Factor IX variant polypeptides for administration to soft tissues - Patent Application 20070122999

Abstract

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

Description

[Technical Field]

[0001] Related application data This application claims priority to European Patent Application No. EP22200505.0, filed October 10, 2022, entitled "Factor IX variant polypeptides for administration to soft tissue," the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the contents of which are incorporated herein by reference in their entirety.

[0003] Technical Field The present invention relates to factor IX (FIX) variant polypeptides for administration to soft tissue, such as skin tissue (including subcutaneous administration) or mucosal tissue (e.g., gastrointestinal mucosal tissue), and their use in therapy. In particular, FIX variant polypeptides are described herein that have increased hemostatic effect when administered to soft tissue compared to wild-type FIX. [Background technology]

[0004] Human coagulation FIX plays a key role in the formation of blood clots. FIX has been used to prevent and treat bleeding disorders such as hemophilia B. Maintaining adequate levels of FIX activity in plasma is crucial to preventing bleeding in patients with hemophilia B. Lack of FIX activity, if left untreated, can cause serious damage to patients, even leading to death.

[0005] It is generally accepted in the art that increasing the circulation time of FIX protein in plasma is important for maintaining adequate levels of FIX activity and for achieving hemostasis. Hemostasis is the mechanism that stops bleeding from blood vessels. Thus, current treatments for hemophilia B include intravenous administration of FIX proteins with extended plasma half-lives, including IDELVION®, ALPROLIX®, and REBYNIN®.

[0006] Gene therapy is currently being investigated for the treatment of hemophilia B. The current approach uses an adeno-associated virus (AAV) vector to deliver a FIX transgene (Reference 1). This approach has already been utilized in clinical trials, where a significant reduction in annualized bleeding rates was observed through 5 years of treatment with an AAV expressing a highly active variant of FIX (Reference 2). However, certain patients may be better candidates for FIX protein replacement therapy rather than gene therapy. Summary of the Invention [Problem to be solved by the invention]

[0007] In recent years, the relevance of an extravascular FIX reservoir has emerged as a concept that may contribute to hemostasis. The extravascular FIX reservoir refers to non-circulating FIX that is bound outside of plasma in extravascular spaces, such as the vascular endothelium or subendothelial extracellular matrix. Understanding the balance between extravascular and circulating FIX opens up new possibilities for identifying novel and improved strategies for treating hemophilia B. The present invention is based on the surprising realization that the hemostatic efficacy of FIX can be improved when administered specifically to soft tissues (including subcutaneous administration) by using certain FIX variant polypeptides with reduced binding to the extracellular matrix. [Means for solving the problem]

[0008] The present invention provides suitable FIX variant polypeptides that have increased hemostatic efficacy when administered specifically to soft tissues, e.g., subcutaneously. The present invention is particularly suitable for use with FIX variant polypeptides, such as the K5A variant, that have reduced binding to extracellular matrix compared to wild-type FIX. The K5A variant, for example, has been described previously (Reference 3), but the surprising effect of improved hemostatic efficacy when administered specifically to soft tissues was not demonstrated. As shown herein, the inventors have advantageously identified that these FIX variant polypeptides more readily enter the circulation when administered to soft tissues. The examples demonstrate that this results in higher levels of bioavailable FIX in plasma, which leads to significantly improved hemostatic efficacy, compared to administering wild-type FIX by the same route or the same variants administered intravenously, a particularly surprising finding. Thus, these FIX variant polypeptides are particularly useful for treating and preventing bleeding disorders, such as hemophilia B, when administered to soft tissues.

[0009] Thus, 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 comprising administering the FIX variant polypeptide 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. SEQ ID NO: 1 is an example of a wild-type FIX polypeptide sequence as referred to herein and below.

[0010] As an alternative to any of the aspects and embodiments described herein that use a FIX variant polypeptide containing the amino acid alanine at position corresponding to position 5 of wild-type factor IX ("K5A"), the FIX variant polypeptide may instead contain the amino acid lysine at position corresponding to position 10 of wild-type factor IX ("V10K"). Both variants have been shown to have reduced binding to the extracellular matrix when compared to wild-type FIX (Reference 4).

[0011] The present invention also provides a method for treating or preventing a bleeding disorder in a subject, comprising administering to a soft tissue in the subject a therapeutically or prophylactically effective amount of a 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.

[0012] The present invention further provides the use of a FIX variant polypeptide in the manufacture 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 in the subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0013] The present invention further provides a FIX variant polypeptide for treating or preventing a bleeding disorder, wherein the FIX variant polypeptide is to be administered to soft tissue in a subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0014] In some embodiments, the bleeding disorder is hemophilia B (also known as congenital factor IX deficiency).

[0015] In some embodiments, the FIX variant polypeptide further comprises the amino acid lysine at the position corresponding to position 10 of wild-type factor IX (i.e., in addition to the amino acid alanine at the position corresponding to position 5 of wild-type factor IX).

[0016] In some embodiments, the FIX variant polypeptide further comprises the amino acid leucine at a position corresponding to position 338 of wild-type factor IX (i.e., in addition to the amino acid alanine at a position corresponding to position 5 of wild-type factor IX, and optionally the amino acid lysine at a position corresponding to position 10 of wild-type factor IX).

[0017] In other embodiments, the FIX variant polypeptide further comprises an amino acid other than arginine (e.g., 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 in combination with the amino acid histidine at a position corresponding to position 410 of wild-type factor IX (i.e., in addition to the amino acid alanine at a position corresponding to position 5 of wild-type factor IX and, optionally, the amino acid lysine at a position corresponding to position 10 of wild-type factor IX). In certain such embodiments, the FIX variant polypeptide comprises a valine at a position corresponding to position 338 of wild-type factor IX and the amino acid histidine at a position corresponding to position 410 of wild-type factor IX.

[0018] In some embodiments, the FIX variant polypeptide further comprises the amino acid tyrosine at a position corresponding to position 318 of wild-type factor IX, the amino acid glutamic acid at a position corresponding to position 338 of wild-type factor IX, and the amino acid arginine at a position corresponding to position 343 of wild-type factor IX (i.e., in addition to the amino acid alanine at a position corresponding to position 5 of wild-type factor IX, and optionally the amino acid lysine at a position corresponding to position 10 of wild-type factor IX). The Factor IX variant polypeptide may 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 entire length of SEQ ID NO:1.

[0019] In any of the embodiments described herein, the Factor IX variant polypeptide may have the sequence of SEQ ID NO: 1, except for the substitutions specified herein (e.g., SEQ ID NO: 1 in which the lysine at position 5 is replaced with alanine).

[0020] In some embodiments, the FIX variant polypeptide comprises albumin, including variants and derivatives thereof, albumin family polypeptides, including variants and derivatives thereof, immunoglobulins without an antigen-binding domain (e.g., only the Fc portion), or a half-life enhancing moiety such as polyethylene glycol.

[0021] In some embodiments, the FIX variant polypeptide further comprises a cleavable peptide linker between the FIX variant polypeptide and the half-life enhancing moiety.

[0022] 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 for use in a method for treating or preventing a bleeding disorder, comprising administering the FIX variant polypeptide subcutaneously, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0023] In an alternative embodiment, the FIX variant polypeptide is administered to gastrointestinal tissue using an oral drug delivery device, for example, the FIX variant polypeptide is for use in a method of treating or preventing a bleeding disorder comprising administering a FIX variant polypeptide to gastrointestinal tissue using an oral drug delivery device, wherein the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0024] Another aspect of the present invention provides a pharmaceutical composition comprising a FIX variant polypeptide for use in a method for treating or preventing a bleeding disorder 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.

[0025] The present invention also provides a method for treating or preventing a bleeding disorder in a subject, comprising administering to soft tissue in the subject a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a 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.

[0026] The present invention further 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 in the subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0027] The present invention further 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 a subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0028] It is clear that FIX variant polypeptides containing further mutations as disclosed herein (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. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of those skilled in the art, and such techniques are fully explained in the literature.

[0030] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers, whether native or modified by intervening procedures; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the invention are based, for example, on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can exist as a single chain or as two or more associated chains.

[0031] The percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same comparing the two sequences. The percentage sequence identity is calculated as the percentage of identical amino acids in the aligned sequences. A sequence that "has" (or "has") x% sequence identity to another sequence means that the sequence is x% identical to that other sequence.

[0032] The term "wild-type factor IX" refers to a factor IX polypeptide sequence that exists in nature and has FIX activity typical of native FIX, such as that found in normal human plasma. The sequence has not been artificially modified relative to the sequence of the naturally occurring polypeptide sequence. This means that none of the amino acids in the naturally occurring polypeptide sequence have been replaced with different amino acids. SEQ ID NO: 1 is an example of a wild-type polypeptide sequence, but the term also encompasses functional fragments, truncated forms, and the like, as exemplified below. For example, the term includes polypeptides with modified N- or C-termini, including deletions or additions of terminal amino acids, as long as those polypeptides substantially retain the activity of wild-type factor IX. The term also includes any naturally occurring polymorphic variants of factor IX. For example, a common naturally occurring polymorphic variant occurring at a frequency of 33% is a factor IX polypeptide presenting an alanine (A) at the position corresponding to position T148 in SEQ ID NO: 1. This T148A polymorphic variant is represented by SEQ ID NO: 20. Thus, any reference to SEQ ID NO: 1 herein may also refer to SEQ ID NO: 20. These polymorphic variants occur naturally in the general population, but at least some of them have been associated with phenotypic effects, for example T148A, which has been described in the literature (Reference 56).

[0033] The terms "FIX variant polypeptide," "FIX variant," "variant," "FIX polypeptide," and the like are used interchangeably herein and all refer to FIX variant polypeptides unless expressly stated otherwise. FIX variant polypeptides include full-length FIX proteins or fragments of FIX proteins that are biologically active, i.e., the polypeptides are 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). Variants differ from the corresponding positions in wild-type factor IX at one or more amino acid positions, i.e., the variants have one or more amino acid substitutions relative to the corresponding positions in wild-type factor IX. 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.

[0034] For the avoidance of any doubt, all FIX variant polypeptides described herein have FIX clotting activity, e.g., may have the clotting activity of wild-type FIX or even have higher clotting activity than wild-type FIX; clotting activity can be measured by standard assays known to those skilled in the art.

[0035] Factor IX variant polypeptides are also derived from wild-type factor IX, including the signal and / or propeptide as set forth in SEQ ID NO:3. SEQ ID NO:3 includes both the signal peptide (aa 1-28) and the propeptide (aa 29-46). The polypeptide of SEQ ID NO:3 is known in the art as the precursor of human factor IX, or as prepropeptide factor IX. Factor IX having the propeptide but lacking the signal peptide is also known as propeptide factor IX. An exemplary polynucleotide coding sequence encoding the polypeptide of SEQ ID NO:3 is set forth in SEQ ID NO:4.

[0036] Variant factor IX polypeptides may also be derived from one or more functional fragments of wild-type factor IX, for example, from activated factor IX, which contains two fragments of factor IX (the intervening "activation peptide" present in SEQ ID NO: 1 is missing). SEQ ID NOs: 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 human factor IX isoform 2, which lacks a stretch of 38 aa at positions 47-84 of SEQ ID NO: 1.

[0037] Alternatively, the Factor IX variant polypeptide is derived from a truncation or fusion of wild-type Factor IX.

[0038] The term "derived from the polypeptide sequence of wild-type Factor IX" (or similar expressions) means that the Factor IX variant polypeptide has a degree of sequence identity with the wild-type Factor IX polypeptide when the two sequences are aligned. For example, the Factor IX variant polypeptide may have at least 70% or so sequence identity to SEQ ID NO: 1, as discussed above. The Factor IX variant polypeptide is biologically active, i.e., capable of activating Factor X (i.e., generating Factor Xa).

[0039] The Factor IX variant polypeptides are provided as "isolated" or "purified" polypeptides. The terms may refer to polypeptides produced by expression of the isolated nucleic acid molecules of the present invention. Alternatively, the terms may refer to proteins that have been sufficiently separated from other proteins with which they are naturally associated (e.g., so as to exist in "substantially pure" form). "Isolated" does not imply artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with essential activity and that may be present due, for example, to incomplete purification or the addition of stabilizers.

[0040] Unless otherwise specified, "FIX protein" or "FIX polypeptide" herein refers to the weight of the FIX portion of 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).

[0041] The terms "administration" or "administering" or "administered" are used interchangeably herein. Unless otherwise specified, the term administration refers to administration to soft tissue.

[0042] The terms "treatment," "therapy," and "treating" are used interchangeably herein and refer to therapeutic measures that cure, slow, alleviate symptoms, and / or halt the progression of a diagnosed pathological condition or disorder. The terms "treatment," "therapy," and "treating" may also include prophylaxis, unless otherwise specified. The terms "treatment," "therapy," and "treating" also include on-demand treatment. A disorder is treated or prevented when administration of a factor IX variant polypeptide as described herein to a subject (e.g., a human with factor IX deficiency such as hemophilia B) results in a therapeutic or prophylactic effect. This means that the plasma level of factor IX activity in the subject increases, at least temporarily, after treatment, as measured using at least one factor IX assay. 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). The increase may be clinically significant, for example, a reduction in the frequency or intensity of bleeding events.

[0043] By "therapeutically effective amount" is meant that administration of that amount of Factor IX variant polypeptide to a subject, either in a single dose or as part of a series, is effective for treatment. By "prophylactically effective amount" is meant that administration of that amount of Factor IX variant polypeptide to a subject, either in a single dose or as part of a series, is effective for prevention. Such methods are effective in treating or preventing disorders in which procoagulant activity is required (e.g., to prevent, reduce, or inhibit bleeding), including, but not limited to, hemophilia, particularly hemophilia B.

[0044] The terms "reduced binding" or "reduced binding" refer to a factor IX variant polypeptide that exhibits reduced FIX binding to the extracellular matrix compared to wild-type FIX, and include FIX variants that do not exhibit binding to the extracellular matrix. FIX binding to the extracellular matrix can be determined by various known biological assays, such as competitive binding assays as described in (Reference 4).

[0045] For the avoidance of doubt, FIX variant polypeptides with reduced binding for use in the present invention retain FIX clotting activity, and may, for example, have the clotting activity of wild-type FIX, or even have higher clotting activity than wild-type FIX. Clotting activity is assessed by assays known in the art. Also, any reference to a method of treatment comprising administering a FIX variant polypeptide to a subject encompasses the FIX variant polypeptide for use in said method of treatment, as well as the use of the FIX variant polypeptide in said method of treatment, and the use of the FIX variant polypeptide in the manufacture of a medicament for treating disease.

[0046] The term "subject" refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, dogs, cats, rabbits, rodents, etc. The subject is preferably a human. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0047] The term "pharmaceutically acceptable" refers to a substance approved or approvable by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans.

[0048] 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, does not destroy its pharmacological activity when administered in a dose sufficient to deliver a therapeutic effect, and is non-toxic. Generally, those skilled in the art and the US FDA consider pharmaceutically acceptable excipients, carriers, or adjuvants to be inactive ingredients of any formulation.

[0049] The term "substantially pure" refers to a preparation that contains at least 75% by weight of Factor IX variant polypeptide, particularly at least 80%, at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98%, or 99% by weight, e.g., 90-99% or more by weight of Factor IX variant polypeptide. Purity is measured by methods appropriate for the compound of interest (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0050] "Comprising" encompasses "including" as well as "consisting," "consisting of," and / or "consisting essentially of," e.g., a composition "comprising" X may consist exclusively of X, or may include something additional, e.g., X+Y. Also, whenever embodiments are described herein using the language "consisting essentially of," it is understood that otherwise similar embodiments described with the term "consisting of" are also provided.

[0051] The term "about" in relation to a numerical value x is optional and means, for example, x±10%.

[0052] The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. Where appropriate, the word "substantially" is omitted from the definition of the invention.

[0053] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or for either meaning.

[0054] As used herein, the verb "to comprise" and its conjugations are used in an open-ended sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. Additionally, the verb "to consist" may, where appropriate, be replaced with "consisting essentially of," which means that the products defined herein may include additional component(s) other than those specifically identified that do not alter the distinctive characteristics of the invention.

[0055] Unless otherwise indicated, processes or methods involving multiple steps may include additional steps at the beginning or end of the method, or may include additional intervening steps, and steps may be combined, omitted, or performed in a different order where appropriate.

[0056] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0057] All patents and references cited herein are hereby incorporated by reference in their entirety.

[0058] Various embodiments of the present invention are described herein. It will be understood that the features specified in each embodiment may be combined with other specified features to provide further embodiments. In particular, embodiments highlighted herein as suitable, exemplary, or preferred may be combined with each other (except to the extent they are mutually exclusive).

[0059] Factor IX (FIX) variant polypeptides The present invention relates to the use of FIX variant polypeptides that have reduced binding to the extracellular matrix relative to wild-type FIX for use in therapy by administering the FIX variant polypeptide to soft tissue (e.g., subcutaneous tissue).

[0060] Extravascular FIX The concept of extravascular FIX was first reported in 1983, when it was shown that FIX could bind to endothelial cells [Reference 7]. Subsequently, in 1987, Stern et al. demonstrated that large amounts of FIX can be present in the extravascular space and that a rapid, reversible equilibrium exists between plasma and extravascular FIX [Reference 8]. Subsequent studies demonstrated direct binding of FIX to endothelial cells. In vitro experiments have shown that the zymogen form of FIX reversibly binds to vascular endothelium [References 9, 10, and 11] and, in some cases, platelets [Reference 12].

[0061] Experiments in hemophilia B (HB) mice have shown that FIX occupies extravascular reservoirs and can provide hemostatic protection for more than 7 days, while not being detectable in plasma (Ref. 13). This study also estimated that these extravascular reservoirs contain significantly more FIX than the circulating FIX. In an attempt to characterize the phenomenon of the extravascular FIX reservoir, Cheung et al. reported that mutations in the vitamin K-dependent γ-carboxyglutamic acid (Gla) domain of FIX at residue 5 (lysine) or residue 10 (valine) strongly affected their interaction with endothelial cells. Specifically, single-point mutations at residue 5 of the FIX molecule from lysine to alanine (FIXK5A) or arginine (FIXK5R) resulted in changes in endothelial cell binding affinity (Ref. 3). The FIXK5R variant was shown to have higher binding affinity for endothelial cells than wild-type FIX (FIXWT) in vitro, whereas 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, and possibly collagen IV specifically, is a FIX binding site on endothelial cells. Subsequent in vivo studies in HB mice found that HB mice injected with FIXK5R provided better hemostatic protection than wild-type FIX in a saphenous vein bleeding model. In contrast, HB mice injected with FIXK5A showed reduced coagulation (Reference 14). Based on this, the authors proposed that collagen IV binding by FIX provides a longer-lasting extravascular reservoir of FIX and therefore better hemostatic protection (see also References 15 and 16).

[0062] Thus, it was not clear from these studies that FIX variant polypeptides with reduced binding to the extracellular matrix, such as the K5A variant, are useful for treating bleeding disorders, let alone that they can provide increased hemostatic efficacy when administered to soft tissues. The inventors have realized that when FIX is specifically administered to soft tissues (e.g., subcutaneously), FIX variant polypeptides with reduced binding to the extracellular matrix actually provide increased hemostatic protection. For example, the Examples demonstrate that FIX variant polypeptides with reduced binding to the extracellular matrix (e.g., the K5A variant) have a higher hemostatic efficacy after subcutaneous administration compared to wild-type FIX. Without wishing to be bound by any particular theory, it is hypothesized that this higher hemostatic efficacy after subcutaneous administration is due to the fact that these FIX variant polypeptides interact less strongly with the extracellular matrix present at the administration site, such as collagen IV in the extravascular space, and are therefore more easily released into the plasma circulation after subcutaneous administration. Surprisingly, the absence of an extravascular reservoir of bound FIX does not appear to adversely affect the hemostatic efficacy of these FIX variants, such as the K5A variant, when they are administered subcutaneously, in contrast to the effects previously described for variants when other routes of administration (e.g., intravenous) are used.

[0063] Therefore, the FIX variant polypeptides for use in the present invention have reduced binding to extracellular matrices 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 position 5 of wild-type factor IX, the amino acid lysine at 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 position 5 of wild-type factor IX, or FIX variant polypeptides containing an amino acid with a positively charged side chain at position 10 of wild-type factor IX, as long as they retain FIX clotting activity, for example, they have the clotting activity of wild-type FIX, or may even have higher clotting activity than wild-type FIX; 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.

[0064] In some embodiments, the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX, but does not comprise the amino acid lysine at the position corresponding to position 10 of wild-type factor IX (alternatively, valine may be used at position 10).

[0065] FIX variant polypeptides for use in the present invention can also contain two or more mutations (e.g., at positions 5 and 10) that reduce 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 a positively charged side chain at the position corresponding to position 10 of wild-type factor IX.

[0066] Thus, 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 comprising administering the FIX variant polypeptide 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.

[0067] The present invention also provides a method for treating or preventing a disease or disorder in a subject, comprising administering to a soft tissue in the subject a therapeutically or prophylactically effective amount of a 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.

[0068] The present invention further provides the use of a FIX variant polypeptide in the manufacture 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 the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0069] The present invention further 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 a subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0070] Additional factor IX mutations In further embodiments, the FIX variant polypeptides for use in the present invention may also contain additional mutations compared to wild-type factor IX, which may increase clotting activity (e.g., increase specific activity) relative to wild-type factor IX. Such variant polypeptides are also referred to herein as "highly active" FIX polypeptides or highly active FIX variant polypeptides. Other terms, such as "superactive" FIX variants, are used interchangeably in the art. These variants optionally retain the biological function of factor IX, i.e., the variants are capable of generating factor Xa after the factor IX variant polypeptide is converted to its activated form (factor IXa) by cleavage of the activation peptide. The variants are capable of generating factor Xa with higher activity than wild-type FIX. Activation cleavage of factor IX can be achieved in vitro, for example, 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.).

[0071] Exemplary high activity factor IX variant polypeptides contain a leucine (L) at the position corresponding to position 338 in wild-type factor IX, which normally has an arginine (R) at that 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 typically at least about 5-8 times higher than that of wild-type factor IX.

[0072] Thus, in some embodiments, a factor IX (FIX) variant polypeptide for use in the present invention comprises the amino acid alanine at position 5 of wild-type factor IX and leucine at position 338 of wild-type factor IX. Other exemplary highly active factor IX variants are E410H, E410K, R338V, and R338L+E410K, as well as those described in Reference 18, which comprise, for example, the amino acid H at position 410 of wild-type factor IX and an amino acid other than R at position 338 of wild-type factor IX, for example, an amino acid selected from the group consisting of V, T, and W at position 338 of wild-type factor IX, such as R338V+E410H, R338T+E410H, R338W+E410H, and R338L+E410H. Another useful variant is R318Y+R338E+T343R.

[0073] Thus, in some embodiments, a 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, an amino acid selected from valine, threonine and tryptophan at the position corresponding to position 338 of wild-type factor IX, and the amino acid histidine at the position corresponding to position 410 of wild-type factor IX. In a specific embodiment, a 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 valine at the position corresponding to position 338 of wild-type factor IX, and the amino acid histidine at the position corresponding to position 410 of wild-type factor IX.

[0074] As mentioned above, a further highly active factor IX variant for use in the present invention is the darcinonacog alfa variant (also known as CB 2679d), see SEQ ID NO: 19. Darcinonacog alfa has three amino acid substitutions in two loops within the FIX protein. Based on the numbering of the mature FIX sequence, (1) R318Y, located in the "150-loop," stabilizes activated FIX (FIXa), interacts directly with the substrate factor X (FX), and provides resistance to antithrombin; (2) R338E and (3) T343R, both located in the "170-loop," significantly enhance the affinity for the cofactor, activated factor VIIIa (FVIIIa), and increase the catalytic activity of FIXa. R318Y / R338E / T343R refers to R150Y / R170E / T175R of the classical chymotrypsin numbering [Reference 19] and R364Y / R384E / T389R of the Human Genome Variation Society (HGVS) nomenclature including the 46 amino acid propeptide [Reference 20]. Thus, in some embodiments, a FIX variant polypeptide for use in the present invention comprises the amino acid alanine at position corresponding to position 5 of wild-type factor IX, the amino acid tyrosine at position corresponding to position 318 of wild-type factor IX, the amino acid glutamic acid at position corresponding to position 338 of wild-type factor IX, and the amino acid arginine at position corresponding to position 343 of wild-type factor IX.

[0075] Further exemplary highly active Factor IX variant polypeptides include those listed in Table 1 below. (Reference 21)

[0076] [Table 1]

[0077] The numbering in Table 1 refers to the position in the mature FIX protein (SEQ ID NO: 1) without the propeptide sequence. Activity was determined using a one-stage clotting assay.

[0078] One skilled in the art can identify and validate these and other highly active Factor IX variant polypeptides by determining the specific (molar) activity of the Factor IX polypeptide using methods known in the art and comparing that activity to wild-type Factor IX.

[0079] The Factor IX variant polypeptide is derived from the Factor IX polypeptide sequence of any mammalian species. In certain embodiments, the Factor IX variant polypeptide is derived from a Factor IX polypeptide sequence of human origin. 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 sequence and / or nucleotide sequence of wild-type human Factor IX.

[0080] The Factor IX variant polypeptide according to the invention is derived from mature (i.e. excluding the signal peptide and propeptide) wild-type Factor IX, for example of human origin, the amino acid sequence of which is shown in SEQ ID NO: 1. The polypeptide sequence is "isoform 1" of human Factor IX.

[0081] Route of administration The Examples demonstrate that FIX variant polypeptides containing the amino acid alanine at position 5 of wild-type factor IX have a greater hemostatic effect than wild-type FIX when administered to subcutaneous tissue. Without wishing to be bound by any particular theory, it is hypothesized that the reason these FIX variant polypeptides have a greater hemostatic effect after subcutaneous administration is because they bind less strongly to the extracellular matrix and are therefore released more rapidly from the extracellular space into the circulation. Therefore, based on these data, it is plausible that the disclosed FIX variant polypeptides also have a greater hemostatic effect than wild-type FIX when administered more generally to soft tissue.

[0082] Therefore, the factor IX (FIX) variant polypeptide for use in the present invention is administered to soft tissue. The term is understood by those skilled in the art. For example, soft tissue administration is defined by the FDA as administration to any soft tissue (https: / / www.fda.gov / drugs / data-standards-manual-monographs / route-administration). Soft tissue is any tissue in the body that is not hardened by the process of ossification or calcification, such as bone and teeth. In one embodiment, the soft tissue excludes muscle tissue. In another embodiment, the soft tissue excludes 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).

[0083] The FIX variant polypeptide is intended for administration to a subject, such as an animal, usually a human subject.

[0084] The methods and uses described herein do not include intravenous administration of FIX variant polypeptides. 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.

[0085] In some embodiments, the methods and uses described herein do not involve intramuscular administration of a FIX variant polypeptide.

[0086] Administration into soft tissue immediately exposes the FIX variant polypeptide to components in the extracellular space between cells (known as interstitial space).For example, after administration of FIX variant polypeptide into soft tissue, at least a portion of the FIX variant polypeptide is delivered directly to the extracellular space, and another portion of the FIX variant polypeptide is delivered to or taken up by cells, and then the FIX variant polypeptide is secreted from the cells into the extracellular space.Therefore, the methods and uses described herein are different from gene-based (e.g., viral or non-viral vector) approaches in which a nucleic acid sequence encoding FIX is administered (e.g., into muscle tissue) and the FIX polypeptide is produced intracellularly.

[0087] In a preferred embodiment, the soft tissue is skin tissue. For the purposes of this disclosure, skin comprises 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 subcutaneous tissue. For the avoidance of doubt, in the context of this disclosure, reference to "administration into the skin" or "administration into skin tissue" includes subcutaneous administration (administration into the subcutaneous tissue). Furthermore, subcutaneous administration, which is characterized as administration "under" or "below" or "below side" (or synonyms) of the skin, is also encompassed by the present invention.

[0088] Thus, in some embodiments, the FIX variant polypeptide is administered to skin tissue. In some embodiments, the FIX variant polypeptide is administered to subcutaneous tissue (tissue under the skin), to dermal tissue, or to epidermal tissue. Thus, administration may be subcutaneous, intradermal, topical (e.g., on the skin), or transdermal (e.g., via transdermal injection or absorption). In a preferred embodiment, the FIX variant polypeptide is administered subcutaneously.

[0089] In a preferred embodiment, the factor IX (FIX) variant polypeptide for use in the invention comprises the amino acid alanine at position corresponding to position 5 of wild-type factor IX and is administered subcutaneously. For example, the invention provides a method of treating or preventing disease in a subject comprising subcutaneously administering to the subject an effective amount of a FIX variant polypeptide comprising the amino acid alanine at position corresponding to position 5 of wild-type factor IX.

[0090] The present invention also provides a Factor IX (FIX) variant polypeptide for use in a method of treating or preventing disease in a subject 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.

[0091] Also provided is the use of a Factor IX variant polypeptide in the manufacture 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 the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type Factor IX.

[0092] The present invention also provides a 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.

[0093] In some embodiments, the FIX variant polypeptide is administered to a mucosal tissue, such as a gastrointestinal mucosal tissue. In some embodiments, the FIX variant polypeptide is administered enterally (through the human digestive tract). Examples of enteral administration include oral, sublingual, gastric, and rectal administration.

[0094] FIX variant polypeptides can be administered by injection into the mucosal tissue of the gastrointestinal tract using an orally ingestible drug delivery device (also known as an applicator) that autonomously positions itself to engage with and inject the drug into the GI tissue. Exemplary drug delivery devices are described in Reference 30. Exemplary devices include SOMA (Self-Directing Millimeter-Scale Applicator) (Reference 31), BIONDD™ (Reference 32), and RaniPill™ (References 33 and 34). In some embodiments, FIX variant polypeptides are administered by injection into the mucosal tissue of the stomach, for example, using a BIONDD™ device. BIONDD™ is designed to insert a drug-loaded biodegradable spike into the stomach wall. BIONDD™ consists of a capsule that adheres to and delivers the drug to the stomach tissue.

[0095] Bleeding disorders In a preferred embodiment, the Factor IX variant polypeptide described herein is for treating or preventing bleeding disorders.Bleeding disorders can be any disorders that require a procoagulant (for example, to prevent, reduce or inhibit bleeding).An exemplary bleeding disorder is hemophilia, particularly hemophilia B.

[0096] Accordingly, the present invention provides a FIX variant polypeptide for use in a method for treating or preventing a bleeding disorder comprising administering the FIX variant polypeptide 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.

[0097] The present invention also provides a method for treating or preventing a bleeding disorder in a subject, comprising administering to a soft tissue in the subject a therapeutically or prophylactically effective amount of a 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.

[0098] The present invention further provides the use of a FIX variant polypeptide in the manufacture 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 in the subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0099] The present invention further provides a FIX variant polypeptide for treating or preventing a bleeding disorder, wherein the FIX variant polypeptide is to be administered to soft tissue in a subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0100] Treating or preventing can include on-demand control of bleeding episodes, perioperative management of bleeding, and / or routine prophylaxis to prevent or reduce the frequency of bleeding episodes. For example, treating can include on-demand control of bleeding episodes or perioperative management of bleeding. Prevention can include preventing bleeding episodes or reducing the frequency of bleeding episodes.

[0101] The subject is typically a human. The subject may be an adult or a child. The subject may have a basal (without prophylaxis or treatment) plasma factor IX activity of 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1-5%, or 1% or less compared to the plasma factor IX activity of a healthy subject. In certain embodiments, the subject is a pediatric subject (child), for example, 18 years of age or younger. In one embodiment, the subject is ineligible for FIX gene therapy.

[0102] 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.

[0103] In one embodiment, the FIX polypeptide is administered in a composition that does not contain an antithrombotic agent (eg, heparin).

[0104] Bleeding disorders include hemophilia (hemophilia A, hemophilia B, hemophilia A and B patients with inhibitory antibodies; particularly hemophilia B), deficiency of at least one clotting factor (e.g., Factor VII, IX, X, XI, V, XII, II, and / or von Willebrand factor; particularly Factor IX), combined FV / FVIII deficiency, vitamin K epoxide reductase CI deficiency, gamma-carboxylase deficiency; excessive anticoagulation associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC); bleeding associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, and storage pool deficiency.

[0105] In a preferred embodiment, the method or use is for the treatment or prevention of bleeding in a subject with hemophilia B, also known in the art as congenital factor IX deficiency.

[0106] One way to express factor IX activity in plasma is as a percentage of normal human plasma. Another way to express factor IX activity in plasma is in international units (IU) relative to the international standard for factor IX in plasma. One IU of factor IX activity in plasma is equal to the amount of factor IX in 1 mL of normal human plasma.

[0107] One method for confirming efficacy of prophylaxis or treatment is to measure plasma factor IX activity in a 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 less than 1%, or 1% to 5%, or 5 to 40% of normal human plasma, to a peak level, e.g., greater than 15%, 20%, 25%, greater than 30%, greater than 35%, greater than 40%, greater than 50%, or greater than 60% of normal human plasma, e.g., from less than 5% to greater than 5%, e.g., 5 to 40%) indicates prophylactic or therapeutic efficacy. Factor IX levels of 5 to 10% of normal human serum have been targeted in clinical trials to achieve bleeding control during prophylaxis.

[0108] A prophylactic or therapeutic effect is also achieved when factor IX activity after prophylaxis or treatment is sufficient to prevent, reduce or inhibit bleeding.

[0109] Factor IX activity after prophylaxis or treatment may result in a trough of at least 15-40% or even be outside the pathological range (e.g., greater than 40% of peak levels in normal human serum).

[0110] Factor IX activity can be measured using any factor IX activity assay known to those skilled in the art, for example, using an aPTT assay (a decrease in 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].

[0111] The Factor IX variant polypeptides for use in the present invention may have a higher specific molar activity than the corresponding wild-type Factor IX polypeptide when administered to a subject in vivo. Such high-activity variants are described above. For example, the percent increase in plasma Factor IX activity (e.g., measured using an in vitro aPTT-based one-stage clotting assay) may be higher with the Factor IX variant polypeptides described herein compared to the same molar amount of the corresponding wild-type Factor IX polypeptide. Another way to explain this is that the aPTT time in serum samples after administration of the Factor IX variant polypeptides described herein is shorter compared to the same molar amount of the corresponding wild-type Factor IX polypeptide.

[0112] Production of Factor IX Variant Polypeptides Factor IX variant polypeptides for use in the present invention can be produced using standard techniques well known to those of skill in the art. For example, a wild-type factor IX cDNA sequence (e.g., SEQ ID NO: 2) is 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 position corresponding to position 5 of wild-type factor IX (which encodes lysine (K) at that position). For purposes of recombinant protein production, the N-terminal leader peptide can be based on the native factor IX leader peptide (as shown in SEQ ID NO: 3) or alternatives known to those of skill in the art. The cDNA sequence is inserted into an expression plasmid suitable for expressing the recombinant factor IX variant polypeptide. This is usually performed using mammalian cells (e.g., HEK for transient expression or CHO cell lines for stable expression), although other types of cells capable of producing glycosylated, correctly folded proteins can also be used. The recombinant factor IX variant polypeptide is then purified, e.g., using anion exchange chromatography.

[0113] The Factor IX variant polypeptide may be combined with other medicinal agents and / or a pharmaceutically acceptable carrier.

[0114] Fusions and conjugates Factor IX variant polypeptides for use in the present invention may also be provided as part of a fusion with another moiety, for example with albumin (eg attached via a cleavable linker).

[0115] The Factor IX variant polypeptide is provided in a fusion with or conjugated to one or more further moieties. The one or more further moieties are usually 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, for example a linker comprising a fragment of a polypeptide sequence derived from Factor IX but which does not itself have the function of Factor IX, may be such "one or more further moieties", i.e., a linker without the function of Factor IX may not be part of the Factor IX moiety but may be part of a molecule comprising the Factor IX moiety.

[0116] Half-Life Enhancing Moieties and Linkers In exemplary embodiments, the FIX variant polypeptide is linked to a half-life-enhancing moiety. The half-life-enhancing moiety may 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), such as an Fc fragment, e.g., an IgG Fc, such as an IgG1 Fc. Alternatively, the HLEP may be a C-terminal peptide of human chorionic gonadotropin (CTP). The HLEP may also be an unstructured recombinant polypeptide (e.g., XTEN). Such molecules are also referred to in the art as fusion polypeptides.

[0117] The FIX variant polypeptide is linked to the HLEP via a cleavable linker, particularly a cleavable peptide linker. Typically, the cleavable linker is cleavable by the same protease that activates factor IX. Therefore, such a cleavable linker provides a high molar specific activity of the fusion polypeptide.

[0118] The FIX variant polypeptide may also be PEGylated, ie, one or more polyethylene glycol moieties are conjugated to the FIX variant polypeptide using methods known in the art.

[0119] A FIX variant polypeptide for use in the present invention may 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 may be of the same type. The half-life-enhancing moieties may be of different types. For example, a FIX variant polypeptide is linked to an XTEN (e.g., XTEN72) and further to an Fc domain (e.g., human IgG1 Fc).

[0120] Preferably, the half-life-enhancing moiety is capable of extending the half-life of the FIX variant polypeptide in vivo (in plasma) by at least about 25% compared to the unfused FIX variant polypeptide. Preferably, the half-life-enhancing moiety is capable of extending the half-life of the FIX variant polypeptide in vivo (in plasma) by at least about 50%, more preferably by more than 100%. In vivo half-life is generally determined as the terminal half-life or β-half-life.

[0121] albumin As used herein, "albumin" collectively refers to an albumin polypeptide or amino acid sequence, or to an albumin fragment, variant, or analog having one or more functional (biological) activities of albumin. In particular, "albumin" may refer to human albumin (HA) or a fragment thereof, particularly the mature form of human albumin as set forth herein in SEQ ID NO: 5. Albumin may also be derived from other species, particularly other vertebrates. The albumin portion of the fusion polypeptide may comprise the full length of the HA sequence as set forth in SEQ ID NO: 5, or may comprise one or more fragments thereof capable of stabilizing or extending the therapeutic activity of a Factor IX variant polypeptide. Such fragments may be 10 or more amino acids long, or may comprise about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA sequence, or may comprise some or all of a specific domain of HA. These and other suitable albumin portions (including variants) are described in Reference 36.

[0122] Members of structurally related families of the albumin family are also used as HLEPs. For example, alpha-fetopolypeptide (AFP, Reference 35) is a member of the albumin family and is also used to enhance the half-life of factor IX variant polypeptides. 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 are also used.

[0123] In embodiments using the albumin HLEP, the albumin is typically provided as a genetic fusion with the Factor IX moiety, meaning that a single cDNA molecule encodes the Factor IX moiety and the albumin moiety, optionally with an intervening sequence encoding a linker, such as a cleavable linker.

[0124] Immunoglobulin Immunoglobulins (Ig) or fragments thereof can also be used as HLEPs. Examples of suitable immunoglobulins include IgG or IgG fragments such as the Fc region. The Fc region can be an Fc domain (e.g., two polypeptide chains each comprising a hinge region (or part of the hinge region), a CH2 region, and a CH3 region). Thus, the Factor IX variant polypeptide is fused to the Fc domain directly or via a linker. In embodiments using a linker, the linker can be cleavable.

[0125] Monomers, dimers, and hybrids are all encompassed. For example, a Factor IX variant polypeptide may be a heterodimer comprising two polypeptide chains, wherein the first chain comprises a Factor IX moiety linked to the hinge region (or a portion of the hinge region), CH2, and CH3 regions of an immunoglobulin (e.g., IgG1), and the second chain comprises the hinge region (or a portion of the hinge region), CH2, and CH3 regions of an immunoglobulin (e.g., IgG1).

[0126] In another embodiment, the Factor IX variant polypeptide is a homodimer comprising two polypeptide chains, wherein each chain comprises a Factor IX moiety linked to the hinge region (or part of the hinge region), CH2 region, and CH3 region of an immunoglobulin (e.g., IgG1).

[0127] In a further embodiment, the Factor IX variant polypeptide is a monomer comprising a Factor IX moiety linked to the hinge region (or part of the hinge region), CH2 region and CH3 region of an immunoglobulin (eg, IgG1).

[0128] Other examples of suitable Factor IX IgG Fc fusion molecule constructs can be found, for example, in ref.

[0129] An exemplary Fc polypeptide (derived from a human IgG1 Fc domain) is set forth in SEQ ID NO: 6. Another exemplary Fc polypeptide (derived from a human IgG1 Fc domain) is set forth in SEQ ID NO: 7.

[0130] In any of these embodiments, the Factor IX portion is linked to the Fc portion directly or via a linker. In embodiments that use a linker, the linker can be cleavable or non-cleavable. In certain embodiments, the linker is cleavable. An exemplary cleavable linker is shown in SEQ ID NO:8.

[0131] An exemplary Fc portion is the Fc portion of eftrenonacog alfa (Alprolix®). See also references 40, 41, or 42.

[0132] C-terminal peptide (CTP) of human chorionic gonadotropin Another exemplary half-life-enhancing moiety is the C-terminal peptide of human chorionic gonadotropin (CTP). CTP is based on a 31 amino acid long naturally occurring peptide and is the C-terminal peptide of the beta chain of human chorionic gonadotropin (hCG).

[0133] One or more units of CTP can be fused to the Factor IX moiety. One or more units of CTP can be fused to the N-terminus and / or C-terminus of Factor IX, preferably to the C-terminus.

[0134] In one embodiment, the Factor IX variant polypeptide is a CTP-modified Factor IX comprising a Factor IX variant polypeptide as described herein linked with three to five CTPs, optionally attached to the C-terminus of the Factor IX variant polypeptide. In a specific embodiment, three tandem units of CTP are attached to the Factor IX variant polypeptide, optionally at the C-terminus of the Factor IX variant polypeptide.

[0135] In any of these embodiments, at least one of the CTPs is attached to the Factor IX moiety via a linker. The linker may be a peptide bond. The linker is cleavable.

[0136] 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.

[0137] Other suitable CTP sequences and related methods are known to those skilled in the art; for example, see references 43, 44 or 45.

[0138] Recombinant polypeptides of undefined structure Another exemplary half-life-enhancing moiety is an unstructured recombinant polypeptide. An example of such an unstructured recombinant polypeptide is XTEN; see, e.g., Reference 46. Thus, in one embodiment, the factor IX variant polypeptide is a factor IX variant polypeptide fused to at least one XTEN. The XTEN is 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 is inserted between two adjacent amino acids in the activation peptide of factor IX at a position where the inserted XTEN does not interfere with cleavage of the activation peptide during coagulation. Alternatively, the XTEN is fused to the C-terminus and / or N-terminus of factor IX, preferably the C-terminus. The XTEN is fused to the C-terminus and / or N-terminus (preferably the C-terminus) of factor IX via a linker, e.g., a cleavable linker. The linker is cleavable by thrombin.

[0139] A preferred XTEN is XTEN72. An exemplary XTEN72 sequence is set forth in SEQ ID NO: 14. An alternative XTEN sequence is set forth in SEQ ID NO: 15. Other suitable sequences and methods are disclosed, for example, in references 47, 48 or 49.

[0140] In a specific embodiment, the Factor IX variant polypeptide comprises XTEN72 linked to an activation peptide of Factor IX, wherein the Factor IX moiety is further linked to a human IgG1 Fc domain at the C-terminus of the Factor IX moiety.

[0141] PEGylation Another exemplary half-life enhancing moiety is polyethylene glycol (PEG).GlycoPEGylation is within the scope of the term "PEGylation" as used herein.For example, a PEG moiety of about 40 kDa is covalently attached to the Factor IX variant polypeptide, for example, via a specific N-linked glycan in the activation peptide.

[0142] An example of a glycoPEG moiety is nonacogbeta pegol (Refixia®) (see also Reference 50), in which on average one non-reducing end of the glycan at N157 or N167 (numbered according to SEQ ID NO: 1) of factor IX is linked via an amino group to neuraminic acid conjugated to two PEG polymers (the total average molecular weight of the polymers is approximately 42 kDa).

[0143] Pegylation of Factor IX polypeptides is also taught, for example, in refs.

[0144] Linker Factor IX variant polypeptides comprising a half-life-enhancing moiety may employ a cleavable linker, particularly a proteolytically cleavable linker. The linker is generally disposed between the Factor IX polypeptide moiety and the half-life-enhancing moiety. The linker can release the Factor IX moiety upon cleavage by a protease of the coagulation cascade, such as a protease that can convert Factor IX to its activated form, e.g., FXIa or VIIa / tissue factor (TF). Cleavable linkers are particularly useful when the HLEP is albumin.

[0145] While it is desirable to enhance the in vivo half-life of factor IX, particularly when using superactive factor IX variant polypeptides, it is also desirable to limit the half-life of factor IX upon activation to reduce the risk of prothrombotic effects. Thus, in some embodiments, a cleavable linker links the factor IX variant polypeptide to the half-life-enhancing moiety, thereby providing a factor IX variant polypeptide with a longer half-life relative to the unfused polypeptide. However, when bleeding occurs and the coagulation cascade is initiated, proteases in the coagulation cascade activate the factor IX variant polypeptide, e.g., with increased specific activity relative to the corresponding wild-type factor IX. Simultaneously, the linker is cleaved, and the activated factor IX variant polypeptide is released from the half-life-enhancing moiety, thereby reducing the risk of prothrombotic effects due to any increased prolongation of factor IX activity.

[0146] The linker may be a fragment of Factor IX, preferably a fragment involved in Factor IX activation. For example, the linker may comprise such a fragment of the Factor IX sequence extended by an N-terminal residue such as a proline residue. An exemplary cleavable linker is shown in SEQ ID NO: 8. Other cleavable linkers are described in Reference 36.

[0147] A Factor IX variant polypeptide linked to a half-life enhancing moiety via an intervening cleavable linker may have a molar specific activity that is at least 25% higher than a corresponding molecule having a non-cleavable linker (e.g., GGGGGGV, SEQ ID NO: 16), as measured in at least one coagulation-related assay (examples of which are known to those of skill in the art), such as the aPTT one-stage assay. Preferably, a Factor IX variant polypeptide linked to a half-life enhancing moiety via an intervening cleavable linker has a molar specific activity that is increased by at least 50%, more preferably at least 100%, compared to a corresponding molecule without the cleavable linker.

[0148] Thus, in one embodiment, a 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 to increase the coagulation activity of FIX (e.g., R338L)), wherein FIX is linked to a half-life-enhancing moiety (e.g., albumin) as described herein, optionally via a cleavable linker as described herein.

[0149] Pharmaceutical Composition The FIX variant polypeptide can be provided as a pharmaceutical composition. The pharmaceutical composition is formulated with a pharmaceutically acceptable carrier. Thus, the present invention also provides a pharmaceutical composition comprising a factor IX (FIX) variant polypeptide for use in a method for treating or preventing a bleeding disorder, 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.

[0150] The present invention also provides a method for treating or preventing a bleeding disorder in a subject, comprising administering to soft tissue in the subject a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a 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.

[0151] The present invention further 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 in the subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0152] The present invention further 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 a subject, and the FIX variant polypeptide comprises the amino acid alanine at the position corresponding to position 5 of wild-type factor IX.

[0153] Pharmaceutical compositions are intended for administration to a subject, such as an animal, usually a human subject.

[0154] Pharmaceutical compositions are pharmaceutically acceptable and will usually include a suitable carrier. A thorough discussion of pharmaceutically acceptable carriers is available in reference 54. Compositions are preferably sterile and free of pyrogens and / or preservatives.

[0155] Thus, the Factor IX variant polypeptide is provided in a buffered liquid form, e.g., a citrate buffer, optionally containing stabilizers and / or bulking agents. An exemplary pharmaceutical composition for use in the present invention comprises 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% to 0.024% polysorbate 80, 18 to 29 g / L mannitol, 7 to 12 g / L sucrose, hydrochloric acid to adjust the pH to 6.6 to 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.

[0156] Alternatively, the Factor IX variant polypeptide in the composition is lyophilized and reconstituted with a liquid diluent, for example, sterile water for injection, prior to administration. Typical excipients in compositions comprising lyophilized Factor IX variant polypeptide include trisodium citrate dihydrate, polysorbate 80, mannitol, sucrose, and / or hydrochloric acid.

[0157] In some embodiments, the composition is suitable for administration to soft tissue, for example subcutaneous administration, optionally after reconstitution or dilution.

[0158] The compositions may be prophylactic (to prevent bleeding) or therapeutic (to treat bleeding). [Brief explanation of the drawings]

[0159] [Figure 1] Pharmacokinetic profile of FIX variant polypeptide in HB mice after intravenous administration. Samples were collected at various time points up to 336 hours post-administration. The LLOQ (<1.6 pmol / mL) refers to the lowest concentration of rFIX antigen that could be reliably detected based on ELISA. Values ​​below the LLOQ were not plotted. Plotted time points: 0 (time of injection) and up to 168 hours. Each data point represents the mean ± SD of n = 3–5 mice, with α indicating n = 1. In the rFIXK5R group, only 1 of 3 animals (α) had detectable levels at 48 hours, and in the rFIXWT group, only 1 of 5 animals (α) had detectable levels at 72 and 168 hours. [Figure 2]Pharmacokinetic profile of FIX variant polypeptide in HB mice after subcutaneous administration. Samples were collected at various time points up to 336 hours post-administration. The LLOQ (<1.6 pmol / mL) refers to the lowest concentration of rFIX antigen that can be reliably detected based on ELISA. Values ​​below the LLOQ were not plotted. Plotted time points: 0 (time of injection) and up to 72 hours. Each data point represents the mean ± SD of n = 3–5 mice, with α indicating n = 1. Only 1 of 3 animals (α) had detectable levels at 24 hours in the rFIXK5R group and at 72 hours in the rFIXWT group. [Figure 3-1] Livers from HB mice (n=3) after intravenous administration of rFIX (25 nmol / kg) or saline control. (A) Quantitative analysis of FIX-positive liver sections collected at 5 min (0.08 h), 24 h, 72 h, and 120 h after treatment with rFIX protein (samples from each group were prepared and imaged in parallel under identical conditions) was performed using ZEN software. Each bar represents the median ± 95% CI of two to three sampled livers. Bars represent rFIXWT, rFIXK5A, and rFIXK5R, respectively. At each time point, groups were compared using a one-way ANOVA test to determine p-values. ns is not significant, ***p<0.001. (B) Representative images from liver sections stained with DAPI for nuclei and rhodamine for FIX. (C) Top image: Liver from an HB mouse treated with saline buffer as a control for the specificity of FIX staining. Bottom image: Liver section from the rFIXK5A treatment group at 5 minutes. Portal triads are demarcated by dashed lines: 1 - hepatic portal vein branch, 2 - hepatic artery branch, and bile duct branch. FIX-positive areas are indicated by arrows. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 4]Pharmacokinetic profile of fusion FIX variant polypeptides in HB mice after intravenous administration. HB mice (n = 3) were injected with rFIXWT-FP, rFIXK5A-FP, and rFIXK5R-FP at an rFIX antigen dose of 200 IU / kg via the lateral tail vein. Samples were collected at various time points up to 168 h post-administration. Blood collection time points up to 168 h are 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. [Figure 5] Pharmacokinetic profile of fusion FIX variant polypeptides in HB mice after subcutaneous administration. HB mice (n=3) were injected in the neck with rFIX antigen doses of 200 IU / kg with rFIXWT-FP, rFIXK5A-FP, and rFIXK5R-FP. Samples were collected at various time points up to 168 hours post-administration. Blood collection time points up to 168 hours are plotted on the x-axis. 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. In the rFIXWT and rFIXK5A groups, only 1 of 3 animals (α) had detectable levels at 168 hours. [Figure 6-1]Comparison of hemostatic effects in the tail clip bleeding model after subcutaneous administration of vehicle control, rFIXWT, rFIXK5A, and rFIXK5R. Group sizes were n = 9–10 animals, and treatment groups were compared with the vehicle group for statistical analysis. (A) Blood loss normalized to body weight (grams) is shown in a scatter plot. Each bar represents the median. (B) Bleeding incidence, as determined by the time to cessation of bleeding over 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) (Adjusted) P values ​​are summarized in the table below the respective graphs (in Figure 6B) for each time point and are highlighted in bold if significant (p < 0.05). Statistical analysis of blood loss was performed using a one-way ANOVA test followed by Dunnett's post hoc test. BI is an abbreviation for bleeding incidence, and statistical analysis of BI was performed using the log-rank (Mantel-Cox) test. Vehicle at 24 and 168 hours was extracted from historical data (under similar conditions). [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 7-1] Comparison of hemostatic efficacy at various time points (0.25-336 hours post-dose) after intravenous administration of vehicle control, rFIXWT, rFIXK5A, and rFIXK5R in hemophilia B mice in a tail clip bleeding model. Group sizes were n = 8-10 animals, and treatment groups were compared to the vehicle group for statistical analysis. (A) Blood loss normalized to body weight (grams) is shown in a scatter plot. Each point represents the median. (B) The efficacy of rFIX to stop bleeding over 30 minutes is plotted using a Kaplan-Meier curve. (C) (Adjusted) P values ​​are summarized in the table below each graph at each time point and are highlighted in bold if significant (p < 0.05). Statistics for blood loss parameters were performed using a one-way ANOVA test followed by Dunnett's post-hoc test. BI is an abbreviation for bleeding incidence, and statistical analysis was performed using the log-rank (Mantel-Cox) method. [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 8] Exposure of rFIX in the plasma of HB mice after the tail clip model (intravenous administration). HB mice were intravenously administered rFIXWT, rFIXK5A, and rFIXK5R. At the end of each experiment, blood was collected from the injured animals for measurement of antigen levels (FIX:Ag, solid line) and activity levels (FIX:C, dashed line). Antigen levels at 24 h in the rFIXWT group are unavailable because blood samples were not collected (†). Each symbol represents the median ± 95% CI for 8–10 animals. The LLOQs for FIX:Ag (6.25 mIU / mL) and FIX:C (100 mIU / mL) are indicated by black dashed lines on the left and right y-axes, respectively. FIX:Ag levels were undetectable in plasma at 336 h; the LLOQs were different at this time point, 25 mIU / mL for all three proteins (*). Values ​​below the LLOQ were plotted as 100 mIU / mL for FIX:C or 6.25 mIU / mL for FIX:Ag. [Example]

[0160] The following examples are provided to illustrate various embodiments of the present invention. The examples are illustrative and are not intended to limit the invention in any way. Example 1

[0161] Subcutaneous and intravenous administration of wild-type FIX and FIX variant polypeptides in a mouse model of hemophilia The pharmacokinetic profile of FIX variant polypeptides was analyzed when administered subcutaneously or intravenously using a mouse model of hemophilia (55), herein referred to as the "HB mouse."

[0162] Recombinant FIX variant polypeptides modified with alanine (rFIXK5A) or arginine (rFIXK5R) at position 5 of the Gla domain were tested. The wild-type FIX polypeptide used was the commercially available rFIXWT product (BeneFIX®). HB mice were injected with rFIXWT, rFIXK5A, and rFIXK5R at a dose of 25 nmol / kg intravenously via the lateral tail vein or subcutaneously in the neck. Blood samples were collected at several time points: 5 min, 2 h, 6 h, 24 h, 48 h, 72 h, 120 h, and 144 h (6 days), 168 h (7 days), 240 h (10 days), and 336 h (14 days). Blood samples for plasma generation were collected retroorbitally at all time points and terminally by vena cava puncture under deep anesthesia (65 mg / kg ketamine, 13 mg / kg xylazine, and 2 mg / kg acepromazine, mixed in the same syringe and given i.p.).

[0163] The pharmacokinetic profile of the FIX polypeptide was determined by measuring rFIX antigen levels at various time points.

[0164] As shown in Figure 1, overall, the plasma exposure of rFIX after intravenous administration was comparable for all proteins (rFIXWT, rFIXK5A, and rFIXK5R). However, focusing on the early time points (Figure 1 insert), it is clear that rFIXK5A clearance was monophasic, whereas rFIXWT and rFIXK5R had biphasic profiles. The biphasic profiles suggested that rFIXWT and rFIXK5R were distributed to the extravascular compartment at a faster rate during the early phase.

[0165] Interestingly, the K5A mutation had a positive effect on plasma exposure when administered subcutaneously compared with rFIXWT and rFIXK5R (Figure 2). The area under the plasma drug concentration-time curve (which reflects the actual plasma exposure of FIX protein) was clearly higher for rFIXK5A than for rFIXK5R. Collectively, these results suggest that after subcutaneous administration, rFIXK5A enters the circulation more readily, whereas release of rFIXK5R from the subcutaneous site of injection into the circulation is slower and less efficient. While not wishing to be bound by any particular theory, it is likely that the reason rFIXK5A enters the circulation more readily is because the variant binds extracellular components to a weaker extent.

[0166] Non-compartmental PK analysis Plasma levels of FIX antigen after intravenous administration were used for non-compartmental PK analysis as summarized in Table 2.

[0167] [Table 2]

[0168] The total exposure after intravenous injection, showing an area under the curve (AUC) from time 0 to the last measurable concentration (AUC_0-last) of approximately 1560 h*pmol / mL, was clearly higher for rFIXK5A, followed by lower AUC levels for rFIXWT (approximately 981 h*pmol / mL) and rFIXK5R (approximately 955 h*pmol / mL). The predicted maximum concentration (Cmax_pred) of rFIX was determined to be highest for the rFIXK5A group (approximately 268 pmol / mL), followed by rFIXWT (approximately 233 pmol / mL) and rFIXK5R (approximately 203 pmol / mL).

[0169] In general, rFIXK5A reached a Cmax_pred that was approximately 32% higher than rFIXK5R, and the total exposure over time was approximately 63% and 65% higher for rFIXK5A compared with rFIXK5R (AUC_0-last and AUC_0-inf; Table 2). Compared with rFIXWT and rFIXK5R, rFIXK5A exhibited the shortest terminal half-life (approximately 4 hours), the lowest systemic clearance (approximately 16 mL / h / mg), and the lowest mean residence time (MRT) (approximately 4.68 hours) (Table 2). However, as shown in Table 2, rFIXK5A had the highest in vivo recovery (approximately 42.9%), whereas rFIXWT (37.2%) and rFIXK5A (32.4%) exhibited slightly lower in vivo recovery (IVR).

[0170] The apparent volume of distribution in the central compartment (Vc), the volume of distribution at steady state (Vss), and the volume of distribution during the terminal elimination phase (Vz) were smallest for rFIXK5A, suggesting a low tissue distribution of rFIXK5A. The fraction of the dose absorbed by the extravascular compartment can only be estimated (indirectly) based on plasma level measurements.

[0171] These results also suggested that lower levels of rFIXK5A were associated with the extravascular compartment compared with rFIXWT and rFIXK5R. Example 2

[0172] Tissue absorption of FIX variant polypeptides Samples from the livers of HB mice treated intravenously and subcutaneously in Example 1 were collected at 0.08, 24, and 72 hours after administration, as shown in Figure 3A. Samples were processed, and quantification of FIX immunostaining was performed on at least six independent sections. Representative images of these samples are provided in Figure 3B.

[0173] Subcutaneously treated HB mice had a low signal-to-noise ratio, which made quantification of FIX immunostaining unreliable.

[0174] The specificity of FIX staining was confirmed in liver sections from HB mice previously treated with saline buffer (human FIX-negative samples). These sections served as negative controls and were stained under identical conditions (including primary and secondary antibodies) as liver samples from mice previously treated with rFIX. Only samples from animals previously treated with human rFIX showed a signal (Figure 3C).

[0175] In the intravenous group, all three rFIX proteins were detected in the liver with similar mean fluorescence intensities at 15 minutes after intravenous injection. However, at 24 hours after intravenous injection, only rFIXK5R was detected in liver sections with a strong and robust signal, whereas faint signals were detected in the rFIXK5A and rFIXWT treatment groups under the same conditions (Figure 3B). This data indicates that rFIXK5A and rFIXWT were cleared from the liver at a faster rate than rFIXK5R. Example 3

[0176] Subcutaneous and intravenous administration of fusion FIX variant polypeptides in a mouse model FIX variant polypeptides modified with alanine (rFIXK5A-FP) and arginine (rFIXK5R-FP) at position 5 of the Gla domain fused to albumin were tested and compared to rFIXWT-FP (IDELVION®).

[0177] HB mice were injected with a dose of 200 IU / kg based on antigen concentration (21 nmol / kg for rFIXWT-FP, 15 nmol / kg for rFIXK5A-FP, and 19 nmol / kg for rFIXK5R-FP). The antigen concentration of each protein (rFIX:Ag) was previously determined using ELISA against rFIXWT-FP with known concentrations (one-stage coagulation potency, FIX:C). Measured concentrations (FIX:Ag) of rFIXK5A-FP (840 IU / mL), rFIXK5R-FP (722 IU / mL), and rFIXWT-FP (229 IU / mL) were diluted as necessary and administered intravenously via a lateral vein or subcutaneously in the neck. Blood samples were collected from the saphenous vein at several time points: 5 min, 2 h, 8 h, 16 h, 24 h, 32 h, 72 h, 96 h, 120 h (5 days), 144 h (6 days), and 168 h (7 days). Samples from the saphenous vein were collected in EDTA tubes (Sarstedt Microvette CB 300 DI-Kalium-EDTA). The subcutaneous and intravenous pharmacokinetic profiles of rFIXWT-FP, rFIXK5A-FP, and rFIXK5R-FP were determined by measuring rFIX antigen levels (FIX:Ag) at various time points.

[0178] When administered intravenously, rFIXK5R-FP was rapidly cleared from plasma in the early phase, albeit at a slower rate than the non-fusion protein, likely due to the half-life-extending effect of albumin. rFIXK5A-FP showed slower elimination from plasma and a more linear initial distribution phase.

[0179] Overall, the progression of the curves is very similar for rFIXK5R-FP and rFIXWT-FP, while rFIXK5A-FP shows linear clearance from the blood at early time points, with plasma concentrations declining more rapidly at later time points (>96 h) compared to the other two proteins.

[0180] When administered subcutaneously, rFIXK5R-FP had the lowest bioavailability and exhibited the lowest Cmax and AUC (Figure 5). These results were consistent with the observations made with the non-fused equivalents (Figure 2). Examination of the subcutaneous profile revealed lower AUC_0-last (approximately 9h*IU / mL), AUC_inf (approximately 12h*IU / mL), and Cmax (approximately 0.1 IU / mL) for rFIXK5R-FP compared to rFIXWT-FP (approximately 31h*IU / mL; approximately 31h*IU / mL; approximately 0.6 IU / mL) and rFIXK5A-FP (approximately 32h*IU / mL; approximately 34h*IU / mL; approximately 0.8 IU / mL).

[0181] Non-compartmental PK analysis Plasma levels of rFIX (rFIX:Ag) after intravenous and subcutaneous administration were used in a non-compartmental model as summarized in Table 3 for intravenous administration and Table 4 for subcutaneous administration.

[0182] [Table 3]

[0183] Table 3 shows that total exposure after intravenous injection was comparable between rFIXK5A-FP and rFIXWT-FP, with AUC_0-last of approximately 49 h*IU / mL and approximately 48 h*IU / mL, respectively, followed by the lower concentration of rFIXK5R-FP (approximately 32 h*IU / mL) (Figure 4). Maximum rFIX concentrations (Cmax) were predicted to be similar for rFIXK5R-FP (approximately 2.7 IU / mL), rFIXWT-FP (approximately 2.8 IU / mL), and rFIXK5A (approximately 2.3 IU / mL). rFIXK5R-FP exhibited the longest half-life (approximately 115 hours), followed by rFIXK5A-FP (approximately 74 hours) and rFIXWT-FP (approximately 43 hours). Systemic clearance and mean residence time (MRT) were higher for rFIXK5R-FP (approximately 5 mL / h / kg and approximately 80 hours) but comparable between rFIXK5A-FP (approximately 4 mL / h / kg and approximately 35 hours) and rFIXWT-FP (approximately 4 mL / h / kg and approximately 38 hours). As shown in Table 3, the in vivo recovery of rFIXK5A-FP was reduced (approximately 47%), while rFIXWT-FP (54%) and rFIXK5A-FP (54%) showed slightly higher in vivo recovery (IVR).

[0184] [Table 4]

[0185] The bioavailability (BA) of rFIXK5R-FP was lower (approximately 31%) than rFIXK5A-FP (66%) and rFIXWT-FP (approximately 63%) when administered subcutaneously (Table 4).

[0186] These results suggest that after subcutaneous administration, rFIXK5R-FP (stronger binding in the extravascular space) may remain attached to the site of injection for longer, resulting in lower plasma levels and bioavailability.

[0187] In general, the area under the curve (AUC) of the albumin fusion proteins was higher compared to the non-fusion protein, as expected. The albumin fusion extends the time that rFIX can circulate in the body before being excreted. Example 4

[0188] In vivo efficacy of subcutaneously and intravenously administered FIX variant polypeptides The hemostatic effects of FIX variant polypeptides were evaluated in HB mice. Efficacy studies were performed using unfused rFIX protein to allow for better comparison with published literature data on the role of extravascular FIX.

[0189] Subcutaneous administration The hemostatic effects of rFIX proteins (rFIXWT, rFIXK5A, and rFIXK5R) were evaluated in a tail clip model at 24, 72, and 168 hours after subcutaneous injection of rFIX protein (FIX:C 50 IU / kg). Plasma exposure at the end of the experiment showed no detectable rFIX antigen levels in the samples (FIX:Ag, less than 12.5 mIU / mL), and only low levels of rFIX activity (FIX:C) were detectable 24 hours after subcutaneous administration in a coagulation activity assay. FIX:C activity levels were close to the LLOQ, indicating that negligible, if any, amounts of FIX were detectable in the circulation at the end of the experimental procedure.

[0190] Interestingly, subcutaneous administration of rFIXWT and rFIXK5R impaired the efficacy of these two rFIX proteins, as no significant differences in blood loss or bleeding incidence were observed between the vehicle and rFIXWT or rFIXK5R-treated groups (Figure 6). Despite being dosed at the same clotting activity, only rFIXK5A demonstrated statistical significance in blood loss at 24 hours (Figure 6C). The efficacy of rFIXK5 also decreased over time, with no significant effect on blood loss at 72 hours.

[0191] The greatest reduction in bleeding incidence after subcutaneous administration was observed for rFIXWT and rFIXK5A proteins at 72 hours (vehicle: 90%, rFIXWT: 60%, rFIXK5A: 60%, rFIXK5R: 80%, Figures 6B and 6C). Overall, only rFIXK5A demonstrated statistically significant efficacy after subcutaneous administration. The efficacy of rFIXWT and rFIXK5R was inferior to that of rFIXK5A, despite providing hemostatic protection.

[0192] Intravenous administration The hemostatic effect of rFIX protein was evaluated by monitoring total blood loss and bleeding incidence at 15 min, 24 h, 72 h, 168 h, and 336 h after intravenous injection. The results of blood loss and bleeding incidence are shown in Figure 7A and Figure 7B, respectively. For statistical analysis, the rFIX molecule sets (rFIXWT, rFIXK5A, and rFIXK5R) were compared individually at each time point, and p values ​​are summarized in Figure 7C. Treatment of HB mice with rFIX significantly protected the animals from total blood loss up to 24 h after treatment (vehicle: approximately 10 μL / g BW; rFIX-treated: approximately 1 μL / g BW, Figure 7A). As shown in Figure 7A, total blood loss was comparable between the rFIX-treated groups up to 168 h. However, at 336 hours post-administration, only animals treated with rFIXK5R had a nearly 50% reduction in blood loss compared to the vehicle group (vehicle: BW approx. 18 μL / g; rFIXK5R: BW approx. 9 μL / g), although this was not statistically significant.

[0193] Significant effects in protecting against bleeding incidence were observed for all rFIX molecules at 15 min posttreatment. However, at later time points, the potency of individual rFIX proteins decreased over time at different rates. More specifically, statistically significant effects on reducing bleeding incidence were lost at 24 h posttreatment for rFIXK5A, 72 h for rFIXWT, and 168 h for rFIXK5R (Figure 7C). It is noteworthy that both blood loss and bleeding incidence parameters must be considered in context, as these parameters reflect different aspects of hemostasis. For example, bleeding incidence (Figure 7B), or bleeding time, which indicates the time it takes for a mouse to stop bleeding, reflects complete occlusion of the blood vessel. On the other hand, a non-occlusive thrombus may reduce blood loss (Figure 7A), but the animal may continue to bleed throughout the observation period. rFIX antigen and activity were detectable in plasma in the 15-min group, and no detectable rFIX protein was present in the circulation after 24 h (Figure 8). Therefore, the hemostatic effect observed at 24 h and beyond could be partially attributed to extravascular FIX. The two-fold difference between FIX:Ag and FIX:C values ​​in mIU / mL can be explained by assay differences. The calibration curve for the one-stage coagulation assay (OSCA; FIX:C) was based on standard human plasma (SHP), and the injection solution was used as the standard in the ELISA. Furthermore, matrix effects using OSCA (normal ranges of other plasma proteins and activation of plasma proteins involved in the intrinsic pathway of the coagulation cascade) cannot be excluded due to the conditions under which the mouse plasma samples were collected (after transection of major peripheral arteries and veins, leading to massive bleeding and consumption of coagulation factors).

[0194] Generally, all proteins were comparable in their ability to reduce blood loss after intravenous administration.Although statistically not significant, rFIXK5R showed the longest efficacy in reducing blood loss compared with the vehicle group until 14 days, even when rFIX was not detected in the circulation.This result suggests that there is rFIX that is not circulating but is accessible to the injury site.The incidence of bleeding over blood loss suggests that rFIXK5A provides good protection, but this ability is lost very quickly, demonstrating that rFIXK5A may not be easily accessible to the injury site 24 hours after intravenous administration, and therefore restricts the growth of stable clots, which require both FIX circulating in the thrombus area and FIX that is easily accessible at the site to achieve complete vascular occlusion.

[0195] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

[0196] array

[0197] SEQ ID NO:1 - Human wild-type FIX polypeptide [ka]

[0198] SEQ ID NO:2 - Coding sequence for human wild-type FIX polypeptide

[0199] SEQ ID NO:3 - Human wild-type factor IX containing signal and propeptide MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSY ECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGG EDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0200] SEQ ID NO: 4 - Coding sequence of human wild-type factor IX including signal and propeptide

[0201] SEQ ID NO:5 - Human albumin DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0202] SEQ ID NO: 6 - Fc polypeptide EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0203] SEQ ID NO: 7 - Fc polypeptide DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0204] SEQ ID NO:8 - cleavable linker PVSQTSKLTRAETVFP

[0205] SEQ ID NO:9 - K5A human FIX variant polypeptide [ka]

[0206] SEQ ID NO:10 - R338L human FIX variant polypeptide YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSA DNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCG GSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWG RVFHKGRSALVLQYLRVPLVDRATCLLSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0207] SEQ ID NO: 11 - CTP sequence SSSSKAPPPS

[0208] SEQ ID NO: 12 - CTP sequence DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL

[0209] SEQ ID NO: 13 - CTP sequence SSSSKAPPPSLPSPSRLPGPSDTPILPQ

[0210] SEQ ID NO: 14 - XTEN72 sequence GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGASS

[0211] SEQ ID NO: 15 - XTEN sequence GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS

[0212] SEQ ID NO: 16 - Non-cleavable linker GGGGGGV

[0213] SEQ ID NO: 17 - Light chain human activated factor IX YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTR

[0214] SEQ ID NO: 18 - Heavy chain human activated factor IX VVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTN IFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

[0215] SEQ ID NO: 19 - R318Y / R338E / T343R (darcinonacog) human FIX variant polypeptide [ka]

[0216] SEQ ID NO: 20 - T148A human FIX polymorphic variant YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSA DNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCG GSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWG RVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

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Claims

1. 1. A Factor IX (FIX) variant polypeptide for use in a method of treating or preventing a bleeding disorder 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. 2. The FIX variant polypeptide for use according to claim 1, wherein the bleeding disorder is hemophilia B.

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

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

5. 4. The FIX variant polypeptide for use according to any one of claims 1 to 3, wherein the FIX variant polypeptide further comprises 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, and the amino acid histidine at the position corresponding to position 410 of wild-type factor IX.

6. 4. The FIX variant polypeptide for use according to any one of claims 1 to 3, wherein the FIX variant polypeptide further comprises the amino acid tyrosine at the position corresponding to position 318 of wild-type factor IX, the amino acid glutamic acid 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.

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

9. 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. 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 claims 1 to 10, wherein the soft tissue is skin tissue or gastrointestinal tissue.

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

13. 13. The FIX variant polypeptide for use according to claim 11 or claim 12, wherein the method comprises administering the FIX variant polypeptide subcutaneously.

14. 12. The FIX variant polypeptide for use according to claim 11, wherein the method comprises administering the FIX variant polypeptide to gastrointestinal tissue using an oral drug delivery device.

15. 1. A pharmaceutical composition comprising a Factor IX (FIX) variant polypeptide for use in a method for treating or preventing a bleeding disorder 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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