WO2023143559A1 - Tfpi binding polypeptides and uses thereof - Google Patents

Tfpi binding polypeptides and uses thereof Download PDF

Info

Publication number
WO2023143559A1
WO2023143559A1 PCT/CN2023/073679 CN2023073679W WO2023143559A1 WO 2023143559 A1 WO2023143559 A1 WO 2023143559A1 CN 2023073679 W CN2023073679 W CN 2023073679W WO 2023143559 A1 WO2023143559 A1 WO 2023143559A1
Authority
WO
WIPO (PCT)
Prior art keywords
tfpi
polypeptide
binding
tcdb4
amino acid
Prior art date
Application number
PCT/CN2023/073679
Other languages
French (fr)
Inventor
Liang TAO
Yanyan LI
Jianhua Luo
Qi Yang
Original Assignee
Westlake University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Westlake University filed Critical Westlake University
Publication of WO2023143559A1 publication Critical patent/WO2023143559A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention generally relates to polypeptides that bind to TFPI, nucleic acids encoding the polypeptides, compositions comprising the polypeptides and uses thereof.
  • Tissue Factor Pathway Inhibitor-1 (referred to as TFPI) is a 43 kDa serine protease inhibitor comprising three Kunitz-type inhibitor domains. It regulates tissue factor-induced coagulation via factor Xa-dependent feedback inhibition of the tissue factor-factor VIIa complex.
  • Kunitz domain 1 of TFPI binds FVIIa and Kunitz domain 2 binds FXa, enabling the inhibitor to form a quaternary FXa-TFPI-FVIIa-TF complex that blocks activity of the TF/FVIIa extrinsic complex.
  • TFPI binding of FXa downregulates the common pathway of the coagulation cascade, during which FXa converts prothrombin to thrombin (Audu et al., Anesth. Analg., 103 (4) , 841-845 (2006) ) .
  • TFPI is a promising target for restoring thrombin generation. Severe bleeding disorders, such as hemophilia, result from disruption of the blood coagulation cascade. There is currently no cure for hemophilia and other clotting diseases. Factor replacement therapy is the most common treatment for blood coagulation disorders. However, blood clotting factors typically are cleared from the bloodstream shortly after administration. In addition, therapeutic efficacy of factor replacement therapy can diminish drastically upon formation of inhibitory antibodies. Approximately 30%of patients with severe hemophilia A develop inhibitory antibodies that neutralize Factor VIII (FVIII) (Peerlinck and Hermans, Haemophilia, 12, 579-590 (2006) ) . Few therapeutic options exist for patients with anti-Factor antibodies.
  • FVIII Factor VIII
  • compositions and methods for treating blood coagulation disorders addresses the need by providing an anti-TFPI polypeptide and methods and uses thereof.
  • isolated polypeptides that bind TFPI, wherein the isolated polypeptides are derived from TcdB (such as TcdB4 or TcdB2) .
  • the isolated polypeptides block TFPI’s inhibitory action on the blood coagulation cascade, thereby enhancing thrombin formation.
  • the isolated polypeptide of the invention binds TFPI-1 (e.g., TFPI-1 ⁇ ) and, optionally, improves TFPI-regulated thrombin generation in the absence of FVIII, FIX, and/or FXI.
  • a composition e.g., a pharmaceutical composition
  • TFPI-1 e.g., TFPI-1 ⁇
  • FIX FIX
  • FXI FXI-regulated thrombin generation in the absence of FVIII, FIX, and/or FXI.
  • (c) a variant of (a) or (b) that retains the binding to TFPI, wherein the amino acid sequence of the variant is at least 80%, at least 85%, at least 90%, at least 95%or at least 99%identical to the amino acid sequence of (a) or (b) .
  • the TcdB4 protein is a native TcdB4 protein comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 1.
  • the TcdB2 protein is a native TcdB2 protein comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 2.
  • the isolated polypeptide comprises:
  • (c) N-terminal and/or C-terminal truncated amino acid sequence of the amino acid sequence of (a) or (b) , for example, truncated by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) ; or
  • N-terminal and/or C-terminal extended amino acid sequence of the amino acid sequence of (a) or (b) for example, extended by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) at corresponding positions of SEQ ID NO: 1 or 2.
  • the isolated polypeptide comprises one or more of the following residues corresponding to SEQ ID NO: 1: E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 and Y1510.
  • the isolated polypeptide further comprises one or more of the following residues corresponding to SEQ ID NO: 1: L1434, K1435, M1438, K1597, S1598, L1599 and K1600 as shown in SEQ ID NO: 1.
  • the variant of the functional fragment comprises at least one mutation (e.g. substitution, insertion or deletion) compared to corresponding positions of SEQ ID NO: 3 or 4, in order to improve the binding to TFPI.
  • mutation e.g. substitution, insertion or deletion
  • the isolated polypeptide binds to TFPI with a dissociation constant of less than 10 ⁇ M, less than 1 ⁇ M, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM or less than 5 nM, as measured by bio-layer interferometry (BLI) assay.
  • a dissociation constant of less than 10 ⁇ M, less than 1 ⁇ M, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM or less than 5 nM, as measured by bio-layer interferometry (BLI) assay.
  • the isolated polypeptide specifically binds to the K2 domain but not K1 domain of TFPI.
  • the isolated polypeptide comprises:
  • (c) N-terminal and/or C-terminal truncated amino acid sequence of the amino acid sequence of (a) or (b) , for example, truncated by 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid.
  • the isolated polypeptide comprises at least one mutation (e.g. substitution, insertion or deletion) compared to corresponding wild-type sequence as set forth in SEQ ID NO: 1 or 2.
  • the isolated polypeptide as disclosed herein inhibits TFPI activity and binds to TFPI with a dissociation constant of less than 10 ⁇ M.
  • the isolated polypeptide as disclosed herein is operably linked to a moiety that enhances the half-life of the isolated polypeptide.
  • the isolated polypeptide is conjugated to a polyethylene glycol (PEG) moiety, human serum albumin (HSA) , an antibody or fragment thereof, hydroxyethyl starch, a multimer comprising proline, alanine, serine, or a combination thereof (PASylation) , or a C12-C18 fatty acid.
  • PEG polyethylene glycol
  • HSA human serum albumin
  • PASylation a multimer comprising proline, alanine, serine, or a combination thereof
  • fusion protein comprising the isolated polypeptide fused to a heterogenous polypeptide.
  • the heterogenous polypeptide is an IgG Fc portion, such as human IgG1, IgG2, IgG3 or IgG4 Fc potion, and optionally comprising a hinge region.
  • an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the isolated polypeptide or the fusion protein as disclosed herein.
  • a vector comprising the nucleic acid molecule as disclosed herein.
  • a host cell comprising the nucleic acid molecule or the vector as disclosed herein.
  • composition comprising the isolated polypeptide as disclosed herein and a pharmaceutically acceptable carrier.
  • a method of inhibiting TFPI activity comprising contacting the TFPI protein with an isolated polypeptide as described herein.
  • provided herein are methods of using the isolated polypeptide or the nucleic acid molecule encoding the isolated polypeptide of the invention, e.g. for treating TFPI related or TFPI mediated disorders, such as blood coagulation disorder.
  • provided herein is a method of enhancing thrombin formation in a subject in need thereof (such as a clotting factor-deficient subject) , comprising administering to the subject the isolated polypeptide as provided herein in an amount effective to enhance thrombin formation.
  • provided herein is a method for increasing blood clot formation in a subject in need thereof, and a method of treating a blood coagulation disorder in a subject in need thereof.
  • the methods comprise administering to the subject an isolated polypeptide as provided herein in an amount effective to enhance blood clot formation, or an amount effective to treat the blood coagulation disorder in the subject.
  • the method is performed after it has been determined that a subject is at risk for developing a blood coagulation disorder (e.g., a deficiency in a clotting factor (e.g., FVIII, FIX, or FXI) is detected) or after a blood coagulation disorder (e.g., hemophilia A, hemophilia B, or hemophilia C) is detected.
  • a blood coagulation disorder e.g., hemophilia A, hemophilia B, or hemophilia C
  • the polypeptide as disclosed herein is administered to protect, in whole or in part, against excessive blood loss during injury or surgery.
  • the isolated polypeptide or the nucleic acid molecule encoding the isolated polypeptide of the invention for use as a medicament. Also provided is the isolated polypeptide or the nucleic acid molecule encoding the isolated polypeptide of the invention for use in enhancing thrombin formation, increasing blood clot formation and/or treating a blood coagulation disorder in a subject in need thereof.
  • provided herein is use of the isolated polypeptide of the invention in the manufacture of a medicament for enhancing thrombin formation, increasing blood clot formation and/or treating a blood coagulation disorder in a subject in need thereof.
  • provided herein is a method for targeting a cell displaying TFPI.
  • provided herein is a method for treating or diagnosing a subject suffering from a disease or at risk of suffering from a TFPI related or TFPI mediated disease.
  • provided herein is a method of purifying TFPI.
  • a method of identifying a TFPI-binding compound is provided herein.
  • TFPI is a cellular receptor for TcdB4
  • TFPI K1+K2 protects the HeLa CSPG4 -/- cells from TcdB4 but not TcdB1 as measured by the cell rounding assay.
  • TcdB4 specifically binds to TFPI.
  • TFPI K1+K2 -Fc protects the HeLa CSPG4 -/- cells from TcdB4 but not TcdB1 as measured by the cell rounding assay.
  • TcdB4-TFPI complex Cryo-EM structure of the TcdB4-TFPI complex.
  • GTD glucosyltransferase domain
  • CPD cysteine protease domain
  • DRBD transmembrane delivery and receptor-binding domain
  • CROPs combined repetitive oligopeptides domain of TcdB4 are shown in wheat, pink, light blue, green, respectively.
  • the TFPI is bound at the periphery of TcdB4 through direct interactions between the Kunitz-2 domain (cyan) of TFPI and the DRBD of TcdB4.
  • TFPI K2 is anchored on a hydrophobic surface of TcdB4 DRBD through two epitopes: loop1 (residues 131-138) and loop2 (residues 155-162) .
  • TcdB contains two classes of RBIs recognizing either FZDs or TFPI.
  • TcdB4 1285-1834 Point mutations on TcdB4 1285-1834 were examined in pull-down assays, using TFPI K2 -Fc as bait. Bound TcdB4 1285-1834 mutants were co-precipitated with TFPI K2 -Fc using the Protein A resin and detected by immunoblot analysis.
  • TcdB2 binds to TFPI K2 .
  • TFPI refers to Tissue Factor Pathway Inhibitor, a multivalent Kunitz-type serine protease inhibitor that regulates tissue factor-induced coagulation via factor Xa-dependent feedback inhibition of the tissue factor–factor VIIa complex.
  • TFPI has two major isoforms, TFPI ⁇ and TFPI ⁇ , which are also produced by all mammals (Wood et al., 2014) .
  • TFPI ⁇ contains three tandem Kunitz-type protease inhibitory (Kunitz-1, Kunitz-2, and Kunitz-3, or K1, K2, and K3) domains followed by a basic carboxyterminal (C-terminal) region.
  • TFPI ⁇ lacks the K3 and basic C-terminal domains of TFPI ⁇ . Instead, it contains a C-terminal signal peptide that directs cleavage and attachment of a GPI anchor (Broze and Girard, 2012; Girard et al., 2012; Maroney and Mast, 2015; Zhang et al., 2003) .
  • An exemplary sequence of mouse TFPI ⁇ is shown in SEQ ID NO: 10.
  • Exemplary sequences of human TFPI ⁇ and TFPI ⁇ are shown in SEQ ID NO: 11 and 12, respectively.
  • TcdB or “Clostridium difficile toxin B” is an exotoxin produced by pathogenic C. difficile.
  • TcdB is a member of the large clostridial toxin (LCT) family, which enters target cells via receptor-mediated endocytosis and glucosylates small GTPase proteins, leading to cytoskeletal dysfunction and eventual cell death (Aktories et al., 2017; Chandrasekaran and Lacy, 2017; Voth and Ballard, 2005) .
  • TcdB has three major domains: a catalytically active N-terminal domain, a centrally located translocation domain and a C-terminal receptor binding domain.
  • TcdB has 4 variants, TcdB1, TcdB2, TcdB3 and TcdB4, among them TcdB2 and TcdB4 do not recognize Frizzled proteins (FZDs) but as characterized herein, recognize TFPI.
  • FZDs Frizzled proteins
  • specific binding or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as between TcdB4 and TFPI.
  • high affinity refers to an interaction having a dissociation constant (Kd) of 1 x 10 -7 M or less, more preferably 5 x 10 -8 M or less, even more preferably 1x10 -8 M or less, even more preferably 5 x 10 -9 M, e.g. as measured by bio-layer interferometry (BLI) assay.
  • Kd dissociation constant
  • the term “functional fragment” in relation to full length TcdB4 or TcdB2 refers to a fragment of wild-type TcdB4 or TcdB2 that retains the function of binding to TFPI.
  • the binding between the functional fragment and TFPI may be approximately lower, equivalent or higher compared to the binding between full-length wild-type TcdB4 or TcdB2 and TFPI, preferably the binding is higher compared to full-length wild-type TcdB2.
  • a functional fragment comprising a residue corresponding to E1433 of SEQ ID NO: 1 means the functional fragment, which is derived from SEQ ID NO: 1, comprises the Glu (E) amino acid residue at the position corresponding to 1433 of SEQ ID NO: 1.
  • the functional fragment may also comprise the same residues of SEQ ID NO: 1 that are close to E1433, e.g. comprise amino acids 1431-1606 of SEQ ID NO: 1.
  • the functional fragment comprising the E1433 amino acid residue may be mutated at positions close to E1433, e.g. the residue (s) close to E1433 in the functional fragment is/are different from those in SEQ ID NO: 1.
  • the residue (s) close to E1433 in the functional fragment is/are different from those in SEQ ID NO: 1.
  • a numbering of the amino acid is related, it refers to the numbering according to SEQ ID NO: 1 or SEQ ID NO: 2, depending on the context.
  • treating and “treatment” refers to any reduction in the severity and/or onset of symptoms associated with a TFPI related or TFPI mediated disorder, such as blood coagulation disorder. Accordingly, “treating” and “treatment” includes therapeutic and prophylactic measures.
  • a TFPI related or TFPI mediated disorder such as blood coagulation disorder.
  • treating and “treatment” includes therapeutic and prophylactic measures.
  • any degree of protection from, or amelioration of, a blood coagulation disorder or symptom associated therewith is beneficial to a subject, such as a human patient.
  • the quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and/or delaying the appearance of symptoms.
  • Polypeptides comprising functional fragments of TcdB or variants thereof
  • the polypeptides disclosed herein retain the binding to TFPI and/or have the capability to inhibit TFPI function (referred to as TFPI-inhibitory polypeptides) , wherein the polypeptides comprise or consist of functional fragments of TcdB (especially TcdB4) , or highly homologous sequences of the fragments.
  • the polypeptides may comprise the receptor-binding domain (DRBD) of TcdB2 or TcdB4.
  • the polypeptides comprise N-terminal truncated fragments of the receptor-binding domain (DRBD) of TcdB4, specifically, truncated by 1-400 amino acids, e.g. no more than 400 amino acids, no more than 350 amino acids, no more than 300 amino acids, no more than 250 amino acids, no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids or no more than 10 amino acids from the N-terminal of the receptor-binding domain (DRBD) of TcdB4 or TcdB2.
  • DRBD receptor-binding domain
  • the polypeptides comprise C-terminal truncated fragments of the receptor-binding domain (DRBD) of TcdB4, specifically, truncated by 1-400 amino acids, e.g. no more than 400 amino acids, no more than 350 amino acids, no more than 300 amino acids, no more than 250 amino acids, no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids or no more than 10 amino acids from the C-terminal of the receptor-binding domain (DRBD) of TcdB4 or TcdB2.
  • DRBD receptor-binding domain
  • the N-terminal and/or C-terminal truncated fragments as described above could retain the binding to TFPI or have the capability to inhibit TFPI function.
  • a polypeptide as disclosed herein comprises a functional fragment of TcdB2 that has improved binding to TFPI by including a S1496I substitution.
  • polypeptides may comprise:
  • TcdB4 protein as shown in SEQ ID NO: 1 or TcdB2 protein as shown in SEQ ID NO: 2 that retains the binding to TFPI;
  • a variant of the functional fragment that retains the binding to TFPI, wherein the amino acid sequence of the variant is at least 80%identical to the amino acid sequence of the functional fragment.
  • the functional fragment is derived from TcdB4 and comprises an amino acid sequence having at least 80%, at least 85%, or at least 90%identity to the amino acid sequence as shown in SEQ ID NO: 3 or 4.
  • the functional fragment comprises or consists of positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 1.
  • the functional fragment comprises or consists of N-terminal and/or C-terminal truncated amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 1, for example, truncated by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) .
  • the functional fragment comprises or consists of amino acid sequence from positions 842-1834 of SEQ ID NO: 1 or 2 (i.e. SEQ ID NO: 13 or 14, respectively) .
  • the functional fragment comprises or consists of N-terminal and/or C-terminal extended amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 1, for example, extended by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) .
  • the extended amino acids may be substantially the same with corresponding positions in SEQ ID NO: 1.
  • the functional fragment is derived from TcdB2 and comprises an amino acid sequence having at least 80%, at least 85%, or at least 90%identity to the amino acid sequence as shown in SEQ ID NO: 5 or 6.
  • the functional fragment comprises or consists of positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 2.
  • the functional fragment comprises or consists of N-terminal and/or C-terminal truncated amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 2, for example, truncated by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) .
  • the functional fragment comprises or consists of N-terminal and/or C-terminal extended amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 2, for example, extended by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid (s) .
  • the extended amino acids may be substantially the same with corresponding positions in SEQ ID NO: 2. Since the binding between TcdB2 and TFPI is lower than that of TcdB4 and TFPI, it is preferred one or more mutations are included in the functional fragment derived from TcdB2 to improve the binding affinity.
  • the polypeptide comprises a variant of the functional fragment derived from TcdB2, and the variant comprises a S1496I substitution (the numbering is according to SEQ ID NO: 2) .
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215: 403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402.
  • the default parameters of the respective programs can be used. See www. ncbi. nlm. nih. gov .
  • variant herein is meant a polypeptide comprising one or more amino acid substitutions, amino acid deletions, or amino acid additions to a parent amino acid sequence.
  • Variants include, but are not limited to, polypeptides having an amino acid sequence that is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to any of the amino acid sequences provided herein while retaining the ability to bind TFPI and/or inhibit TFPI activity.
  • the polypeptide comprises one or more of the following residues corresponding to SEQ ID NO: 1: E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 and Y1510.
  • the polypeptide may comprise a fragment of SEQ ID NO: 1 from positions 1433 to 1510.
  • the polypeptide further comprises one or more of the following residues corresponding to SEQ ID NO: 1: L1434, K1435, M1438, K1597, S1598, L1599 and K1600 as shown in SEQ ID NO: 1.
  • the polypeptide may comprise a fragment of SEQ ID NO: 1 from positions 1433 to 1600.
  • the functional fragment comprises or consists of SEQ ID NO: 7, 4 or 3 (corresponding to positions 1431-1606, 1285-1834, and 841-1834 of SEQ ID NO: 1, respectively) . In some embodiments, the functional fragment comprises or consists of SEQ ID NO: 6 or 5 (corresponding to positions 1285-1834, and 841-1834 of SEQ ID NO: 2, respectively) .
  • the polypeptides as disclosed herein comprise 20 amino acids or more, 30 amino acids or more, 40 amino acids or more, 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, 150 amino acids or more, 200 amino acids or more, 250 amino acids or more, 200 amino acids or more, 350 amino acids or more, 400 amino acids or more, 450 amino acids or more, 500 amino acids or more, 550 amino acids or more, 600 amino acids or more, 650 amino acids or more, 700 amino acids or more, 750 amino acids or more, or 800 amino acids or more of wildtype full length TcdB4 or TcdB2, preferably from the DRBD domain of TcdB4 or TcdB2.
  • the polypeptide comprises 100-300 amino acid residues (e.g., 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 26, 270, 280, 290, 300 amino acid residues) in the DRBD domain of TcdB4 or TcdB2.
  • the polypeptide comprises positions 1431-1606 in the DRBD domain, which are likely at the receptor-binding interface (RBI) of TcdB.
  • polypeptide described herein comprising one or more extensions or deletions is suitable in the context of the invention so long as the polypeptide binds TFPI and, optionally, blocks TFPI inhibition of the coagulation cascade.
  • the polypeptide of the invention comprises one or more amino acid substitutions (with reference to any of the amino acid sequences provided herein) that do not destroy (or even improve) the ability of the polypeptide to bind and/or inhibit TFPI.
  • Amino acid substitutions include, but are not limited to, those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on a polypeptide.
  • the substitution is a conservative substitution, wherein an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined within the art, and include amino acids with basic side chains (e.g., lysine, arginine, and histidine) , acidic side chains (e.g., aspartic acid and glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan) , beta-branched side chains (e.g., threonine, valine, and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine) .
  • basic side chains e.g., lysine, arginine,
  • TFPI-inhibitory polypeptides comprising atypical, non-naturally occurring amino acids, which are well known in the art.
  • non-naturally occurring amino acids include ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2, 4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, y-amino butyric acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, a fluoro-amino acid, a 3-methyl amino acid, ⁇ -C-methyl amino acid, a N-methyl amino acid, 2-amino-isobutyric acid, ⁇ -homoglutamatic acid, ⁇
  • the invention further includes polypeptide variants comprising one or more amino acids inserted within an amino acid sequence provided herein and/or attached to the N-terminus or C-terminus.
  • the polypeptide further comprises one or more amino acids that facilitate synthesis, handling, or use of the polypeptide, including, but not limited to, one or two lysines at the N-terminus and/or C-terminus to increase solubility of the polypeptide, or one or two methionine at the N-terminus and/or C-terminus, and it is expected such modifications are not to change the function of the polypeptide.
  • “Derivatives” are included in the invention and include TFPI-binding or TFPI-inhibitory polypeptides that have been chemically modified in some manner distinct from addition, deletion, or substitution of amino acids.
  • a polypeptide of the invention provided herein is chemically bonded with polymers, lipids, other organic moieties, and/or inorganic moieties. Examples of polypeptide and protein modifications are given in Hermanson, Bioconjugate Techniques, Academic Press, (1996) .
  • the TFPI-binding polypeptides described herein optionally comprise a functional group that facilitates conjugation to another moiety (e.g., a peptide moiety) .
  • Exemplary functional groups include, but are not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, epoxide, oxirane, carbonate, arylating agent, imidoester, carbodiimide, anhydride, alkyl halide derivatives (e.g., haloacetyl derivatives) , maleimide, aziridine, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents (e.g., pyridyl disulfides or TNB thiol) , diazoalkane, carboyldiimadazole, N, N′-Disuccinyl carbonate, N-Hydroxysuccinimidyl chloroformate, and hydrazine derivatives.
  • Maleimide is useful, for example, for generating a TFPI-bind
  • the invention includes TFPI-binding polypeptides covalently modified to include one or more water soluble polymer attachments.
  • a water soluble polymer (or other chemical moiety) is attached to any amino acid residue, although attachment to the N-or C-terminus is preferred in some embodiments.
  • a polymer is attached to the polypeptide via one or more amino acids or building blocks that offer functional groups that facilitate polymer attachment.
  • JBT2315 comprises a C-terminal cysteine (position X4021 with respect to formula (XI) ) , which facilitates the addition of, e.g., a maleimide polyethylene glycol (PEG) .
  • PEG polyethylene glycol
  • Useful polymers include, but are not limited to, PEG (e.g., PEG approximately 40 kD, 30 kD, 20 kD, 10, kD, 5 kD, or 1 kD in size) , polyoxyethylene glycol, polypropylene glycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch, cellulose, poly- (N-vinyl pyrrolidone) -polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polysialic acid (PSA) , polyoxyethylated polyols (e.g., glycerol) and poly
  • the polypeptide of the invention is a PEGylated polypeptide.
  • PEG moieties are available in different shapes, e.g., linear or branched.
  • water soluble polymer attachments see U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337.
  • moieties useful for improving polypeptide half life or stability include, for instance, albumin (optionally modified to allow conjugation to the inventive polypeptide) , fatty acid chains (e.g., C12-C18 fatty acid, such as a C14 fatty acid) , an antibody or fragment thereof (e.g., an Fc portion of an antibody) , and proline-alanine-serine multimers.
  • albumin optionally modified to allow conjugation to the inventive polypeptide
  • fatty acid chains e.g., C12-C18 fatty acid, such as a C14 fatty acid
  • an antibody or fragment thereof e.g., an Fc portion of an antibody
  • proline-alanine-serine multimers e.g., proline-alanine-serine multimers.
  • a polypeptide derivative in another aspect, includes a targeting moiety specific for a particular cell type, tissue, and/or organ.
  • the polypeptide is linked to one or more chemical moieties that facilitate purification, detection, multimerization, binding with an interaction partner, and characterization of polypeptide activity.
  • An exemplary chemical moiety is biotin.
  • Other moieties suitable for conjugation to the TFPI-binding polypeptide of the invention include, but are not limited to, a photosensitizer, a dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex, an enzyme, a toxin, and a cytotoxic agent.
  • Photosensitizers include, e.g., Photofrin, Visudyne, Levulan, Foscan, Metvix, Cysview TM , Laserphyrin, Antrin, Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200 ALA, and Amphinex. If desired, a His tag, a FLAG tag, a strep tag, or a myc tag is conjugated to the polypeptide.
  • polypeptides of the invention are acylated at the N-terminal amino acid of the polypeptide.
  • polypeptides of the invention are amidated at the C-terminal amino acid of the polypeptide.
  • polypeptides of the invention are acylated at the N-terminal amino acid of the polypeptide and are amidated at the C-terminal amino acid of the polypeptide.
  • Derivatives also include peptides comprising modified or non-proteinogenic amino acids or a modified linker group (see, e.g., Grant, Synthetic Peptides: A User's Guide, Oxford University Press (1992) ) .
  • Modified amino acids include, for example, amino acids wherein the amino and/or carboxyl group is replaced by another group.
  • Non-limiting examples include modified amino acids incorporating thioamides, ureas, thioureas, acylhydrazides, esters, olefines, sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acid amides, benzodiazepines and other aromatic or non-aromatic heterocycles (see Estiarte et al., Burgers Medicinal Chemistry, 6th edition, Volume 1, Part 4, John Wiley &Sons, New York (2002) ) .
  • Modified amino acids are often connected to the peptide with at least one of the above mentioned functional groups instead of an amide bond.
  • Non-proteinogenic amino acids include, but are not limited, to ⁇ -alanine (Bal) , norvaline (Nva) , norleucine (Nle) , 4-aminobutyric acid ( ⁇ -Abu) , 2-aminoisobutyric acid (Aib) , 6-aminohexanoic acid ( ⁇ -Ahx) , ornithine (Orn) , hydroxyproline (Hyp) , taurine, sarcosine, citrulline (Cit) , cysteic acid (Coh) , cyclohexylalanine (Cha) , methioninesulfoxide (Meo) , methioninesulfone (Moo) , homoserinemethylester (Hsm) , propargylglycine (Eag) , 5-fluorotryptophan (5Fw) , 6-fluorotryptophan (6Fw) , 3′, 4′-dimethoxy
  • non-proteinogenic amino acids are provided in Table 2. These and other non-proteinogenic amino acids may exist as D-or L-isomers.
  • modified linkers include, but are not limited to, the flexible linker 4, 7, 10-trioxa-1, 13-tridecanediamine (Ttds) , glycine, 6-aminohexanoic acid, beta-alanine (Bal) , pentynoic acid (Pyn) , and combinations of Ttds, glycine, 6-aminohexanoic acid and Bal.
  • Suitable fusion proteins include, but are not limited to, proteins comprising the TFPI-inhibitory polypeptide linked to one or more polypeptides, polypeptide fragments, or amino acids not generally recognized to be part of the protein sequence.
  • a fusion polypeptide comprises the entire amino acid sequences of two or more polypeptides or, alternatively, comprises portions (fragments) of two or more polypeptides.
  • a fusion protein optionally includes all or part of any suitable polypeptide comprising a desired biological activity/function.
  • the TFPI-inhibitory polypeptide is operably linked to, for instance, one or more of the following: a polypeptide with long circulating half life, a marker protein, a polypeptide that facilitates purification of the TFPI-inhibitory polypeptide, a polypeptide sequence that promotes formation of multimeric proteins, or a fragment of any of the foregoing.
  • Suitable fusion partners include, but are not limited to, a His tag, a FLAG tag, a strep tag, and a myc tag.
  • the TFPI-inhibitor polypeptide is fused to one or more entities that enhance the half life of the polypeptide.
  • Half life can be increased by, e.g., increasing the molecular weight of the TFPI-binding polypeptide to avoid renal clearance and/or incorporating a ligand for the nFc receptor-mediated recycling pathway.
  • the TFPI-binding polypeptide is fused to or chemically conjugated to (as described further below) an albumin polypeptide or a fragment thereof (e.g., human serum albumin (HSA) or bovine serum albumin (BSA) ) .
  • the albumin fragment comprises 10%, 25%, 50%, or 75%of the full length albumin protein.
  • the TFPI-binding polypeptide comprises an albumin binding domain or fatty acid that binds albumin when administered in vivo.
  • suitable fusion partners include, but are not limited to, a proline-alanine-serine multimer (PASylation) and an antibody or fragment thereof (e.g., an Fc portion of an antibody) .
  • two or more TFPI-inhibitory peptides are fused together, linked by a multimerization domain, or attached via chemical linkage to generate a TFPI-inhibitory polypeptide complex.
  • the TFPI-inhibitor peptides may be the same or different.
  • TFPI-binding polypeptides of the invention can be made in a variety of ways.
  • the polypeptides are synthesized by solid phase synthesis techniques including those described in Merrifield, J. Am. Chem. Soc., 85, 2149 (1963) ; Davis et al., Biochem. Intl., 10, 394-414 (1985) ; Larsen et al., J. Am. Chem. Soc., 115, 6247 (1993) ; Smith et al., J. Peptide Protein Res., 44, 183 (1994) ; O'Donnell et al., J. Am. Chem.
  • the TFPI-binding polypeptide (e.g., the TFPI-inhibitory polypeptide) is expressed recombinantly by introducing a nucleic acid encoding the TFPI-binding polypeptide into host cells, which are cultured to express the polypeptide.
  • Such polypeptides can be purified from the cell culture using standard protein purification techniques.
  • the invention also encompasses a nucleic acid comprising a nucleic acid sequence encoding the TFPI-binding polypeptide of the invention.
  • Methods of preparing DNA and/or RNA molecules are well known in the art.
  • a DNA/RNA molecule encoding the TFPI-binding polypeptide provided herein is generated using chemical synthesis techniques and/or using polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the TFPI-binding polypeptide coding sequence is incorporated into an expression vector.
  • any of a number of expression vectors known in the art are suitable in the context of the invention, such as, but not limited to, plasmids, plasmid-liposome complexes, and viral vectors.
  • nucleic acid is operably linked to one or more regulatory sequences, such as a promoter, activator, enhancer, cap signal, polyadenylation signal, or other signal involved with the control of transcription or translation.
  • any of the the TFPI-binding polypeptide of the invention or nucleic acids encoding the polypeptides also is provided in a composition (e.g., a pharmaceutical composition) .
  • the polypeptide is formulated with a physiologically-acceptable (i.e., pharmacologically-acceptable) carrier, buffer, excipient, or diluent, as described further herein.
  • the polypeptide is in the form of a physiologically acceptable salt, which is encompassed by the invention.
  • physiologically acceptable salts means any salts that are pharmaceutically acceptable. Some examples of appropriate salts include acetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate.
  • the composition comprises one or more additional pharmaceutically-effective agents.
  • the poly peptide provided herein optionally inhibits at least one TFPI-1 (e.g., TFPI-1 ⁇ or TFPI-1 ⁇ ) activity such as, but not limited to, an activity that downregulates the blood coagulation cascade.
  • TFPI-1 e.g., TFPI-1 ⁇ or TFPI-1 ⁇
  • a proposed mechanism of inhibition may involve preventing formation of the quaternary TF-FVIIA-FXA-TFPI complex.
  • the polypeptide may inhibit binding (competitively or allosterically) of TFPI to FXa (e.g., inhibit binding of TFPI Kunitz domain 2 to Factor Xa or interrupt binding of TFPI Kunitz domain 1 to an exosite of Factor Xa) , the TF/FVIIa complex (e.g., inhibit binding of TFPI Kunitz domain 1 to the TF/FVIIa complex) , TF alone, and/or FVIIa alone.
  • TFPI activity diminished, TF and FVIIa are free to activate FX which, in turn, enhances conversion of prothrombin to thrombin.
  • the polypeptide of the invention that binds Kunitz domain 1 interferes with TFPI-mediated inhibition of FXa.
  • the invention provides a method of, e.g., inhibiting TFPI-mediated downregulation of the extrinsic and/or common pathway of the coagulation cascade and/or enhancing FXa-mediated conversion of prothrombin to thrombin, by administering to a subject the polypeptide described herein that binds Kunitz domain 1.
  • the polypeptide of the invention exhibits TFPI antagonistic activity in model and/or plasmatic systems.
  • An exemplary model system for determining TFPI-inhibitory activity is the extrinsic tenase assay, which tests the ability of candidate polypeptides to restore extrinsic complex-mediated FX activation in the presence of TFPI (which is a natural inhibitor of the FX activation reaction) (see, e.g., Lindhout et al., Thromb. Haemost., 74, 910-915 (1995) ) .
  • FXa inhibition assay Another model system for characterizing TFPI-inhibitory activity is the FXa inhibition assay, wherein FXa activity is measured in the presence of TFPI (see Sprecher et al., PNAS, 91, 3353-3357 (1994) ) .
  • the polypeptide of the invention enhances FX activation in the presence of TFPI with a half maximal effective concentration (EC50) of less than or equal to 1 ⁇ 10 -4 M, less than or equal to 1 ⁇ 10 -5 M, less than or equal to 1 ⁇ 10 -6 M, or less than or equal to 1 ⁇ 10 -7 M.
  • EC50 half maximal effective concentration
  • TFPI-antagonist activity is characterized in a plasma-based assay.
  • Thrombin formation is triggered in plasma substantially lacking FVIII or FIX activity (e.g., the residual coagulation factor activity is lower than 1%) in the presence of a candidate polypeptide.
  • Thrombin formation can be detected using a fluorogenic or chromogenic substrate.
  • a system for measuring thrombin activity is provided by Thrombinoscope BV (Maastricht, The Netherlands) .
  • Prothrombin conversion is measured using, e.g., a Thrombograph TM (Thermo Scientific, Waltham, Mass.
  • the TFPI-inhibitory polypeptide increases the amount of peak thrombin generated during the assay and/or decreases the time required to achieve peak thrombin formation.
  • the polypeptide enhances thrombin formation in the absence of Factor VIII to at least about 2%, at least about 3%, at least about 5%, at least about 7%, or at least about 10%of the level of thrombin formation in normal plasma, i.e., in the presence of physiological levels of Factor VIII.
  • the polypeptide is administered to an animal model of thrombin deficiency or hemophilia to characterize TFPI inhibitory activity in vivo.
  • animal model of thrombin deficiency or hemophilia to characterize TFPI inhibitory activity in vivo.
  • in vivo models include for example, mice administered anti-FVIII antibodies to induce hemophilia A (Tranholm et al., Blood, 102, 3615-3620 (2003) ) ; coagulation factor knock-out models such as, but not limited to, FVIII knock-out mice (Bi et al., Nat.
  • polypeptides bind TFPI from any source including, but not limited to, mouse, rat, rabbit, dog, cat, cow, horse, pig, guinea pig, and primate.
  • the polypeptide binds human TFPI.
  • the polypeptide binds TFPI from more than one species (i.e., the polypeptide is cross-reactive among multiple species) .
  • the polypeptide binds TFPI with a dissociation constant (KD) of less than or equal to 1 ⁇ 10 -4 M, less than or equal to 1 ⁇ 10 -5 M, less than or equal to 1 ⁇ 10 -6 M, or less than or equal to 1 ⁇ 10 -7 M.
  • KD dissociation constant
  • Affinity may be determined using, for example and without limitation, any one, two, or more of a variety of techniques, such as affinity ELISA assay, a competitive ELISA assay, and/or surface plasmon resonance (BIAcore TM ) assay.
  • IC50 surface plasmon resonance
  • the polypeptide of the invention optionally demonstrates an IC50 of less than or equal to about 50,000 nM.
  • the polypeptide demonstrates an IC50 of less than or equal to about 10,000 nM, such as an IC50 of less than or equal to about 5,000 nM, less than or equal to about 1,000 nM, or less than or equal to about 500 nM.
  • the polypeptide demonstrates an IC50 of less than or equal to about 250 nM, less than or equal to about 100 nM, less than or equal to about 50 nM, or less than or equal to about 10 nM.
  • Affinity may also be determined by a kinetic method or an equilibrium/solution method. Such methods are described in further detail herein or known in the art.
  • Another suitable assay for characterizing the inventive polypeptides is a koff assay, which examines a peptide's release from TFPI.
  • the koff assay result is not the dissociation rate constant, but a percentage of competitor peptide blocked from TFPI binding by a test peptide after an incubation period with TFPI.
  • An exemplary koff assay includes the following steps: 1) incubation of a TFPI-coated microtiter plate with an amount of test peptide resulting in approximately 90%TFPI occupation; 2) removal of unbound test peptide; 3) addition of a biotinylated tracer (i.e., competitor) peptide that competes with the test peptide for binding to TFPI; 4) incubation for a period of time during which binding sites released by the test peptide is occupied by the tracer; 5) removal of unbound tracer and test peptide; and 6) detection of bound tracer by a chromogenic reaction using streptavidin-horseradish peroxidase conjugate. The resulting signal is indicative of binding sites freed by the test peptide.
  • a test peptide that does not dissociate from TFPI during the incubation period yields a weaker signal compared to an analyte that dissociates completely.
  • the term “specifically binds” refers to the ability of a polypeptide to bind TFPI with greater affinity than it binds to an unrelated control protein that is not TFPI.
  • the polypeptide may bind to TFPI with an affinity that is at least, 5, 10, 15, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for a control protein.
  • the polypeptide binds TFPI with greater affinity than it binds to an “anti-target, ” a protein or other naturally occurring substance in humans to which binding of the polypeptide might lead to adverse effects.
  • an “anti-target, ” a protein or other naturally occurring substance in humans to which binding of the polypeptide might lead to adverse effects.
  • TFPI-inhibitory polypeptides exert their activity in the blood stream and/or at the endothelium, plasma proteins represent potential anti-targets.
  • Proteins containing Kunitz domains (KDs) are potential anti-targets because KDs of different proteins share a significant similarity.
  • the TFPI-binding polypeptide demonstrates one or more desired characteristics described herein, and the amino acid sequence of a polypeptide can be modified to optimize binding, stability, and/or activity, if desired.
  • An exemplary TFPI-binding polypeptide binds TFPI with a KD of less than or equal to 20 nM and/or exhibits a binding affinity for TFPI that is at least 100 times greater than the binding affinity for an anti-target.
  • the TFPI-binding polypeptide enhances FX activation in the presence of TFPI with an EC50 (as measured using any suitable assay, such as the assays described here) of less than or equal to 50 nM and/or enhances thrombin formation in the absence of Factor VIII to at least about 20% (e.g., 40%) of the level of thrombin formation in plasma containing physiological levels of Factor VIII.
  • the TFPI-binding polypeptide achieves a desired level of plasma stability (e.g., 50%or more of a dose remains in plasma after 12 hours) and/or demonstrates a desired half life in vivo (e.g., at least two, three, four, five, six, seven, eight, nine, or ten hours) .
  • the TFPI-binding polypeptide exhibits a desired level of bioavailability, such as a desired level of bioavailability following subcutaneous administration (e.g., greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, or 50%) and/or demonstrates a desired level of TFPI-inhibitory activity at a given dose in vivo.
  • TFPI-binding polypeptides are used to detect and/or quantify TFPI in vivo or in vitro.
  • An exemplary method of detecting and/or quantifying TFPI in a sample comprises (a) contacting a sample with a TFPI-binding polypeptide of the invention, and (b) detecting binding of the TFPI-binding polypeptide to TFPI.
  • the invention further includes a method for targeting biological structures (including, but not limited to, cell surfaces and endothelial lining) where TFPI is located.
  • the method comprises contacting the biological structure (e.g., including, without limitation, a cell displaying TFPI on the cell surface) with a TFPI-binding polypeptide described herein, optionally conjugated to a moiety that adds additional functionality to the polypeptide.
  • the moiety can be a dye (such as a fluorescence dye) , a radionuclide or a radionuclide-containing complex, a protein (e.g., an enzyme, a toxin, or an antibody) or a cytotoxic agent.
  • the polypeptide is linked or conjugated to an effector moiety that facilitates polypeptide detection and/or purification and/or comprises therapeutic properties.
  • the TFPI-binding polypeptide or polypeptide conjugate is administered to a mammal to target a TFPI-displaying cell within the mammal.
  • the method further comprises detecting binding of the TFPI-binding polypeptide to TFPI. The method is useful for therapy and diagnosis of disease where TFPI is a suitable diagnostic marker or TFPI-expressing cells are a target for a therapeutic approach.
  • Polypeptide-TFPI complexes are directly or indirectly detected.
  • Detection moieties are widely used in the art to identify biological substances and include, for example, dye (e.g., fluorescent dye) , radionuclides and radionuclide-containing complexes, and enzymes.
  • polypeptide-TFPI binding is detected indirectly.
  • the polypeptide is optionally contacted with an interaction partner that binds the polypeptide of invention without significantly interfering with polypeptide-TFPI binding, and the interaction partner is detected.
  • Exemplary interaction partners include, but are not limited to, antibodies, antigen-binding antibody fragments, anticalins and antibody mimetics, aptamers, streptavidin, avidin, neutravidin, and aptamers.
  • the interaction partner comprises a detection moiety to facilitate detection of an interaction partner-peptide complex.
  • the TFPI-binding polypeptide is, in some embodiments, modified to facilitate binding of an interaction partner.
  • the TFPI-binding polypeptide is conjugated to biotin, which is bound by an interaction partner comprising streptavidin.
  • An exemplary interaction partner comprises strepavidin fused to horseradish peroxidase, which is detected in, e.g., an ELISA-like assay.
  • the TFPI-binding polypeptide is modified to include an antibody epitope, and binding of the corresponding antibody to the polypeptide-TFPI complex is detected. Methods of detecting, e.g., antibodies and fragments thereof, are well understood in the art.
  • Polypeptide-TFPI complexes and interaction partner-peptide complexes are identified using any of a number of methods, such as, but not limited to, biochemical assays (e.g., enzymatic assays) , spectroscopy (e.g., detection based on optical density, fluorescence, FRET, BRET, TR-FRET, fluorescence polarization, electrochemoluminescence, or NMR) , positron emission tomography (PET) , and single Photon Emission Computed Tomography (SPECT) .
  • biochemical assays e.g., enzymatic assays
  • spectroscopy e.g., detection based on optical density, fluorescence, FRET, BRET, TR-FRET, fluorescence polarization, electrochemoluminescence, or NMR
  • PET positron emission tomography
  • SPECT single Photon Emission Computed Tomography
  • Detectable moieties that facilitate fluorescence detection of polypeptide-TFPI complexes or interaction partner-peptide complexes include, but are not limited to, fluorescein, Alexa 350, Marina Blue TM , Cascade Yellow TM , Alexa 405, Pacific Blue TM , Pacific Orange TM , Alexa 430, Alexa 488, Oregon 488, Alexa 500, Oregon 514, Alexa 514, Alexa 532, Alexa 555, Tetramethylrhodamine, Alexa 546, Rhodamine B, Rhodamine Red TM -X, Alexa 568, Alexa 594, Texas Texas Alexa 610, Alexa 633, Alexa 635, Alexa 647, Alexa 660, Alexa 680, Alexa 700, Alexa 750, B-Phycoerythrin, R-Phycoerythrin, Allophycocyanin, Cy3, Cy5, TAMRA, and fluorescent proteins (GFP and derivatives thereof) .
  • TFPI-binding polypeptide comprising a fluorescent detection moiety
  • JBT2454 FAM-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 4171)
  • JBT2454 FAM-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 4171)
  • 5 6-carboxyfluoresceine is labeled with 5, 6-carboxyfluoresceine.
  • Radioactive labels also are used to detect biological materials (e.g., TFPI, TFPI-binding polypeptides, or TFPI-binding polypeptide-TFPI complexes) , and, in some instances, are attached to polypeptides or interaction partners using a chelator, such as (but not limited to) EDTA (ethylene diamine tetra-acetic acid) , DTPA (diethylene triamine pentaacetic acid) , CDTA (cyclohexyl 1, 2-diamine tetra-acetic acid) , EGTA (ethyleneglycol-O, O′-bis (2-aminoethyl) -N, N, N′, N′-tetra-acetic) , HBED (N, N-bis (hydroxybenzyl) -ethylenediamine-N, N′-diacetic acid) , TTHA (triethylene tetramine hexa-acetic acid) , DOTA (1, 4, 7, 10-te
  • radioactive labels examples include ⁇ 99m>Tc, ⁇ 203>Pb, ⁇ 66>Ga, ⁇ 67>Ga, ⁇ 68>Ga, ⁇ 72>As, ⁇ 111>In, ⁇ 113m>In, ⁇ 97>Ru, ⁇ 62>Cu, ⁇ 64>Cu, ⁇ 52>Fe, ⁇ 52m>Mn, ⁇ 51>Cr, ⁇ 186>Re, ⁇ 188>Re, ⁇ 77>As, ⁇ 90>Y, ⁇ 67>Cu, ⁇ 169>Er, ⁇ 117m>Sn, ⁇ 121>Sn, ⁇ 127>Te, ⁇ 142>Pr, ⁇ 143>Pr, ⁇ 198>Au, ⁇ 199>Au, ⁇ 149>Tb, ⁇ 161>Tb, ⁇ 109>Pd, ⁇ 165>Dy, ⁇ 149>Pm, ⁇ 151>Pm, ⁇ 153>Sm, ⁇ 157>Gd, ⁇ 166>Ho, ⁇ 172
  • Paramagnetic metals also are detectable moieties that are suitable for attachment to TFPI-binding polypeptides or interaction partners, optionally via chelator complex.
  • Examples of paramagnetic metals include, for example, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho, and Er.
  • the TFPI-binding polypeptides themselves, are, in some aspects, modified to include one or more amino acids with detectable substituents or nuclides.
  • the TFPI-binding polypeptide comprises at least one amino acid comprising a detectable isotope (e.g., 13C, 14C, 35S, 3H, 18O or 15N) , and/or an amino acid that is halogenated with, e.g., ⁇ 123>I, ⁇ 124>I, ⁇ 125>I, ⁇ 131>I, ⁇ 75>Br, ⁇ 76>Br, ⁇ 77>Br or ⁇ 82>Br.
  • Amino acids suitable for halogenation include, but are not limited to, tyrosine and tryptophan.
  • the invention also provides a method for diagnosing a subject suffering from a disease or disorder, or at risk of suffering from a disease or disorder, wherein the disease or disorder is associated with or caused by aberrant TFPI activity.
  • the method comprises administering to the subject the TFPI-binding polypeptide and detecting the TFPI-peptide complex.
  • the polypeptide is conjugated to a detectable moiety, and the method comprises detecting the detectable moiety. Exemplary detectable moieties are described herein.
  • the method comprises administering to the subject a TFPI-binding polypeptide interaction partner that binds the TFPI-binding polypeptide, and detecting the interaction partner.
  • the interaction partner comprises or is conjugated to a detectable moiety, and the detectable moiety is detected.
  • the presence of the detectable moiety indicates the presence of TFPI, thereby allowing diagnosis of a disease or disorder associated with TFPI (e.g., a disease or disorder which (i) can be treated by inhibiting TFPI or (ii) comprises symptoms which can be ameliorated or prevented by inhibiting TFPI) .
  • a disease or disorder associated with TFPI e.g., a disease or disorder which (i) can be treated by inhibiting TFPI or (ii) comprises symptoms which can be ameliorated or prevented by inhibiting TFPI
  • a biological sample is obtained from the subject, contacted with the TFPI-binding polypeptide as described herein, and TFPI-peptide complexes are detected.
  • the polypeptides of the invention bind TFPI and, therefore, are useful for purifying TFPI or recombinant TFPI from a biological sample (e.g., a biological fluid, such as serum) , fermentation extract, tissue preparations, culture medium, and the like.
  • a biological sample e.g., a biological fluid, such as serum
  • the invention includes methods of using the TFPI-binding polypeptide in the commercial production of TFPI or in a method of characterizing TFPI molecules.
  • the invention includes a method of purifying TFPI. The method comprises contacting a sample containing TFPI with a polypeptide as defined herein under conditions appropriate to form a complex between TFPI and the polypeptide; removing the complex from the sample; and, optionally, dissociating the complex to release TFPI.
  • the polypeptide is immobilized to a support, e.g., a solid support, to facilitate recovery of TFPI.
  • a support e.g., a solid support
  • the polypeptide is immobilized to chromatography stationary phase (e.g., silica, affinity chromatography beads, or chromatography resins) , a sample comprising TFPI is applied to the stationary phase such that TFPI-peptide complexes are formed, the remainder of the sample is removed from the stationary phase, and TFPI is eluted from the stationary phase.
  • the polypeptides of the invention are, in one aspect, suitable for use in affinity chromatography techniques.
  • a method of enhancing thrombin formation in a clotting factor-deficient subject also comprises administering to the subject a polypeptide provided herein under conditions effective to inhibit TFPI.
  • the TFPI-inhibitory polypeptide is administered in an amount and under conditions effective to enhance thrombin formation in the subject.
  • clotting factor-deficient is meant that the subject suffers from a deficiency in one or more blood factors required for thrombin formation, such as FVIII, FIX, or FXI. Indeed, in one embodiment, the subject is deficient in FVIII. Alternatively or in addition, the subject is deficient in Factor IX. Clotting factor deficiencies are identified by examining the amount of factor in a clinical sample.
  • Practitioners classify hemophilia according to the magnitude of clotting factor deficiency. Subjects suffering from mild hemophilia have approximately 5%to 30%of the normal amount (1 U/ml) of Factor VIII or Factor IX. Moderate hemophilia is characterized by approximately 1%to 5%of normal Factor VIII, Factor IX, or Factor XI levels, while subjects suffering from severe hemophilia have less than 1%of the normal amount of Factor VIII, Factor IX, or Factor XI. Deficiencies can be identified indirectly by activated partial thromboplastin time (APTT) testing.
  • APTT activated partial thromboplastin time
  • APTT testing measures the length of time required for a blood clot to form, which is longer for patients with Factor VIII Deficiency (hemophilia A) , Factor IX Deficiency (hemophilia B) , and Factor XI Deficiency (hemophilia C) compared to patients with normal clotting factor levels. Almost 100%of patients with severe and moderate Factor VIII deficiency can be diagnosed with an APTT.
  • the invention further includes enhancing thrombin formation in a subject that does not suffer from a clotting factor deficiency.
  • the method comprises administering to a subject (e.g., a subject comprising normal, physiological levels of clotting factor) a polypeptide provided herein under conditions effective to enhance thrombin formation.
  • the TFPI-inhibitory polypeptide is used for increasing blood clot formation in a subject.
  • the method of increasing blood clot formation comprises administering to the subject a polypeptide described herein in an amount and under conditions effective to increase blood clot formation. It will be appreciated that the method need not completely restore the coagulation cascade to achieve a beneficial (e.g., therapeutic) effect. Any enhancement or increase in thrombin or blood clot formation that reduces the onset or severity of symptoms associated with clotting factor deficiencies is contemplated. Methods of determining the efficacy of the method in promoting thrombin formation and blood clotting are known in the art and described herein.
  • the invention further includes a method of treating a blood coagulation disorder in a subject, the method comprising administering to the subject one or more TFPI-inhibitory polypeptides, such as any one or more of the polypeptides described herein, in an amount and under conditions effective to treat the blood coagulation disorder in the subject.
  • the polypeptide is a recombinant or synthetic polypeptide that inhibits TFPI activity.
  • Coagulation disorders include bleeding disorders caused by deficient blood coagulation factor activity and deficient platelet activity.
  • Blood coagulation factors include, but are not limited to, Factor V (FV) , FVII, FVIII, FIX, FX, FXI, FXIII, FII (responsible for hypoprothrombinemia) , and von Willebrand's factor.
  • Factor V Factor V
  • FVII Factor VIII
  • FIX Factor I
  • FXI Factor II
  • FXIII Factor VIII
  • FII von Willebrand's factor.
  • Factor deficiencies are caused by, for instance, a shortened in vivo-half life of the factor, altered binding properties of the factor, genetic defects of the factor, and a reduced plasma concentration of the factor.
  • Coagulation disorders can be congenital or acquired.
  • Coagulation disorders also stem from development of inhibitors or autoimmunity (e.g., antibodies) against clotting factors.
  • the coagulation disorder is hemophilia A.
  • the coagulation disorder is hemophilia B or hemophilia C.
  • Platelet disorders are caused by deficient platelet function or abnormally low platelet number in circulation. Low platelet count may be due to, for instance, underproduction, platelet sequestration, or uncontrolled patent destruction.
  • Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other drug therapy, radiation therapy, surgery, accidental blood loss, and other disease conditions.
  • Exemplary disease conditions that involve thrombocytopenia are: aplastic anemia; idiopathic or immune thrombocytopenia (ITP) , including idiopathic thrombocytopenic purpura associated with breast cancer; HIV-associated ITP and HIV-related thrombotic thrombocytopenic purpura; metastatic tumors which result in thrombocytopenia; systemic lupus erythematosus, including neonatal lupus syndrome splenomegaly; Fanconi's syndrome; vitamin B12 deficiency; folic acid deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronic liver disease; myelodysplastic syndrome associated with thrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute profound thrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmune thrombocytopenia, including maternal alloimmune thrombocytopenia
  • Platelet disorders also include, but are not limited to, Von Willebrand Disease, paraneoplastic platelet dysfunction, Glanzman's thrombasthenia, and Bernard-Soulier disease.
  • Additional bleeding disorders amenable to treatment with a TFPI-inhibitory polypeptide include, but are not limited to, hemorrhagic conditions induced by trauma; a deficiency in one or more contact factors, such as FXI, FXII, prekallikrein, and high molecular weight kininogen (HMWK) ; vitamin K deficiency; a fibrinogen disorder, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia; and alpha2-antiplasmin deficiency.
  • contact factors such as FXI, FXII, prekallikrein, and high molecular weight kininogen (HMWK)
  • HMWK high molecular weight kininogen
  • fibrinogen disorder including afibrin
  • the TFPI-inhibitory polypeptide is used to treat excessive bleeding, such as excessive bleeding caused by surgery, trauma, intracerebral hemorrhage, liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, hypothermia, menstruation, pregnancy, and Dengue hemorrhagic fever. All of the above are considered “blood coagulation disorders” in the context of the disclosure.
  • the TFPI-inhibitory polypeptide of the invention is used to reverse the effects (in whole or in part) of one or more anticoagulants in a subject.
  • anticoagulants include, for instance, heparin; coumarin derivatives, such as warfarin or dicumarol; TFPI; AT III; lupus anticoagulant; nematode anticoagulant polypeptide (NAPc2) ; FVIIa inhibitors; active-site blocked FVIIa (FVIIai) ; active-site blocked FIXa (FIXai) ; FIXa inhibitors; FXa inhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906) ; active-site blocked FXa (FXai) ; inhibitors of FVa or FVIIIa, including activated protein C (APC) and soluble thrombomodulin; thrombin inhibitors, including
  • the invention provides a polypeptide for use in a method for the treatment of a subject, such as a method for the treatment of a disease where the inhibition of TFPI is beneficial.
  • the disease or disorder is a blood coagulation disorder.
  • the subject is suffering from a disease or disorder or is at risk from suffering from a disease or disorder (or adverse biological event, such as excessive blood loss) .
  • the method comprises administering to the subject the polypeptide of the invention in an amount and under conditions effective to treat or prevent, in whole or in part, the disease or disorder.
  • the invention further provides a polypeptide for use in the manufacture of a medicament.
  • the polypeptide can be used in the manufacture of a medicament for the treatment of a blood coagulation disorder, as described in detail herein.
  • nucleic acid comprising a nucleic acid sequence encoding a TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) of the invention.
  • a nucleic acid in one aspect, is provided instead of, or in addition to, a TFPI-inhibitory polypeptide.
  • Expression vectors, nucleic acid regulatory sequences, administration methods, and the like, are further described herein and in U.S. Patent Publication No. 20030045498.
  • a particular administration regimen for a particular subject will depend, in part, upon the TFPI-inhibitory polypeptide of the invention used, the amount of TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) administered, the route of administration, the particular ailment being treated, considerations relevant to the recipient, and the cause and extent of any side effects.
  • the amount of polypeptide administered to a subject e.g., a mammal, such as a human
  • the conditions of administration e.g., timing of administration, route of administration, dosage regimen
  • the method comprises administering, e.g., from about 0.1 ⁇ g/kg to about 100 mg/kg or more, depending on the factors mentioned above.
  • the dosage may range from 1 ⁇ g/kg up to about 75 mg/kg; or 5 ⁇ g/kg up to about 50 mg/kg; or 10 ⁇ g/kg up to about 20 mg/kg.
  • the dose comprises about 0.5 mg/kg to about 20 mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of polypeptide.
  • the TFPI-inhibitory polypeptide is administered to treat an acute condition (e.g., bleeding caused by surgery or trauma, or factor inhibitor/autoimmune episodes in subjects receiving coagulation replacement therapy) for a relatively short treatment period, e.g., one to 14 days.
  • an acute condition e.g., bleeding caused by surgery or trauma, or factor inhibitor/autoimmune episodes in subjects receiving coagulation replacement therapy
  • a relatively short treatment period e.g., one to 14 days.
  • a physiologically-acceptable composition such as a pharmaceutical composition comprising a polypeptide described herein
  • a pharmaceutical composition comprising a polypeptide described herein
  • a particular route can provide a more immediate and more effective reaction than another route.
  • a pharmaceutical composition is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation.
  • a composition comprising a TFPI-inhibitory polypeptide is administered intravenously, intraarterially, or intraperitoneally to introduce the polypeptide of the invention into circulation.
  • Non-intravenous administration also is appropriate, particularly with respect to low molecular weight therapeutics.
  • a pharmaceutical composition comprising the TFPI-inhibitory polypeptide orally, topically, sublingually, vaginally, rectally, pulmonary; through injection by intracerebral (intra-parenchymal) , intracerebroventricular, intramuscular, intra-ocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, or enteral means; by sustained release systems; or by implantation devices.
  • the TFPI-inhibitory polypeptide is administered regionally via intraarterial or intravenous administration feeding a region of interest, e.g., via the femoral artery for delivery to the leg.
  • the polypeptide is incorporated into a microparticle as described in, for example, U.S. Pat. Nos. 5,439,686 and 5,498,421, and U.S.
  • composition is administered via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated.
  • the device in one aspect is implanted into any suitable tissue, and delivery of the desired molecule is in various aspects via diffusion, timed-release bolus, or continuous administration.
  • the TFPI-inhibitory polypeptide is administered directly to exposed tissue during surgical procedures or treatment of injury, or is administered via transfusion of blood procedures.
  • Therapeutic delivery approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,399,363.
  • the TFPI-binding polypeptide in one embodiment is formulated into a physiologically-acceptable composition
  • a carrier i.e., vehicle, adjuvant, buffer, or diluent
  • the particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the polypeptide, and by the route of administration.
  • Physiologically-acceptable carriers are well known in the art.
  • Illustrative pharmaceutical forms suitable for injectable use include without limitation sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat.
  • a pharmaceutical composition comprising a polypeptide provided herein is optionally placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions.
  • such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents that may be necessary to reconstitute the pharmaceutical composition.
  • the TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) of the invention is administered in combination with other substances and/or other therapeutic modalities to achieve an additional or augmented biological effect.
  • Co-treatments include, but are not limited to, plasma-derived or recombinant coagulation factors, hemophilia prophylaxis treatments, immunosuppressants, plasma factor-inhibiting antibody antagonists (i.e., anti-inhibitors) , antifibrinolytics, antibiotics, hormone therapy, anti-inflammatory agents (e.g., Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or steroidal anti-inflammatory substances) , procoagulants, and pain relievers.
  • NSAIDs Non-Steroidal Anti-Inflammatory Drugs
  • the method is an adjunct therapy to traditional replacement factor treatment regimens involving administration of, e.g., FXIII, FXII, FXI (e.g., (Laboratoire francais du Fraction réelle et des Biotechnologies, Les Ulis, France) and FXI concentrate (BioProducts Laboratory, Elstree, Hertfordshire, UK) ) , FX, FIX (e.g., Coagulation Factor IX (Wyeth, Madison, N.J. ) ; SD (Grifols, Los Angeles, Calif. ) ; (CSL Behring, King of Prussia, Pa. ) ; BEBULIN-VH TM (Baxter, Deerfield, Ill.
  • the subject also receives FEIBA VH Immuno TM (Baxter BioScience, Vienna, Austria) , which is a freeze-dried sterile human plasma fraction with Factor VIII inhibitor bypassing activity.
  • FEIBA VH Immuno TM contains approximately equal units of Factor VIII inhibitor bypassing activity and Prothrombin Complex Factors (Factors II, VII, IX, and X and protein C) .
  • Other exemplary co-treatments include, but are not limited to, prekallikrein, high molecular weight kininogen (HMWK) , Von Willebrand's factor, Tissue Factor, and thrombin.
  • the TFPI-inhibitory polypeptide is co-formulated with one or more different TFPI-inhibitory polypeptides.
  • administration of the TFPI-binding polypeptide allows a reduction in the dose of co-therapeutic required to achieve a desired biological response.
  • the invention thus includes administering to a subject a TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) of the invention (or multiple TFPI-inhibitory polypeptides) , in combination with one or more additionally suitable substances (s) , each being administered according to a regimen suitable for that medicament.
  • Administration strategies include concurrent administration (i.e., substantially simultaneous administration) and non-concurrent administration (i.e., administration at different times, in any order, whether overlapping or not) of the TFPI-inhibitory polypeptide and one or more additionally suitable agents (s) . It will be appreciated that different components are optionally administered in the same or in separate compositions, and by the same or different routes of administration.
  • the polypeptide of the invention is conjugated to a moiety, e.g., a therapeutic or diagnostic moiety, such as the detection moieties and co-treatments described above.
  • a moiety e.g., a therapeutic or diagnostic moiety, such as the detection moieties and co-treatments described above.
  • the polypeptide is administered in combination with an interaction partner (e.g., an antibody, antibody fragment, anticalin, aptamer, or spiegelmer) that (a) binds the polypeptide and (b) is therapeutically active and/or is linked to a moiety that provides additional functionality to the interaction partner (e.g., a therapeutic, diagnostic, or detection agent) .
  • an interaction partner e.g., an antibody, antibody fragment, anticalin, aptamer, or aptamer
  • Suitable moieties include, but are not limited to, photosensitizers, dyes, radionuclides, radionuclide-containing complexes, enzymes, toxins, antibodies, antibody fragments, and cytotoxic agents, and, in some instances, the moiety possesses therapeutic activity (i.e., achieves an advantageous or desired biological effect) .
  • the polypeptide conjugates or polypeptide-interaction partner pair is suitable for use in any of the methods described herein, such as methods of treating a subject suffering from a disease or disorder or at risk of suffering from a disease or disorder.
  • Binding of a test compound to the TFPI binding site defined herein is detected using any of a number methods, including the detection methods described herein.
  • An exemplary method for detecting binding employs nuclear magnetic resonance (NMR) to recognize chemical shifts at amino acid residues within the TFPI binding site. Chemical shifts at TFPI amino acid positions 28-30, 32, 46, 47, and 55, and optionally positions 27, 31, 36-38, and 44, denotes interaction of the test compound with these amino acid contact points on TFPI.
  • NMR data obtained from the KD1-test compound complex is compared to NMR data obtained from free KD1 polypeptide. Use of NMR to detect binding between a test compound and TFPI KD1 is further described in the Examples.
  • binding of a test compound to the TFPI-binding site defined herein is determined indirectly by detecting alterations in the ability of TFPI KD1 to interact with its natural binding partners, e.g., FVIIa or FXa.
  • the method comprises contacting the polypeptide comprising TFPI KD1 with FVIIa in the presence of the test compound under conditions that allow binding of KD1 to FVIIa, and KD1-FVIIa binding is compared with KD1-FVIIa binding in the absence of the test compound.
  • the method comprises contacting the polypeptide comprising TFPI KD1 with FXa in the presence of the test compound under conditions that allow binding of KD1 to FXa, and comparing KD1-FXa binding in the presence of the test compound with KD1-FXa binding in the absence of the test compound.
  • the polypeptide comprising KD1 also comprises KD2, and the method comprises contacting the polypeptide with FXa in the presence of a test compound under conditions that allow binding of KD2 to FXa, and KD2-FXa binding is compared with KD2-FXa binding in the absence of the test compound.
  • a decrease in KD1-FVIIa binding, KD1-FXa binding, or KD2-FXa binding in the presence of the test compound indicates that the test compound is a TFPI-binding compound.
  • the method optionally comprises contacting KD1 and/or KD2 to FVIIa and/or FXa in the absence of the test compound as a reference for comparing binding in the presence of the test compound.
  • KD binding to FVIIa or FXa is determined and/or quantified using any suitable method for detecting protein-protein interactions, such as the methods described herein using detectable labels. Binding of the test compound to the TFPI binding site is, alternatively, detected using an enzymatic assay.
  • FVIIa or FXa enzymatic activity is a suitable surrogate for evaluating binding of the proteins to TFPI KD1 or KD2; test compounds that bind the TFPI-binding site defined herein inhibit TFPI activity, resulting in increased FVIIa and FXa activity.
  • Enzymatic assays for evaluating FVIIa or FXa activity are described in detail herein.
  • the invention further includes compounds identified as TFPI-binding compounds in the methods of the invention, as well as compositions comprising one or more identified compounds.
  • Methods for isolating or purifying a compound, such as TFPI-binding compound (e.g., a TFPI-binding polypeptide) identified as described herein are known in the art and described above.
  • TFPI-binding compounds identified as described herein are TFPI inhibitors that downregulate or ablate one or more TFPI activities.
  • the invention includes a method for purifying a compound that inhibits FXa activity.
  • the method comprises contacting a polypeptide comprising TFPI KD1 with a compound under conditions that allow formation of compound-KD1 complexes, removing unbound compound, and dissociating the compound-KD1 complexes to release the compound, which binds TFPI.
  • a TFPI inhibitor identified and/or purified as described herein for the manufacture of a medicament such as a medicament for treating a blood coagulation disorder, is provided, as well as a method for treating a subject suffering from a disease or at risk of suffering from a disease comprising administering the TFPI inhibitor to the subject.
  • a method of inhibiting human TFPI comprises contacting human TFPI with an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55.
  • Another aspect of the invention includes a method for treating a subject suffering from a disease or at risk of suffering from a disease. The method comprises administering to the subject an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55.
  • the human TFPI binding site is defined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55, such as a binding site defined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55.
  • Any inhibitor that contacts the TFPI binding site defined herein and inhibits (downregulates or ablates) one or more TFPI activity is suitable for use in the context of the method.
  • the TFPI inhibitor is, optionally, a TFPI-binding polypeptide, such as a TFPI-binding polypeptide having the characteristics described herein.
  • SEQ ID NO: 1 full-length TcdB4 protein from Clostridioides difficile strain 8864
  • SEQ ID NO: 2 full-length TcdB2 protein from Clostridioides difficile strain R20291
  • SEQ ID NO: 7 (receptor-binding interface of TcdB4, residues 1431-1606 of SEQ ID NO: 1) :
  • SEQ ID NO: 12 (human TFPI ⁇ ) :
  • SEQ ID NO: 15 (K2 domain of human TFPI)
  • mice Male and female, 6-8 weeks were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd. (Shanghai, China) .
  • the Tfpi ⁇ KO mice were generated in the Laboratory Animal Resources Center of Westlake University. Mice were housed with food and water without limitation and monitored under the care of full-time staff.
  • DNA fragments encoding TcdB4 1-841 , TcdB4 842-1834 , TcdB4 1801-2367 , TcdB4 1285-1834 , and TcdB2 1285-1834 were PCR amplified and cloned into a pET28a vector with a HA-His tag introduced to their C-terminus.
  • Genes encoding human TFPI ⁇ , TFPI ⁇ , TFPI K1 -GPI, TFPI K2 -GPI, TFPI2 K1 -GPI, TFPI2 K2 -GPI, AMBP K3 -GPI, and mouse Tfpi ⁇ were codon-optimized, synthesized by Genscript (Nanjing, China) , and cloned into a PLVX-IRES-Cherry vector.
  • DNA fragments encoding TFPI K1+K2 , TFPI K1 , TFPI K2 , Tfpi K1 , Tfpi K2 , CSPG4 R1 , and TFPI K2 -CSPG4 R1 were PCR amplified and cloned into a pCAG or PHLsec vector with a FLAG, Fc-FLAG, Fc-His, or GFP-His tag fused to their C-terminus.
  • TcdB4 1-841 , TcdB4 842-1834 , TcdB4 1801-2367 , TcdB4 1285-1834 , and TcdB2 1285-1834 were expressed in E. coli BL21 (DE3) and purified as His-tagged proteins.
  • TFPI ⁇ -GFP Recombinant TFPI ⁇ -GFP, TFPI K1+K2 -GFP, TFPI K1+K2 TFPI K1+K2 -Fc, TFPI K1 -Fc, TFPI K2 -Fc, Tfpi K1 -Fc, Tfpi K2 -Fc, CSPG4 R1 , and TFPI K2 -CSPG4 R1 were expressed in 293F cells and purified as His or FLAG-tagged proteins.
  • HeLa CRISPR/Cas9 genome-wide KO library was generated as previously described (Tao et al., 2019; Tao et al., 2016) .
  • the GeCKO v2 library is composed of two sub-libraries (A and B) and contains six gRNAs targeting each gene.
  • 293T cells were used to package the lentiviruses. 48 hours post-transfection, the supernatant of the 293T culture was collected.
  • the HeLa-Cas9 cells were then transduced with the lentiviral library at a multiplicity of infection (MOI) of 0.3 and selected with 2.5 ug/mL puromycin for 4 days.
  • MOI multiplicity of infection
  • each CRISPR sub-library at least 6.7 ⁇ 10 7 cells were plated onto 15-cm cell culture dishes to ensure sufficient gRNA coverage.
  • the cell library was then added with TcdB4 of indicated concentrations and cultured for 18 hours.
  • the plates were then washed with phosphate buffer saline (PBS) to remove loosely attached cells.
  • PBS phosphate buffer saline
  • the remaining cells were cultured with the toxin-free medium and allowed to grow to ⁇ 70%confluence and subjected to the next round of screen.
  • Three rounds of screens were performed with increasing concentrations of TcdB4 (0.045, 0.15, and 0.45 pM, respectively) .
  • Cells from the final round of the screen were collected, and their genomic DNA was extracted using the Blood and Cell Culture DNA mini kit (Qiagen) .
  • DNA fragments containing the gRNA sequences were amplified by PCR using primers lentiGP1_F (SEQ ID NO: 8 AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG) and lentiGP-3_R (SEQ ID NO: 9 ATGAATACTGCCATTTGTCTCAAGATCTAGTTACGC) .
  • NGS Next-Generation Sequencing
  • Pull-down assays were performed using Protein A agarose beads (Thermo Fisher Scientific) . Briefly, Fc-tagged TFPI domains were mixed with full-length TcdB4 or TcdB4 fragments of indicated concentrations in 1 ml of PBS. The mixtures were incubated at 4°C for 30 minutes and co-precipitated by Protein A agarose beads. Beads were washed, pelleted, boiled in SDS sample buffer, and subjected to SDS-PAGE or immunoblot analysis.
  • the binding affinities between recombinant TcdB4 and human/mouse TFPI domains were measured by BLI assay using the Octet RED96 system (ForteBio) . All proteins were diluted in PBS.
  • Fc-tagged human/mouse TFPI domains (10 ⁇ g/mL) were immobilized onto Dip and Read Anti-human IgG-Fc biosensors (ForteBio) and balanced with PBS. The biosensors were then exposed to full-length TcdB4, TcdB4 1285-1834 , or TcdB2 1285-1834 , followed by washing (dissociation) with PBS. Binding affinities (K d ) were calculated using the Data Analysis software (ForteBio) .
  • TFPI K2 -Fc (10 ⁇ g /mL) was immobilized onto Dip and Read Anti-human IgG-Fc biosensors and balanced with HNBSACa buffer (50mM HEPES, 100mM NaCl, 5mM CaCl2, 0.1%BSA, pH 7.3) .
  • HNBSACa buffer 50mM HEPES, 100mM NaCl, 5mM CaCl2, 0.1%BSA, pH 7.3
  • the loaded biosensors were first exposed to 100 nM FXa, balanced again with HNBSACa, and then exposed to 300 nM TcdB4 1285-1834 .
  • the loaded biosensors were first exposed to 300 nM TcdB4 1285-1834 , balanced with HNBSACa, and then exposed to 100 nM FXa. All biosensors were then washed with HNBSACa.
  • Example 1 Genome-wide CRISPR-Cas9 screen identifies TFPI as a factor for TcdB4
  • a genome-wide CRISPR-Cas9 screen was conducted in HeLa cells.
  • cells were transduced with a genome-wide gRNA library (GeCKO v2) targeting 19, 052 human genes (Sanjana et al., 2014) using lentiviruses and subjected to three rounds of selection with increasing concentration of TcdB4 ( Figure 1A) .
  • the gRNAs sequences from the surviving cell population were decoded by next-generation sequencing (NGS) .
  • NGS next-generation sequencing
  • the identified genes were assessed based on fold-enrichment of gRNA reads, NGS reads per gene, and the number of unique gRNAs.
  • TFPI is known to regulate the tissue factor-dependent pathway of blood coagulation and mainly exists on the cell membrane and in the extracellular space (Broze and Girard, 2012; Wood et al., 2014) .
  • TFPI has two major isoforms, TFPI ⁇ and TFPI ⁇ .
  • TFPI ⁇ contains three tandem Kunitz-type protease inhibitory (K1, K2, and K3) domains followed by a basic carboxyterminal (C-terminal) region.
  • TFPI ⁇ lacks the K3 and basic C-terminal domains of TFPI ⁇ , and instead contains a C-terminal signal peptide that directs cleavage and attachment of a GPI anchor ( Figure 2A) .
  • TFPI ⁇ is a GPI-anchored protein, which is in line with the screen result that multiple GPI anchor biosynthesis genes were targeted. Because TcdB4 also binds CSPG4 (yet inefficiently) for cellular entry and CSPG4 is highly expressed in the HeLa cells (Gupta et al., 2017; Tao et al., 2016) , HeLa CSPG4 -/- cells were utilized to minimize the effect of CSPG4 when studying the roles of TFPI in HeLa cells.
  • TcdB4 interacts with the Kunitz-2 domain of TFPI
  • TcdB4 interacts with the Kunitz-1 and Kunitz-2 domains of TFPI (TFPI K1+K2 ) .
  • TFPI K1+K2 recombinant TFPI K1+K2 fused with human Fc fragment
  • Figure 3A recombinant TFPI K1+K2 fused with human Fc fragment
  • Figure 3B and 4A recombinant TFPI K1+K2 fused with human Fc fragment
  • TFPI K1+K2 effectively protected the cells from TcdB4 but failed to alleviate the intoxication of TcdB1 ( Figure 3B and 4A) , supporting the idea that TcdB4 specifically binds TFPI K1+K2 .
  • TcdB4 but not TcdB1 was bound to the recombinant TFPI K1+K2 -Fc and co-precipitated by Protein A beads ( Figure 4B) .
  • Kunitz domains are small, disulfide-rich, and ⁇ / ⁇ fold structural domains that function as protease inhibitors (Ascenzi et al., 2003) . Kunitz domains can be found in many proteins including TFPI, TFPI2, Alpha-1-Microglobulin/Bikunin Precursor (AMBP) , and Amyloid Precursor Protein (APP) .
  • ABP Alpha-1-Microglobulin/Bikunin Precursor
  • APP Amyloid Precursor Protein
  • TcdB4 binds to TFPI K1 or TFPI K2 and the binding selectivity.
  • GPI-anchored TFPI K1 , TFPI K2 , TFPI2 K1 , or AMBP K3 in the HeLa CSPG4 -/- /TFPI -/- cells and then tested their sensitivities to TcdB4.
  • the result showed that transient transfection of only GPI-anchored TFPI K2 could restore the susceptibility of CSPG4 -/- /TFPI -/- cells ( Figure 3D) , suggesting TcdB4 specifically recognizes the Kunitz-2 domain of TFPI.
  • TFPI K2 -Fc, but not TFPI K1 -Fc protected the HeLa CSPG4 -/- cells in the competition experiment ( Figure 4C) .
  • TcdB2-TFPI interaction reference sequence from R20291, an ST01/RT027 strain
  • TcdB2 and TcdB2 1285-1834 bound to Fc-tagged mouse Tfpi K2 and human TFPI K2 with respective K d of ⁇ 0.2 and 0.4 ⁇ M ( Figure 12B-12D) .
  • TcdB ( ⁇ 270 kDa) is one of the largest bacterial toxins composed of four functional domains, including a glucosyltransferase domain (GTD) , a cysteine protease domain (CPD) , a transmembrane delivery and receptor-binding domain (DRBD) , and a C-terminal combined repetitive oligopeptides (CROPs) domain ( Figure 6A) .
  • GTD glucosyltransferase domain
  • CPD cysteine protease domain
  • DRBD transmembrane delivery and receptor-binding domain
  • CROPs C-terminal combined repetitive oligopeptides
  • TcdB4 The overall architecture of TcdB4 is similar to the previously determined structure of TcdB (Chen et al., 2019; Simeon et al., 2019) .
  • the CPD and GTD form an integrated region while the CROPs domain module curve around the core region by forming like a big hook ( Figure 6B) .
  • the Kunitz-2 domain of TFPI is bound to the convex edge of the DRBD (residues 841-1834) largely through two flexible loops in TFPI: loop-1 (residues 131-138) and loop-2 (residues 155-162) (Figure 6B and 6C) .
  • Each loop engages TcdB4 via an extensive network composed of hydrogen bonds and hydrophobic interactions (Figure 6D) .
  • the interaction between TcdB4 DRBD and TFPI K2 was further validated by the pulldown assay (Figure 10A) .
  • Class I and II The phylogenetic tree is clustered into two major branches (denoted as Class I and II) .
  • Class I RBIs derive from TcdB1, TcdB3, and TcdB5, which prefer to bind FZDs.
  • Class II RBIs were composed of sequences from TcdB2, TcdB4, TcdB6, and TcdB7 (Figure 11A) , mainly existing in clade 2 C. difficile ( Figure 11B) .
  • Albeit FZD2 and TFPI are structurally very different, they both engage the same hydrophobic interface located on the convex edge of TcdB RBI ( Figure 11C) .
  • residues including but not limited to L1434, M1438, L1494, and Y1510 (positions in TcdB4; residues in TcdB1 shift to the left by one position in the alignment) , are shared by Class I and Class II RBIs and contribute to both FZD and TFPI interactions.
  • the interaction is bridged by a palmitoleic acid (PAM) with its tail protruding into a hydrophobic pocket in TcdB1.
  • PAM palmitoleic acid
  • TcdB4-TFPI complex the side chain of TFPI R135 is deeply embedded in the same pocket but forms multiple hydrogen bonds with adjacent residues including E1433, D1467, and E1469 from TcdB4 ( Figure 11D) .
  • F1597 in TcdB1 is a critical residue stabilizing the middle part of PAM and interacts with the nearby F130 in FZD2 (Chen et al., 2018; Peng et al., 2019) , while a Phe to Ser substitution mimicking TcdB2 and TcdB4 abolishes FZD binding (Henkel et al., 2020) .
  • TcdB4 forms a close hydrogen bond with t he nearby R140 in TFPI ( Figure 11E) .
  • an F1598S substitution for TcdB may impair FZD-binding but contribute to TFPI-binding.
  • Phe is conserved in all Class I RBIs while Ser is conserved in all Class II RBIs at this position.
  • TcdB2 The potential TFPI-binding interface in TcdB2 resembles TcdB4 ( Figure 11F) , providing a structural clue that TcdB2 may also recognize TFPI.
  • the HeLa CSPG4 -/- /TFPI -/- cells are more resistant to TcdB2 than its parental CSPG4 -/- cells ( Figure 11G) , while its susceptibility to TcdB2 could be restored by ectopic expressing a GPI-anchored Tfpi K2 ( Figure 11H) .
  • Tfpi K2 -Fc effectively protected the HeLa CSPG4 -/- cells from TcdB2 intoxication ( Figure 11I and 12A) .
  • Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature 538, 350-355.10.1038/nature19799

Abstract

The invention provides polypeptides that bind Tissue Factor Pathway Inhibitor (TFPI), including TFPI-inhibitory polypeptides, and compositions thereof. The polypeptides may be used to inhibit a TFPI, enhance thrombin formation in a clotting factor-deficient subject, increase blood clot formation in a subject, treat a blood coagulation disorder in a subject, purify TFPI, and identify a TFPI-binding compound, among others.

Description

TFPI binding polypeptides and uses thereof
CROSS-REFERENCE
This application claims priority to International Patent Application No. PCT/CN2022/075170, filed on January 30, 2022. The entire contents of the application are incorporated herein by reference.
Sequence Listing
The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.
Field
The invention generally relates to polypeptides that bind to TFPI, nucleic acids encoding the polypeptides, compositions comprising the polypeptides and uses thereof.
Background
Tissue Factor Pathway Inhibitor-1 (referred to as TFPI) is a 43 kDa serine protease inhibitor comprising three Kunitz-type inhibitor domains. It regulates tissue factor-induced coagulation via factor Xa-dependent feedback inhibition of the tissue factor-factor VIIa complex. Kunitz domain 1 of TFPI binds FVIIa and Kunitz domain 2 binds FXa, enabling the inhibitor to form a quaternary FXa-TFPI-FVIIa-TF complex that blocks activity of the TF/FVIIa extrinsic complex. TFPI binding of FXa downregulates the common pathway of the coagulation cascade, during which FXa converts prothrombin to thrombin (Audu et al., Anesth. Analg., 103 (4) , 841-845 (2006) ) .
TFPI is primarily produced by endothelial cells, megakaryocytes, monocytes, and smooth muscle cells (Maroney and Mast, 2015; Wood et al., 2014) . In human intestines, TFPI is highly expressed in endothelial cells and glandular cells (Uhlen et al., 2015) . TFPI regulates the tissue factor-dependent pathway of blood coagulation and mainly exists on the cell membrane and in the extracellular space (Broze and Girard, 2012; Wood et al., 2014) .
TFPI is a promising target for restoring thrombin generation. Severe bleeding disorders, such as hemophilia, result from disruption of the blood coagulation cascade. There is currently no cure for hemophilia and other clotting diseases. Factor replacement therapy is the most common treatment for blood coagulation disorders. However, blood clotting factors typically are cleared from the bloodstream shortly after administration. In addition, therapeutic efficacy of factor replacement therapy can diminish drastically upon formation of inhibitory antibodies.  Approximately 30%of patients with severe hemophilia A develop inhibitory antibodies that neutralize Factor VIII (FVIII) (Peerlinck and Hermans, Haemophilia, 12, 579-590 (2006) ) . Few therapeutic options exist for patients with anti-Factor antibodies.
Thus, there exists a need for compositions and methods for treating blood coagulation disorders. The invention addresses the need by providing an anti-TFPI polypeptide and methods and uses thereof.
Summary
In one aspect, provided herein are isolated polypeptides that bind TFPI, wherein the isolated polypeptides are derived from TcdB (such as TcdB4 or TcdB2) . In some embodiments, the isolated polypeptides block TFPI’s inhibitory action on the blood coagulation cascade, thereby enhancing thrombin formation.
In some embodiments, the isolated polypeptide of the invention binds TFPI-1 (e.g., TFPI-1α) and, optionally, improves TFPI-regulated thrombin generation in the absence of FVIII, FIX, and/or FXI. A composition (e.g., a pharmaceutical composition) comprising the isolated polypeptide also is provided.
In one aspect, provided herein is an isolated polypeptide that binds to TFPI, wherein the isolated polypeptide comprises or is:
(a) a functional fragment of TcdB4 protein that retains the binding to TFPI;
(b) a functional fragment of TcdB2 protein that retains the binding to TFPI; or
(c) a variant of (a) or (b) that retains the binding to TFPI, wherein the amino acid sequence of the variant is at least 80%, at least 85%, at least 90%, at least 95%or at least 99%identical to the amino acid sequence of (a) or (b) .
In some embodiments, the TcdB4 protein is a native TcdB4 protein comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the TcdB2 protein is a native TcdB2 protein comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 2.
In some embodiments, the isolated polypeptide comprises:
(a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%or at least 99%identity to the amino acid sequence as shown in SEQ ID NO: 3 or 4;
(b) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%or at least 99%identity to the amino acid sequence as shown in SEQ ID NO: 5 or 6;
(c) N-terminal and/or C-terminal truncated amino acid sequence of the amino acid sequence of (a) or (b) , for example, truncated by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) ; or
(d) N-terminal and/or C-terminal extended amino acid sequence of the amino acid sequence of (a) or (b) , for example, extended by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) at corresponding positions of SEQ ID NO: 1 or 2.
In some embodiments, the isolated polypeptide comprises one or more of the following residues corresponding to SEQ ID NO: 1: E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 and Y1510.
In some embodiments, the isolated polypeptide further comprises one or more of the following residues corresponding to SEQ ID NO: 1: L1434, K1435, M1438, K1597, S1598, L1599 and K1600 as shown in SEQ ID NO: 1.
In some embodiments, the functional fragment comprises or consists of any of SEQ ID NOs: 3-7 and 13-14.
In some embodiments, the variant of the functional fragment comprises at least one mutation (e.g. substitution, insertion or deletion) compared to corresponding positions of SEQ ID NO: 3 or 4, in order to improve the binding to TFPI.
In some embodiments, the isolated polypeptide binds to TFPI with a dissociation constant of less than 10 μM, less than 1 μM, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM or less than 5 nM, as measured by bio-layer interferometry (BLI) assay.
In some embodiments, the isolated polypeptide specifically binds to the K2 domain but not K1 domain of TFPI.
In some embodiments, the isolated polypeptide comprises:
(a) an amino acid sequence having at least 90%identity to the amino acid sequence set forth in positions 1285-1834 of SEQ ID NO: 1;
(b) an amino acid sequence having at least 90%identity to the amino acid sequence set forth in positions 1285-1834 of SEQ ID NO: 2; or
(c) N-terminal and/or C-terminal truncated amino acid sequence of the amino acid sequence of (a) or (b) , for example, truncated by 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid.
In some embodiments, the isolated polypeptide comprises at least one mutation (e.g. substitution, insertion or deletion) compared to corresponding wild-type sequence as set forth in SEQ ID NO: 1 or 2.
In some embodiments, the isolated polypeptide as disclosed herein inhibits TFPI activity and binds to TFPI with a dissociation constant of less than 10 μM.
In some embodiments, the isolated polypeptide as disclosed herein is operably linked to a moiety that enhances the half-life of the isolated polypeptide. For example, the isolated polypeptide is conjugated to a polyethylene glycol (PEG) moiety, human serum albumin (HSA) ,  an antibody or fragment thereof, hydroxyethyl starch, a multimer comprising proline, alanine, serine, or a combination thereof (PASylation) , or a C12-C18 fatty acid.
In one aspect, provided herein is a fusion protein, comprising the isolated polypeptide fused to a heterogenous polypeptide.
In some embodiments, the heterogenous polypeptide is an IgG Fc portion, such as human IgG1, IgG2, IgG3 or IgG4 Fc potion, and optionally comprising a hinge region.
In one aspect, provided herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the isolated polypeptide or the fusion protein as disclosed herein.
In one aspect, provided herein is a vector comprising the nucleic acid molecule as disclosed herein.
In one aspect, provided herein is a host cell comprising the nucleic acid molecule or the vector as disclosed herein.
In one aspect, provided herein is a pharmaceutical composition comprising the isolated polypeptide as disclosed herein and a pharmaceutically acceptable carrier.
In one aspect, provided herein is a method of inhibiting TFPI activity comprising contacting the TFPI protein with an isolated polypeptide as described herein.
In one aspect, provided herein are methods of using the isolated polypeptide or the nucleic acid molecule encoding the isolated polypeptide of the invention, e.g. for treating TFPI related or TFPI mediated disorders, such as blood coagulation disorder.
In some embodiments, provided herein is a method of enhancing thrombin formation in a subject in need thereof (such as a clotting factor-deficient subject) , comprising administering to the subject the isolated polypeptide as provided herein in an amount effective to enhance thrombin formation.
In some embodiments, provided herein is a method for increasing blood clot formation in a subject in need thereof, and a method of treating a blood coagulation disorder in a subject in need thereof. The methods comprise administering to the subject an isolated polypeptide as provided herein in an amount effective to enhance blood clot formation, or an amount effective to treat the blood coagulation disorder in the subject.
In some embodiments, the method is performed after it has been determined that a subject is at risk for developing a blood coagulation disorder (e.g., a deficiency in a clotting factor (e.g., FVIII, FIX, or FXI) is detected) or after a blood coagulation disorder (e.g., hemophilia A, hemophilia B, or hemophilia C) is detected. In some embodiments, the polypeptide as disclosed herein is administered to protect, in whole or in part, against excessive blood loss during injury or surgery.
Unless explicitly indicated to the contrary, the description provided herein with respect to  one isolated polypeptide of the invention or method of the invention applies to each and every isolated polypeptide of the invention and method of the invention, respectively.
In one aspect, provided herein is the isolated polypeptide or the nucleic acid molecule encoding the isolated polypeptide of the invention for use as a medicament. Also provided is the isolated polypeptide or the nucleic acid molecule encoding the isolated polypeptide of the invention for use in enhancing thrombin formation, increasing blood clot formation and/or treating a blood coagulation disorder in a subject in need thereof.
In one aspect, provided herein is use of the isolated polypeptide of the invention in the manufacture of a medicament for enhancing thrombin formation, increasing blood clot formation and/or treating a blood coagulation disorder in a subject in need thereof.
In one aspect, provided herein is a method for targeting a cell displaying TFPI.
In one aspect, provided herein is a method for treating or diagnosing a subject suffering from a disease or at risk of suffering from a TFPI related or TFPI mediated disease.
In one aspect, provided herein is a method of purifying TFPI.
In one aspect, provided herein is a method of identifying a TFPI-binding compound.
The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings set forth herein.
Description of the Figures
Figure 1. CRISPR-Cas9 screen identifies host factors for TcdB4
(A) Schematic diagram of the screening process using TcdB4 on HeLa cells transduced with GeCKO v2 lentiviral library.
(B) Genes identified from the third round of the screen (R3) are ranked and plotted. The x-axis shows the number of NGS reads per gene. The y axis represents the number of unique gRNAs for each targeted gene.
Figure 2. TFPI is a cellular receptor for TcdB4
(A) Schematic illustrations of TFPIα and TFPIβ, two major isoforms of TFPI.
(B) Transient transfection of TFPIα or TFPIβ restored TcdB4 entry into HeLa CSPG4-/-/TFPI-/- cells (0.14 nM TcdB4, 3 hours) . Scale bar, 50 μm.
(C) Quantification of the cell rounding in (G) . Error bars (n=6) indicate mean ± SD.
(D) HeLa WT or TFPI-/- cells transfected with TFPIα, TFPIβ, or mock-transfected were incubated with TcdB4 (10 nM) on ice for 10 minutes, washed by PBS, lysed, and subjected to  immunoblot analysis. Cell-surface bound TcdB4 was detected by an anti-TcdB polyclonal antibody. Actin is a loading control.
Figure 3. TcdB4 interacts with the Kunitz-2 domain of TFPI
(A) Schematic illustrations of TFPIK1+K2-Fc, TFPIK1-Fc, and TFPIK2-Fc.
(B) TFPIK1+K2 protects the HeLa CSPG4-/-cells from TcdB4 but not TcdB1 as measured by the cell rounding assay.
(C) Phylogenic tree of closely related Kunitz domains from TFPI, TFPI2, and AMBP.
(D) Ectopic expression of GPI-anchored TFPIK2 (TFPIK2-GPI) restored TcdB4 entry into CSPG4-/-/TFPI-/-cells.
(E) Characterization of TcdB4 binding to Fc-tagged TFPIK1 or TFPIK2 using the BLI assay (see Figure S4 for Kd analysis) . The Human IgG Fc fragment was served as a negative control.
Figure 4. TcdB4 specifically binds to TFPI.
(A) TFPIK1+K2-Fc protects the HeLa CSPG4-/-cells from TcdB4 but not TcdB1 as measured by the cell rounding assay.
(B) The pull-down assay showed that TcdB4, but not TcdB1, binds to Fc-tagged TFPIK1+K2.
(C) Purified TFPIK2-Fc, but not TFPIK2-Fc, protects CSPG4-/-cells from TcdB4 as measured by the cell rounding assay. Scale bar represents 50 μm.
Figure 5. Kinetic analysis of TcdB4-TFPI interaction.
(A) Representative binding curves of TcdB4 to Fc-tagged human TFPIK1+K2 were examined by BLI assays.
(B) Representative binding curves of TcdB4 to Fc-tagged human TFPIK2.
(C) Representative binding curves of TcdB4 to Fc-tagged mouse TfpiK2.
Figure 6. Cryo-EM structure of TcdB4-TFPI complex
(A) Schematic diagrams of the domain organization of full-length TcdB4 and TFPIK1+K2 used for Cryo-EM structure determination.
(B) Cryo-EM structure of the TcdB4-TFPI complex. The glucosyltransferase domain (GTD) , cysteine protease domain (CPD) , transmembrane delivery and receptor-binding domain (DRBD) , and combined repetitive oligopeptides (CROPs) domain of TcdB4 are shown in wheat, pink, light blue, green, respectively. The TFPI is bound at the periphery of TcdB4 through direct interactions between the Kunitz-2 domain (cyan) of TFPI and the DRBD of TcdB4.
(C) Zoomed-in view of the interaction between TcdB4DRBD (light purple) and TFPIK2 (Cyan) . TFPIK2 is anchored on a hydrophobic surface of TcdB4DRBD through two epitopes: loop1 (residues 131-138) and loop2 (residues 155-162) .
(D) Close-up views on the loop1-TcdB4 (left panel) and loop2-TcdB4 (right panel) binding interface.
(E) Mutations in TcdB41285-1834 disrupting its interactions with Fc-tagged TFPIK2 were demonstrated by a pulldown assay.
(F) Preloading TcdB41285-1834 to TFPIK2 impeded the subsequent binding of FXa. The TFPIK2-Fc loaded biosensors were first exposed to 300 nM TcdB41285-1834 or control buffer, balanced, and then exposed to 100 nM FXa.
Figure 7. Purification and functional characterization of TcdB4 and TFPI.
(A) Size exclusion chromatography profile of TFPI and SDS-PAGE of the peak fraction shown in the inset.
(B) Size exclusion chromatography profile of TcdB4 and SDS-PAGE of the peak fraction shown in the inset.
(C) Size exclusion chromatography profile of TcdB4-TFPI complex and SDS-PAGE of the peak fraction shown in the inset.
Figure 8. Image processing for the single-particle cryo-EM data.
(A) Flow chart for cryo-EM data processing. For details, see ‘Data processing’ in the Methods section.
(B) Representative cryo-EM images. Some single-particles for TcdB4-TFPI are highlighted by cyan circles.
(C) 2D class averages of cryo-EM particle images of TcdB4.
Figure 9. Single-particle cryo-EM analysis of TcdB4.
(A) Angular distribution of particle images included in the final 3D reconstruction.
(B) Local resolution of the final cryo-EM map of TcdB4.
(C) Fourier shell correlation (FSC) curve of TcdB4 with indicated resolution at FSC=0.5.
(D) FSC curve of TcdB4 between the atomic model and the final map with indicated resolution at FSC=0.5 (black) ; FSC curve between half map 1 (red) or half map 2 (green) and the atomic model refined against half map 1.
Figure 10. TcdB4 and FXa competitively bind to TFPI.
(A) Fc-tagged TFPIK2 binds to TcdB4842-1834, but not to TcdB41-841 and TcdB41801-2367 shown by the immunoblot analysis.
(B) Comparison between the structures of TFPI-FXa/Trypsin (PDB: 1TFX and 1FAX, left panel) and TFPI-TcdB4 complex (right panel) . Their interfaces are marked by red circles.
(C) Preloading FXa to TFPIK2 impeded subsequent binding of TcdB41285-1834. The TFPIK2-Fc loaded biosensors were first exposed to 100 nM FXa or control buffer, balanced, and then exposed to 300 nM TcdB41285-1834.
Figure 11. TcdB contains two classes of RBIs recognizing either FZDs or TFPI.
(A) The schematic illustration of the RBI in TcdB (upper panel) and a phylogenic split network  covering RBIs from known TcdB sequences (lower panel) .
(B) A maximum-likelihood tree was built for 110 MLST types to separate five major C. difficile clades (inner ring: clade 1 in light blue, clade 2 in purple, clade 3 in yellow, clade 4 in green, clade 5 in orange, and gray for other cryptic clades) . Different RBI classes in each MLST type are marked as the outer ring.
(C) Structural comparison between the TcdB1-FZD2 complex (PDB: 6C0B, left panel) and the TcdB4-TFPI complex (right panel) . Only DRBDs (residues 1285-1804) of TcdB1 (grey) and TcdB4 (light purple) are shown.
(D) Close-up view on the TcdB1-FZD2 (left panel) and TcdB4-TFPI (right panel) interfaces. A PAM molecule is inserted into a hydrophobic channel formed by residues from both FZD2 and TcdB1, stabilizing the TcdB1-FZD2 interaction. Key residues and PAM are shown as stick models.
(E) F1597 in TcdB1 stabilizes the middle part of PAM, while S1598 (corresponding residue) in TcdB4 forms a close hydrogen bond with R140 from TFPI.
(F) Superposition of RBIs from TcdB2 (green, PDB: 6OQ5) and that from TcdB4-TFPI complex (light purple) .
(G) Sensitivity of the HeLa CSPG4-/-and CSPG4-/-/TFPI-/-cells to TcdB2 were measured using the cell-rounding assay, and their CR50 are plotted in a bar chart. (***P<0.001)
(H) Ectopic expression of GPI-anchored TfpiK2 (TfpiK2-GPI) restored TcdB2 entry into the CSPG4-/-/TFPI-/-cells. Scale bar represents 50 μm.
(I) Protection from TcdB2 using mouse TfpiK2-Fc on HeLa CSPG4-/-cells was quantified by the cytopathic cell-rounding assay over time. Data (n=6) are presented as mean ± SD.
(J) Point mutations on TcdB41285-1834 were examined in pull-down assays, using TFPIK2-Fc as bait. Bound TcdB41285-1834 mutants were co-precipitated with TFPIK2-Fc using the Protein A resin and detected by immunoblot analysis.
Figure 12. TcdB2 binds to TFPIK2.
(A) The HeLa CSPG4-/-cells were exposed to 100 pM TcdB2 with or without Fc-tagged mouse TfpiK2. Cells were then incubated at 37 ℃ and the percentage of cell rounding was examined. Representative bright-field images of cell rounding at 4 hours post-exposure to the toxin were shown. Scale bar represents 100 μm.
(B) Characterization of TcdB2 binding to Fc-tagged TFPIK1 or TFPIK2 using the BLI assay.
(C) Representative binding curves of TcdB21285-1834 to Fc-tagged human TFPIK2.
(D) Representative binding curves of TcdB21285-1834 to Fc-tagged mouse TfpiK2.
Figure 13. S1496 is an important residue affecting TcdB2-TFPI interaction.
(A) Sequence alignment between the RBIs of TcdB2 and TcdB4. Non-conserved residues are  marked in red, similar residues are marked in blue. Residues that contribute to the TFPI binding are denoted by green triangles.
(B) Representative binding curves of TcdB21285-1834 S1496I mutant to Fc-tagged mouse Tfpi.
(C) Representative binding curves of TcdB21285-1834 S1496I mutant to Fc-tagged human TFPI.
Detailed description
All publications cited in this specification are herein incorporated by reference as though fully set forth. If certain content of a reference cited herein contradicts or is inconsistent with the present disclosure, the present disclosure controls.
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.
In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings generally understood by a person skilled in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present disclosure, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the Specification as a whole.
As used herein, the singular terms “a, ” “an, ” and “the” include the plural reference unless the context clearly indicates otherwise.
It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those of skills in the art.
Unless the context requires otherwise, the terms “comprise, ” “comprises, ” and “comprising, ” or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.
Definitions
As used herein, the term “TFPI” refers to Tissue Factor Pathway Inhibitor, a multivalent Kunitz-type serine protease inhibitor that regulates tissue factor-induced coagulation via factor Xa-dependent feedback inhibition of the tissue factor–factor VIIa complex. In humans, TFPI has two major isoforms, TFPIα and TFPIβ, which are also produced by all mammals (Wood et al., 2014) . TFPIα contains three tandem Kunitz-type protease inhibitory (Kunitz-1, Kunitz-2, and Kunitz-3, or K1, K2, and K3) domains followed by a basic carboxyterminal (C-terminal) region. TFPIβ lacks the K3 and basic C-terminal domains of TFPIα. Instead, it contains a C-terminal  signal peptide that directs cleavage and attachment of a GPI anchor (Broze and Girard, 2012; Girard et al., 2012; Maroney and Mast, 2015; Zhang et al., 2003) . An exemplary sequence of mouse TFPIβ is shown in SEQ ID NO: 10. Exemplary sequences of human TFPIα and TFPIβ are shown in SEQ ID NO: 11 and 12, respectively.
As used herein, the term “TcdB” or “Clostridium difficile toxin B” is an exotoxin produced by pathogenic C. difficile. TcdB is a member of the large clostridial toxin (LCT) family, which enters target cells via receptor-mediated endocytosis and glucosylates small GTPase proteins, leading to cytoskeletal dysfunction and eventual cell death (Aktories et al., 2017; Chandrasekaran and Lacy, 2017; Voth and Ballard, 2005) . TcdB has three major domains: a catalytically active N-terminal domain, a centrally located translocation domain and a C-terminal receptor binding domain. TcdB has 4 variants, TcdB1, TcdB2, TcdB3 and TcdB4, among them TcdB2 and TcdB4 do not recognize Frizzled proteins (FZDs) but as characterized herein, recognize TFPI.
The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as between TcdB4 and TFPI. The term “high affinity” as used herein, refers to an interaction having a dissociation constant (Kd) of 1 x 10-7 M or less, more preferably 5 x 10-8 M or less, even more preferably 1x10-8 M or less, even more preferably 5 x 10-9 M, e.g. as measured by bio-layer interferometry (BLI) assay.
As used herein, the term “functional fragment” in relation to full length TcdB4 or TcdB2 refers to a fragment of wild-type TcdB4 or TcdB2 that retains the function of binding to TFPI. The binding between the functional fragment and TFPI may be approximately lower, equivalent or higher compared to the binding between full-length wild-type TcdB4 or TcdB2 and TFPI, preferably the binding is higher compared to full-length wild-type TcdB2.
The terms “polypeptide” and “peptide” can be used exchangeably herein and refers to an amino acid sequence of at least 2 amino acids, and generally more than 10 amino acids. Polypeptide complexes with more than one chains are also included.
As used herein, the term “corresponding to” in relation to full length wild-type TcdB4 or TcdB2 means the residue at the corresponding numbering position of full length wild-type TcdB4 or TcdB2 as set forth in SEQ ID NO: 1 or 2, respectively. For example, a functional fragment comprising a residue corresponding to E1433 of SEQ ID NO: 1 means the functional fragment, which is derived from SEQ ID NO: 1, comprises the Glu (E) amino acid residue at the position corresponding to 1433 of SEQ ID NO: 1. In some embodiments, the functional fragment may also comprise the same residues of SEQ ID NO: 1 that are close to E1433, e.g. comprise amino acids 1431-1606 of SEQ ID NO: 1. In some other embodiments, the functional fragment comprising the E1433 amino acid residue may be mutated at positions close to E1433, e.g. the  residue (s) close to E1433 in the functional fragment is/are different from those in SEQ ID NO: 1. Throughout the specification, where a numbering of the amino acid is related, it refers to the numbering according to SEQ ID NO: 1 or SEQ ID NO: 2, depending on the context.
As used herein, “treating” and “treatment” refers to any reduction in the severity and/or onset of symptoms associated with a TFPI related or TFPI mediated disorder, such as blood coagulation disorder. Accordingly, “treating” and “treatment” includes therapeutic and prophylactic measures. One of ordinary skill in the art will appreciate that any degree of protection from, or amelioration of, a blood coagulation disorder or symptom associated therewith is beneficial to a subject, such as a human patient. The quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and/or delaying the appearance of symptoms.
Polypeptides comprising functional fragments of TcdB or variants thereof
In certain embodiments, the polypeptides disclosed herein retain the binding to TFPI and/or have the capability to inhibit TFPI function (referred to as TFPI-inhibitory polypeptides) , wherein the polypeptides comprise or consist of functional fragments of TcdB (especially TcdB4) , or highly homologous sequences of the fragments. For example, the polypeptides may comprise the receptor-binding domain (DRBD) of TcdB2 or TcdB4.
In some embodiments, the polypeptides comprise N-terminal truncated fragments of the receptor-binding domain (DRBD) of TcdB4, specifically, truncated by 1-400 amino acids, e.g. no more than 400 amino acids, no more than 350 amino acids, no more than 300 amino acids, no more than 250 amino acids, no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids or no more than 10 amino acids from the N-terminal of the receptor-binding domain (DRBD) of TcdB4 or TcdB2. Additionally or alternatively, the polypeptides comprise C-terminal truncated fragments of the receptor-binding domain (DRBD) of TcdB4, specifically, truncated by 1-400 amino acids, e.g. no more than 400 amino acids, no more than 350 amino acids, no more than 300 amino acids, no more than 250 amino acids, no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids or no more than 10 amino acids from the C-terminal of the receptor-binding domain (DRBD) of TcdB4 or TcdB2. As would be understood by a person skilled in the art, the N-terminal and/or C-terminal truncated fragments as described above could retain the binding to TFPI or have the capability to inhibit TFPI function.
By the term “retain the binding to TFPI” it is meant the binding between the functional fragment (or a variant thereof) and TFPI is substantially the same, higher, or slightly lower compared to the binding between full-length TcdB and TFPI. In some embodiments, a  polypeptide as disclosed herein comprises a functional fragment of TcdB2 that has improved binding to TFPI by including a S1496I substitution.
Specifically, the polypeptides may comprise:
a functional fragment of TcdB4 protein as shown in SEQ ID NO: 1 or TcdB2 protein as shown in SEQ ID NO: 2 that retains the binding to TFPI; or
a variant of the functional fragment that retains the binding to TFPI, wherein the amino acid sequence of the variant is at least 80%identical to the amino acid sequence of the functional fragment.
In some embodiments, the functional fragment is derived from TcdB4 and comprises an amino acid sequence having at least 80%, at least 85%, or at least 90%identity to the amino acid sequence as shown in SEQ ID NO: 3 or 4. For example, the functional fragment comprises or consists of positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 1. In some further embodiments, the functional fragment comprises or consists of N-terminal and/or C-terminal truncated amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 1, for example, truncated by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) . For example, the functional fragment comprises or consists of amino acid sequence from positions 842-1834 of SEQ ID NO: 1 or 2 (i.e. SEQ ID NO: 13 or 14, respectively) . In some further embodiments, the functional fragment comprises or consists of N-terminal and/or C-terminal extended amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 1, for example, extended by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) . The extended amino acids may be substantially the same with corresponding positions in SEQ ID NO: 1.
In some embodiments, the functional fragment is derived from TcdB2 and comprises an amino acid sequence having at least 80%, at least 85%, or at least 90%identity to the amino acid sequence as shown in SEQ ID NO: 5 or 6. For example, the functional fragment comprises or consists of positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 2. In some further embodiments, the functional fragment comprises or consists of N-terminal and/or C-terminal truncated amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 2, for example, truncated by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid (s) . In some further embodiments, the functional fragment comprises or consists of N-terminal and/or C-terminal extended amino acid sequence from positions 841-1834, 1285-1834, or 1431-1606 of SEQ ID NO: 2, for example, extended by 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid (s) . The extended amino acids may be substantially the same with corresponding positions in SEQ ID NO: 2. Since the binding between TcdB2 and TFPI is lower than that of TcdB4 and TFPI, it is preferred one or more  mutations are included in the functional fragment derived from TcdB2 to improve the binding affinity. For example, certain mutations may be introduced into the functional fragment derived from TcdB2 to make it more similar to a functional fragment of TcdB4, i.e. certain residues in the functional fragment derived from TcdB2 are mutated to residues at corresponding positions in TcdB4. In one example, the polypeptide comprises a variant of the functional fragment derived from TcdB2, and the variant comprises a S1496I substitution (the numbering is according to SEQ ID NO: 2) .
The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
Additionally or alternatively, the protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www. ncbi. nlm. nih. gov.
By “variant” herein is meant a polypeptide comprising one or more amino acid substitutions, amino acid deletions, or amino acid additions to a parent amino acid sequence. Variants include, but are not limited to, polypeptides having an amino acid sequence that is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to any of the amino acid sequences provided herein while retaining the ability to bind TFPI and/or inhibit TFPI activity.
In some embodiments, the polypeptide comprises one or more of the following residues corresponding to SEQ ID NO: 1: E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435,  M1438, V1492, L1494, I1496, L1489, P1506 and Y1510. Specifically, the polypeptide may comprise a fragment of SEQ ID NO: 1 from positions 1433 to 1510.
In some embodiments, the polypeptide further comprises one or more of the following residues corresponding to SEQ ID NO: 1: L1434, K1435, M1438, K1597, S1598, L1599 and K1600 as shown in SEQ ID NO: 1. Specifically, the polypeptide may comprise a fragment of SEQ ID NO: 1 from positions 1433 to 1600.
In some embodiments, the functional fragment comprises or consists of SEQ ID NO: 7, 4 or 3 (corresponding to positions 1431-1606, 1285-1834, and 841-1834 of SEQ ID NO: 1, respectively) . In some embodiments, the functional fragment comprises or consists of SEQ ID NO: 6 or 5 (corresponding to positions 1285-1834, and 841-1834 of SEQ ID NO: 2, respectively) .
Functional fragments and derivatives of TcdB
In one aspect, the polypeptides as disclosed herein comprise 20 amino acids or more, 30 amino acids or more, 40 amino acids or more, 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, 150 amino acids or more, 200 amino acids or more, 250 amino acids or more, 200 amino acids or more, 350 amino acids or more, 400 amino acids or more, 450 amino acids or more, 500 amino acids or more, 550 amino acids or more, 600 amino acids or more, 650 amino acids or more, 700 amino acids or more, 750 amino acids or more, or 800 amino acids or more of wildtype full length TcdB4 or TcdB2, preferably from the DRBD domain of TcdB4 or TcdB2. In various embodiments, the polypeptide comprises 100-300 amino acid residues (e.g., 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 26, 270, 280, 290, 300 amino acid residues) in the DRBD domain of TcdB4 or TcdB2. In some embodiments, the polypeptide comprises positions 1431-1606 in the DRBD domain, which are likely at the receptor-binding interface (RBI) of TcdB. However, it is also contemplated that a polypeptide described herein comprising one or more extensions or deletions is suitable in the context of the invention so long as the polypeptide binds TFPI and, optionally, blocks TFPI inhibition of the coagulation cascade.
Optionally, the polypeptide of the invention comprises one or more amino acid substitutions (with reference to any of the amino acid sequences provided herein) that do not destroy (or even improve) the ability of the polypeptide to bind and/or inhibit TFPI.
Amino acid substitutions include, but are not limited to, those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on a polypeptide. In some embodiments, the substitution is a conservative substitution, wherein an amino acid residue  is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art, and include amino acids with basic side chains (e.g., lysine, arginine, and histidine) , acidic side chains (e.g., aspartic acid and glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan) , beta-branched side chains (e.g., threonine, valine, and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine) .
It will be appreciated, however, that a practitioner is not limited to creating conservative substitutions so long as the resulting polypeptide retains the ability to downregulate, in whole or in part, TFPI activity. The invention also embraces TFPI-inhibitory polypeptides comprising atypical, non-naturally occurring amino acids, which are well known in the art. Exemplary non-naturally occurring amino acids include ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2, 4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, y-amino butyric acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, a fluoro-amino acid, a 3-methyl amino acid, α-C-methyl amino acid, a N-methyl amino acid, 2-amino-isobutyric acid, β-homoglutamatic acid, β-homophenylalanine, β-homolysine, β-homoleucine, β-homoasparagine, β-homoglutamine, β-homoarginine, β-homoserine, β-homotyrosine, β-homoaspartic acid, β-homovaline, β-homoasparagin, (S) -cyclohexylalanine, (S) -citrullin, (S) -2, 4-diaminobutyric acid, (S) -2, 4-diaminobutyric acid, (S) -diaminopropionic acid, (S) -2-propargylglycine, (S) -N (omega) -nitro-arginine, L-homophenylalanine, S) -homo-arginine, (S) -homo-citrulline, (S) -homo-cysteine, (S) -2-amino-5-methyl-hexanoic acid, (S) -homo-lysine, (S) -norleucine, (S) -N-methylalanine, (S) -N-methyl-aspartic acid, (S) -N-methyl-glutamic acid, (S) -N-methyl-phenylalanine, N-methyl-glycine, (S) -N-methyl-lysine, (S) -N-methyl-leucine, (S) -N-methyl-arginine, (S) -N-methyl-serine, (S) -N-methyl-valine, (S) -N-methyl-tyrosine, (S) -2-amino-pentanoic acid, (S) -2-pyridyl-alanine, (S) -ornithine, L-phenylglycin, 4-phenyl-butyric acid and selenomethionine. The individual amino acids may have either L or D stereochemistry when appropriate, although the L stereochemistry is typically employed for all of the amino acids in the polypeptide.
The invention further includes polypeptide variants comprising one or more amino acids inserted within an amino acid sequence provided herein and/or attached to the N-terminus or C-terminus. In some embodiments, the polypeptide further comprises one or more amino acids that facilitate synthesis, handling, or use of the polypeptide, including, but not limited to, one or  two lysines at the N-terminus and/or C-terminus to increase solubility of the polypeptide, or one or two methionine at the N-terminus and/or C-terminus, and it is expected such modifications are not to change the function of the polypeptide.
“Derivatives” are included in the invention and include TFPI-binding or TFPI-inhibitory polypeptides that have been chemically modified in some manner distinct from addition, deletion, or substitution of amino acids. In this regard, a polypeptide of the invention provided herein is chemically bonded with polymers, lipids, other organic moieties, and/or inorganic moieties. Examples of polypeptide and protein modifications are given in Hermanson, Bioconjugate Techniques, Academic Press, (1996) . The TFPI-binding polypeptides described herein optionally comprise a functional group that facilitates conjugation to another moiety (e.g., a peptide moiety) . Exemplary functional groups include, but are not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, epoxide, oxirane, carbonate, arylating agent, imidoester, carbodiimide, anhydride, alkyl halide derivatives (e.g., haloacetyl derivatives) , maleimide, aziridine, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents (e.g., pyridyl disulfides or TNB thiol) , diazoalkane, carboyldiimadazole, N, N′-Disuccinyl carbonate, N-Hydroxysuccinimidyl chloroformate, and hydrazine derivatives. Maleimide is useful, for example, for generating a TFPI-binding polypeptide that binds with albumin in vivo.
Derivatives are prepared in some situations to increase solubility, stability, absorption, or circulating half life. Various chemical modifications eliminate or attenuate any undesirable side effect of the agent. In one aspect, the invention includes TFPI-binding polypeptides covalently modified to include one or more water soluble polymer attachments. A water soluble polymer (or other chemical moiety) is attached to any amino acid residue, although attachment to the N-or C-terminus is preferred in some embodiments. Optionally, a polymer is attached to the polypeptide via one or more amino acids or building blocks that offer functional groups that facilitate polymer attachment. For example, JBT2315 comprises a C-terminal cysteine (position X4021 with respect to formula (XI) ) , which facilitates the addition of, e.g., a maleimide polyethylene glycol (PEG) . Useful polymers include, but are not limited to, PEG (e.g., PEG approximately 40 kD, 30 kD, 20 kD, 10, kD, 5 kD, or 1 kD in size) , polyoxyethylene glycol, polypropylene glycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch, cellulose, poly- (N-vinyl pyrrolidone) -polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polysialic acid (PSA) , polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of any of the foregoing. In one aspect, the polypeptide of the invention is a PEGylated polypeptide. PEG moieties are available in different shapes, e.g., linear or branched. For further discussion of water soluble polymer attachments, see U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and  4,179,337. Other moieties useful for improving polypeptide half life or stability are described herein and include, for instance, albumin (optionally modified to allow conjugation to the inventive polypeptide) , fatty acid chains (e.g., C12-C18 fatty acid, such as a C14 fatty acid) , an antibody or fragment thereof (e.g., an Fc portion of an antibody) , and proline-alanine-serine multimers.
In another aspect, a polypeptide derivative includes a targeting moiety specific for a particular cell type, tissue, and/or organ. Alternatively, the polypeptide is linked to one or more chemical moieties that facilitate purification, detection, multimerization, binding with an interaction partner, and characterization of polypeptide activity. An exemplary chemical moiety is biotin. Other moieties suitable for conjugation to the TFPI-binding polypeptide of the invention include, but are not limited to, a photosensitizer, a dye, a fluorescence dye, a radionuclide, a radionuclide-containing complex, an enzyme, a toxin, and a cytotoxic agent. Photosensitizers include, e.g., Photofrin, Visudyne, Levulan, Foscan, Metvix, CysviewTM, Laserphyrin, Antrin, Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200 ALA, and Amphinex. If desired, a His tag, a FLAG tag, a strep tag, or a myc tag is conjugated to the polypeptide.
In addition, in one aspect, the polypeptides of the invention are acylated at the N-terminal amino acid of the polypeptide. In another aspect, the polypeptides of the invention are amidated at the C-terminal amino acid of the polypeptide. In a still further aspect, the polypeptides of the invention are acylated at the N-terminal amino acid of the polypeptide and are amidated at the C-terminal amino acid of the polypeptide.
Derivatives also include peptides comprising modified or non-proteinogenic amino acids or a modified linker group (see, e.g., Grant, Synthetic Peptides: A User's Guide, Oxford University Press (1992) ) . Modified amino acids include, for example, amino acids wherein the amino and/or carboxyl group is replaced by another group. Non-limiting examples include modified amino acids incorporating thioamides, ureas, thioureas, acylhydrazides, esters, olefines, sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acid amides, benzodiazepines and other aromatic or non-aromatic heterocycles (see Estiarte et al., Burgers Medicinal Chemistry, 6th edition, Volume 1, Part 4, John Wiley &Sons, New York (2002) ) . Modified amino acids are often connected to the peptide with at least one of the above mentioned functional groups instead of an amide bond. Non-proteinogenic amino acids include, but are not limited, to β-alanine (Bal) , norvaline (Nva) , norleucine (Nle) , 4-aminobutyric acid (γ-Abu) , 2-aminoisobutyric acid (Aib) , 6-aminohexanoic acid (ε-Ahx) , ornithine (Orn) , hydroxyproline (Hyp) , taurine, sarcosine, citrulline (Cit) , cysteic acid (Coh) , cyclohexylalanine (Cha) , methioninesulfoxide (Meo) , methioninesulfone (Moo) , homoserinemethylester (Hsm) , propargylglycine (Eag) ,  5-fluorotryptophan (5Fw) , 6-fluorotryptophan (6Fw) , 3′, 4′-dimethoxyphenyl-alanine (Ear) , 3′, 4′-difluorophenylalanine (Dff) , 4′-fluorophenyl-alanine (Pff) , 1-naphthyl-alanine (1Ni) , 1-methyltryptophan (1Mw) , penicillamine (Pen) , homoserine (Hse) , t-butylglycine, t-butylalanine, phenylglycine (Phg) , benzothienylalanine (Bta) , L-homo-cysteine (Hcy) , N-methyl-phenylalanine (Nmf) , 2-thienylalanine (Thi) , 3, 3-diphenylalanine (Ebw) , homophenylalanine (Hfe) and S-benzyl-L-cysteine (Ece) . The structures of many of the non-proteinogenic amino acids are provided in Table 2. These and other non-proteinogenic amino acids may exist as D-or L-isomers. Examples of modified linkers include, but are not limited to, the flexible linker 4, 7, 10-trioxa-1, 13-tridecanediamine (Ttds) , glycine, 6-aminohexanoic acid, beta-alanine (Bal) , pentynoic acid (Pyn) , and combinations of Ttds, glycine, 6-aminohexanoic acid and Bal.
TcdB fusion protein
Suitable fusion proteins include, but are not limited to, proteins comprising the TFPI-inhibitory polypeptide linked to one or more polypeptides, polypeptide fragments, or amino acids not generally recognized to be part of the protein sequence. In some embodiments, a fusion polypeptide comprises the entire amino acid sequences of two or more polypeptides or, alternatively, comprises portions (fragments) of two or more polypeptides. In addition to all or part of the TFPI-inhibitory polypeptides described herein, a fusion protein optionally includes all or part of any suitable polypeptide comprising a desired biological activity/function. Indeed, in some embodiments, the TFPI-inhibitory polypeptide is operably linked to, for instance, one or more of the following: a polypeptide with long circulating half life, a marker protein, a polypeptide that facilitates purification of the TFPI-inhibitory polypeptide, a polypeptide sequence that promotes formation of multimeric proteins, or a fragment of any of the foregoing. Suitable fusion partners include, but are not limited to, a His tag, a FLAG tag, a strep tag, and a myc tag.
Optionally, the TFPI-inhibitor polypeptide is fused to one or more entities that enhance the half life of the polypeptide. Half life can be increased by, e.g., increasing the molecular weight of the TFPI-binding polypeptide to avoid renal clearance and/or incorporating a ligand for the nFc receptor-mediated recycling pathway. In one embodiment, the TFPI-binding polypeptide is fused to or chemically conjugated to (as described further below) an albumin polypeptide or a fragment thereof (e.g., human serum albumin (HSA) or bovine serum albumin (BSA) ) . The albumin fragment comprises 10%, 25%, 50%, or 75%of the full length albumin protein. Alternatively or in addition, the TFPI-binding polypeptide comprises an albumin binding domain or fatty acid that binds albumin when administered in vivo. Other suitable fusion partners include,  but are not limited to, a proline-alanine-serine multimer (PASylation) and an antibody or fragment thereof (e.g., an Fc portion of an antibody) .
In some embodiments, two or more TFPI-inhibitory peptides are fused together, linked by a multimerization domain, or attached via chemical linkage to generate a TFPI-inhibitory polypeptide complex. The TFPI-inhibitor peptides may be the same or different. Thus, the invention provides a homo-dimer (i.e., a dimer comprising two identical TFPI-binding peptides) , a homo-multimer (i.e., a complex comprising three or more identical TFPI-binding peptides) , a hetero-dimer (i.e., a dimer comprising two different TFPI-binding peptides) , and heteromultimer (i.e., a complex comprising three or more TFPI-binding peptides, wherein at least two of the TFPI-binding peptides are different) comprising or consisting of any of the peptides described herein, optionally attached by one or more linkers.
Methods for producing the polypeptides
TFPI-binding polypeptides of the invention (including TFPI inhibitory polypeptides) can be made in a variety of ways. In some embodiments, the polypeptides are synthesized by solid phase synthesis techniques including those described in Merrifield, J. Am. Chem. Soc., 85, 2149 (1963) ; Davis et al., Biochem. Intl., 10, 394-414 (1985) ; Larsen et al., J. Am. Chem. Soc., 115, 6247 (1993) ; Smith et al., J. Peptide Protein Res., 44, 183 (1994) ; O'Donnell et al., J. Am. Chem. Soc., 118, 6070 (1996) ; Stewart and Young, Solid Phase Peptide Synthesis, Freeman (1969) ; Finn et al., The Proteins, 3rd ed., vol. 2, pp. 105-253 (1976) ; and Erickson et al., The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976) . Alternatively, the TFPI-binding polypeptide (e.g., the TFPI-inhibitory polypeptide) is expressed recombinantly by introducing a nucleic acid encoding the TFPI-binding polypeptide into host cells, which are cultured to express the polypeptide. Such polypeptides can be purified from the cell culture using standard protein purification techniques.
Nucleic Acids encoding the polypeptides
The invention also encompasses a nucleic acid comprising a nucleic acid sequence encoding the TFPI-binding polypeptide of the invention. Methods of preparing DNA and/or RNA molecules are well known in the art. In one aspect, a DNA/RNA molecule encoding the TFPI-binding polypeptide provided herein is generated using chemical synthesis techniques and/or using polymerase chain reaction (PCR) . If desired, the TFPI-binding polypeptide coding sequence is incorporated into an expression vector. One of ordinary skill in the art will appreciate that any of a number of expression vectors known in the art are suitable in the context of the invention, such as, but not limited to, plasmids, plasmid-liposome complexes, and viral vectors. Any of these expression vectors are prepared using standard recombinant DNA techniques  described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) , and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley &Sons, New York, N.Y. (1994) . Optionally, the nucleic acid is operably linked to one or more regulatory sequences, such as a promoter, activator, enhancer, cap signal, polyadenylation signal, or other signal involved with the control of transcription or translation.
Any of the the TFPI-binding polypeptide of the invention or nucleic acids encoding the polypeptides also is provided in a composition (e.g., a pharmaceutical composition) . In this regard, the polypeptide is formulated with a physiologically-acceptable (i.e., pharmacologically-acceptable) carrier, buffer, excipient, or diluent, as described further herein. Optionally, the polypeptide is in the form of a physiologically acceptable salt, which is encompassed by the invention. “Physiologically acceptable salts” means any salts that are pharmaceutically acceptable. Some examples of appropriate salts include acetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate. If desired, the composition comprises one or more additional pharmaceutically-effective agents.
Properties of the polypeptides
The polypeptide provided herein optionally inhibits at least one TFPI-1 (e.g., TFPI-1α or TFPI-1β) activity such as, but not limited to, an activity that downregulates the blood coagulation cascade. Without being bound by any specific mechanism of action, a proposed mechanism of inhibition may involve preventing formation of the quaternary TF-FVIIA-FXA-TFPI complex. The polypeptide may inhibit binding (competitively or allosterically) of TFPI to FXa (e.g., inhibit binding of TFPI Kunitz domain 2 to Factor Xa or interrupt binding of TFPI Kunitz domain 1 to an exosite of Factor Xa) , the TF/FVIIa complex (e.g., inhibit binding of TFPI Kunitz domain 1 to the TF/FVIIa complex) , TF alone, and/or FVIIa alone. With TFPI activity diminished, TF and FVIIa are free to activate FX which, in turn, enhances conversion of prothrombin to thrombin. Surprisingly, in some embodiments, the polypeptide of the invention that binds Kunitz domain 1 interferes with TFPI-mediated inhibition of FXa. Thus, the invention provides a method of, e.g., inhibiting TFPI-mediated downregulation of the extrinsic and/or common pathway of the coagulation cascade and/or enhancing FXa-mediated conversion of prothrombin to thrombin, by administering to a subject the polypeptide described herein that binds Kunitz domain 1.
In one aspect, the polypeptide of the invention exhibits TFPI antagonistic activity in model and/or plasmatic systems. An exemplary model system for determining TFPI-inhibitory activity is the extrinsic tenase assay, which tests the ability of candidate polypeptides to restore extrinsic  complex-mediated FX activation in the presence of TFPI (which is a natural inhibitor of the FX activation reaction) (see, e.g., Lindhout et al., Thromb. Haemost., 74, 910-915 (1995) ) . Another model system for characterizing TFPI-inhibitory activity is the FXa inhibition assay, wherein FXa activity is measured in the presence of TFPI (see Sprecher et al., PNAS, 91, 3353-3357 (1994) ) . Optionally, the polypeptide of the invention enhances FX activation in the presence of TFPI with a half maximal effective concentration (EC50) of less than or equal to 1×10-4 M, less than or equal to 1×10-5 M, less than or equal to 1×10-6 M, or less than or equal to 1×10-7 M.
In one aspect, TFPI-antagonist activity is characterized in a plasma-based assay. Thrombin formation is triggered in plasma substantially lacking FVIII or FIX activity (e.g., the residual coagulation factor activity is lower than 1%) in the presence of a candidate polypeptide. Thrombin formation can be detected using a fluorogenic or chromogenic substrate. A system for measuring thrombin activity is provided by Thrombinoscope BV (Maastricht, The Netherlands) . Prothrombin conversion is measured using, e.g., a ThrombographTM (Thermo Scientific, Waltham, Mass. ) , and the resulting data is compiled into a Calibrated Automatic Thrombogram generated by ThrombinoscopeTM software available from Thrombinoscope BV. In certain embodiments, the TFPI-inhibitory polypeptide increases the amount of peak thrombin generated during the assay and/or decreases the time required to achieve peak thrombin formation. In further embodiments, the polypeptide enhances thrombin formation in the absence of Factor VIII to at least about 2%, at least about 3%, at least about 5%, at least about 7%, or at least about 10%of the level of thrombin formation in normal plasma, i.e., in the presence of physiological levels of Factor VIII. In various aspects, the polypeptide is administered to an animal model of thrombin deficiency or hemophilia to characterize TFPI inhibitory activity in vivo. Such in vivo models are known in the art and include for example, mice administered anti-FVIII antibodies to induce hemophilia A (Tranholm et al., Blood, 102, 3615-3620 (2003) ) ; coagulation factor knock-out models such as, but not limited to, FVIII knock-out mice (Bi et al., Nat. Genet., 10 (1) , 119-121 (1995) ) and FIX knock-out mice (Wang et al., PNAS, 94 (21) , 11563-66 (1997) ) ; induced hemophilia-A in rabbits (Shen et al., Blood, 42 (4) , 509-521 (1973) ) ; and Chapel Hill HA dogs (Lozier et al., PNAS, 99, 12991-12996 (2002) ) .
Various polypeptides bind TFPI from any source including, but not limited to, mouse, rat, rabbit, dog, cat, cow, horse, pig, guinea pig, and primate. In some embodiments, the polypeptide binds human TFPI. Optionally, the polypeptide binds TFPI from more than one species (i.e., the polypeptide is cross-reactive among multiple species) . In certain aspects, the polypeptide binds TFPI with a dissociation constant (KD) of less than or equal to 1×10-4 M, less than or equal to 1×10-5M, less than or equal to 1×10-6 M, or less than or equal to 1×10-7 M. Affinity may be determined using, for example and without limitation, any one, two, or more of a variety of  techniques, such as affinity ELISA assay, a competitive ELISA assay, and/or surface plasmon resonance (BIAcoreTM) assay. When characterized using a competitive (IC50) ELISA assay, the polypeptide of the invention optionally demonstrates an IC50 of less than or equal to about 50,000 nM. For example, the polypeptide demonstrates an IC50 of less than or equal to about 10,000 nM, such as an IC50 of less than or equal to about 5,000 nM, less than or equal to about 1,000 nM, or less than or equal to about 500 nM. In one aspect, the polypeptide demonstrates an IC50 of less than or equal to about 250 nM, less than or equal to about 100 nM, less than or equal to about 50 nM, or less than or equal to about 10 nM. Affinity may also be determined by a kinetic method or an equilibrium/solution method. Such methods are described in further detail herein or known in the art.
Another suitable assay for characterizing the inventive polypeptides is a koff assay, which examines a peptide's release from TFPI. The koff assay result is not the dissociation rate constant, but a percentage of competitor peptide blocked from TFPI binding by a test peptide after an incubation period with TFPI. An exemplary koff assay includes the following steps: 1) incubation of a TFPI-coated microtiter plate with an amount of test peptide resulting in approximately 90%TFPI occupation; 2) removal of unbound test peptide; 3) addition of a biotinylated tracer (i.e., competitor) peptide that competes with the test peptide for binding to TFPI; 4) incubation for a period of time during which binding sites released by the test peptide is occupied by the tracer; 5) removal of unbound tracer and test peptide; and 6) detection of bound tracer by a chromogenic reaction using streptavidin-horseradish peroxidase conjugate. The resulting signal is indicative of binding sites freed by the test peptide. A test peptide that does not dissociate from TFPI during the incubation period yields a weaker signal compared to an analyte that dissociates completely.
As with all binding agents and binding assays, one of skill in the art recognizes that the various moieties to which a binding agent should not detectably bind in order to be biologically (e.g., therapeutically) effective would be exhaustive and impractical to list. Therefore, the term “specifically binds” refers to the ability of a polypeptide to bind TFPI with greater affinity than it binds to an unrelated control protein that is not TFPI. For example, the polypeptide may bind to TFPI with an affinity that is at least, 5, 10, 15, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for a control protein. In some embodiments, the polypeptide binds TFPI with greater affinity than it binds to an “anti-target, ” a protein or other naturally occurring substance in humans to which binding of the polypeptide might lead to adverse effects. Several classes of polypeptides or proteins are potential anti-targets. Because TFPI-inhibitory polypeptides exert their activity in the blood stream and/or at the endothelium, plasma proteins represent potential anti-targets. Proteins containing Kunitz domains (KDs) are potential  anti-targets because KDs of different proteins share a significant similarity. Tissue Factor Pathway Inhibitor-2 (TFPI-2) is highly similar to TFPI-1α and, like TFPI-1α, contains KDs (Sprecher et al., PNAS, 91, 3353-3357 (1994) ) . Thus, in one aspect, the polypeptide of the invention binds to TFPI with an affinity that is at least 5, 10, 15, 25, or 50 times greater than the affinity for an anti-target, such as TFPI-2.
Optionally, the TFPI-binding polypeptide demonstrates one or more desired characteristics described herein, and the amino acid sequence of a polypeptide can be modified to optimize binding, stability, and/or activity, if desired. An exemplary TFPI-binding polypeptide binds TFPI with a KD of less than or equal to 20 nM and/or exhibits a binding affinity for TFPI that is at least 100 times greater than the binding affinity for an anti-target. Alternatively or in addition, the TFPI-binding polypeptide enhances FX activation in the presence of TFPI with an EC50 (as measured using any suitable assay, such as the assays described here) of less than or equal to 50 nM and/or enhances thrombin formation in the absence of Factor VIII to at least about 20% (e.g., 40%) of the level of thrombin formation in plasma containing physiological levels of Factor VIII. Alternatively or in addition, the TFPI-binding polypeptide achieves a desired level of plasma stability (e.g., 50%or more of a dose remains in plasma after 12 hours) and/or demonstrates a desired half life in vivo (e.g., at least two, three, four, five, six, seven, eight, nine, or ten hours) . Alternatively or in addition, the TFPI-binding polypeptide exhibits a desired level of bioavailability, such as a desired level of bioavailability following subcutaneous administration (e.g., greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, or 50%) and/or demonstrates a desired level of TFPI-inhibitory activity at a given dose in vivo.
Applications
The invention further includes a method of inhibiting TFPI. The method comprises contacting TFPI with a TFPI-binding polypeptide as described herein. Any degree of TFPI-activity inhibition is contemplated. For example, a TFPI-inhibitory polypeptide reduces TFPI-inhibition of the extrinsic pathway at least about 5% (e.g., at least about 10%, at least about 25%, or at least about 30%) . In some embodiments, the TFPI-inhibitory polypeptide reduces TFPI activity within the extrinsic pathway at least about 50%, at least about 75%, or at least about 90%compared to TFPI activity in the absence of the polypeptide.
In one aspect of the invention, TFPI-binding polypeptides are used to detect and/or quantify TFPI in vivo or in vitro. An exemplary method of detecting and/or quantifying TFPI in a sample comprises (a) contacting a sample with a TFPI-binding polypeptide of the invention, and (b) detecting binding of the TFPI-binding polypeptide to TFPI.
The invention further includes a method for targeting biological structures (including, but  not limited to, cell surfaces and endothelial lining) where TFPI is located. The method comprises contacting the biological structure (e.g., including, without limitation, a cell displaying TFPI on the cell surface) with a TFPI-binding polypeptide described herein, optionally conjugated to a moiety that adds additional functionality to the polypeptide. The moiety can be a dye (such as a fluorescence dye) , a radionuclide or a radionuclide-containing complex, a protein (e.g., an enzyme, a toxin, or an antibody) or a cytotoxic agent. For example, the polypeptide is linked or conjugated to an effector moiety that facilitates polypeptide detection and/or purification and/or comprises therapeutic properties. In one aspect, the TFPI-binding polypeptide or polypeptide conjugate is administered to a mammal to target a TFPI-displaying cell within the mammal. Optionally, the method further comprises detecting binding of the TFPI-binding polypeptide to TFPI. The method is useful for therapy and diagnosis of disease where TFPI is a suitable diagnostic marker or TFPI-expressing cells are a target for a therapeutic approach.
Polypeptide-TFPI complexes are directly or indirectly detected. Detection moieties are widely used in the art to identify biological substances and include, for example, dye (e.g., fluorescent dye) , radionuclides and radionuclide-containing complexes, and enzymes. In some aspects, polypeptide-TFPI binding is detected indirectly. In this regard, the polypeptide is optionally contacted with an interaction partner that binds the polypeptide of invention without significantly interfering with polypeptide-TFPI binding, and the interaction partner is detected. Exemplary interaction partners include, but are not limited to, antibodies, antigen-binding antibody fragments, anticalins and antibody mimetics, aptamers, streptavidin, avidin, neutravidin, and spiegelmers. Optionally, the interaction partner comprises a detection moiety to facilitate detection of an interaction partner-peptide complex. The TFPI-binding polypeptide is, in some embodiments, modified to facilitate binding of an interaction partner. For example, in one aspect, the TFPI-binding polypeptide is conjugated to biotin, which is bound by an interaction partner comprising streptavidin. An exemplary interaction partner comprises strepavidin fused to horseradish peroxidase, which is detected in, e.g., an ELISA-like assay. Alternatively, the TFPI-binding polypeptide is modified to include an antibody epitope, and binding of the corresponding antibody to the polypeptide-TFPI complex is detected. Methods of detecting, e.g., antibodies and fragments thereof, are well understood in the art.
Polypeptide-TFPI complexes and interaction partner-peptide complexes are identified using any of a number of methods, such as, but not limited to, biochemical assays (e.g., enzymatic assays) , spectroscopy (e.g., detection based on optical density, fluorescence, FRET, BRET, TR-FRET, fluorescence polarization, electrochemoluminescence, or NMR) , positron emission tomography (PET) , and single Photon Emission Computed Tomography (SPECT) . Detectable moieties that facilitate fluorescence detection of polypeptide-TFPI complexes or interaction  partner-peptide complexes include, but are not limited to, fluorescein, Alexa350, Marina BlueTM, Cascade YellowTM, Alexa405, Pacific BlueTM, Pacific OrangeTM, Alexa430, Alexa488, Oregon488, Alexa500, Oregon514, Alexa 514, Alexa532, Alexa555, Tetramethylrhodamine, Alexa546, Rhodamine B, Rhodamine RedTM-X, Alexa568, Alexa594, TexasTexas Alexa610, Alexa633, Alexa635, Alexa647, Alexa 660, Alexa680, Alexa700, Alexa750, B-Phycoerythrin, R-Phycoerythrin, Allophycocyanin, Cy3, Cy5, TAMRA, and fluorescent proteins (GFP and derivatives thereof) . An example of a TFPI-binding polypeptide comprising a fluorescent detection moiety is JBT2454 (FAM-Ttds-FQSKpNVHVDGYFERL-Aib-AKL-NH2 (SEQ ID NO: 4171) ) , which is labeled with 5, 6-carboxyfluoresceine.
Radioactive labels also are used to detect biological materials (e.g., TFPI, TFPI-binding polypeptides, or TFPI-binding polypeptide-TFPI complexes) , and, in some instances, are attached to polypeptides or interaction partners using a chelator, such as (but not limited to) EDTA (ethylene diamine tetra-acetic acid) , DTPA (diethylene triamine pentaacetic acid) , CDTA (cyclohexyl 1, 2-diamine tetra-acetic acid) , EGTA (ethyleneglycol-O, O′-bis (2-aminoethyl) -N, N, N′, N′-tetra-acetic) , HBED (N, N-bis (hydroxybenzyl) -ethylenediamine-N, N′-diacetic acid) , TTHA (triethylene tetramine hexa-acetic acid) , DOTA (1, 4, 7, 10-tetraazacyclododecane-N, N′, N″, N′″-tetra-acetic acid) , HEDTA (hydroxyethyldiamine triacetic acid) , or TETA (1, 4, 8, 11-tetra-azacyclotetradecane-N, N, N″, N″-tetra-acetic acid) . Examples of radioactive labels include <99m>Tc, <203>Pb, <66>Ga, <67>Ga, <68>Ga, <72>As, <111>In, <113m>In, <97>Ru, <62>Cu, <64>Cu, <52>Fe, <52m>Mn, <51>Cr, <186>Re, <188>Re, <77>As, <90>Y, <67>Cu, <169>Er, <117m>Sn, <121>Sn, <127>Te, <142>Pr, <143>Pr, <198>Au, <199>Au, <149>Tb, <161>Tb, <109>Pd, <165>Dy, <149>Pm, <151>Pm, <153>Sm, <157>Gd, <166>Ho, <172>Tm, <169>Yb, <175>Yb, <177>Lu, <105>Rh and <111>Ag. Paramagnetic metals also are detectable moieties that are suitable for attachment to TFPI-binding polypeptides or interaction partners, optionally via chelator complex. Examples of paramagnetic metals include, for example, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho, and Er.
TFPI-binding polypeptides, themselves, are, in some aspects, modified to include one or more amino acids with detectable substituents or nuclides. In this regard, in one embodiment, the TFPI-binding polypeptide comprises at least one amino acid comprising a detectable isotope (e.g., 13C, 14C, 35S, 3H, 18O or 15N) , and/or an amino acid that is halogenated with, e.g., <123>I, <124>I, <125>I, <131>I, <75>Br, <76>Br, <77>Br or <82>Br. Amino acids suitable for halogenation include, but are not limited to, tyrosine and tryptophan.
The invention also provides a method for diagnosing a subject suffering from a disease or disorder, or at risk of suffering from a disease or disorder, wherein the disease or disorder is associated with or caused by aberrant TFPI activity. The method comprises administering to the subject the TFPI-binding polypeptide and detecting the TFPI-peptide complex. In some instances, the polypeptide is conjugated to a detectable moiety, and the method comprises detecting the detectable moiety. Exemplary detectable moieties are described herein. In other instances, the method comprises administering to the subject a TFPI-binding polypeptide interaction partner that binds the TFPI-binding polypeptide, and detecting the interaction partner. If desired, the interaction partner comprises or is conjugated to a detectable moiety, and the detectable moiety is detected. The presence of the detectable moiety indicates the presence of TFPI, thereby allowing diagnosis of a disease or disorder associated with TFPI (e.g., a disease or disorder which (i) can be treated by inhibiting TFPI or (ii) comprises symptoms which can be ameliorated or prevented by inhibiting TFPI) . If administration of the polypeptide to the subject is not desired, a biological sample is obtained from the subject, contacted with the TFPI-binding polypeptide as described herein, and TFPI-peptide complexes are detected.
The polypeptides of the invention bind TFPI and, therefore, are useful for purifying TFPI or recombinant TFPI from a biological sample (e.g., a biological fluid, such as serum) , fermentation extract, tissue preparations, culture medium, and the like. The invention includes methods of using the TFPI-binding polypeptide in the commercial production of TFPI or in a method of characterizing TFPI molecules. For example, the invention includes a method of purifying TFPI. The method comprises contacting a sample containing TFPI with a polypeptide as defined herein under conditions appropriate to form a complex between TFPI and the polypeptide; removing the complex from the sample; and, optionally, dissociating the complex to release TFPI. Exemplary conditions appropriate to form a complex between TFPI and the polypeptide are disclosed in the Examples, and such conditions can be easily modified to dissociate the TFPI-peptide complex. In some embodiments, the polypeptide is immobilized to a support, e.g., a solid support, to facilitate recovery of TFPI. For example, in one embodiment, the polypeptide is immobilized to chromatography stationary phase (e.g., silica, affinity chromatography beads, or chromatography resins) , a sample comprising TFPI is applied to the stationary phase such that TFPI-peptide complexes are formed, the remainder of the sample is removed from the stationary phase, and TFPI is eluted from the stationary phase. In this regard, the polypeptides of the invention are, in one aspect, suitable for use in affinity chromatography techniques.
A method of enhancing thrombin formation in a clotting factor-deficient subject also is provided. The method comprises administering to the subject a polypeptide provided herein under conditions effective to inhibit TFPI. In this regard, the TFPI-inhibitory polypeptide is  administered in an amount and under conditions effective to enhance thrombin formation in the subject. By “clotting factor-deficient” is meant that the subject suffers from a deficiency in one or more blood factors required for thrombin formation, such as FVIII, FIX, or FXI. Indeed, in one embodiment, the subject is deficient in FVIII. Alternatively or in addition, the subject is deficient in Factor IX. Clotting factor deficiencies are identified by examining the amount of factor in a clinical sample. Practitioners classify hemophilia according to the magnitude of clotting factor deficiency. Subjects suffering from mild hemophilia have approximately 5%to 30%of the normal amount (1 U/ml) of Factor VIII or Factor IX. Moderate hemophilia is characterized by approximately 1%to 5%of normal Factor VIII, Factor IX, or Factor XI levels, while subjects suffering from severe hemophilia have less than 1%of the normal amount of Factor VIII, Factor IX, or Factor XI. Deficiencies can be identified indirectly by activated partial thromboplastin time (APTT) testing. APTT testing measures the length of time required for a blood clot to form, which is longer for patients with Factor VIII Deficiency (hemophilia A) , Factor IX Deficiency (hemophilia B) , and Factor XI Deficiency (hemophilia C) compared to patients with normal clotting factor levels. Almost 100%of patients with severe and moderate Factor VIII deficiency can be diagnosed with an APTT. The invention further includes enhancing thrombin formation in a subject that does not suffer from a clotting factor deficiency. The method comprises administering to a subject (e.g., a subject comprising normal, physiological levels of clotting factor) a polypeptide provided herein under conditions effective to enhance thrombin formation.
In one aspect, the TFPI-inhibitory polypeptide is used for increasing blood clot formation in a subject. The method of increasing blood clot formation comprises administering to the subject a polypeptide described herein in an amount and under conditions effective to increase blood clot formation. It will be appreciated that the method need not completely restore the coagulation cascade to achieve a beneficial (e.g., therapeutic) effect. Any enhancement or increase in thrombin or blood clot formation that reduces the onset or severity of symptoms associated with clotting factor deficiencies is contemplated. Methods of determining the efficacy of the method in promoting thrombin formation and blood clotting are known in the art and described herein.
The invention further includes a method of treating a blood coagulation disorder in a subject, the method comprising administering to the subject one or more TFPI-inhibitory polypeptides, such as any one or more of the polypeptides described herein, in an amount and under conditions effective to treat the blood coagulation disorder in the subject. In one aspect, the polypeptide is a recombinant or synthetic polypeptide that inhibits TFPI activity. “Coagulation disorders” include bleeding disorders caused by deficient blood coagulation factor activity and deficient platelet activity. Blood coagulation factors include, but are not limited to, Factor V (FV) , FVII, FVIII, FIX, FX, FXI, FXIII, FII (responsible for hypoprothrombinemia) , and von Willebrand's factor.  Factor deficiencies are caused by, for instance, a shortened in vivo-half life of the factor, altered binding properties of the factor, genetic defects of the factor, and a reduced plasma concentration of the factor. Coagulation disorders can be congenital or acquired. Potential genetic defects include deletions, additions and/or substitution within a nucleotide sequence encoding a clotting factor whose absence, presence, and/or substitution, respectively, has a negative impact on the clotting factor's activity. Coagulation disorders also stem from development of inhibitors or autoimmunity (e.g., antibodies) against clotting factors. In one example, the coagulation disorder is hemophilia A. Alternatively, the coagulation disorder is hemophilia B or hemophilia C.
Platelet disorders are caused by deficient platelet function or abnormally low platelet number in circulation. Low platelet count may be due to, for instance, underproduction, platelet sequestration, or uncontrolled patent destruction. Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other drug therapy, radiation therapy, surgery, accidental blood loss, and other disease conditions. Exemplary disease conditions that involve thrombocytopenia are: aplastic anemia; idiopathic or immune thrombocytopenia (ITP) , including idiopathic thrombocytopenic purpura associated with breast cancer; HIV-associated ITP and HIV-related thrombotic thrombocytopenic purpura; metastatic tumors which result in thrombocytopenia; systemic lupus erythematosus, including neonatal lupus syndrome splenomegaly; Fanconi's syndrome; vitamin B12 deficiency; folic acid deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronic liver disease; myelodysplastic syndrome associated with thrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute profound thrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmune thrombocytopenia, including maternal alloimmune thrombocytopenia; thrombocytopenia associated with antiphospholipid antibodies and thrombosis; autoimmune thrombocytopenia; drug-induced immune thrombocytopenia, including carboplatin-induced thrombocytopenia and heparin-induced thrombocytopenia; fetal thrombocytopenia; gestational thrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidental and/or massive blood loss; myeloproliferative disorders; thrombocytopenia in patients with malignancies; thrombotic thrombocytopenia purpura, including thrombotic microangiopathy manifesting as thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in cancer patients; post-transfusion purpura (PTP) ; autoimmune hemolytic anemia; occult jejunal diverticulum perforation; pure red cell aplasia; autoimmune thrombocytopenia; nephropathia epidemica; rifampicin-associated acute renal failure; Paris-Trousseau thrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematologic changes in stomach cancer; hemolytic uremic syndromes (e.g., uremic conditions in childhood) ; and hematologic manifestations related to viral infection including hepatitis A virus and CMV-associated thrombocytopenia. Platelet  disorders also include, but are not limited to, Von Willebrand Disease, paraneoplastic platelet dysfunction, Glanzman's thrombasthenia, and Bernard-Soulier disease. Additional bleeding disorders amenable to treatment with a TFPI-inhibitory polypeptide include, but are not limited to, hemorrhagic conditions induced by trauma; a deficiency in one or more contact factors, such as FXI, FXII, prekallikrein, and high molecular weight kininogen (HMWK) ; vitamin K deficiency; a fibrinogen disorder, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia; and alpha2-antiplasmin deficiency. In one embodiment, the TFPI-inhibitory polypeptide is used to treat excessive bleeding, such as excessive bleeding caused by surgery, trauma, intracerebral hemorrhage, liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, hypothermia, menstruation, pregnancy, and Dengue hemorrhagic fever. All of the above are considered “blood coagulation disorders” in the context of the disclosure.
In one aspect, the TFPI-inhibitory polypeptide of the invention is used to reverse the effects (in whole or in part) of one or more anticoagulants in a subject. Numerous anticoagulants are known in the art and include, for instance, heparin; coumarin derivatives, such as warfarin or dicumarol; TFPI; AT III; lupus anticoagulant; nematode anticoagulant polypeptide (NAPc2) ; FVIIa inhibitors; active-site blocked FVIIa (FVIIai) ; active-site blocked FIXa (FIXai) ; FIXa inhibitors; FXa inhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906) ; active-site blocked FXa (FXai) ; inhibitors of FVa or FVIIIa, including activated protein C (APC) and soluble thrombomodulin; thrombin inhibitors, including hirudin, bivalirudin, argatroban, and ximelagatran; and antibodies or antibody fragments that bind a clotting factor (e.g., FV, FVII, FVIII, FIX, FX, FXIII, FII, FXI, FXII, von Willebrand factor, prekallikrein, or high molecular weight kininogen (HMWK) ) .
In view of the above, the invention provides a polypeptide for use in a method for the treatment of a subject, such as a method for the treatment of a disease where the inhibition of TFPI is beneficial. In one aspect, the disease or disorder is a blood coagulation disorder. The subject is suffering from a disease or disorder or is at risk from suffering from a disease or disorder (or adverse biological event, such as excessive blood loss) . The method comprises administering to the subject the polypeptide of the invention in an amount and under conditions effective to treat or prevent, in whole or in part, the disease or disorder. The invention further provides a polypeptide for use in the manufacture of a medicament. For example, the polypeptide can be used in the manufacture of a medicament for the treatment of a blood coagulation disorder, as described in detail herein.
In some embodiments, it is advantageous to administer to a subject a nucleic acid comprising a nucleic acid sequence encoding a TFPI-binding polypeptide (e.g., TFPI-inhibitory  polypeptide) of the invention. Such a nucleic acid, in one aspect, is provided instead of, or in addition to, a TFPI-inhibitory polypeptide. Expression vectors, nucleic acid regulatory sequences, administration methods, and the like, are further described herein and in U.S. Patent Publication No. 20030045498.
A particular administration regimen for a particular subject will depend, in part, upon the TFPI-inhibitory polypeptide of the invention used, the amount of TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) administered, the route of administration, the particular ailment being treated, considerations relevant to the recipient, and the cause and extent of any side effects. The amount of polypeptide administered to a subject (e.g., a mammal, such as a human) and the conditions of administration (e.g., timing of administration, route of administration, dosage regimen) are sufficient to affect the desired biological response over a reasonable time frame. Dosage typically depends upon a variety of factors, including the particular TFPI-inhibitory polypeptide employed, the age and body weight of the subject, as well as the existence and severity of any disease or disorder in the subject. The size of the dose also will be determined by the route, timing, and frequency of administration. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art. Purely by way of illustration, in one aspect, the method comprises administering, e.g., from about 0.1 μg/kg to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 1 μg/kg up to about 75 mg/kg; or 5 μg/kg up to about 50 mg/kg; or 10 μg/kg up to about 20 mg/kg. In certain embodiments, the dose comprises about 0.5 mg/kg to about 20 mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of polypeptide. Given the chronic nature of many blood coagulation disorders, it is envisioned that a subject will receive the TFPI-inhibitory polypeptide over a treatment course lasting weeks, months, or years, and may require one or more doses daily or weekly. In other embodiments, the TFPI-inhibitory polypeptide is administered to treat an acute condition (e.g., bleeding caused by surgery or trauma, or factor inhibitor/autoimmune episodes in subjects receiving coagulation replacement therapy) for a relatively short treatment period, e.g., one to 14 days.
Suitable methods of administering a physiologically-acceptable composition, such as a pharmaceutical composition comprising a polypeptide described herein, are well known in the art. Although more than one route can be used to administer a polypeptide, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition is applied or instilled into body cavities, absorbed  through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation. In one aspect, a composition comprising a TFPI-inhibitory polypeptide is administered intravenously, intraarterially, or intraperitoneally to introduce the polypeptide of the invention into circulation. Non-intravenous administration also is appropriate, particularly with respect to low molecular weight therapeutics. In certain circumstances, it is desirable to deliver a pharmaceutical composition comprising the TFPI-inhibitory polypeptide orally, topically, sublingually, vaginally, rectally, pulmonary; through injection by intracerebral (intra-parenchymal) , intracerebroventricular, intramuscular, intra-ocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, or enteral means; by sustained release systems; or by implantation devices. If desired, the TFPI-inhibitory polypeptide is administered regionally via intraarterial or intravenous administration feeding a region of interest, e.g., via the femoral artery for delivery to the leg. In one embodiment, the polypeptide is incorporated into a microparticle as described in, for example, U.S. Pat. Nos. 5,439,686 and 5,498,421, and U.S. Patent Publications 2003/0059474, 2003/0064033, 2004/0043077, 2005/0048127, 2005/0170005, 2005/0142205, 2005/142201, 2005/0233945, 2005/0147689.2005/0142206, 2006/0024379, 2006/0260777, 2007/0207210, 2007/0092452, 2007/0281031, and 2008/0026068. Alternatively, the composition is administered via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device in one aspect is implanted into any suitable tissue, and delivery of the desired molecule is in various aspects via diffusion, timed-release bolus, or continuous administration. In other aspects, the TFPI-inhibitory polypeptide is administered directly to exposed tissue during surgical procedures or treatment of injury, or is administered via transfusion of blood procedures. Therapeutic delivery approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,399,363.
To facilitate administration, the TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) in one embodiment is formulated into a physiologically-acceptable composition comprising a carrier (i.e., vehicle, adjuvant, buffer, or diluent) . The particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the polypeptide, and by the route of administration. Physiologically-acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include without limitation sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468) . Injectable formulations are further described in, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers. eds., pages 238-250  (1982) , and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986) ) . A pharmaceutical composition comprising a polypeptide provided herein is optionally placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents that may be necessary to reconstitute the pharmaceutical composition.
When appropriate, the TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) of the invention is administered in combination with other substances and/or other therapeutic modalities to achieve an additional or augmented biological effect. Co-treatments include, but are not limited to, plasma-derived or recombinant coagulation factors, hemophilia prophylaxis treatments, immunosuppressants, plasma factor-inhibiting antibody antagonists (i.e., anti-inhibitors) , antifibrinolytics, antibiotics, hormone therapy, anti-inflammatory agents (e.g., Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or steroidal anti-inflammatory substances) , procoagulants, and pain relievers. In one aspect, the method is an adjunct therapy to traditional replacement factor treatment regimens involving administration of, e.g., FXIII, FXII, FXI (e.g., (Laboratoire francais du Fractionnement et des Biotechnologies, Les Ulis, France) and FXI concentrate (BioProducts Laboratory, Elstree, Hertfordshire, UK) ) , FX, FIX (e.g., Coagulation Factor IX (Wyeth, Madison, N.J. ) ; SD (Grifols, Los Angeles, Calif. ) ;  (CSL Behring, King of Prussia, Pa. ) ; BEBULIN-VHTM (Baxter, Deerfield, Ill. ) ; SD (Grifols, Los Angeles, Calif. ) ; or PROPLEX TTM (Baxter, Deerfield, Ill. ) ) , FVIII (e.g., ADVATETM (Baxter, Deerfield, Ill. ) ; FS (CSL Behring, King of Prussia, Pa. ) ;  (Wyeth, Madison, N.J. ) , XYNTHATM (Wyeth, Madison, N.J. ) , andFS (Bayer, Pittsburgh, Pa. ) ; (Grifols, Los Angeles, Calif. ) ; HEMOPHIL MTM (Baxter, Deerfield, Ill. ) ; (Talecris Biotherapeutics-USA, Research Triangle Park, N.C. ) ; or MONARC-MTM (Baxter, Deerfield, Ill. ) ) , FVIIa (e.g., FVIIa (Novo Nordisk, Princeton, N.J. ) and FVII concentrate (Baxter Bioscience, Vienna, Austria, or BioProducts Laboratory, Elstree, Hertfordshire, UK) ) , FV, FVa, FII, and/or FIII, to a subject. In some instances, the subject also receives FEIBA VH ImmunoTM (Baxter BioScience, Vienna, Austria) , which is a freeze-dried sterile human plasma fraction with Factor VIII inhibitor bypassing activity. FEIBA VH ImmunoTM contains approximately equal units of Factor VIII inhibitor bypassing activity and Prothrombin Complex Factors (Factors II, VII, IX, and X and protein C) . Other exemplary co-treatments include, but are not limited to, prekallikrein, high molecular weight kininogen (HMWK) , Von Willebrand's factor, Tissue Factor, and thrombin. Alternatively or in addition, the  TFPI-inhibitory polypeptide is co-formulated with one or more different TFPI-inhibitory polypeptides. In one aspect, administration of the TFPI-binding polypeptide allows a reduction in the dose of co-therapeutic required to achieve a desired biological response.
The invention thus includes administering to a subject a TFPI-binding polypeptide (e.g., TFPI-inhibitory polypeptide) of the invention (or multiple TFPI-inhibitory polypeptides) , in combination with one or more additionally suitable substances (s) , each being administered according to a regimen suitable for that medicament. Administration strategies include concurrent administration (i.e., substantially simultaneous administration) and non-concurrent administration (i.e., administration at different times, in any order, whether overlapping or not) of the TFPI-inhibitory polypeptide and one or more additionally suitable agents (s) . It will be appreciated that different components are optionally administered in the same or in separate compositions, and by the same or different routes of administration.
In some embodiments, the polypeptide of the invention is conjugated to a moiety, e.g., a therapeutic or diagnostic moiety, such as the detection moieties and co-treatments described above. Alternatively or in addition, the polypeptide is administered in combination with an interaction partner (e.g., an antibody, antibody fragment, anticalin, aptamer, or spiegelmer) that (a) binds the polypeptide and (b) is therapeutically active and/or is linked to a moiety that provides additional functionality to the interaction partner (e.g., a therapeutic, diagnostic, or detection agent) . Suitable moieties include, but are not limited to, photosensitizers, dyes, radionuclides, radionuclide-containing complexes, enzymes, toxins, antibodies, antibody fragments, and cytotoxic agents, and, in some instances, the moiety possesses therapeutic activity (i.e., achieves an advantageous or desired biological effect) . The polypeptide conjugates or polypeptide-interaction partner pair is suitable for use in any of the methods described herein, such as methods of treating a subject suffering from a disease or disorder or at risk of suffering from a disease or disorder.
Binding of a test compound to the TFPI binding site defined herein is detected using any of a number methods, including the detection methods described herein. An exemplary method for detecting binding employs nuclear magnetic resonance (NMR) to recognize chemical shifts at amino acid residues within the TFPI binding site. Chemical shifts at TFPI amino acid positions 28-30, 32, 46, 47, and 55, and optionally positions 27, 31, 36-38, and 44, denotes interaction of the test compound with these amino acid contact points on TFPI. To determine the presence or absence of chemical shifts at particular amino acids resulting from test compound binding, NMR data obtained from the KD1-test compound complex is compared to NMR data obtained from free KD1 polypeptide. Use of NMR to detect binding between a test compound and TFPI KD1 is further described in the Examples.
Alternatively, binding of a test compound to the TFPI-binding site defined herein is determined indirectly by detecting alterations in the ability of TFPI KD1 to interact with its natural binding partners, e.g., FVIIa or FXa. In this regard, the method comprises contacting the polypeptide comprising TFPI KD1 with FVIIa in the presence of the test compound under conditions that allow binding of KD1 to FVIIa, and KD1-FVIIa binding is compared with KD1-FVIIa binding in the absence of the test compound. Alternatively or in addition, the method comprises contacting the polypeptide comprising TFPI KD1 with FXa in the presence of the test compound under conditions that allow binding of KD1 to FXa, and comparing KD1-FXa binding in the presence of the test compound with KD1-FXa binding in the absence of the test compound. Optionally, the polypeptide comprising KD1 also comprises KD2, and the method comprises contacting the polypeptide with FXa in the presence of a test compound under conditions that allow binding of KD2 to FXa, and KD2-FXa binding is compared with KD2-FXa binding in the absence of the test compound. A decrease in KD1-FVIIa binding, KD1-FXa binding, or KD2-FXa binding in the presence of the test compound (compared to KD1-FVIIa binding, KD1-FXa binding, or KD2-FXa binding in the absence of the test compound) indicates that the test compound is a TFPI-binding compound. The method optionally comprises contacting KD1 and/or KD2 to FVIIa and/or FXa in the absence of the test compound as a reference for comparing binding in the presence of the test compound.
KD binding to FVIIa or FXa is determined and/or quantified using any suitable method for detecting protein-protein interactions, such as the methods described herein using detectable labels. Binding of the test compound to the TFPI binding site is, alternatively, detected using an enzymatic assay. FVIIa or FXa enzymatic activity is a suitable surrogate for evaluating binding of the proteins to TFPI KD1 or KD2; test compounds that bind the TFPI-binding site defined herein inhibit TFPI activity, resulting in increased FVIIa and FXa activity. Enzymatic assays for evaluating FVIIa or FXa activity are described in detail herein.
The invention further includes compounds identified as TFPI-binding compounds in the methods of the invention, as well as compositions comprising one or more identified compounds. Methods for isolating or purifying a compound, such as TFPI-binding compound (e.g., a TFPI-binding polypeptide) identified as described herein are known in the art and described above. In some aspects, TFPI-binding compounds identified as described herein are TFPI inhibitors that downregulate or ablate one or more TFPI activities. In one embodiment, the invention includes a method for purifying a compound that inhibits FXa activity. The method comprises contacting a polypeptide comprising TFPI KD1 with a compound under conditions that allow formation of compound-KD1 complexes, removing unbound compound, and dissociating the compound-KD1 complexes to release the compound, which binds TFPI. Use of  a TFPI inhibitor identified and/or purified as described herein for the manufacture of a medicament, such as a medicament for treating a blood coagulation disorder, is provided, as well as a method for treating a subject suffering from a disease or at risk of suffering from a disease comprising administering the TFPI inhibitor to the subject.
In addition, a method of inhibiting human TFPI is provided, wherein the method comprises contacting human TFPI with an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55. Another aspect of the invention includes a method for treating a subject suffering from a disease or at risk of suffering from a disease. The method comprises administering to the subject an inhibitor that binds human TFPI at a binding site defined by amino acid residues Phe28, Lys29, Ala30, Asp32, Ile46, Phe47, and Ile55. In one aspect, the human TFPI binding site is defined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ile38, Ile46, Phe47, and Ile55, such as a binding site defined by amino acid residues Ala27, Phe28, Lys29, Ala30, Asp31, Asp32, Lys36, Ala37, Ile38, Phe44, Ile46, Phe47, and Ile55. Any inhibitor that contacts the TFPI binding site defined herein and inhibits (downregulates or ablates) one or more TFPI activity is suitable for use in the context of the method. The TFPI inhibitor is, optionally, a TFPI-binding polypeptide, such as a TFPI-binding polypeptide having the characteristics described herein.
Sequence Summary
SEQ ID NO: 1 (full-length TcdB4 protein from Clostridioides difficile strain 8864) :

(underlined part is SEQ ID NO: 3, positions 841-1834 of SEQ ID NO: 1; bolded part is SEQ ID NO: 4, positions 1285-1834 of SEQ ID NO: 1; SEQ ID NO: 13 is positions 842-1834 of SEQ ID NO: 1)
SEQ ID NO: 2 (full-length TcdB2 protein from Clostridioides difficile strain R20291) :

(underlined part is SEQ ID NO: 5, positions 841-1834 of SEQ ID NO: 2; bolded part is SEQ ID NO: 6, positions 1285-1834 of SEQ ID NO: 2; SEQ ID NO: 14 is positions 842-1834 of SEQ ID NO: 2)
SEQ ID NO: 7 (receptor-binding interface of TcdB4, residues 1431-1606 of SEQ ID NO: 1) :
SEQ ID NO: 10 (mouse TFPIβ) :
SEQ ID NO: 11 (human TFPIα) :
SEQ ID NO: 12 (human TFPIβ) :
SEQ ID NO: 15 (K2 domain of human TFPI)
Examples
The invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention.
Methods and Materials
Cell Lines
All cell lines were cultured in DMEM medium plus 10%fetal bovine serum (FBS) and 1%penicillin-streptomycin in a humidified atmosphere of 95%air and 5%CO2 at 37 ℃. HeLa and 293T cells were tested negative for mycoplasma contamination and authenticated via STR profiling (Shanghai Biowing Biotechnology Co. LTD, Shanghai, China) .
Mice
All the animal procedures reported herein were approved by the Institutional Animal Care and Use Committee at Westlake University (IACUC Protocol #19-010-TL) . C57BL/6J mice (male and female, 6-8 weeks) were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd. (Shanghai, China) . The Tfpiβ KO mice were generated in the Laboratory Animal Resources Center of Westlake University. Mice were housed with food and water without limitation and monitored under the care of full-time staff.
cDNA Constructs
DNA fragments encoding TcdB41-841, TcdB4842-1834, TcdB41801-2367, TcdB41285-1834, and TcdB21285-1834 were PCR amplified and cloned into a pET28a vector with a HA-His tag introduced to their C-terminus. Genes encoding human TFPIα, TFPIβ, TFPIK1-GPI, TFPIK2-GPI, TFPI2K1-GPI, TFPI2K2-GPI, AMBPK3-GPI, and mouse Tfpiβ were codon-optimized, synthesized by Genscript (Nanjing, China) , and cloned into a PLVX-IRES-Cherry vector. DNA fragments encoding TFPIK1+K2, TFPIK1, TFPIK2, TfpiK1, TfpiK2, CSPG4R1, and TFPIK2-CSPG4R1 were PCR amplified and cloned into a pCAG or PHLsec vector with a FLAG, Fc-FLAG, Fc-His, or GFP-His tag fused to their C-terminus.
Recombinant Proteins
Recombinant full-length TcdB proteins were expressed in Bacillus subtilis SL401 as described previously (Shen et al., 2020) . TcdB41-841, TcdB4842-1834, TcdB41801-2367, TcdB41285-1834, and TcdB21285-1834 were expressed in E. coli BL21 (DE3) and purified as His-tagged proteins. Recombinant TFPIα-GFP, TFPIK1+K2-GFP, TFPIK1+K2 TFPIK1+K2-Fc, TFPIK1-Fc, TFPIK2-Fc, TfpiK1-Fc, TfpiK2-Fc, CSPG4R1, and TFPIK2-CSPG4R1 were expressed in 293F cells and purified as His or FLAG-tagged proteins.
Genome-wide CRISPR Screen
HeLa CRISPR/Cas9 genome-wide KO library was generated as previously described (Tao et al., 2019; Tao et al., 2016) . The GeCKO v2 library is composed of two sub-libraries (A and B) and contains six gRNAs targeting each gene. 293T cells were used to package the lentiviruses. 48 hours post-transfection, the supernatant of the 293T culture was collected. The HeLa-Cas9  cells were then transduced with the lentiviral library at a multiplicity of infection (MOI) of 0.3 and selected with 2.5 ug/mL puromycin for 4 days. For each CRISPR sub-library, at least 6.7×107 cells were plated onto 15-cm cell culture dishes to ensure sufficient gRNA coverage. The cell library was then added with TcdB4 of indicated concentrations and cultured for 18 hours. The plates were then washed with phosphate buffer saline (PBS) to remove loosely attached cells. The remaining cells were cultured with the toxin-free medium and allowed to grow to ～70%confluence and subjected to the next round of screen. Three rounds of screens were performed with increasing concentrations of TcdB4 (0.045, 0.15, and 0.45 pM, respectively) . Cells from the final round of the screen were collected, and their genomic DNA was extracted using the Blood and Cell Culture DNA mini kit (Qiagen) . DNA fragments containing the gRNA sequences were amplified by PCR using primers lentiGP1_F (SEQ ID NO: 8 AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG) and lentiGP-3_R (SEQ ID NO: 9 ATGAATACTGCCATTTGTCTCAAGATCTAGTTACGC) . The Next-Generation Sequencing (NGS) was performed by a commercial vendor (Novogene) .
Pull-Down Assays
Pull-down assays were performed using Protein A agarose beads (Thermo Fisher Scientific) . Briefly, Fc-tagged TFPI domains were mixed with full-length TcdB4 or TcdB4 fragments of indicated concentrations in 1 ml of PBS. The mixtures were incubated at 4℃ for 30 minutes and co-precipitated by Protein A agarose beads. Beads were washed, pelleted, boiled in SDS sample buffer, and subjected to SDS-PAGE or immunoblot analysis.
Bio-Layer Interferometry Assays
The binding affinities between recombinant TcdB4 and human/mouse TFPI domains were measured by BLI assay using the Octet RED96 system (ForteBio) . All proteins were diluted in PBS. In brief, Fc-tagged human/mouse TFPI domains (10 μg/mL) were immobilized onto Dip and Read Anti-human IgG-Fc biosensors (ForteBio) and balanced with PBS. The biosensors were then exposed to full-length TcdB4, TcdB41285-1834, or TcdB21285-1834, followed by washing (dissociation) with PBS. Binding affinities (Kd) were calculated using the Data Analysis software (ForteBio) .
To analyze sequential bindings of FXa and TcdB4 to TFPI, TFPIK2-Fc (10 μg /mL) was immobilized onto Dip and Read Anti-human IgG-Fc biosensors and balanced with HNBSACa buffer (50mM HEPES, 100mM NaCl, 5mM CaCl2, 0.1%BSA, pH 7.3) . The loaded biosensors were first exposed to 100 nM FXa, balanced again with HNBSACa, and then exposed to 300 nM TcdB41285-1834. Alternatively, the loaded biosensors were first exposed to 300 nM TcdB41285-1834,  balanced with HNBSACa, and then exposed to 100 nM FXa. All biosensors were then washed with HNBSACa.
Example 1: Genome-wide CRISPR-Cas9 screen identifies TFPI as a factor for TcdB4
To identify the receptor for TcdB4, a genome-wide CRISPR-Cas9 screen was conducted in HeLa cells. In brief, cells were transduced with a genome-wide gRNA library (GeCKO v2) targeting 19, 052 human genes (Sanjana et al., 2014) using lentiviruses and subjected to three rounds of selection with increasing concentration of TcdB4 (Figure 1A) . The gRNAs sequences from the surviving cell population were decoded by next-generation sequencing (NGS) . The identified genes were assessed based on fold-enrichment of gRNA reads, NGS reads per gene, and the number of unique gRNAs.
We focused on the top enriched genes with multiple unique gRNAs targeted. In addition to UGP2 and CSPG4, which are two expected top hits, TFPI unexpectedly stood out from the screen with five different gRNAs targeted (Figure 1B) . TFPI is known to regulate the tissue factor-dependent pathway of blood coagulation and mainly exists on the cell membrane and in the extracellular space (Broze and Girard, 2012; Wood et al., 2014) .
Example 2: TcdB4 binds to both TFPI isoforms
In humans, TFPI has two major isoforms, TFPIα and TFPIβ. TFPIα contains three tandem Kunitz-type protease inhibitory (K1, K2, and K3) domains followed by a basic carboxyterminal (C-terminal) region. TFPIβ lacks the K3 and basic C-terminal domains of TFPIα, and instead contains a C-terminal signal peptide that directs cleavage and attachment of a GPI anchor (Figure 2A) .
TFPIβ is a GPI-anchored protein, which is in line with the screen result that multiple GPI anchor biosynthesis genes were targeted. Because TcdB4 also binds CSPG4 (yet inefficiently) for cellular entry and CSPG4 is highly expressed in the HeLa cells (Gupta et al., 2017; Tao et al., 2016) , HeLa CSPG4-/-cells were utilized to minimize the effect of CSPG4 when studying the roles of TFPI in HeLa cells.
The result shows that HeLa CSPG4-/-/TFPI-/-cells were more resistant to TcdB4 than CSPG4-/-cells, while the susceptibility of CSPG4-/-/TFPI-/-cells could be restored by transient transfection of TFPIβ (Figure 2B and 2C) . In addition, transient transfection of TFPIα also restored the susceptibility of CSPG4-/-/TFPI-/-cells to TcdB4 (Figure 2B and 2C) , indicating TFPIα could also mediate the entry of TcdB4.
Consistently, immunoblot analysis showed that overexpression of either TFPIα or TFPIβ mediated robust binding of TcdB4 on the cell surface (Figure 2D) . Collectively, these data  demonstrate that membrane attached TFPI, in both α and β isoforms, serve as cellular receptors for TcdB4.
Example 3: TcdB4 interacts with the Kunitz-2 domain of TFPI
Because both TFPIα and TFPIβ can mediate the binding/entry of TcdB4, it was conjectured that TcdB4 interacts with the Kunitz-1 and Kunitz-2 domains of TFPI (TFPIK1+K2) . Thus, recombinant TFPIK1+K2 fused with human Fc fragment (Figure 3A) were expressed and purified and the competition assay on the HeLa CSPG4-/-cells was performed. TFPIK1+K2 effectively protected the cells from TcdB4 but failed to alleviate the intoxication of TcdB1 (Figure 3B and 4A) , supporting the idea that TcdB4 specifically binds TFPIK1+K2. Also, TcdB4 but not TcdB1 was bound to the recombinant TFPIK1+K2-Fc and co-precipitated by Protein A beads (Figure 4B) .
Kunitz domains are small, disulfide-rich, and α/β fold structural domains that function as protease inhibitors (Ascenzi et al., 2003) . Kunitz domains can be found in many proteins including TFPI, TFPI2, Alpha-1-Microglobulin/Bikunin Precursor (AMBP) , and Amyloid Precursor Protein (APP) . Phylogenic analysis showed that the primary sequence of TFPIK2 is most closely related to TFPIK1 with 67.9%sequence similarity, followed by TFPI2K1 (64.1%similarity) , whereas TFPIK3 is less similar to either TFPIK1 (56.6%similarity) or TFPIK2 (58.5%similarity) (Figure 3C) .
To determine whether TcdB4 binds to TFPIK1 or TFPIK2 and the binding selectivity, we expressed GPI-anchored TFPIK1, TFPIK2, TFPI2K1, or AMBPK3 in the HeLa CSPG4-/-/TFPI-/- cells and then tested their sensitivities to TcdB4. The result showed that transient transfection of only GPI-anchored TFPIK2 could restore the susceptibility of CSPG4-/-/TFPI-/-cells (Figure 3D) , suggesting TcdB4 specifically recognizes the Kunitz-2 domain of TFPI. Consistently, TFPIK2-Fc, but not TFPIK1-Fc, protected the HeLa CSPG4-/-cells in the competition experiment (Figure 4C) .
The binding kinetics between TFPIK1+K2-Fc and TcdB4 was further quantified using the bio-layer interferometry (BLI) assay, which revealed a high affinity with a dissociation constant (Kd) of ～13 nM (Figure 5A) . BLI assay also showed that TcdB4 bound to TFPIK2-Fc with a Kd of ～6 nM, but not to TFPIK1-Fc or human Fc fragment (Figure 3E and 5B) . Similarly, TcdB4 bound to Fc-tagged mouse TFPIK2 with a Kd of ～4 nM (Figure 5C) .
BLI analysis was also performed to detect the TcdB2-TFPI interaction (reference sequence from R20291, an ST01/RT027 strain) and observed that TcdB2 and TcdB21285-1834 bound to Fc-tagged mouse TfpiK2 and human TFPIK2 with respective Kd of ～0.2 and 0.4 μM (Figure 12B-12D) .
In addition, previous studies showed that the K2 domain of TFPI binds to the trypsin-like  substrates such as Factor Xa (FXa) (Brandstetter et al., 1996; Burgering et al., 1997) . Structurally, the K2 domain of TFPI interacts with both FXa and TcdB4 through the same loops (Figure 10B) . Using the BLI assay, we further demonstrated that the prebound of TcdB41285-1834 to TFPI blocked the binding of FXa and vice versa (Figure 6F and 10C) , indicating TcdB4 or its fragments such as TcdB41285-1834 may serve as a potential target for treating blood coagulation related disorders.
Example 4: Cryo-EM structure of TcdB4-TFPI complex
TcdB (～270 kDa) is one of the largest bacterial toxins composed of four functional domains, including a glucosyltransferase domain (GTD) , a cysteine protease domain (CPD) , a transmembrane delivery and receptor-binding domain (DRBD) , and a C-terminal combined repetitive oligopeptides (CROPs) domain (Figure 6A) . To further characterize the detailed interaction between TcdB4 and TFPI, the full-length TcdB4 (as shown in SEQ ID NO: 1) were mixed with TFPIK1+K2 and the complex was then isolated by size-exclusion FPLC. The complex fractions were confirmed by SDS-PAGE and concentrated for Cryo-EM sample preparation (Figure 7) . The two-dimensional class averages of the TcdB4-TFPI complex showed clear structural features (Figure 8) . The final maps of the full-length TcdB4 core region resolution at and the TcdB4-TFPI complex at resolution under neutral pH conditions reveal most side-chain densities and secondary structural elements (Figure 6B and 9) .
The overall architecture of TcdB4 is similar to the previously determined structure of TcdB (Chen et al., 2019; Simeon et al., 2019) . The CPD and GTD form an integrated region while the CROPs domain module curve around the core region by forming like a big hook (Figure 6B) . The Kunitz-2 domain of TFPI is bound to the convex edge of the DRBD (residues 841-1834) largely through two flexible loops in TFPI: loop-1 (residues 131-138) and loop-2 (residues 155-162) (Figure 6B and 6C) . Each loop engages TcdB4 via an extensive network composed of hydrogen bonds and hydrophobic interactions (Figure 6D) . The interaction between TcdB4DRBD and TFPIK2 was further validated by the pulldown assay (Figure 10A) .
Furthermore, point mutations at L1599, K1435, L1434, V1492, L1494, M1438, and D1468 in TcdB4 abolish the TFPI-binding ability, suggesting that these positions are important for TFPI receptor recognition (Figure 6E) .
Example 5: Determination of receptor-binding specificities of TcdB variants
The resolved structure of TcdB4-TFPI, together with the previously reported structure of TcdB1-FZD2 (Chen et al., 2018) , revealed an evolutionarily functional region in TcdB for receptor recognition. To predict the receptor specificities of divergent TcdB sequences, we  performed a phylogenetic analysis of the receptor-binding interface (RBI) of TcdB (residues 1431-1606, Figure 11A) .
The phylogenetic tree is clustered into two major branches (denoted as Class I and II) . Class I RBIs derive from TcdB1, TcdB3, and TcdB5, which prefer to bind FZDs. Class II RBIs were composed of sequences from TcdB2, TcdB4, TcdB6, and TcdB7 (Figure 11A) , mainly existing in clade 2 C. difficile (Figure 11B) . A small number of RBIs from TcdB2 and TcdB4 were found either in Class I or as outliers, possibly due to the historically frequent recombination events among TcdB variants (Knight et al., 2021; Mansfield et al., 2020; Shen et al., 2020) .
Albeit FZD2 and TFPI are structurally very different, they both engage the same hydrophobic interface located on the convex edge of TcdBRBI (Figure 11C) . Several residues, including but not limited to L1434, M1438, L1494, and Y1510 (positions in TcdB4; residues in TcdB1 shift to the left by one position in the alignment) , are shared by Class I and Class II RBIs and contribute to both FZD and TFPI interactions. In the TcdB1-FZD2 complex, the interaction is bridged by a palmitoleic acid (PAM) with its tail protruding into a hydrophobic pocket in TcdB1. In the TcdB4-TFPI complex, the side chain of TFPI R135 is deeply embedded in the same pocket but forms multiple hydrogen bonds with adjacent residues including E1433, D1467, and E1469 from TcdB4 (Figure 11D) . F1597 in TcdB1 is a critical residue stabilizing the middle part of PAM and interacts with the nearby F130 in FZD2 (Chen et al., 2018; Peng et al., 2019) , while a Phe to Ser substitution mimicking TcdB2 and TcdB4 abolishes FZD binding (Henkel et al., 2020) . Remarkably, S1598 in TcdB4 forms a close hydrogen bond with t he nearby R140 in TFPI (Figure 11E) . Thus, an F1598S substitution for TcdB may impair FZD-binding but contribute to TFPI-binding. Accordantly, Phe is conserved in all Class I RBIs while Ser is conserved in all Class II RBIs at this position.
The potential TFPI-binding interface in TcdB2 resembles TcdB4 (Figure 11F) , providing a structural clue that TcdB2 may also recognize TFPI. As expected, the HeLa CSPG4-/-/TFPI-/-cells are more resistant to TcdB2 than its parental CSPG4-/-cells (Figure 11G) , while its susceptibility to TcdB2 could be restored by ectopic expressing a GPI-anchored TfpiK2 (Figure 11H) . In addition, TfpiK2-Fc effectively protected the HeLa CSPG4-/-cells from TcdB2 intoxication (Figure 11I and 12A) . We also performed BLI analysis to detect the TcdB2-TFPI interaction (reference sequence from R20291, an ST01/RT027 strain) and observed that TcdB2 bound to Fc-tagged mouse TfpiK2 and human TFPIK2 with respective Kd of ～0.2 and 0.4 μM (Figure 12B-12D) .
There are a few different residues between TcdB2 and TcdB4 within the RBI (Figure 13A) . To interrogate the residues in TcdB2 that cause reduced binding of TFPI, we generated a series of TcdB41285-1834 mutants by replacing the residues with the corresponding ones in TcdB2. The  pulldown experiment showed that I1496S is a key mutation attenuating the TFPI binding, while substitutions P1506L, Y1510C, and K1597N/K1600Q slightly reduce the interaction (Figure 11J) . On the other hand, an S1496I substitution within TcdB21285-1834 enhanced its binding affinity to both human and mouse TFPI (Figure 13B and 13C) .
References:
1. Broze, G.J., Jr., and Girard, T.J. (2012) . Tissue factor pathway inhibitor: structure-function. Front Biosci (Landmark Ed) 17, 262-280.3926 [pii] 10.2741/3926.
2. Maroney, S.A., Ellery, P.E., and Mast, A.E. (2010) . Alternatively spliced isoforms of tissue factor pathway inhibitor. Thromb. Res. 125 Suppl 1, S52-56. 10.1016/j. thromres. 2010.01.038 S0049-3848 (10) 00091-5 [pii] .
3. Maroney, S.A., and Mast, A.E. (2015) . New insights into the biology of tissue factor pathway inhibitor. J Thromb Haemost 13 Suppl 1, S200-207. 10.1111/jth. 12897.
4. Mast, A.E. (2016) . Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein. Atertio. Thromb. Vasc. Biol. 36, 9-14. 10.1161/ATVBAHA. 115.305996 ATVBAHA. 115.305996 [pii] .
5. Chen, P., Zeng, J., Liu, Z., Thaker, H., Wang, S., Tian, S., Zhang, J., Tao, L., Gutierrez, C.B., Xing, L., et al. (2021) . Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection. Nat Commun 12, 3748. 10.1038/s41467-021-23878-310.1038/s41467-021-23878-3 [pii] .
6. Sanjana, N.E., Shalem, O., and Zhang, F. (2014) . Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783-784.10.1038/nmeth. 3047.
7. Wood, J.P., Ellery, P.E., Maroney, S.A., and Mast, A.E. (2014) . Biology of tissue factor pathway inhibitor. Blood 123, 2934-2943.10.1182/blood-2013-11-512764 blood-2013-11-512764 [pii] .
8. Gupta, P., Zhang, Z., Sugiman-Marangos, S.N., Tam, J., Raman, S., Julien, J.P., Kroh, H.K., Lacy, D.B., Murgolo, N., Bekkari, K., et al. (2017) . Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants. J Biol Chem 292, 17290-17301. 10.1074/jbc. M117.806687
9. Tao, L., Zhang, J., Meraner, P., Tovaglieri, A., Wu, X., Gerhard, R., Zhang, X., Stallcup, W.B., Miao, J., He, X., et al. (2016) . Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature 538, 350-355.10.1038/nature19799
10. Knight, D.R., Imwattana, K., Kullin, B., Guerrero-Araya, E., Paredes-Sabja, D., Didelot, X., Dingle, K.E., Eyre, D.W., Rodriguez, C., and Riley, T.V. (2021) . Major genetic discontinuity and novel toxigenic species in Clostridioides difficile taxonomy. Elife 10. 10.7554/eLife. 64325
11. Mansfield, M.J., Tremblay, B.J., Zeng, J., Wei, X., Hodgins, H., Worley, J., Bry, L., Dong, M., and Doxey, A.C. (2020) . Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B. PLoS Pathog 16, e1009181. 10.1371/journal. ppat. 1009181
12. Shen, E., Zhu, K., Li, D., Pan, Z., Luo, Y., Bian, Q., He, L., Song, X., Zhen, Y., Jin, D., and Tao, L. (2020) . Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B. Communications Biology 3, 347. 10.1038/s42003-020-1078-y10.1038/s42003-020-1078-y [pii] .
13. Chen, S., McMullan, G., Faruqi, A.R., Murshudov, G.N., Short, J.M., Scheres, S.H., and Henderson, R. (2013) . High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24-35. 10.1016/j. ultramic. 2013.06.004
14. Peng, Z., Simeon, R., Mitchell, S.B., Zhang, J., Feng, H., and Chen, Z. (2019) . Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027. mSphere 4. e00596-19 [pii]
15. Henkel, D., Tatge, H., Schottelndreier, D., Tao, L., Dong, M., and Gerhard, R. (2020) . Receptor Binding Domains of TcdB from Clostridioides difficile for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive. Toxins (Basel) 12. E736 [pii]
16. Uhlen, M., Fagerberg, L., Hallstrom, B.M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, A., Kampf, C., Sjostedt, E., Asplund, A., et al. (2015) . Proteomics. Tissue-based map of the human proteome. Science 347, 1260419. 10.1126/science. 1260419
17. Ascenzi, P., Bocedi, A., Bolognesi, M., Spallarossa, A., Coletta, M., De Cristofaro, R., and Menegatti, E. (2003) . The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) : a milestone protein. Curr Protein Pept Sci 4, 231-251. 10.2174/1389203033487180.
18. Chen, P., Lam, K.H., Liu, Z., Mindlin, F.A., Chen, B., Gutierrez, C.B., Huang, L., Zhang, Y., Hamza, T., Feng, H., et al. (2019) . Structure of the full-length Clostridium difficile toxin B. Nat Struct Mol Biol 26, 712-719. 10.1038/s41594-019-0268-0
19. Simeon, R., Jiang, M., Chamoun-Emanuelli, A.M., Yu, H., Zhang, Y., Meng, R., Peng, Z., Jakana, J., Zhang, J., Feng, H., and Chen, Z. (2019) . Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B. PLoS Biol 17, e3000311.10.1371/journal. pbio. 3000311
20. Zhang, J., Piro, O., Lu, L., and Broze, G. J., Jr. (2003) . Glycosyl phosphatidylinositol anchorage of tissue factor pathway inhibitor. Circulation 108, 623-627. 10.1161/01. CIR. 0000078642.45127.7B 01. CIR. 0000078642.45127.7B [pii] .

Claims (23)

  1. An isolated polypeptide that binds to Tissue Factor Pathway Inhibitor (TFPI) , wherein the polypeptide comprises:
    a functional fragment of wild type TcdB4 protein or TcdB2 protein that retains the binding to TFPI; or
    a variant of the functional fragment that retains the binding to TFPI, wherein the amino acid sequence of the variant is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of the functional fragment.
  2. The isolated polypeptide of claim 1, wherein the wild type TcdB4 protein is as shown in SEQ ID NO: 1, and/or the wild type TcdB2 protein is as shown in SEQ ID NO: 2.
  3. The isolated polypeptide of claim 2, wherein the polypeptide comprises:
    (a) an amino acid sequence having at least 80%, at least 85%, or at least 90%identity to the amino acid sequence as shown in SEQ ID NO: 3 or 4;
    (b) an amino acid sequence having at least 80%, at least 85%, or at least 90%identity to the amino acid sequence as shown in SEQ ID NO: 5 or 6;
    (c) N-terminal and/or C-terminal truncated amino acid sequence of the amino acid sequence of (a) or (b) , for example, truncated by 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid; or
    (d) N-terminal and/or C-terminal extended amino acid sequence of the amino acid sequence of (a) or (b) , for example, extended by 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5 , 4, 3, 2 or 1 amino acid at corresponding positions of SEQ ID NO: 1 or 2.
  4. The isolated polypeptide of any of claims 1-3, wherein the polypeptide comprises one or more of the following residues corresponding to SEQ ID NO: 1: E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 and Y1510.
  5. The isolated polypeptide of claim 4, wherein the polypeptide further comprises one or more of the following residues corresponding to SEQ ID NO: 1: L1434, K1435, M1438, K1597, S1598, L1599 and K1600 as shown in SEQ ID NO: 1.
  6. The isolated polypeptide of any of the preceding claims, wherein the functional fragment comprises or consists of any of SEQ ID NOs: 3-7 and 13.
  7. The isolated polypeptide of any of the preceding claims, wherein the variant of the functional fragment comprises at least one mutation (e.g. substitution, insertion or deletion) in order to improve the binding to TFPI.
  8. The isolated polypeptide of any of the preceding claims, which binds to TFPI with a dissociation constant of less than 10 μM, less than 1 μM, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM or less than 5 nM, as measured by bio-layer interferometry (BLI) assay.
  9. The isolated polypeptide of any of the preceding claims, which specifically binds to the K2 domain but not K1 domain of TFPI.
  10. A fusion protein, comprising the isolated polypeptide of any of claims 1-9 fused to a heterogenous polypeptide, such as a heterogenous polypeptide that enhances the half-life of the fusion protein.
  11. The fusion protein of claim 10, wherein the heterogenous polypeptide is an IgG Fc portion, optionally comprising a hinge region.
  12. A conjugate, comprising the polypeptide of any of claims 1-9 conjugated to a moiety, such as polyethylene glycol (PEG) moiety, a cytotoxic moiety, a small molecule drug, human serum albumin (HSA) , an antibody or fragment thereof, hydroxyethyl starch, a multimer comprising proline, alanine, serine, or a combination thereof (PASylation) , or a C12-C18 fatty acid.
  13. An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the isolated polypeptide of any of claims 1-9 or the fusion protein of claim 10.
  14. A vector comprising the nucleic acid molecule of claim 13.
  15. A host cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14.
  16. A pharmaceutical composition comprising the isolated polypeptide of any of claims 1-9 and a pharmaceutically acceptable carrier.
  17. A method for detecting TFPI in a sample, the method comprising contacting the sample with the isolated polypeptide of any of claims 1-9.
  18. A method for treating a subject suffering from a TFPI-related or TFPI-mediated disorder or being at risk of suffering from such a disorder, the method comprising administering to the subject the isolated polypeptide of any of claims 1-9 or the pharmaceutical composition of claim 16.
  19. The method of claim 18, wherein the TFPI-related or TFPI-mediated disorder is a blood coagulation disorder, such as hemophilia.
  20. A method for purifying TFPI, wherein the method comprises
    a) contacting a sample containing TFPI with the polypeptide of any of claims 1-8 under conditions appropriate to form a complex between TFPI and the polypeptide;
    b) removing the complex from the sample; and
    c) optionally, dissociating the complex to release TFPI.
  21. A method of inhibiting or antagonizing TFPI activity in a subject, the method comprising administering the isolated polypeptide of any of claims 1-9 or the pharmaceutical composition of claim 16 to the subject.
  22. Use of the isolated polypeptide of any of claims 1-9 in the manufacture of a medicament for treating or preventing a blood coagulation disorder.
  23. A kit, wherein the kit comprises a container comprising the polypeptide of any of claims 1-9.
PCT/CN2023/073679 2022-01-30 2023-01-29 Tfpi binding polypeptides and uses thereof WO2023143559A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2022/075170 2022-01-30
CN2022075170 2022-01-30

Publications (1)

Publication Number Publication Date
WO2023143559A1 true WO2023143559A1 (en) 2023-08-03

Family

ID=87470701

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/073679 WO2023143559A1 (en) 2022-01-30 2023-01-29 Tfpi binding polypeptides and uses thereof

Country Status (1)

Country Link
WO (1) WO2023143559A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110318356A1 (en) * 2008-12-22 2011-12-29 Novo Nordisk A/S Antibodies Against Tissue Factor Pathway Inhibitor
US20120028901A1 (en) * 2010-03-19 2012-02-02 Baxter International Inc. Tfpi inhibitors and methods of use
US20150044250A1 (en) * 2012-01-27 2015-02-12 Intervet Inc. Vaccines against clostridium difficile comprising recombinant toxins
US20150093389A1 (en) * 2012-04-04 2015-04-02 The Secretary Of State For Health Clostridium difficile antigens

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110318356A1 (en) * 2008-12-22 2011-12-29 Novo Nordisk A/S Antibodies Against Tissue Factor Pathway Inhibitor
US20120028901A1 (en) * 2010-03-19 2012-02-02 Baxter International Inc. Tfpi inhibitors and methods of use
US20150044250A1 (en) * 2012-01-27 2015-02-12 Intervet Inc. Vaccines against clostridium difficile comprising recombinant toxins
US20150093389A1 (en) * 2012-04-04 2015-04-02 The Secretary Of State For Health Clostridium difficile antigens

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AKTORIES KLAUS: "Another surprise in receptor binding of C. difficile toxins", THE INNOVATION, vol. 3, no. 4, 1 July 2022 (2022-07-01), pages 100261, XP093081023, ISSN: 2666-6758, DOI: 10.1016/j.xinn.2022.100261 *
CHEN PENG; LAM KWOK-HO; LIU ZHENG; MINDLIN FRANK A.; CHEN BAOHUA; GUTIERREZ CRAIG B.; HUANG LAN; ZHANG YONGRONG; HAMZA THERWA; FEN: "Structure of the full-lengthClostridium difficiletoxin B", NATURE STRUCTURAL & MOLECULAR BIOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 26, no. 8, 15 July 2019 (2019-07-15), New York , pages 712 - 719, XP036852000, ISSN: 1545-9993, DOI: 10.1038/s41594-019-0268-0 *
DATABASE Protein 26 March 2021 (2021-03-26), ANONYMOUS : "glycosylating toxin TcdB [Clostridioides difficile]", XP093081021, retrieved from NCBI Database accession no. WP_009888442.1 *
DATABASE UniProtKB/Swiss-Prot 29 September 2021 (2021-09-29), ANONYMOUS : "RecName: Full=Toxin B {ECO:0000303", XP093081930, Database accession no. Q9EXR0 *
LUO JIANHUA; YANG QI; ZHANG XIAOFENG; ZHANG YUANYUAN; WAN LI; ZHAN XIECHAO; ZHOU YAO; HE LIUQING; LI DANYANG; JIN DAZHI; ZHEN YING: "TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile", CELL, ELSEVIER, AMSTERDAM NL, vol. 185, no. 6, 17 March 2022 (2022-03-17), Amsterdam NL , pages 980, XP086998459, ISSN: 0092-8674, DOI: 10.1016/j.cell.2022.02.010 *
TIAN SONGHAI, XIONG XIAOZHE, ZENG JI, WANG SIYU, TREMBLAY BENJAMIN JEAN-MARIE, CHEN PENG, CHEN BAOHUA, LIU MIN, CHEN PENGSHENG, SH: "Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants", NATURE COMMUNICATIONS, vol. 13, no. 1, 9 November 2022 (2022-11-09), pages 6786, XP093081024, DOI: 10.1038/s41467-022-33964-9 *

Similar Documents

Publication Publication Date Title
US10201586B2 (en) TFPI inhibitors and methods of use
KR102165021B1 (en) Tfpi inhibitors and methods of use
US10800816B2 (en) TFPI inhibitors and methods of use
WO2023143559A1 (en) Tfpi binding polypeptides and uses thereof
US10124033B2 (en) Peptides and methods of use
EA041584B1 (en) PEPTIDES INHIBITING PROTEIN S ACTIVITY AND THEIR USE FOR THE TREATMENT OF BLOOD COAGULATION DISTURBANCES

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23746436

Country of ref document: EP

Kind code of ref document: A1