Modified factor Xa polypeptides and methods of use - Patents.com

Abstract

Modified factor Xa polypeptides are provided that have functional activity and reduced affinity to apixaban. Nucleic acids encoding the modified factor Xa polypeptides and vectors, and host cells containing the nucleic acids, are also provided. Methods of reducing or inhibiting bleeding, such as in subjects treated with direct oral anticoagulants, are provided. Provided herein are factor Xa polypeptides that contain one or more amino acid substitutions at factor Xa subsites S1, S4, or both, and have at least 300% enzymatic activity of unmodified factor Xa in the presence of apixaban, an IC for apixaban that is at least 50-fold higher than unmodified factor Xa, and a 5-fold higher IC for apixaban than unmodified factor Xa. 50 The present invention is a modified factor Xa polypeptide having a nucleotide sequence, a nucleotide sequence, a nucleotide value, or both.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 339,230, filed May 6, 2022, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to modified factor Xa polypeptides and methods of their use, particularly to reduce or inhibit the effects of direct oral anticoagulants that inhibit factor Xa. [Background technology]

[0003] Direct oral anticoagulants (DOACs), which directly inhibit factor Xa (FXa), have revolutionized the prevention of systemic embolism and stroke. Five DOACs have been approved. One of them (dabigatran) inhibits factor IIa, while four (rivaroxaban, apixaban, edoxaban, and betrixaban) are FXa inhibitors. DOACs are widely prescribed for the prevention of systemic embolism and stroke, for nonvalvular atrial fibrillation, and for the treatment of venous thromboembolism. Although FXa inhibitors have shown a favorable risk-benefit profile in the prevention and / or treatment of thrombotic events, these agents are also associated with acute massive bleeding. More than 100,000 cases of DOAC-associated massive bleeding occur annually in the United States and the European Union and are difficult to treat. The only currently approved specific reversal agent of FXa inhibitors, andexanet alfa (Andexxa®), is a bioengineered inactive variant of human FXa. Andexxa® has been shown to bind and capture FXa inhibitors.Andexxa® and FXa have comparable affinity to DOACs, and as a result, the recommended initial low-dose intravenous (IV) bolus is 400 mg at a target rate of 30 mg / min, followed by IV infusion at 4 mg / min for up to 120 minutes, and the recommended initial high-dose IV bolus is 800 mg at a target rate of 30 mg / min, followed by IV infusion at 8 mg / min for up to 120 minutes.Risks associated with Andexxa® include arterial and venous thrombosis, myocardial infarction, ischemic stroke, cardiac arrest, and sudden death. Summary of the Invention [Means for solving the problem]

[0004] There remains an unmet need for safe and effective DOAC antagonists. The design of functional factor Xa analogs with low DOAC affinity provides an alternative antagonistic strategy. Disclosed herein is a functional factor Xa analog with low affinity for apixaban, the most widely prescribed factor Xa inhibitor.

[0005] Provided herein are factor Xa antibodies that contain one or more amino acid substitutions in factor Xa subsites S1, S4, or both, and have an enzymatic activity of at least 300% of unmodified factor Xa in the presence of apixaban, an IC20 to apixaban that is at least 50-fold higher than unmodified factor Xa. 50 In certain embodiments, the unmodified factor Xa polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In additional embodiments, the modified factor Xa polypeptide does not include the signal peptide and propeptide of factor Xa (e.g., does not include amino acids 1-40 of SEQ ID NO: 1).

[0006] In certain embodiments provided herein, the modified Factor Xa polypeptide includes one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, where amino acid numbering corresponds to SEQ ID NO:1. In some examples, the modified Factor Xa polypeptide comprises one of W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In other embodiments, the modified Factor Xa polypeptide comprises one of W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has an amino acid sequence having at least 95% sequence identity to amino acids 3-450 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or comprises or consists of an amino acid sequence of amino acids 3-450 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

[0007] Also provided are nucleic acids encoding the disclosed modified Factor Xa polypeptides. In some embodiments, the nucleic acid encodes a polypeptide having W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs:3, 5, 7, 9, 11, 13, and 15, or comprises or consists of the nucleotide sequence of any one of SEQ ID NOs:3, 5, 7, 9, 11, 13, and 15. In other embodiments, the nucleic acid encodes one of W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317 and has a nucleotide sequence having at least 95% sequence identity to, or comprising, nucleotides 7-1350 of any one of SEQ ID NOs:3, 5, 7, 9, 11, 13, and 15. Also provided are vectors comprising nucleic acids encoding the disclosed modified Factor Xa polypeptides and host cells comprising the nucleic acids or vectors.

[0008] In certain embodiments, a composition is provided that includes a disclosed modified factor Xa polypeptide and a pharma- ceutically acceptable carrier.

[0009] Also provided is a method of treating or inhibiting bleeding in a subject receiving direct oral anticoagulant therapy. The method comprises administering to the subject an effective amount of the disclosed modified factor Xa polypeptide, thereby treating or inhibiting bleeding. In some embodiments, the subject is receiving the DOAC apixaban, rivaroxaban, edoxaban, or betrixaban. In one example, the subject is receiving apixaban. In some examples, the modified factor Xa polypeptide or composition is administered intravenously to the subject. In some examples, the modified factor Xa polypeptide or composition is administered to the subject at a dose of about 0.1-10 mg / kg. Treatment with the modified factor Xa polypeptide may reduce bleeding time by at least 10% compared to a control, reduce blood loss by at least 10% compared to a control, or both.

[0010] The foregoing and other features of the present disclosure will become more apparent from the following detailed description of various embodiments, which proceeds with reference to the accompanying figures. [Brief description of the drawings]

[0011] [Figure 1-1]Figures 1A-1H show the design and functional characterization of low affinity FXa variants. Figure 1A illustrates the computational strategy using RosettaDesign and homology insertion. Figure 1B shows models of FXa variants 61 (upper panel) and 70 (lower panel) showing mutations that affect apixaban binding. Variants 61 and 70 and apixaban are shown as stick models. Figure 1C shows the location of the variants on the FXa heavy chain region (catalytic domain). Variants 69 and 70 are located in the same region between H311 and T318. Figure 1D shows the location of the variants represented as the "surface" where apixaban occupies the binding pocket. Figure 1E shows the S1 and S4 subsites of the binding pocket. Figure 1F is a graph showing the chromogenic substrate cleavage rate in a pure buffer system by different concentrations of FXa variants. FXa HTI (commercially available plasma-derived FXa) was used to evaluate homemade FXa (FXa WJ) and determine whether activity was comparable. Figure 1G is a graph showing the rate of chromogenic substrate cleavage by 0.5 μg of FXa variants in the absence (V0) or presence (Vi) of increasing apixaban concentrations. IC50 ± SD values ​​were obtained from fitted curves. Figure 1H shows side-by-side 1H NMR spectra of stoichiometric amounts of apixaban and factor VII (FVII) (top), a negative control sample; apixaban (middle), a reference spectrum; and rFXa:apixaban (bottom), a test sample at a stoichiometric ratio. The top spectrum is the result of overlay of the unchanged ligand and protein signals. The reference NMR spectrum (middle) contains only sharp signals, as expected in the NMR spectrum of a small molecule. However, despite the presence of small molecules in the sample, the 1H NMR spectrum of the test sample (bottom) shows only broadened NMR signals, indicating strong binding of apixaban to rFXa. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above.

[0012] [Diagram 2] Figures 2A and 2B show the chromogenic substrate cleavage rates by different amounts of FXa variants in the absence (V0) or presence (Vi) of increasing apixaban concentrations. IC50 ± SD values ​​were obtained from curve fitting. Variants are as described in Tables 1 and 2.

[0013] [Figure 3-1] Figures 3A-3E show the effect of variants 61 and 70 on the in vitro and in vivo procoagulant potential in the presence of apixaban (Apix or A). Figure 3A shows the effect of FXa variants on thrombin generation in plasma spiked with apixaban. Thrombin peak height (TPH) values ​​were obtained by titration of the indicated concentrations of FXa variants in normal pooled plasma spiked with 1 μM apixaban. Experimental design of the mouse tail cut model (Figure 3B) and readout parameters: bleeding time (Figure 3C), blood loss (Figure 3D), and bleeding profile (Figure 3E). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above.

[0014] [Figure 4-1] Figures 4A-4D show the effect of variants 61 and 70 and andexanet alfa (Andx) in the presence of apixaban (APX) on the procoagulant potential in vitro and in vivo. Figure 4A shows the experimental design. For the indicated treatments, Figure 4B shows the bleeding time, Figure 4C shows the blood loss, and Figure 4D shows the bleeding profile. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0015] [Diagram 5] FIG. 5 shows tissue factor pathway inhibitor (TFPI) activity in response to FXa, 4 nM variants 61 or 70, and 100 nM andexanet alpha (Andx). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Sequence Listing Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 CFR § 1.822. In at least some cases, only a single strand of each nucleic acid sequence is shown, but the complementary strand is understood as encompassed by any reference to the represented strand.

[0017] SEQ ID NO:1 is the amino acid sequence of an exemplary Factor X preproprotein (signal peptide, amino acids 1-31; propeptide, amino acids 32-40; Gla domain, amino acids 41-85; EGF-like domain 1, amino acids 86-122; EGF-like domain 2, amino acids 125-165; activation peptide, amino acids 179-234; catalytic domain, amino acids 235-488): [ka]

[0018] SEQ ID NO:2 is the amino acid sequence of FXa variant 61: [ka]

[0019] SEQ ID NO:3 is the nucleic acid sequence encoding FXa variant 61: [ka] [ka]

[0020] SEQ ID NO:4 is the amino acid sequence of FXa variant 70: [ka]

[0021] SEQ ID NO:5 is the nucleic acid sequence encoding FXa variant 70: [ka]

[0022] SEQ ID NO:6 is the amino acid sequence of FXa variant 63: [ka]

[0023] SEQ ID NO:7 is the nucleic acid sequence encoding FXa variant 63: [ka] [ka]

[0024] SEQ ID NO:8 is the amino acid sequence of FXa variant 65: [ka]

[0025] SEQ ID NO:9 is the nucleic acid sequence encoding FXa variant 65: [ka]

[0026] SEQ ID NO: 10 is the amino acid sequence of FXa variant 68: [ka] [ka]

[0027] SEQ ID NO:11 is the nucleic acid sequence encoding FXa variant 68: [ka]

[0028] SEQ ID NO:12 is the amino acid sequence of FXa variant 69: [ka]

[0029] SEQ ID NO: 13 is the nucleic acid sequence encoding FXa variant 69: [ka] [ka]

[0030] SEQ ID NO:14 is the amino acid sequence of FXa variant 73: [ka]

[0031] SEQ ID NO: 15 is the nucleic acid sequence encoding FXa variant 73: [ka]

[0032] Detailed Description Apixaban is the most prescribed DOAC in the United States and has proven to be an important drug for the prevention and treatment of thromboembolic disorders. However, safe and effective reversals to control anticoagulant-associated bleeding remain an unmet need. Described herein is the rational design of reversals for apixaban and other DOACs. The rationale was the design of FXa variants that bind with low affinity to apixaban but retain sufficient potency to antagonize bleeding caused by apixaban use. The design of FXa variants followed a hybrid approach. Rosetta-based computational biology was combined with biochemistry and structural biology knowledge to iteratively redesign FXa variants. In particular, the binding affinity of apixaban to two comprehensively characterized FXa variants was reduced by orders of magnitude. These variants retained sufficient potency to antagonize the anticoagulant effect of apixaban in an in vivo mouse model. The doses of apixaban used in the mouse model studies described herein are comparable to those used in the clinic, and the amount of variant FXa used translates to approximately 70 mg for a 70 kg adult, which is significantly lower than the low or high doses of andexanet alfa (Andexxa®), the only approved reversal agent that specifically targets FXa inhibitors, including apixaban.

[0033] I. Terminology Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published in 2017 by Jones & Bartlett Learning. As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "a polypeptide" encompasses a single or multiple polypeptides and can be considered equivalent to the expression "at least one polypeptide". As used herein, the term "comprises" means "includes". Furthermore, it should be understood that any and all base or amino acid sizes, as well as all molecular weights and molecular mass values ​​given for nucleic acids or polypeptides, are approximate and are provided for illustrative purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described herein.In case of conflict, the present specification, including explanations of terms, will control.Furthermore, the materials, methods, and examples are only illustrative and are not intended to be limiting.

[0034] In order to facilitate description of the various embodiments, the following explanations of terms are provided:

[0035] Administration: Providing or giving an agent, such as a therapeutic agent (e.g., a polypeptide composition), to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraductal, sublingual, rectal, transdermal, nasal, intravaginal, and inhalation routes.

[0036] Direct oral anticoagulant (DOAC): an anticoagulant compound that directly inhibits thrombin (factor IIa) or factor Xa. Currently approved DOACs include the factor IIa inhibitor dabigatran (PRADAXA®) and the factor Xa inhibitors apixaban (ELIQUIS®), rivaroxaban (XARELTO®), and edoxaban (LIXIANA®). DOACs are clinically approved to reduce the risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation, to treat deep vein thrombosis or pulmonary embolism and reduce the risk of recurrent deep vein thrombosis or pulmonary embolism, to prevent deep vein thrombosis in patients who have undergone hip or knee replacement surgery, and to reduce the risk of major thrombotic vascular events (such as myocardial infarction or ischemic stroke) in patients with peripheral arterial disease.

[0037] The most serious side effect of DOAC therapy is uncontrollable bleeding that is difficult to treat. A specific reversal agent of apixaban and rivaroxaban is currently available (Andexanet alfa (ANDEXXA®)). However, this reversal agent carries significant risks, including arterial and venous thrombosis, myocardial infarction, ischemic stroke, cardiac arrest, and sudden death.

[0038] Factor Xa: Factor X is the vitamin K-dependent X clotting factor of the blood coagulation cascade. It undergoes multiple processing steps before its preprotein is converted to the mature two-chain form by excision of the tripeptide RKR. The two chains of the factor are held together by one or more disulfide bonds; the light chain contains two EGF-like domains, and the heavy chain contains a catalytic domain that is structurally homologous to those of other hemostatic serine proteases. The mature factor (factor Xa) is activated by cleavage of an activation peptide by factor IXa (in the intrinsic pathway) or by factor VIIa (in the extrinsic pathway). The activated factor then undergoes conversion to factor Va, Ca, and β-terminal cleavage during blood clotting. 2+ , and in the presence of phospholipids converts prothrombin to thrombin.

[0039] The sequence of factor X is known. Exemplary human factor X nucleic acid and amino acid sequences include GenBank Accession Nos. NM_000504.4 and NP_000495.1, respectively (both of which are incorporated by reference herein as present in the GenBank database as of May 6, 2022). An exemplary factor X preproprotein amino acid sequence is SEQ ID NO: 1. An exemplary wild-type (e.g., unmodified) factor Xa amino acid sequence is amino acids 41-488 of SEQ ID NO: 1.

[0040] Heterologous: A heterologous protein, polypeptide, or nucleic acid refers to a protein, polypeptide, or nucleic acid derived from a different source or species. A heterologous protein or polypeptide can also refer to a protein or polypeptide that has an amino acid sequence that is different from a naturally occurring protein or polypeptide. Similarly, a heterologous nucleic acid refers to a nucleic acid that has a nucleotide sequence that is different from a naturally occurring nucleic acid molecule.

[0041] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) is substantially separated or purified from other chromosomal and extrachromosomal DNA and RNA, proteins, and other biological components in the cells or tissues of an organism in which it occurs, or in the organism itself, such as the cells. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins. Isolated does not require absolute purity and can include proteins, peptides, or nucleic acid molecules that are at least 50% isolated, e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0042] Pharmaceutically acceptable carriers: Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23 rd Edition (2021) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, molecules or agents (e.g., polypeptides).

[0043] Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain injectable fluids, which include pharma- ceutically and physiologically acceptable fluids as vehicles, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc. For solid compositions (e.g., in the form of powder, pill, tablet, or capsule), non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0044] Polypeptide, Peptide, or Protein: A polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acids are alpha amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0045] Conservative substitution in a polypeptide is the replacement of one amino acid residue in a protein sequence with a different amino acid residue that has similar biochemical properties. Typically, conservative substitution has little or no effect on the activity of the resulting polypeptide. For example, a protein or peptide that includes one or more conservative substitutions (e.g., not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions) retains the structure and function of the corresponding protein or peptide that does not have the conservative substitution. A polypeptide can be generated to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes the polypeptide, for example, using standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be easily selected by testing protein activity or binding affinity (such as thrombin cleavage activity or apixaban binding affinity).

[0046] Examples of conservative substitutions are shown below. [Table 3-1] [Table 3-2]

[0047] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone, for example a sheet or helix, in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains.

[0048] In general, the substitutions expected to produce the greatest changes in the properties of a protein will be non-conservative, e.g., (a) the substitution of a hydrophilic residue, e.g., seryl or threonyl, for (or being substituted by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) the substitution of a cysteine ​​or proline for (or being substituted by) any other residue; (c) the substitution of a residue having a positively charged side chain, e.g., lysyl, arginyl, or histadyl, for (or being substituted by) a negatively charged residue, e.g., glutamyl or aspartyl; or (d) the substitution of a residue having a bulky side chain, e.g., phenylalanine, for (or being substituted by) one having no side chain, e.g., glycine.

[0049] Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that does not occur in nature or that is made by the artificial combination of two otherwise separate segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques. The term "recombinant" also encompasses nucleic acids and proteins that have been altered only by the addition, substitution, or deletion of portions of a naturally occurring nucleic acid molecule or protein.

[0050] Subject: A living multicellular vertebrate organism, a species that includes human and non-human mammals. In certain embodiments herein, the subject is a human, veterinary, or experimental subject.

[0051] Therapeutically effective amount or effective amount: An amount of an agent, such as a nucleic acid, polypeptide, or other therapeutic agent, sufficient to prevent, treat, reduce, and / or ameliorate the symptoms and / or underlying causes of a disorder or disease. In some embodiments, an "effective amount" is an amount sufficient to reduce or antagonize factor Xa inhibition (e.g., by a DOAC) in a subject and / or treat or inhibit bleeding due to DOAC therapy.

[0052] Treating or ameliorating a condition: "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to a reduction in the number or severity of the signs or symptoms of a disease or pathological condition.

[0053] Vector: A vector is a nucleic acid molecule that allows the insertion of a foreign nucleic acid without interfering with the vector's ability to replicate and / or integrate in a host cell. A vector can include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic components. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of one or more inserted genes.

[0054] II. Modified Factor Xa Polypeptides and Nucleic Acids The present disclosure describes the design of modified factor Xa polypeptides. In some embodiments, the modified factor Xa polypeptide has an enzymatic activity of at least about 300% (e.g., at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 450%, or more, e.g., about 300-450%, about 325-375%, about 350-400%, about 375-425%, or about 400-450%) of unmodified factor Xa in the presence of apixaban. In some examples, the enzymatic activity of factor Xa is measured using a thrombin generation assay. An exemplary thrombin generation assay is described in Example 1, although other thrombin generation assays are available and can be used by one of skill in the art.

[0055] In other embodiments, the modified factor Xa polypeptide has an IC50 value for apixaban that is at least about 50-fold higher (e.g., at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 125-fold, at least about 150-fold, at least about 175-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold higher, or more, e.g., about 50-100-fold higher, about 75-150-fold higher, about 125-175-fold higher, about 150-200-fold higher, about 200-250-fold higher, about 225-275-fold higher, about 250-300-fold higher, about 300-350-fold higher, or about 350-400-fold higher) compared to unmodified factor Xa. These polypeptides can be used to antagonize or reduce the effect of a DOAC (such as apixaban) when necessary, such as in the case of bleeding (such as uncontrolled or heavy bleeding) in a subject treated with DOAC therapy.

[0056] In certain embodiments, the modified factor Xa polypeptides disclosed herein include a substitution of at least one amino acid (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more amino acids) compared to a wild-type or unmodified factor Xa polypeptide. In certain examples, the unmodified factor Xa polypeptide is amino acids 41-488 of SEQ ID NO: 1. In particular examples, the factor Xa polypeptide is a human factor Xa polypeptide.

[0057] Factor Xa consists of four major subsites, designated S1, S2, S3 and S4 (see, e.g., Zacconi, in Anticoagulant Drugs, Ed. Bozic-Mijovski, Intech Open, 2018, pp. 11-37; Hsu et al., J. Biol. Chem. 283:12343-12353, 2008). In certain embodiments, the disclosed modified factor Xa polypeptides include one or more amino acid substitutions in the S4 subsite (S4 binding pocket) of factor Xa. In other embodiments, the disclosed modified factor Xa polypeptides include one or more amino acid substitutions in the S1 subsite (S1 binding pocket) of factor Xa. In further embodiments, the disclosed modified factor Xa polypeptides include one or more amino acid substitutions in each of the S1 and S4 subsites.

[0058] In certain embodiments, modified Factor Xa polypeptides include polypeptides having one or more of the amino acid substitutions listed in Table 1. In some examples, modified Factor Xa polypeptides include one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317 (amino acid numbering corresponding to the Factor Xa amino acid sequence of SEQ ID NO:1). In certain embodiments, the modified Factor Xa polypeptide comprises one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some examples, the modified Factor Xa polypeptide has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In other embodiments, the modified Factor Xa polypeptide comprises one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 3-450 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or comprises or consists of the amino acid sequence of amino acids 3-450 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. Table 1. Exemplary modified Factor Xa polypeptides [Table 1] *Numbering corresponds to the position in the Factor X preproprotein (e.g., SEQ ID NO: 1)

[0059] Additional minor modifications of the modified factor Xa polypeptides provided herein are also contemplated. Such modifications may result in polypeptides with substantially equivalent activity and / or binding affinity when compared to the corresponding starting polypeptide. Such modifications may be deliberate, for example, by site-directed mutagenesis, or may be naturally occurring. All of the polypeptides produced by these modifications are encompassed herein. Thus, specific, non-limiting examples are conservative variants of modified factor Xa polypeptides (such as conservative amino acid substitutions, e.g., one or more conservative amino acid substitutions, e.g., 1-10 conservative substitutions, 2-5 conservative substitutions, 4-9 conservative substitutions, e.g., 1, 2, 5, or 10 conservative substitutions). In other examples, a protein can include one or more non-conservative substitutions (e.g., 1-10 non-conservative substitutions, 2-5 non-conservative substitutions, 4-9 non-conservative substitutions, e.g., 1, 2, 5 or 10 non-conservative substitutions), so long as the polypeptide retains similar thrombin cleavage activity and / or apixaban affinity as the starting polypeptide.

[0060] The disclosed polypeptides can be prepared by chemical synthesis or isolated by methods including preparative chromatography and immunological separation. Polypeptides can also be produced using molecular genetic techniques, such as by inserting a nucleic acid encoding the polypeptide into an expression vector, introducing the expression vector into a host cell (such as E. coli or a mammalian cell), and isolating the polypeptide. In some instances, the protein includes a tag (such as an N-terminal or C-terminal tag), for example, for use in protein purification. Exemplary tags include His-tags, GST tags, antibody recognition sequences (such as Myc-tags or HA-tags), or protein A. In some embodiments, the polypeptides are produced by bacteria (such as E. coli) or mammalian cells that express the polypeptide from an expression vector (such as a vector that includes a constitutive or controllable promoter).

[0061] In some instances, the modified factor Xa polypeptide does not include the signal peptide and propeptide sequences of factor X (e.g., amino acids 1-40 of SEQ ID NO:1). Thus, in some instances, the disclosed modified factor Xa polypeptides do not include a starting methionine (e.g., beginning at amino acid 41 of SEQ ID NO:1). In other instances, the modified factor Xa polypeptides are expressed with an N-terminal tag that is subsequently cleaved prior to use, such that one or more amino acids may remain at the N-terminus of the polypeptide as a result of the cleavage site. In one example, the modified factor Xa polypeptides are expressed with an N-terminal protein A tag that is removed by proteolytic cleavage. In such instances, as a result of the proteolytic cleavage site, the polypeptide includes an N-terminal glycine-proline (GP).

[0062] Also provided are nucleic acid molecules (e.g., DNA, cDNA, RNA, or mRNA) encoding the modified factor Xa polypeptides disclosed herein. Unless otherwise specified, a "nucleic acid molecule encoding a polypeptide" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. For example, a polynucleotide encoding a disclosed modified factor Xa polypeptide includes nucleic acid sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is not altered. In some embodiments, the disclosed polypeptide sequences are reverse translated into codon-optimized DNA using standard methods.

[0063] In some embodiments, the nucleic acid encodes a polypeptide comprising one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some examples, the nucleic acid encodes a polypeptide comprising or consisting of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some examples, the nucleic acid encodes a polypeptide including one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15. In other examples, the nucleic acid has a nucleotide sequence including or consisting of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15.

[0064] In other embodiments, the nucleic acid includes one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and encodes a polypeptide having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acids 3-450 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some examples, the nucleic acid encodes a polypeptide that includes or consists of amino acids 3-450 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some examples, the nucleic acid encodes a polypeptide including one or more amino acid substitutions selected from W439A, NRFTKE→STYVPG at 312-317, G450A, G440A, C415A+C443A, NRFTKE→KNYQRD at 312-317, and W439A+NRFTKE→STYVPG at 312-317, and has a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to nucleotides 7-1350 of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15. In other examples, the nucleic acid has a nucleotide sequence including or consisting of nucleotides 7-1350 of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15.

[0065] Minor modifications of nucleic acids encoding modified factor Xa polypeptide primary amino acid sequences are also contemplated herein. Such modifications to nucleic acids may result in polypeptides with substantially equivalent activity when compared to the corresponding starting polypeptides described herein. Such modifications may be deliberate, for example by site-directed mutagenesis, or may be naturally occurring. All of the nucleic acids produced by these modifications are encompassed herein. Thus, specific, non-limiting examples of modified nucleic acids encoding the disclosed polypeptides are nucleic acids encoding conservative variants of the polypeptides (such as those encoding conservative amino acid substitutions, e.g., one or more conservative amino acid substitutions, e.g., 1-10 conservative substitutions, 2-5 conservative substitutions, 4-9 conservative substitutions, e.g., 1, 2, 5 or 10 conservative substitutions). In other examples, a nucleic acid can encode a polypeptide that includes one or more non-conservative substitutions (e.g., 1-10 non-conservative substitutions, 2-5 non-conservative substitutions, 4-9 non-conservative substitutions, e.g., encoding 1, 2, 5 or 10 non-conservative substitutions), so long as the encoded polypeptide retains similar thrombin cleavage activity and / or apixaban affinity as the starting polypeptide.

[0066] Vectors that include the disclosed nucleic acid molecules are also provided. The DNA sequence encoding the disclosed polypeptides can be expressed in vitro or in vivo by DNA transfer into a suitable host cell. The cell can be prokaryotic or eukaryotic. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. The polynucleotide sequence encoding the disclosed polypeptides can be operably linked to an expression control sequence, such as a heterologous expression control sequence (such as a heterologous promoter). The expression control sequence operably linked to the coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequence. The expression control sequence includes, but is not limited to, one or more suitable promoters, enhancers, transcription terminators, a start codon in front of the protein-coding gene, splicing signals of introns, maintaining the correct reading frame of the gene to allow proper translation of mRNA, and stop codons.

[0067] Hosts can include microbial, yeast, insect, and mammalian organisms. Methods for expressing DNA sequences with eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plants, and animal cells (e.g., mammalian cells, such as human cells). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the growth of mammalian cells in culture are known to those of skill in the art. Examples of commonly used mammalian host cell lines are VERO cells, HeLa cells, CHO cells, HEK293 cells, WI38 cells, BHK cells (such as BHK21 cells), HT-1080 cells, PER.C6 cells, HKB-11 cells, HuH-7 cells, and COS cells, although other cell lines may be used, such as cells engineered to provide higher expression, desirable glycosylation patterns, or other characteristics.

[0068] Transformation of host cells with recombinant DNA can be carried out by techniques known to those skilled in the art. When the host is a prokaryotic organism, such as, but not limited to, E. coli, competent cells capable of DNA uptake can be prepared from cells harvested after logarithmic growth phase and subsequently treated by the CaCl2 method using procedures known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be carried out after forming a protoplast of the host cell, if desired, or by electroporation. When the host is a eukaryotic organism, methods of DNA transfection, such as calcium phosphate co-precipitation, conventional mechanical techniques, e.g., microinjection, electroporation, insertion of a plasmid encapsulated in a liposome, or a viral vector, can be used. Eukaryotic cells can also be transformed with a polynucleotide encoding the disclosed polypeptide and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use eukaryotic viral vectors, such as Simian Virus 40 (SV40) or bovine papilloma virus, to infect or transform eukaryotic cells to express the protein.

[0069] III. Compositions and Methods of Treatment Disclosed herein is The present invention relates to a method for treating or inhibiting bleeding in a subject undergoing or undergoing direct oral anticoagulant therapy using the modified factor Xa polypeptide described herein.In some embodiments, the method comprises administering to the subject an effective amount of the disclosed modified factor Xa polypeptide or a composition comprising an effective amount of the disclosed modified factor Xa polypeptide.In some examples, the subject undergoing DOAC therapy is a subject undergoing factor Xa inhibitor DOAC, such as apixaban, rivaroxaban, edoxaban, betrixaban, darexaban, otamixaban, retaxaban, LY517717, or GW813893.In one embodiment, the subject undergoes treatment with apixaban.

[0070] In some embodiments, treatment with the disclosed modified factor Xa polypeptide or composition reduces or inhibits bleeding, such as uncontrollable bleeding events or hemorrhagic events, in subjects undergoing DOAC therapy.In some instances, the subject has received at least one dose of DOAC within the past 24 hours.In some instances, the subject has undergone or will undergo surgery (such as emergency treatment).In other instances, the subject has undergone injury or trauma (including but not limited to traumatic brain injury, or injury that requires emergency surgery).

[0071] The treatment may reduce bleeding time by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to a control (such as a subject receiving DOAC therapy without treatment with a disclosed modified factor Xa polypeptide or composition). The treatment may reduce blood loss by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to a control (such as a subject receiving DOAC therapy without treatment with a disclosed modified factor Xa polypeptide or composition). In some examples, the treatment reduces bleeding time by about 60% and / or reduces blood loss by about 90%, compared to a control (such as a subject receiving DOAC therapy without treatment with a disclosed modified factor Xa polypeptide).

[0072] In other examples, the treatment provides effective clinical hemostasis in the subject, such as effective hemostasis of non-visible bleeding (e.g., stable hemoglobin levels 48 hours after initial treatment), effective hemostasis of visible bleeding (e.g., no visible bleeding at 4 hours of treatment), effective hemostasis of musculoskeletal bleeding (e.g., reduced pain and swelling within 24 hours), or effective hemostasis in the intracranial space (e.g., stable hematoma or <35% increase compared to baseline within 12 hours). In other examples, effective clinical hemostasis includes no need for further infusion of hemostatic agents, clotting factors, or blood product transfusions up to 48 hours after initial treatment. See, e.g., Khorsand et al., Journal of Thrombosis and Haemostasis 14:211-214, 2015.

[0073] Pharmaceutical compositions comprising the disclosed polypeptides are also provided. The pharmaceutical compositions can comprise one or more modified factor Xa polypeptides and one or more pharma- ceutically acceptable carriers. The pharma-ceutically acceptable carriers are determined in part by the particular composition to be administered and by the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of the pharmaceutical compositions of the present disclosure. Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23rd Edition (2021) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic agents.

[0074] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0075] In some embodiments, an effective amount of a modified polypeptide or composition disclosed herein is administered to a subject. In some examples, an effective amount of a modified Factor Xa polypeptide or composition, such as about 0.05 mg / kg to about 10 mg / kg of the modified polypeptide, is administered. In some examples, the subject is administered about 0.05 mg / kg to about 0.25 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.25 mg / kg to about 0.75 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 1.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2.5 mg / kg to about 7.5 mg / kg, or 5 mg / kg to about 10 mg / kg (e.g., about 0.05 mg / kg, about 0.075 mg / kg, about 0.1 mg / kg, About 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg is administered. In one example, about 1 mg / kg of the modified polypeptide is administered to the subject.

[0076] In other examples, the polypeptide or composition can be provided in a unit dosage form for administration to a subject. The unit dosage form contains a single preselected dose suitable for administration to a subject, two or more preselected unit doses appropriately recorded or measured, and / or a metering mechanism for administering the unit dose or multiples thereof. In some examples, the polypeptide or composition is provided in a unit dosage of about 2.5 mg to about 1.5 g (e.g., about 2.5 mg to about 7.5 mg, about 5 mg to about 15 mg, about 10 mg to about 25 mg, about 20 mg to about 80 mg, about 75 mg to about 100 mg, about 100 mg to about 250 mg, about 200 mg to about 500 mg, about 250 mg to about 800 mg, about 500 mg to about 1 g, about 800 mg to about 1.25 g, or about 1 g to about 1.5 g). In certain examples, the unit dose is about 5 mg, about 10 mg, about 20 mg, about 25 mg, about 40 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 g, about 1.25 g, or about 1.5 g.

[0077] The disclosed modified polypeptide or composition is typically administered parenterally (e.g., intravenously). The skilled artisan can determine the appropriate route of administration. Multiple doses (e.g., 1x, 2x, 3x or more doses) of the polypeptide or composition can be administered, for example, to control ongoing bleeding. A skilled clinician can select the appropriate route and schedule of administration based on clinical studies, the particular subject, the clinical situation, and other factors. EXAMPLES

[0078] The following examples are provided to illustrate particular features of certain embodiments of the present disclosure, but the claims should not be limited to these exemplified features.

[0079] Example 1 material and method RosettaFastRelax: Input structure preparation: The crystal structure of factor Xa (PDB ID: 2P16, after apixaban removal) was used as the starting structure for the all-atom refinement process using the Rosetta Relax protocol. The structure was passed through fixed-backbone Rosetta energy minimization (FastRelax protocol, 1000 decoys were generated) and the highest scoring total energy decoy was used for further design. Another FastRelax protocol was performed with the inhibitor apixaban included in the complex, and similarly the highest scoring total energy decoy was used for further delta energy analysis.

[0080] Rosetta Design: Design of an empty apixaban binding pocket: Two rounds of sequence design were used. The goal of the first round was to identify mutations that were deleterious to activity or overexpression in human cells. The goal of the second round was to minimize protein destabilization through aggressive mutagenesis while maintaining factor Xa activity and inhibiting apixaban binding. The first round of design used the Rosetta design protocol, which designated all residues in the 8 Å surrounding apixaban as "cavity" (except for the catalytic triad required for factor Xa activity), encompassing residues in the binding pocket. The 19 best scoring delta "cavity" designs were identified and their sequence profiles analyzed. The sequence profiles were threaded into the factor Xa apixaban complex structure using the FastRelax protocol (using resfiles) to construct the final model. The final model contained the previously identified mutations, but in the presence of apixaban, to see if they could still fit into the filled binding pocket and to identify clashes with apixaban. Through manual inspection of the models, 21 designs were generated, with or without apixaban, either with or without reverting the mutation. These 21 designs were experimentally validated and tested. Based on the results from these designs, a second round of designs was performed.

[0081] The second round involved using experimental results and the binding pocket locations suggested by Rosetta to create conservative one and two point mutations. This conservative approach focused on introducing small changes, such as focusing on amino acids with smaller side chains, such as alanine and valine, or introducing bulkier side chains, such as tryptophan, compared to their corresponding wild-type amino acids. This conservative approach allowed the identification of one point mutation that could potentially result in a change in apixaban binding, resulting in a total of nine designs for the second round of design.

[0082] Homology-based design: Two proteins were used as homology models to identify potential mutations to include as substitutions starting at positions 90 (PDB numbering based on 2P16), 92, 163, and 173. The crystal structure of human factor VII (PDB ID: 1DAN) was superimposed onto that of human factor Xa (PDB ID: 2P16) and analyzed for differences in amino acid identity. The final insert identity was determined based on structural differences, based on proximity to the binding pocket, length, and insertion location. Human factor VII was selected based on overall structural similarity and previously unpublished NMR results showing no binding to apixaban. This resulted in seven designs during the first round of design using only human factor VII. After experimental expression was achieved, a conservative second round of homology design was selected. The length of the insertion from factor VII was reduced, allowing only one insertion (position 173). In addition, two more designs originating from factor X of Danio rerio (zebrafish) were included. This protein was also selected as a homology model for identification of potential mutations based on sequence similarity to human Factor Xa. The addition of this additional protein and decreasing the length of the Factor VII insert at position 173 resulted in three designs.

[0083] Expression of FX: All variants of recombinant human FX were expressed in human embryonic kidney 293 (HEK293) cells. The expression vector consisted of a CMV promoter, a prolactin signal sequence, and an N-terminal protein-A (PA) tag separated by a PreScission protease cleavage site. Transfection was performed using Lipofectamine 3000 (Invitrogen) with psPAX2, pMD2.G, and transfer plasmids according to the manufacturer's protocol. Undiluted virus was used to transduce HEK293 cells, and a population of GFP-positive cells was sorted using FACS (FACS Aria III cell sorter) 48 hours after transduction. These cells were expanded and finally seeded into adherent cell bioreactors (Cesco Bioengineering, Taiwan) for long-term growth and protein production.

[0084] Purification of FX: The cell supernatant containing PA-FX was centrifuged at 5,000 rpm for 10 min, filtered through a 0.22 μm membrane, and loaded onto an IgG FF column (Cytiva). The column was washed extensively with 20 mM sodium phosphate pH 7, then equilibrated with 25 mM HEPES pH 7.5, 250 mM NaCl, and 5% glycerol. PreScission protease was added to the column at approximately 400 μg per L of supernatant and incubated overnight at 4° C. The cleaved protein was eluted from the column by connecting the IgG column to an FPLC system equilibrated in the buffer used for tag cleavage. A GST column was connected downstream of the IgG to capture the PreScission protease, and the flow-through containing FX was collected. Purified recombinant FX was activated with RVV-X (Haematologic Technologies, Inc.), isolated by size-exclusion chromatography on a Superdex 75 Increase 10 / 300 GL column (Cytiva), and stored at −20°C in PBS containing 50% (v / v) glycerol.

[0085] Chromogenic FXa activity assay: FXa activity assay based on CS11(65)a chromogenic substrate (Biophen) was performed as described below. FXa samples with or without apixaban were diluted in Tris-BSA buffer (pH 7.4) containing 5 mM CaCl2 and mixed with substrate (final concentration [fc] 300 μM) in a 3:2 ratio and dynamically measured by absorbance (410 nm) in a Biotek Synergy H4 microplate reader at 37 °C. Substrate cleavage was calculated for each sample by taking the average rate over a time window between 1 and 2 min.

[0086] Thrombin generation assay: Procoagulant activity of FXa variants was assessed by thrombin generation assay in tissue factor (TF)-induced pooled normal human plasma (Affinity Biologicals Inc, Ontario, Canada) as previously described. FXa variant samples were serially diluted in Tris-BSA buffer (pH 7.4) and mixed with a remineralized plasma mixture containing plasma (50% vol / vol fc), apixaban, platelet substitute in the form of phosphatidylcholine, phosphatidylserine, and sphingomyelin phospholipid vesicles (4 μM fc; Phospholipid-TGT®, Rossix, Mölndal, Sweden), recombinant lipidated TF (5 pM fc; RecombiPlasTin®, Instrumentation Laboratory Company, Lexington, MA, USA), the fluorescent substrate for thrombin ZGGR-AMC (800 μM fc; Bachem, Torrance, CA, USA) and calcium chloride (12.5 mM) using a robotic 96-channel pipettor (ViaFlo 96; Integra Biosciences, Bedford, MA, USA). Fluorescence (product of thrombin substrate consumption) was recorded in kinetic mode every 45 seconds over a period of 1.7 hours using a Tecan Infinite F500 at 37° C. In-house software was used to calculate the calibrated thrombin generation assay (using an internal thrombin calibrator CAT® from Stago, Parsippany, NJ, USA) and calculate the thrombin peak height parameter.

[0087] Tail cut bleeding model: Animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Veterinary Services Division of the US FDA / CBER. Male CD-1 mice (Charles River Laboratories, Kingston, NY, USA) were 5–8 weeks old and weighed 28–34 g. Mice were anesthetized with a mixture of ketamine / xylazine and administered apixaban or sterile DMSO in saline (NaCl, control), followed by two retro-orbital injections of FXa preparations 5 min later. Five minutes later, a 3 mm section of the tail was cut, after which the tail was immersed in a tube with a defined volume of 0.9% NaCl at 37°C and allowed to bleed for 30 min under careful observation. Blood loss was determined by measuring hemoglobin content and expressed as μl per mouse.

[0088] Example 2 In vitro testing of FXa variants Designing functional FXa analogs with low affinity for DOACs provides an alternative antagonistic strategy. In this study, a novel computational approach (Figure 1A) was used to design functional FXa analogs with low affinity for apixaban, the most widely prescribed FXa inhibitor.

[0089] The first round of design used two strategies: (i) the Rosetta design protocol, which specified all residues within 8 Å around apixaban (except the catalytic triad required for factor Xa activity) as the "cavity", encompassing residues within the binding pocket. The 19 top scoring delta "cavity" designs were threaded with the FastRelax protocol (using resfiles) onto the FXa-apixaban complex structure to build the final model. The final model allowed for the identification of clashes with apixaban. After manual inspection of the models, 21 designs were selected. The second round of design involved the use of the experimental results from the first round of designs and focused on identifying single point mutations that could potentially provide the best balance between activity and resistance to apixaban. The second round resulted in nine low affinity FXa variants. (ii) The two proteins were used as homology models, and the inclusion of potential mutations was identified as insertions. The crystal structure of human factor VII (FVII) (PDB ID: 1DAN) was superimposed onto that of human FXa (PDB ID: 2P16) and analyzed for differences in amino acid identity. The final insert identity was based on proximity to the binding pocket and length. FVII was selected for mutational replacement based on the overall structural similarity and NMR results showing that apixaban does not bind to FVII (Figure 1H). Seven FXa variants were selected based on this strategy. Some of these designs failed experimental validation. In the next round of homology-based design, the inserts from factor VII were reduced in length and number. Additionally, amino acid substitutions originating from FX in Danio rerio (zebrafish) were included. Using sequences of two proteins homologous to human FXa that do not provide a binding pocket for apixaban and adjusting the number of substitutions resulted in three homology insertion designs.

[0090] The nine variants were expressed in HEK293T cells, purified, and demonstrated to be functional in the FXa substrate cleavage assay. The inhibition of FXa activity by apixaban (IC 50 ) is shown in Figure 2B. The best IC 50The variants with values ​​(showing minimal inhibition by apixaban) (variant 61 and variant 70) were selected for further characterization.

[0091] The two variants selected for further characterization encompassed a single point mutation variant W215A (variant 61) and an insertion variant H91-STYVPG-T98 (variant 70). Furthermore, Rosetta modeling of variants 61 and 70 in complex with apixaban showed that the altered region of the FXa binding pocket was destabilized, causing disruption of proper docking of apixaban into the active site and partial loss of binding.

[0092] Using thrombin generation assays, we showed that variants 61 and 70 were functional, although less potent than wild-type FXa (Figure 1F). Importantly, however, both variants 61 and 70 had IC values ​​∼350-fold and ∼70-fold higher against apixaban compared to wild-type FXa. 50 These data taken together suggest that at clinically relevant apixaban concentrations, these variant FXa molecules continue to exhibit procoagulant activity.

[0093] The activity and inhibitory concentrations were determined for a larger series of variants (Figures 2A and 2B). The activity of all variants was determined using the rate of chromogenic substrate cleavage in a pure buffer system at different FXa concentrations. The variants were assigned to five hierarchies (Figure 2A). The IC of all variants was 50 was determined using the rate of chromogenic substrate cleavage by a fixed amount of FXa variant in the presence of increasing concentrations of apixaban. The variants were expressed as IC 50 Based on the activity and IC value, five strata were assigned; <5nM (*), 5-50nM (**), 51-150nM (***), 151-250 (****), >251nM (*****). 50After balancing between values ​​(Table 2), we identified six potentially informative variants. The composite score assigned as potentially informative is *****, but no individual score can be less than **. Table 2. Variant activity and IC50 [Table 2-1] [Table 2-2]

[0094] Example 3 In vivo testing of FXa variants To demonstrate the potential in vivo utility of engineered FXa variants to circumvent the effects of apixaban, the activity of wild-type and variant FXa molecules in the presence of a fixed amount (1 μM) of apixaban was measured (Figure 3A). Although the variant FXa molecules were less potent than wild-type FXa, the apparent Km was 3.1 ± 0.4 nM for wild-type FXa, compared to 1105 ± 171 nM for variant 61 and 219 ± 14 nM for variant 70.

[0095] The design of the in vivo study in mice is depicted in Figure 3B. Briefly, either control buffer or apixaban was injected into the vein of mice, followed 5 minutes later by injection of control buffer, variant 61, variant 70, or wild-type FXa (all at 1 mg / kg). The tails of the mice were cut and bleeding was monitored by measuring bleeding time (Figure 3C) and blood loss (Figure 3D). Bleeding time was significantly longer in the group treated with apixaban compared to the control group. This observation is consistent with the mechanism of action of apixaban, i.e., inhibition of coagulation by binding to FXa. This also indicates that the mouse model we used reproduces the clinical challenge associated with the use of apixaban, that is, uncontrolled bleeding. Treatment of mice with either FX or FXa after treatment with apixaban did not significantly reduce bleeding time. However, treatment with either variant 61 or variant 70 significantly reduced bleeding time. Similar results demonstrating that variants 61 and 70 successfully antagonized the anticoagulant effect of apixaban were obtained when measuring blood loss rather than bleeding time (Figure 3D). All bleeding episodes were plotted with the length and number of bleeding episodes for each individual mouse (Figure 3E) to determine the bleeding profile.

[0096] As above, additional experiments were performed in a mouse model, but including andexanet alfa for comparison. The experimental strategy is depicted in Figure 4A. Using an apixaban dose of 4 mg / kg, sufficient to induce bleeding, we demonstrated effective antagonism of bleeding by variants #61 and #70. We observed a significant reduction in bleeding time or blood loss in animals treated with #61 and #70 (Figures 4B and 4C). Injection of wild-type FXa had no significant effect on bleeding time or blood loss. Andexanet alfa at doses of 5 mg / kg and 25 mg / kg showed no hemostatic effect (Andx25 mg / kg is roughly equivalent to a high-dose clinical dosing regimen of 1760 mg). The bleeding graph demonstrated that hemostasis mediated by variants #61 and #70 prevented blood loss through multiple interruptions in the outflow of blood from the tail cut (Figure 4D). Animals treated with apixaban (with or without andexanet alfa or wild-type FXa) experienced a largely uninterrupted outflow of blood (FIG. 4D).

[0097] Tissue factor pathway inhibitor (TFPI) plays a crucial role in controlling the procoagulant activity of tissue factor (TF). Andexanet alfa has been shown to reduce TFPI activity, and since TFPI is the major inhibitor of the TF-FVIIa complex, reducing its activity may lead to thrombus formation. TFPI activity was determined using the ACTICHROME® TFPI assay (BioMedica Diagnostics) according to the manufacturer's protocol. At physiological concentrations, both variants #61 and #70 showed a much smaller response to TFPI compared to andexanet alfa (Figure 5). Thus, these agents are considered to have a minimal risk of thrombus formation.

[0098] It will be apparent that the precise details of the methods or compositions described may be changed or modified without departing from the essence of the described embodiments of this disclosure, and we claim all such modifications and variations that come within the scope and spirit of the following claims.

Claims

1. A modified factor Xa polypeptide, In the Xa factor subsite S1, S4, or both, one or more amino acid substitutions are present. Enzymatic activity of at least 300% of unmodified factor Xa in the presence of apixaban, and IC for apixaban at least 50 times higher than that of unmodified factor Xa. ５０ Having a value, or both Modified Xa factor polypeptide.

2. The modified Xa factor polypeptide according to claim 1, wherein the unmodified Xa factor comprises the amino acid sequence of SEQ ID NO:

1.

3. The modified factor Xa polypeptide according to claim 1, wherein the modified factor Xa polypeptide does not contain the signal peptide and propeptide of factor Xa.

4. The modified Xa factor polypeptide according to claim 1, wherein the one or more of the above amino acid substitutions are selected from 312-317 NRFTKE→STYVPG, W439A, G450A, G440A, C415A+C443A, 312-317 NRFTKE→KNYQRD, and W439A+312-317 NRFTKE→STYVPG, and the amino acid numbering corresponds to SEQ ID NO:

1.

5. The modified factor Xa polypeptide according to claim 4, wherein the modified polypeptide has an amino acid sequence having at least 95% sequence identity with any one of sequence numbers 4, 2, 6, 8, 10, 12, and 14.

6. The modified factor Xa polypeptide according to claim 5, wherein the modified polypeptide comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 4, 2, 6, 8, 10, 12, and 14.

7. The modified factor Xa polypeptide according to claim 4, wherein the modified polypeptide has an amino acid sequence having at least 95% sequence identity with one of the amino acids 3 to 450 of sequence numbers 4, 2, 6, 8, 10, 12, and 14.

8. The modified factor Xa polypeptide according to claim 7, wherein the modified polypeptide comprises an amino acid sequence of 3 to 450 amino acids from any one of SEQ ID NOs: 4, 2, 6, 8, 10, 12, and 14.

9. A nucleic acid encoding the modified factor Xa polypeptide according to claim 1.

10. The nucleic acid according to claim 9, wherein the nucleic acid has a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 5, 3, 7, 9, 11, 13, and 15.

11. The nucleic acid according to claim 10, wherein the nucleic acid contains or consists of any one nucleotide sequence of sequence numbers 5, 3, 7, 9, 11, 13, and 15.

12. The nucleic acid according to claim 9, wherein the nucleic acid has a nucleotide sequence having at least 95% sequence identity with nucleotide 7 to 1350 of any one of sequence numbers 5, 3, 7, 9, 11, 13, and 15.

13. The nucleic acid according to claim 12, wherein the nucleic acid contains or consists of a nucleotide sequence of nucleotides 7 to 1350 of any one of sequence numbers 5, 3, 7, 9, 11, 13, and 15.

14. A vector comprising the nucleic acid according to any one of claims 9 to 13.

15. A host cell comprising the nucleic acid according to any one of claims 9 to 13 or a vector comprising the nucleic acid according to any one of claims 9 to 13.

16. A composition comprising a modified factor Xa polypeptide according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

17. A composition comprising a modified factor Xa polypeptide according to any one of claims 1 to 8, or a composition comprising a modified factor Xa polypeptide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier, for treating or inhibiting bleeding in a patient receiving direct oral anticoagulation therapy.

18. The composition according to claim 17, wherein the direct oral anticoagulant therapy is apixaban, rivaroxaban, edoxaban, or betrixaban.

19. The composition according to claim 18, wherein the direct oral anticoagulant therapy is apixaban.

20. The composition according to claim 17, characterized in that the modified factor Xa polypeptide or composition is administered intravenously.

21. The composition according to claim 17, characterized in that the modified factor Xa polypeptide or composition is administered in a dose of about 0.1 to 10 mg / kg.

22. The composition according to claim 17, wherein administration of the composition reduces bleeding time by at least 10% compared to a control, reduces blood loss by at least 10% compared to a control, or both.

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