeptide with a longer half-life relative to the unfused polypeptide. However, once bleeding occurs and the blood coagulation cascade has been initiated, the protease of the blood coagulation cascade activates the Factor IX variant polypeptide, which has an increased specific activity relative to the corresponding wild-type Factor IX. At the same time, the linker is cleaved, and the activated Factor IX variant polypeptide is released from the half-life enhancing portion, thereby reducing the risk of prothrombotic effects due to any prolonged increase in Factor IX activity.

[0169] The linker can be a fragment of Factor IX, preferably a fragment involved in Factor IX activation. For example, the linker can comprise such a fragment of the Factor IX sequence that is extended from the N-terminal residue (e.g., a proline residue). An exemplary cleavable linker is shown in SEQ ID NO:8. Other cleavable linkers are described in reference 36.

[0170] When measured in at least one blood coagulation-related assay (examples of which are known to those skilled in the art, such as the aPTT one-stage assay), a Factor IX variant polypeptide linked to a half-life enhancing portion via an intervening cleavable linker can have at least 25% higher molar specific activity compared to the corresponding molecule with a non-cleavable linker (e.g., GGGGGGV, SEQ ID NO:16). Preferably, a Factor IX variant polypeptide linked to a half-life enhancing portion via an intervening cleavable linker has at least 50% (more preferably at least 100%) increased molar specific activity compared to the corresponding molecule without a cleavable linker.

[0171] Thus, in one embodiment, the FIX variant polypeptide for use in the present invention comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX (and optionally one or more additional mutations relative to wild-type FIX as described herein to further reduce binding to the extracellular matrix (e.g., V10K) and / or increase the coagulation activity of FIX (e.g., R338L)), wherein said FIX is linked to a half-life enhancing moiety (e.g., albumin) described herein, optionally via a cleavable linker as described herein.

[0172] Pharmaceutical composition

[0173] The FIX variant polypeptide may be provided as a pharmaceutical composition. The pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier. Accordingly, the present invention also provides a pharmaceutical composition comprising a factor IX (FIX) variant polypeptide for use in a method of treating or preventing a bleeding disorder, the method comprising administering the pharmaceutical composition to soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0174] The present invention also provides a method of treating or preventing a bleeding disorder in a subject, the method comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising a FIX variant polypeptide to soft tissue of the subject, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0175] The present invention also provides the use of a pharmaceutical composition comprising a FIX variant polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the pharmaceutical composition is to be administered to soft tissue of the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0176] The present invention also provides a pharmaceutical composition comprising a FIX variant polypeptide for treating or preventing a bleeding disorder, wherein the pharmaceutical composition is to be administered to soft tissue of the subject, and wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0177] The pharmaceutical composition is for administration to a subject, such as an animal, typically a human subject.

[0178] The pharmaceutical composition is pharmaceutically acceptable and generally includes a suitable carrier. A comprehensive discussion of pharmaceutically acceptable carriers is available in reference 54. The composition is preferably sterile, pyrogen-free and / or preservative-free.

[0179] Accordingly, the Factor IX variant polypeptide can be provided in a buffered liquid form, e.g., in a citrate buffer, optionally containing a stabilizer and / or a filler. Exemplary pharmaceutical compositions for use in the present invention comprise a Factor IX variant polypeptide, trisodium citrate dihydrate, polysorbate 80, mannitol, sucrose, hydrochloric acid, and sterile water. In an exemplary formulation, the components are 25 mM trisodium citrate dihydrate, 0.006%-0.024% polysorbate 80, 18-29 g / L mannitol, 7-12 g / L sucrose, hydrochloric acid for adjusting the pH to 6.6-7.2 (e.g., pH 6.8), and sterile water. In a preferred embodiment, the formulation is 30 mmol / L trisodium citrate-2*H2O, 35.5 g / L D-mannitol, 14.0 g / L sucrose, 0.00030 mL / L polysorbate 80, pH 7.0.

[0180] Alternatively, the Factor IX variant polypeptide in the composition is lyophilized but reconstituted with a liquid diluent before administration, e.g., with sterile water for injection. Typical excipients in a composition comprising a lyophilized Factor IX variant polypeptide include trisodium citrate dihydrate, polysorbate 80, mannitol, sucrose, and / or hydrochloric acid.

[0181] In some embodiments, the composition is suitable for administration to soft tissue, e.g., subcutaneous administration, optionally after reconstitution or dilution.

[0182] The composition can be prophylactic (preventing bleeding) or therapeutic (treating bleeding).

[0183] Brief Description of the Drawings

[0184] Figure 1 : Pharmacokinetic curve of FIX variant polypeptide in HB mice after intravenous administration. Samples were collected at different time points up to 336 hours after administration. LLOQ (<1.6 pmol / mL) refers to the lowest concentration of rFIX antigen that can be reliably detected based on ELISA. Data with values ≤ LLOQ were not plotted. Plotted time points: 0 (injection time point) up to 168 hours. Each data point represents the mean ± SD of n = 3-5 mice, and α represents n = 1. In the rFIXK5R group, only 1 out of 3 animals (α) had a detectable level at 48 hours, and in the rFIXWT group, only 1 out of 5 animals (α) had a detectable level at 72 hours and 168 hours.

[0185] Figure 2: Pharmacokinetic curves of FIX variant polypeptides in HB mice after subcutaneous administration. Samples were collected at different time points up to 336 hours after administration. LLOQ (<1.6 pmol / mL) refers to the lowest concentration of rFIX antigen that can be reliably detected based on ELISA. Data with values ≤ LLOQ were not plotted. The time points plotted were from 0 (the injection time point) up to 72 hours. Each data point represents the mean ± SD of n = 3 - 5 mice, and α represents n = 1. At 24 hours in the rFIXK5R group and at 72 hours in the rFIXWT group, only 1 out of 3 animals (α) had a detectable level.

[0186] Figure 3: Liver from HB mice (n = 3) after intravenous administration of rFIX (25 nmol / kg) or saline control. (A) Quantitative analysis of FIX-positive liver sections in ZEN software (samples from each group were prepared and imaged in parallel under the same conditions), and the liver sections were collected at 5 minutes (0.08 hours), 24, 72, and 120 hours after treatment with rFIX protein. Each bar represents the median ± 95% CI of 2 - 3 sampled livers. The bars represent rFIXWT, rFIXK5A, and rFIXK5R, respectively. One-way ANOVA test was used to compare the groups at each time point to determine the p-value. ns, not significant, ***p < 0.001. (B) Representative images of liver sections with nuclei stained with DAPI and FIX stained with rhodamine. (C) Top panel: Liver of HB mice treated with saline buffer as a control for FIX staining specificity. Bottom panel: Liver sections from the 5-minute time point and the rFIXK5A treatment group. The portal triad is demarcated by a dashed line: 1 - branch of the hepatic portal vein, 2 - branch of the hepatic artery, and branch of the bile duct. The FIX-positive area is indicated by an arrow.

[0187] Figure 4 : Pharmacokinetic curves of the fusion FIX variant polypeptides in HB mice after intravenous administration. HB mice (n = 3) were injected via the lateral tail vein with rFIXWT-FP, rFIXK5A-FP, and rFIXK5R-FP at an rFIX antigen dose of 200 IU / kg. Samples were collected at different time points up to 168 hours after administration. The blood collection time points up to 168 hours were plotted on the X-axis. LLOQ refers to the lowest concentration of rFIX:Ag that can be reliably detected based on ELISA. Each point represents the mean ± SD of n = 1 - 3 mice.

[0188] Figure 5: Pharmacokinetic curves of the fusion FIX variant polypeptides in HB mice after subcutaneous administration. HB mice (n = 3) were injected subcutaneously in the neck with rFIXWT-FP, rFIXK5A-FP, and rFIXK5R-FP at an rFIX antigen dose of 200 IU / kg. Samples were collected at different time points up to 168 hours after administration. The blood collection time points up to 168 hours were plotted on the X-axis. The LLOQ refers to the lowest concentration of rFIX:Ag that can be reliably detected based on ELISA. Each data point represents the mean ± SD of n = 1 - 3 mice. At 168 hours in the rFIXWT and rFIXK5A groups, only 1 out of 3 animals (α) had a detectable level.

[0189] Figure 6: Comparison of hemostatic efficacy in the tail clip bleeding model after subcutaneous administration of vehicle control, rFIXWT, rFIXK5A, and rFIXK5R. The group size was n = 9 - 10 animals, and the treatment groups were compared to the vehicle group for statistical analysis. (A) The blood loss normalized to body weight (grams) was plotted in a scatter plot. Each bar represents the median. (B) The bleeding incidence determined by the time to termination of bleeding within a 30-minute observation period was plotted using a Kaplan-Meier curve, and statistical analysis was performed using the log-rank (Mantel-Cox) test. (C) The (adjusted) P-values were summarized in the table below the corresponding graph at each time point ( Figure 6B ) and highlighted in bold if significant (p < 0.05). For blood loss, a one-way ANOVA test followed by Dunnett's post hoc test was used for statistical analysis. BI is the abbreviation for bleeding incidence, and statistical analysis of BI was performed using the log-rank (Mantel-Cox) test. Vehicle at time points 24 hours and 168 hours was extracted from historical data (under similar conditions).

[0190] Figure 7: Comparison of hemostatic efficacy at different time points (0.25 - 336 hours post - administration) after intravenous administration of vehicle control, rFIXWT, rFIXK5A, and rFIXK5R to hemophilia B mice in a tail - clip bleeding model. Group sizes were n = 8 - 10 animals, and for statistical analysis, treatment groups were compared to the vehicle group. (A) Blood loss normalized to body weight (grams) was plotted in a scatter plot. Each point represents the median. (B) The hemostatic efficacy of rFIX within 30 minutes was plotted using a Kaplan - Meier curve. (C) (Adjusted) P - values were summarized in the table below the corresponding plot at each time point and were highlighted in bold if significant (p < 0.05). Statistical analysis of blood loss parameters was performed using a one - way ANOVA test followed by Dunnett's post - hoc test. BI is the abbreviation for bleeding incidence, and statistical analysis was performed using the log - rank (Mantel - Cox).

[0191] Figure 8 : Exposure of rFIX in plasma of HB mice after (intravenous administration) in a tail - clip model. rFIXWT, rFIXK5A, and rFIXK5R were administered intravenously to HB mice. At the end of each experiment, blood was collected from the injured animals for measurement of antigen levels (FIX:Ag, solid lines) and activity levels (FIX:C, dashed lines). The antigen level for the rFIXWT group at 24 hours was not available because no blood samples were collected. Each symbol represents the median ± 95% CI of 8 - 10 animals. The LLOQ for FIX:Ag (6.25 mIU / mL) and FIX:C (100 mIU / mL) are represented by black dashed lines for the left and right Y - axes, respectively. At 336 hours, no detectable FIX:Ag levels were present in plasma, and the LLOQ at this time point was different, 25 mIU / mL for all three proteins (*). Data with values ≤ LLOQ were plotted as 100 mIU / mL for FIX:C or 6.25 mIU / mL for FIX:Ag. Example

[0192] The following examples are provided to illustrate various embodiments of the invention. These examples are illustrative and are not intended to limit the invention in any way.

[0193] Example 1 - Subcutaneous and Intravenous Administration of Wild - type FIX and FIX Variant Polypeptides in a Hemophilia Mouse Model

[0194] A hemophilic murine model (reference 55) was used to analyze the pharmacokinetic profiles of FIX variant polypeptides upon subcutaneous or intravenous administration. This model is referred to herein as "HB mice".

[0195] Recombinant FIX variant polypeptides modified at Gla domain position 5 with alanine (rFIXK5A) or arginine (rFIXK5R) were tested. The wild-type FIX polypeptide used was the commercially available rFIXWT product. HB mice were injected intravenously via the lateral tail vein or subcutaneously in the neck with doses of 25 nmol / kg of rFIXWT, rFIXK5A, and rFIXK5R. Blood samples were collected at the following time points: 5 minutes, 2 hours, 6 hours, 24 hours, 48 hours, 72 hours, 120 hours, and 144 hours (6 days), 168 hours (7 days), 240 hours (10 days), and 336 hours (14 days). Blood samples were collected retro-orbitally at all time points to generate plasma, and finally additionally under deep anesthesia (ketamine 65 mg / kg, xylazine 13 mg / kg, and acepromazine 2 mg / kg, mixed in the same syringe and administered i.p.) by puncturing the vena cava.

[0196] The pharmacokinetic curves of the FIX polypeptides were determined by measuring the rFIX antigen levels at each time point.

[0197] As Figure 1 shown, in general, the plasma exposure of rFIX upon intravenous administration is comparable for all proteins (rFIXWT, rFIXK5A, and rFIXK5R). However, when looking at the early time points ( Figure 1 inset in

[0198] ), it is clear that the clearance rate of rFIXK5A is monoexponential, while rFIXWT and rFIXK5R have a biexponential profile. The biexponential profile indicates that rFIXWT and rFIXK5R distribute to the extravascular compartment at a faster rate during the initial phase. Figure 2 Interestingly, compared to rFIXWT and rFIXK5R, the K5A mutation has a positive effect on plasma exposure upon subcutaneous administration (

[0199] ). The area under the plasma drug concentration-time curve of rFIXK5A (which reflects the actual plasma exposure of the FIX protein) is significantly higher than that of rFIXK5R. These results together suggest that rFIXK5A is more readily taken up into the circulation after subcutaneous administration, while rFIXK5R is released from the subcutaneous injection site into the circulation more slowly and less efficiently. Without being bound by any specific theory, the reason that rFIXK5A is more readily taken up into the circulation seems to be that the variant binds less strongly to extracellular components.

[0200] The plasma levels of FIX antigen after intravenous administration were used for non-compartmental PK analysis, as shown in Table 2.

[0201] Table 2 - Non-compartmental analysis of FIX polypeptides administered intravenously

[0202] PK parameters Unit rFIXWT rFIXK5A rFIXK5R AUC_0 - last h*pmol / mL 981±112 1560±100 955±21 AUC_inf h*pmol / mL 995±111 1590±103 966±22 extrap ％ 1.4±2 1.6±0.3 1.2±0.8 Cmax_pred pmol / mL 233±51 268±17 203±16 Clearance mL / h / kg 25.1±3.0 15.8±1.0 25.9±0.8 t1 / 2_term h 13.8±8 4.24±0.2 8.02±0.3 MRT h 9.55±4 4.68±0.2 7.34±0.4 IVR ％ 37.2±8 42.9±3 32.4±3 Vc mL / kg 107±63 93.6±6 123±9 Vss mL / kg 240±110 73.9±4 190±13 Vz mL / kg 499±285 96.8±7 299±15

[0203] The total exposure after intravenous injection of rFIXK5A was significantly higher, showing the area under the curve (AUC) from time 0 to the last measurable concentration (AUC_0 - last), which was ~1560 h*pmol / mL, followed by lower AUC levels for rFIXWT (~981 h*pmol / mL) and rFIXK5R (~955 h*pmol / mL). The predicted maximum concentration of measured rFIX (Cmax_pred) was highest in the rFIXK5A group (~268 pmol / mL), followed by rFIXWT (~233 pmol / mL) and rFIXK5R (~203 pmol / mL).

[0204] Generally, rFIXK5A reached a Cmax_pred that was approximately 32% higher than that of rFIXK5R, and over time, the total exposure of rFIXK5A was approximately 63% and 65% higher than that of rFIXK5R (AUC_0 - last and AUC_0 - inf; Table 2). Compared with rFIXWT and rFIXK5R, rFIXK5A showed the shortest terminal half - life (~4 hours), the lowest systemic clearance rate (~16 mL / h / mg), and the lowest mean residence time (MRT) (~4.68 hours) (Table 2). However, as shown in Table 2, rFIXK5A had the highest in - vivo recovery rate (~42.9%), while rFIXWT (37.2%) and rFIXK5R (32.4%) showed slightly lower in - vivo recovery rates (IVR).

[0205] rFIXK5A had the lowest apparent volume of distribution in the central compartment (Vc), steady - state (Vss), and terminal elimination phase (Vz), indicating low tissue distribution of rFIXK5A. The dose fraction absorbed into the extravascular compartment could only be estimated indirectly based on plasma level measurements.

[0206] These results again indicate that compared with rFIXWT and rFIXK5R, rFIXK5A has a lower level of binding to the extravascular compartment.

[0207] Example 2 - Tissue absorption of FIX variant polypeptides

[0208] As Figure 3A shown, samples were collected from the livers of HB mice treated intravenously and subcutaneously in Example 1 at 0.08 hours, 24 hours, and 72 hours after administration. The samples were processed and the FIX immunostaining was quantified in at least 6 independent sections. At Figure 3BRepresentative images of these samples are provided.

[0209] The signal-to-noise ratio of the HB mice treated subcutaneously was low, which made the quantification of FIX immunostaining unreliable.

[0210] The specificity of FIX staining was confirmed in liver sections of HB mice previously treated with saline buffer (human FIX-negative samples). These sections were used as negative controls and were stained under the same conditions (including primary and secondary antibodies) as the liver samples of mice previously treated with rFIX. Only samples from animals previously treated with human rFIX showed a signal ( Figure 3C ).

[0211] In the intravenous group, at 15 minutes after injection, similar mean fluorescence intensities of all three rFIX proteins were detected in the liver. However, at 24 hours after intravenous injection, only a strong and robust signal of rFIXK5R was detected in liver sections, while only weak signals were detected under the same conditions in the groups treated with rFIXK5A and rFIXWT ( Figure 3B ). This data shows that rFIXK5A and rFIXWT are cleared from the liver at a faster rate than rFIXK5R.

[0212] Example 3 - Subcutaneous and Intravenous Administration of Fused FIX Variant Polypeptides in a Mouse Model

[0213] FIX variant polypeptides modified with alanine (rFIXK5A-FP) and arginine (rFIXK5R-FP) at position 5 of the Gla domain and fused to albumin were tested and compared with rFIXWT-FP for comparison.

[0214] HB mice were injected at a dose of 200 IU / kg (21 nmol / kg for rFIXWT-FP, 15 nmol / kg for rFIXK5A-FP, and 19 nmol / kg for rFIXK5R-FP) according to the antigen concentration. The antigen concentration (rFIX:Ag) of each protein was determined previously using ELISA against known concentrations of rFIXWT-FP (one-stage clotting potency, FIX:C). The measured concentrations (FIX:Ag) of rFIXK5A-FP (840 IU / mL), rFIXK5R-FP (722 IU / mL), and rFIXWT-FP (229 IU / mL) were diluted as needed and administered intravenously via the lateral vein or subcutaneously in the neck. Blood samples were collected from the saphenous vein at the following time points: 5 minutes, 2 hours, 8 hours, 16 hours, 24 hours, 32 hours, 72 hours, 96 hours, 120 hours (5 days), 144 hours (6 days), and 168 hours (7 days). Samples collected from the saphenous vein were collected into EDTA tubes (Sarstedt Microvette CB 300DI-Potassium-EDTA). The subcutaneous and intravenous pharmacokinetic curves of rFIXWT-FP, rFIXK5A-FP, and rFIXK5R-FP were determined by measuring the rFIX antigen levels (FIX:Ag) at each time point.

[0215] When administered intravenously, rFIXK5R-FP was rapidly eliminated from the plasma in the initial phase, although at a slower rate than the unfused protein, which may be due to the extended half-life effect of albumin. rFIXK5A-FP exhibited a slower rate of elimination from the plasma and a more linear initial distribution phase.

[0216] Overall, the curve profiles of rFIXK5R-FP and rFIXWT-FP were very similar, while rFIXK5A-FP exhibited a linear clearance rate from the blood at the initial time points and a faster decline in plasma concentration compared to the other two proteins at later time points (>96 hours).

[0217] When administered subcutaneously, rFIXK5R-FP had the lowest bioavailability and exhibited the lowest Cmax and the lowest AUC( Figure 5 ). These results were consistent with the observations for the unfused equivalents( Figure 2) The study of the subcutaneous curves revealed that rFIXK5R-FP had lower AUC_0-last (~9 h*IU / mL), AUC_inf (~12 h*IU / mL), and Cmax (~0.1 h*IU / mL) compared to rFIXWT-FP (~31 h*IU / mL; ~31 h*IU / mL; ~0.6 IU / mL) and rFIXK5A-FP (~32 h*IU / mL; ~34 h*IU / mL; ~0.8 IU / mL).

[0218] Non-compartmental PK analysis

[0219] Plasma rFIX levels (rFIX:Ag) after intravenous and subcutaneous administrations were used for the non-compartmental model. Intravenous administration is summarized in Table 3, while subcutaneous administration is summarized in Table 4.

[0220] Table 3 - Non-compartmental analysis of FIX polypeptides administered intravenously

[0221] PK Unit rFIXWT - FP rFIXK5A - FP rFIXK5R - FP AUC_0 - last h*IU / mL 47.6±8.0 49.2±6.2 31.5±4.3 AUC_inf h*IU / mL 49.3±8.1 50.6±5.1 37.4±4.0 extrap ％ 3.5±0.2 3.0±3.1 15.9±2.6 Cmax_pred IU / mL 2.77±0.55 2.33±0.52 2.72±0.51 Clearance mL / h / kg 4.1±0.7 4.0±0.4 5.4±0.6 t1 / 2_term h 42.7±3.9 74.1±59.6 114.9±14.5 MRT h 37.6±3.3 34.5±11.4 80.2±11.8 IVR ％ 54.4±10.9 46.7±10.4 54.4±10.2 Vc mL / kg 75.7±16.8 88.6±19.5 75.2±13.9 Vss mL / kg 156.4±34.5 139.6±57.4 436.4±107.1 Vz mL / kg 254.1±44.0 444.4±382.6 896.5±176.1

[0222] Table 3 shows that the total exposure after intravenous injection between rFIXK5A-FP and rFIXWT-FP was comparable, showing AUC_0-last of ~49 h*IU / mL and ~48 h*IU / mL respectively, followed by lower concentration of rFIXK5R-FP (~32 h*IU / mL)( Figure 4 ) The predicted maximum concentration (Cmax) of rFIX for rFIXK5R-FP (~2.7 IU / mL), rFIXWT-FP (~2.8 IU / mL), and rFIXK5A (~2.3 IU / mL) was similar. rFIXK5R-FP exhibited the longest half-life (~115 h), followed by rFIXK5A-FP (~74 h) and rFIXWT-FP (~43 h). The systemic clearance rate (clearance) and mean residence time (MRT) of rFIXK5R-FP were higher (~5 mL / h / kg and ~80 h), but were comparable between rFIXK5A-FP (~4 mL / h / kg and ~35 h) and rFIXWT-FP (~4 mL / h / kg and ~38 h). As shown in Table 3, the in vivo recovery of rFIXK5A-FP was reduced (~47%), while rFIXWT-FP (54%) and rFIXK5A-FP (54%) showed slightly higher in vivo recoveries (IVR).

[0223] Table 4 - Non-compartmental analysis of FIX polypeptides administered subcutaneously

[0224] PK parameters Unit rFIXWT - FP rFIXK5A - FP rFIXK5R - FP AUC_0 - last h*IU / mL 30.7±1.4 32.0±7.8 8.9±1.0 AUC_inf h*IU / mL 31.0±1.4 33.6±8.3 11.5±1.1 extrap ％ 0.9±0.1 4.7±2.7 22.1±15.8 Cmax IU / mL 0.60±0.12 0.77±0.25 0.14±0.33 Clearance mL / h / kg 6.5±0.3 6.2±1.8 17.5±1.6 t1 / 2_term h 18.0±3.5 46.6±26.6 144.3±164.1 MRT h 42.5±3.0 47.0±6.0 153.2±113.3 BA ％ 62.9±6.6 66.4±11.5 30.8±3.1

[0225] When administered subcutaneously, the bioavailability (BA) of rFIXK5R-FP (~31%) was lower than that of rFIXK5A-FP (66%) and rFIXWT-FP (~63%) (Table 4).

[0226] These results suggest that after subcutaneous administration, rFIXK5R-FP, which binds more strongly in the extravascular space, may adhere at the injection site for a longer time, resulting in lower plasma levels and bioavailability.

[0227] Generally, as expected, the area under the curve (AUC) of albumin fusion proteins was higher than that of unfused proteins. Albumin fusion prolonged the time that rFIX could circulate in the body before being eliminated.

[0228] Example 4 - In Vivo Efficacy of FIX Variant Polypeptides Administered Subcutaneously and Intravenously

[0229] The hemostatic efficacy of FIX variant polypeptides was evaluated in HB mice. An unfused rFIX protein was used for the efficacy study to enable better comparison with published literature data on the extravascular action of FIX.

[0230] Subcutaneous Administration

[0231] The hemostatic efficacy of rFIX proteins (rFIXWT, rFIXK5A, and rFIXK5R) was evaluated at 24 hours, 72 hours, and 168 hours after subcutaneous injection of rFIX proteins (FIX:C 50 IU / kg) in a tail clip model. Plasma exposure at the end of the experiment showed no detectable rFIX antigen levels (FIX:Ag, < 12.5 mIU / mL) in the samples, and only low levels of rFIX activity were detectable at 24 hours after subcutaneous administration in the clotting activity assay (FIX:C). The FIX:C activity level was close to the LLOQ, and only negligible amounts of FIX (if any) could be detected in the circulation at the end of the experimental procedure.

[0232] Interestingly, subcutaneous administration of rFIXWT and rFIXK5R impaired the efficacy of these two rFIX proteins, as there was no significant difference in blood loss or bleeding incidence between the vehicle and rFIXWT or rFIXK5R treatment groups (Figure 6). Although administered at the same clotting activity, only rFIXK5A showed statistical significance in blood loss at 24 hours ( Figure 6C ). The efficacy of rFIXK5A also waned over time and had no significant effect on blood loss after 72 hours.

[0233] The greatest reduction in the incidence of bleeding was observed at 72 hours following subcutaneous administration of rFIXWT and rFIXK5A proteins (vehicle: 90%, rFIXWT: 60%, rFIXK5A: 60%, rFIXK5R: 80%, Figure 6B and Figure 6C ). Overall, only rFIXK5A demonstrated statistically significant efficacy following subcutaneous administration. Despite providing hemostatic protection, the efficacy of rFIXWT and rFIXK5R was poorer compared to rFIXK5A.

[0234] Intravenous administration

[0235] The hemostatic efficacy of rFIX proteins was evaluated at 15 minutes, 24 hours, 72 hours, 168 hours, and 336 hours post i.v. injection by monitoring total blood loss and the incidence of bleeding. The results of blood loss and incidence of bleeding are shown in Figure 7A and Figure 7B respectively. For statistical analysis, the groups of rFIX molecules (rFIXWT, rFIXK5A, and rFIXK5R) were compared separately at each time point, and the p-values are summarized in Figure 7C . Treatment of HB mice with rFIX significantly protected the animals until 24 hours post-treatment in terms of total blood loss (vehicle: ~10 μL / g of BW; rFIX treatment: ~1 μL / g of BW, Figure 7A ). As shown in Figure 7A , the total blood loss among the rFIX treatment groups was comparable until 168 hours. However, at 336 hours post-administration, only in the animals treated with rFIXK5R was the blood loss reduced by nearly 50% compared to the vehicle group, but it was not statistically significant (vehicle: ~18 μL / g of BW; rFIXK5R: ~9 μL / g of BW).

[0236] A significant effect in protecting against the incidence of bleeding was observed for all rFIX molecules at 15 minutes post-treatment. However, at later time points, the efficacy of individual rFIX proteins declined at different rates over time. More specifically, the statistically significant effect on reducing the incidence of bleeding disappeared at 24 hours post-rFIXK5A treatment, 72 hours post-rFIXWT treatment, and 168 hours post-rFIXK5R treatment ( Figure 7C ). It is noteworthy that both parameters of blood loss and incidence of bleeding need to be considered in context, as these parameters reflect different aspects of hemostasis. For example, the incidence of bleeding ( Figure 7B ) or bleeding time (which indicates the time when the mouse stops bleeding) reflects the complete occlusion of blood vessels. On the other hand, non-occlusive thrombi may reduce blood loss ( Figure 7A ), but the animal may continue to bleed throughout the observation period.

[0237] Detectable rFIX antigen and activity were present in the plasma of the 15-minute group, while no detectable rFIX protein was present in the circulation 24 hours later ( Figure 8 ). Thus, the hemostatic efficacy observed at 24 hours and later may be partly attributed to extravascular FIX. The 2-fold difference between FIX:Ag and FIX:C values in mIU / mL can be explained by the differences in the assays. The calibration curve for the one-stage clotting assay (OSCA; FIX:C) is based on standard human plasma (SHP), while the injection solution is used as the standard in the ELISA. In addition, due to the conditions for collecting mouse plasma samples (resulting in massive bleeding and consumption of clotting factors after transection of the major peripheral arteries and veins), matrix effects of using OSCA (normal ranges of other plasma proteins and activation of plasma proteins involved in the intrinsic pathway of the clotting cascade) cannot be excluded.

[0238] Generally, after intravenous administration, all proteins are comparable in their ability to reduce blood loss. Although not statistically significant, compared to the vehicle group, rFIXK5R showed the longest efficacy in reducing blood loss until 14 days even though rFIX was not detected in the circulation. This result indicates the presence of non-circulating but accessible rFIX at the site of injury. In addition to blood loss, the bleeding incidence indicates that rFIXK5A provides good protection but quickly loses this ability, suggesting that after 24 hours post intravenous administration, rFIXK5A may not be accessible at the site of injury, thus limiting the growth of stable clots, the growth of which requires circulating FIX in the thrombus formation area and FIX accessible at this site to achieve complete vascular occlusion.

[0239] It is understood that the present invention has been described only by way of example and modifications can be made while maintaining the scope and spirit of the present invention.

[0240] Sequence

[0241] SEQ ID NO:1 - Human wild-type FIX polypeptide

[0242] YNSG K LEEF VQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKG R SALVLQYLRVPLVDRATCL R STKF T IYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIK E KTKLT

[0243] SEQ ID NO:2 - Coding sequence of human wild-type FIX polypeptide

[0244]

[0245] SEQ ID NO: 3 - Human wild-type factor IX polypeptide containing a signal and a propeptide

[0246] MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0247] SEQ ID NO: 4 - Coding sequence of human wild-type factor IX polypeptide containing a signal and a propeptide

[0248]

[0249] SEQ ID NO:5 - Human Albumin

[0250] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0251] SEQ ID NO:6 - Fc Polypeptide

[0252] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0253] SEQ ID NO:7 - Fc Polypeptide

[0254] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0255] SEQ ID NO:8 - Cleavable linker

[0256] PVSQTSKLTRAETVFP

[0257] SEQ ID NO:9 - K5A human FIX variant polypeptide

[0258] YNSG A LEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0259] SEQ ID NO:10 - R338L human FIX variant polypeptide

[0260] YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLLSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0261] SEQ ID NO:11 - CTP sequence

[0262] SSSSKAPPPS

[0263] SEQ ID NO:12 - CTP sequence

[0264] DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL

[0265] SEQ ID NO:13 - CTP sequence

[0266] SSSSKAPPPSLPSPSRLPGPSDTPILPQ

[0267] SEQ ID NO:14 - XTEN72 sequence

[0268] GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGASS

[0269] SEQ ID NO:15 - XTEN sequence

[0270] GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS

[0271] SEQ ID NO:16 - Non-cleavable linker

[0272] GGGGGGV

[0273] SEQ ID NO:17 - Human activated factor IX light chain

[0274] YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTR

[0275] SEQ ID NO:18 - Human activated factor IX heavy chain

[0276] VVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0277] SEQ ID NO:19 - R318Y / R338E / T343R (Dalcinonacog) human FIX variant polypeptide

[0278] YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGY SALVLQYLRVPLVDRATCL E STKF R IYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0279] SEQ ID NO:20 - T148A human FIX polymorphic variant

[0280] YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

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Claims

1. Factor IX (FIX) variant polypeptide for use in a method of treating or preventing a bleeding disorder, the method comprising administering the FIX variant polypeptide to soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX.

2. The FIX variant polypeptide for use according to claim 1, wherein the bleeding disorder is hemophilia B.

3. The FIX variant polypeptide for use according to claim 1 or claim 2, wherein the FIX variant polypeptide further comprises lysine at a position corresponding to position 10 of wild-type factor IX.

4. The FIX variant polypeptide for use according to any one of claims 1-3, wherein the FIX variant polypeptide further comprises leucine at a position corresponding to position 338 of wild-type factor IX.

5. The FIX variant polypeptide for use according to any one of claims 1-3, wherein the FIX variant polypeptide further comprises an amino acid selected from the group consisting of valine, threonine, and tryptophan at a position corresponding to position 338 of wild-type factor IX, and histidine at a position corresponding to position 410 of wild-type factor IX.

6. The FIX variant polypeptide for use according to claims 1-3, wherein the FIX variant polypeptide further comprises tyrosine at a position corresponding to position 318 of wild-type factor IX, glutamate at a position corresponding to position 338 of wild-type factor IX, and arginine at a position corresponding to position 343 of wild-type factor IX.

7. The FIX variant polypeptide for use according to any one of claims 1-6, wherein the factor IX variant polypeptide has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1 over the full length of SEQ ID NO:

1.

8. The FIX variant polypeptide for use according to any one of claims 1-7, wherein the FIX variant polypeptide comprises a half-life enhancing moiety, specifically, wherein the half-life enhancing moiety is selected from the group consisting of albumin, including its variants and derivatives, polypeptides of the albumin family, including their variants and derivatives, immunoglobulins without antigen-binding domains (e.g., Fc portion), and polyethylene glycol.

9. The FIX variant polypeptide for use according to claim 8, wherein the FIX variant polypeptide further comprises a cleavable peptide linker between the FIX variant polypeptide and the half-life enhancing moiety.

10. The FIX variant polypeptide for use according to claim 8 or claim 9, wherein the half-life enhancing moiety is albumin.

11. The FIX variant polypeptide for use according to any one of the preceding claims, wherein the soft tissue is skin tissue or gastrointestinal tissue.

12. The FIX variant polypeptide for use according to claim 11, wherein the skin tissue is subcutaneous tissue, dermal tissue, or epidermal tissue.

13. The FIX variant polypeptide used according to claim 11 or claim 12, wherein the method comprises subcutaneous administration of the FIX variant polypeptide.

14. The FIX variant polypeptide used according to claim 11, wherein the method comprises using an oral drug delivery device to administer the FIX variant polypeptide into the gastrointestinal tissue.

15. A pharmaceutical composition comprising a factor IX (FIX) variant polypeptide for use in a method of treating or preventing a bleeding disorder, the method comprising administering the FIX variant polypeptide to soft tissue, wherein the FIX variant polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type factor IX.

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