US20050147618A1 - Clotting factor-Fc chimeric proteins to treat hemophilia - Google Patents

Clotting factor-Fc chimeric proteins to treat hemophilia Download PDF

Info

Publication number
US20050147618A1
US20050147618A1 US10/841,819 US84181904A US2005147618A1 US 20050147618 A1 US20050147618 A1 US 20050147618A1 US 84181904 A US84181904 A US 84181904A US 2005147618 A1 US2005147618 A1 US 2005147618A1
Authority
US
United States
Prior art keywords
factor
chimeric protein
clotting
constant region
immunoglobulin
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US10/841,819
Inventor
Daniel Rivera
Robert Peters
Alan Bitonti
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bioverativ Therapeutics Inc
Original Assignee
Syntonix Pharmaceuticals Inc
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
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=33452235&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20050147618(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Syntonix Pharmaceuticals Inc filed Critical Syntonix Pharmaceuticals Inc
Priority to US10/841,819 priority Critical patent/US20050147618A1/en
Publication of US20050147618A1 publication Critical patent/US20050147618A1/en
Assigned to SYNTONIX PHARMACEUTICALS, INC. reassignment SYNTONIX PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RIVERA, DANIEL S., BITONTI, ALAN J., PETERS, ROBERT T.
Assigned to BIOGEN IDEC INC. reassignment BIOGEN IDEC INC. SECURITY AGREEMENT Assignors: SYNTONIX PHARMACEUTICALS, INC.
Assigned to SYNTONIX PHARMACEUTICALS, INC. reassignment SYNTONIX PHARMACEUTICALS, INC. RELEASE & REASSIGNMENT Assignors: BIOGEN IDEC INC.
Priority to US12/949,564 priority patent/US8449884B2/en
Priority to US13/792,889 priority patent/US8815250B2/en
Assigned to BIOGEN IDEC HEMOPHILIA INC. reassignment BIOGEN IDEC HEMOPHILIA INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: SYNTONIX PHARMACEUTICALS, INC.
Priority to US14/334,396 priority patent/US20150044207A1/en
Assigned to BIOVERATIV THERAPEUTICS INC. reassignment BIOVERATIV THERAPEUTICS INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: BIOGEN HEMOPHILIA INC.
Priority to US15/866,203 priority patent/US20180237762A1/en
Priority to US17/239,208 priority patent/US20210348150A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention relates generally to the field of therapeutics for hemostatic disorders. More specifically, the invention relates to a chimeric protein for the treatment of a hemostatic disorder.
  • Hemostatic disorders are characterized by uncontrolled bleeding resulting from the inability or reduced ability to form fibrin clots. Hemostatic disorders can result from a genetic defect or can be acquired as a result of an unrelated medical condition (e.g., cancer and related chemotherapy, autoimmune disease) (Kasper 2000, Hemophilia 6(Supp):13; Cohen et al. 1996, Bailiere's Clinical Hematology 9(2):331). Typically, hemostatic disorders result from the deficiency of a specific blood clotting factor.
  • an unrelated medical condition e.g., cancer and related chemotherapy, autoimmune disease
  • hemostatic disorders include hemophilia A, which results from a deficiency in factor VIII; hemophilia B (Christmas Disease), which results from a deficiency in factor IX; and von Willebrand's disease, which results in a defect in von Willebrand's factor.
  • Von Willebrand factor circulates in association with factor VIII and stabilizes it. It mediates the adherence of platelets to each other and to injured blood vessel walls.
  • factor XI deficiency PTA deficiency
  • factor XII deficiency As well as deficiencies or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XII (Kasper 2000, Hemophilia 6(Supp): 13).
  • Clotting factors act in concert with one another in a coagulation cascade that ultimately results in the formation of a fibrin clot ( FIG. 1 ). These factors can exist in a quiescent state as a proenzyme or zymogen or in an activated enzymatic state when stimulated to form a clot. Stimulation of these factors can occur by two distinct pathways, the intrinsic pathway and the extrinsic pathway.
  • the intrinsic pathway refers to those reactions that lead to clot formation through utilization of factors present only in the plasma.
  • the extrinsic pathway refers to those reactions that lead to clot formation from release of membrane bound tissue factor upon vessel endothelium disruption.
  • Factor VII participates in both pathways cleaving factor X into the activated factor Xa in conjunction with tissue factor in the extrinsic pathway or interacting with factor IXa in the intrinsic pathway. Factor VII acts down stream and independently of factors VII and IX when acting through the extrinsic pathway, thus bypassing the need for these clotting factors. It is, therefore, an attractive therapeutic candidate for treating hemostatic disorders, especially Hemophilia A and B. (U.S. Pat. No. 6,310,183; WO 01/58935).
  • Factor VII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein with a molecular weight of 53 kDa (Broze et al. 1980, J. Biol. Chem. 255:1242).
  • the factor VII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site, Arg152-IIe153, resulting in two chains, heavy (254 amino acids) and light (154 amino acids) linked by a single disulfide bond (Hagen et al. 1986, Proc. Natl. Acad. Sci. (USA) 83:2412).
  • factor VII binds to tissue factor, which then converts factor X to factor Xa.
  • Factor Xa is required to convert prothrombin to thrombin, which converts fibrinogen to fibrin as a final stage in forming a fibrin clot.
  • Factor VII undergoes post-translational modifications, including vitamin K dependent carboxylation resulting in ten ⁇ -carboxyglutamic acid residues in the N-terminal region of the molecule.
  • Other post translational modifications include sugar moiety attachment at two naturally occurring N-linked glycosylation sites at position 145 and 322, respectively, and at two naturally occurring O-linked glycosylation sites at position 52 and 60, respectively (WO 01/58935).
  • clotting factors must be administered parenterally in order to attain effective doses because to date non-invasive methods have not been successful in attaining therapeutic levels. Problems associated with parenteral administration include injection site occlusion, pain and infection. Use of an in-line catheter increases the risk of all of these events. Specific problems associated with the parenteral administration of clotting factors to infants and small children include central venous access. Additionally, parenteral administration of clotting factors runs the risk of precipitating a bleeding episode. These problems are particularly relevant when the patient is undergoing regular prophylactic administration of a clotting factor.
  • Clotting factors such as factor IX and factor VIII must be given frequently and in large doses resulting in the development of inhibitor antibodies against the clotting factor in a significant number of patients (see, e.g., Nilsson 1992, Transfusion Medicine Review 6(4):285; Cohen et al. 1996, Bailiere's Clinical Hematology 9(2):331).
  • One aspect of the invention provides a safer more effective treatment for hemostatic disorders.
  • Another aspect of the invention provides for increased serum half life and increased bioavailability of therapeutics administered through non-invasive means for the treatment of hemostatic disorders thereby reducing the risk of incurring a bleeding episode, infection and injection site occlusion associated with parenteral administration.
  • Another aspect of the invention provides therapy for hemostatic disorders with reduced risk, compared to current therapies, of developing inhibitor antibodies against the clotting factor.
  • Yet another aspect of the invention provides for a prophylactic treatment of a hemostatic disorder.
  • the aspects of the invention provide for a chimeric protein comprised of at least one clotting factor and at least a portion of an immunoglobulin constant region, wherein the clotting factor is capable of promoting blood coagulation and/or fibrin clot formation.
  • Chimeric proteins comprising an Fc portion of an immunoglobulin are known (see, e.g., U.S. Pat. Nos. 6,030,613, 6,086,875, 6,485,726, and PCT Application No. US/02/21335) and while chimeric proteins comprised of mutant clotting factors without clotting activity, and immunoglobulins have been previously described (WO 01/02439), chimeric proteins comprising a clotting factor (i.e., having clotting activity) and at least a portion of an immunoglobulin constant region have not been described.
  • Clotting factor as defined below and used herein refers to any molecule with clotting activity.
  • the invention relates to an improved chimeric protein for treating hemostatic disorders.
  • the invention provides a chimeric protein to treat a hemostatic disorder that can be administered parenterally or non-invasively (e.g., via a pulmonary, nasal, or oral route).
  • the invention thus relates to a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • the invention relates to a method of treating a hemostatic disorder comprising administering a therapeutically effective amount of a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • the invention relates to a method of making a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region; said method comprising transfecting a cell with a DNA construct comprising a first DNA sequence encoding at least one clotting factor operatively linked to a second DNA sequence encoding at least a portion of an immunoglobulin.; culturing said cell under conditions such that the chimeric protein is expressed; and isolating said chimeric protein from said cell.
  • the invention relates to a nucleic acid molecule comprising a sequence encoding at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • the invention relates to a nucleic acid construct comprising a DNA sequence encoding at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • FIG. 1 is a diagram showing the extrinsic and intrinsic pathway of the coagulation cascade.
  • FIG. 2 is a diagram of the inactive and active forms of factor VIIa-Fc.
  • FIG. 3A is the amino acid sequence of a chimeric protein comprising factor VIIa and an Fc fragment of an immunoglobulin (SEQ ID NO:1).
  • FIG. 3B is the amino acid sequence of an Fc fragment of an immunoglobulin (SEQ ID NO:2).
  • FIG. 3C is the nucleic acid sequence of an Fc fragment of an immunoglobulin (SEQ ID NO:3).
  • FIG. 3D is the nucleic acid sequence of a chimeric protein comprising factor VIIa and an Fc fragment of an immunoglobulin (SEQ ID NO:4).
  • FIG. 4 is a diagram of results from a STA-CLOT assay showing factor VIIa-Fc has significant clotting activity.
  • FIG. 5 is a diagram showing factor VIIa-Fc binds to soluble human Fc neonatal receptor (shFcRn).
  • FIG. 6 demonstrates plasma levels over time for orally administered factor VIIa-Fc in neonatal rats.
  • FIG. 7 compares oral uptake in neonatal rats of Factor VIIa-Fc monomer/dimer hybrids comprising two polypeptide chains, each chain comprising at least a portion of an immunoglobulin constant region and wherein factor VII is linked to only one of the two chains versus homodimers comprising two polypeptide chains wherein both chains comprise at least a portion of an immunoglobulin constant region and both chains also comprise factor VII.
  • FIG. 8A demonstrates plasma levels over time of factor VIIa-Fc administered intravenously to minipigs, wherein the factor VIIa-Fc is a monomer/dimer hybrid comprising two polypeptide chains, each chain comprising at least a portion of an immunoglobulin constant region and wherein factor VII is linked to only one of the two chains.
  • FIG. 8B demonstrates clotting activity over time of factor VIIa-Fc administered intravenously to minipigs wherein the factor VIIa-Fc is a monomer/dimer hybrid comprising two polypeptide chains, each chain comprising at least a portion of an immunoglobulin constant region and wherein factor VII is linked to only one of the two chains.
  • FIG. 9A is the amino acid sequence of the chimeric protein Factor IX-Fc. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell and the propeptide (bold) which is recognized by the vitamin K-dependent ⁇ carboxylase which modifies the Factor IX to achieve full activity. The sequence is subsequently cleaved by PACE to yield Factor IX-Fc.
  • FIG. 9B is the nucleic acid sequence of the chimeric protein Factor IX-Fc. Included in the sequence is the signal peptide (underlined) and the propeptide (bold) which is recognized by the vitamin K-dependent ⁇ carboxylase which modifies the Factor IX to achieve full activity. The translated sequence is subsequently cleaved by PACE to yield mature Factor IX-Fc.
  • Seq ID NO FIG. Description 1 3A amino acid sequence of a chimeric protein comprising factor VIIA and an IgG FC fragment 2 3B amino acid sequence of an FC fragment of IgG 3 3C nucleic acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2 4 3D nucleic acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1
  • Affinity tag means a molecule attached to a second molecule of interest, capable of interacting with a specific binding partner for the purpose of isolating or identifying said second molecule of interest.
  • Analogs of, or proteins or peptides or substantially identical to the chimeric proteins of the invention, as used herein, means that a relevant amino acid sequence of a protein or a peptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a given sequence.
  • sequences may be variants derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. Percent identity between two amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al. (1990) J. Mol.
  • BLAST Basic Local Alignment Tool
  • BLAST 2 Sequences program BLASTN, reward for match 2, penalty for mismatch ⁇ 2, open gap and extension gap penalties 5 and 2 respectively, gap x_dropoff 50, expect 10, word size 11, filter ON.
  • program BLASTP program BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11 and 1 respectively, gap x_dropoff 50, expect 10, word size 3, filter ON.
  • Bioavailability means the extent and rate at which a substance is absorbed into a living system or is made available at the site of physiological activity.
  • a chimeric protein refers to any protein comprised of a first amino acid sequence derived from a first source, bonded, covalently or non-covalently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same.
  • a first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity.
  • a chimeric protein can include for example, a protein derived from at least 2 different biological sources.
  • a biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g.
  • a synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences).
  • a chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source.
  • Clotting factor means any molecule, or analog thereof, naturally occurring or recombinantly produced which prevents or decreases the duration of a bleeding episode in a subject with a hemostatic disorder. In other words, it means any molecule having clotting activity.
  • Clotting activity means the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and/or reduces the severity, duration or frequency of hemorrhage or bleeding episode.
  • DNA construct means a DNA molecule, or a clone of such a molecule, either single- or double-stranded that has been modified through human intervention to contain segments of DNA combined in a manner that as a whole would not otherwise exist in nature.
  • DNA constructs contain the information necessary to direct the expression of polypeptides of interest.
  • DNA constructs can include promoters, enhancers and transcription terminators.
  • DNA constructs containing the information necessary to direct the secretion of a polypeptide will also contain at least one secretory signal sequence.
  • a fragment refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, of at least 5 contiguous amino acid residues, of at least 10 contiguous amino acid residues, of at least 15 contiguous amino acid residues, of at least 20 contiguous amino acid residues, of at least 25 contiguous amino acid residues, of at least 40 contiguous amino acid residues, of at least 50 contiguous amino acid residues, of at least 100 contiguous amino acid residues, or of at least 200 contiguous amino acid residues or any deletion or truncation of a protein.
  • Hemostasis means the stoppage of bleeding or hemorrhage; or the stoppage of blood flow through a blood vessel or body part.
  • Hemostatic disorder means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot.
  • Linked refers to a first nucleic acid sequence covalently joined to a second nucleic acid sequence.
  • the first nucleic acid sequence can be directly joined or juxtaposed to the second nucleic acid sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence.
  • Linked as used herein can also refer to a first amino acid sequence covalently joined to a second amino acid sequence.
  • the first amino acid sequence can be directly joined or juxtaposed to the second amino acid sequence or alternatively an intervening sequence can covalently join the first amino acid sequence to the second amino acid sequence.
  • Linked can also refer to a first amino acid sequence non-covalently joined to a second amino acid sequence.
  • Linked as used herein can also refer to a first amino acid sequence covalently joined to a nucleic acid sequence or a small organic or inorganic molecule.
  • Operatively linked means a first nucleic acid sequence linked to a second nucleic acid sequence such that both sequences are capable of being expressed as a biologically active polypeptide.
  • a small inorganic molecule means a molecule containing no carbon atoms and being no larger than 50 kD.
  • a small organic molecule means a molecule containing at least one carbon atom and being no larger than 50 kD.
  • High stringency includes conditions readily determined by the skilled artisan based on, for example, the length of the DNA. Generally, such conditions are defined as hybridization conditions as above, and with washing at approximately 68° C., 0.2 ⁇ SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.
  • Moderate stringency include conditions that can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA.
  • the basic conditions are set forth by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989), and include use of a prewashing solution for the nitrocellulose filters 5 ⁇ SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6 ⁇ SSC at 42° C. (or other similar hybridization solution, such as Stark's solution, in 50% formamide at 42° C.), and washing conditions of 60° C., 0.5 ⁇ SSC, 0.1 % SDS.
  • Polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term does not exclude post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, pegylation, addition of a lipid moiety, or the addition of any organic or inorganic molecule. Included within the definition, are for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids) and polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • Treat, treatment, treating means any of the following: the reduction in severity of a hemostatic disorder; the prophylaxis of one or more symptoms associated with a hemostatic disorder, e.g., a bleeding episode; the reduction in the duration of a disease course of a hemostatic disorder; the amelioration of one or more symptoms associated with a hemostatic disorder; the reduction in duration of a bleeding episode associated with a hemostatic disorder; the reduction in the titer of an inhibitor antibody against a clotting factor; the provision of beneficial effects to a subject with a hemostatic disorder, without necessarily curing the hemostatic disorder.
  • the invention relates generally to improved therapeutics for hemostatic disorders.
  • the invention thus relates to a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • the chimeric proteins of the invention have increased stability and other improved properties when compared to known therapeutic agents having clotting activity. They can also be administered parenterally or non-invasively. Accordingly, the invention provides for improved methods for administering therapeutic agents having clotting activity. While clotting factors presently are generally administered subcutaneously, intramuscularly or intravenously, the chimeric proteins of the invention can be administered using less invasive means such as oral administration, nasal administration, or pulmonary administration. In a specific embodiment, the chimeric proteins of the invention are useful in the prophylactic treatment of hemostatic disorders.
  • the invention also relates generally to improved methods of making therapeutic clotting factors.
  • the invention relates to recombinant methods of producing therapeutic clotting factors with enhanced expression and improved yields.
  • the invention thus relates to methods of making chimeric proteins comprising at least one clotting factor and at least a portion of an immunoglobulin constant region by transfecting a cell with a DNA construct, said construct comprising a DNA sequence encoding at least one clotting factor and a DNA sequence encoding at least a portion of an immunoglobulin constant region; culturing said cell under conditions such that the chimeric protein is expressed by said cell; and isolating said chimeric protein.
  • the invention relates generally to chimeric proteins comprising at least one clotting factor, at least a portion of an immunoglobulin constant region, and optionally a linker.
  • the clotting factor can be linked covalently, or non-covalently with the portion of an immunoglobulin constant region.
  • the portion of an immunoglobulin constant region will have both an N, or an amino terminus, and a C, or carboxy terminus.
  • the chimeric protein of the invention may have a clotting factor linked to the N terminus of the portion of an immunoglobulin constant region.
  • the chimeric protein can optionally comprise at least one linker, thus the clotting factor does not have to be directly linked to the portion of an immunoglobulin constant region.
  • the linker can intervene in between the clotting factor and the portion of an immunoglobulin constant region.
  • the linker can be linked to the N terminus of the portion of an immunoglobulin constant region, or the C terminus of a portion of an immunoglobulin constant region.
  • the linker can be linked to the N terminus of the clotting factor.
  • the invention thus relates to a chimeric protein comprised of at least one clotting factor (X), optionally, a linker (L), and at least a portion of an immunoglobulin constant region (F).
  • a chimeric protein comprised of the formula X-L a -F wherein X is linked at its C terminus to the N terminus of L, and L is linked at its C terminus to the N terminus of F and wherein a is any integer or zero.
  • a is zero
  • X is directly linked at its C terminus to the N terminus of F.
  • a may be 0, 1, 2, 3, 4, 5, 10 or 20.
  • the invention relates to a chimeric protein comprising the amino acid sequence of FIG. 3A (SEQ ID NO:1).
  • the chimeric protein of the invention includes monomers, dimers, as well as higher order multimers.
  • the chimeric protein is a monomer comprising one clotting factor and one portion of an immunoglobulin constant region.
  • the chimeric protein is a dimer comprising two clotting factors and two portions of an immunoglobulin.
  • the two clotting factors are the same.
  • the two clotting factors are different.
  • the two portions of an immunoglobulin are the same.
  • the two portions of an immunoglobulin are different.
  • the chimeric protein is a monomer/dimer hybrid wherein the chimeric protein has a dimeric aspect in that it is comprised of at least a portion of two immunoglobulin constant region polypeptides and a monomeric aspect in that it is comprised of only one clotting factor linked to one of the two immunoglobulin.
  • the invention thus relates to a chimeric protein comprising a first chain and a second chain, wherein said first chain comprises at least a portion of an immunoglobulin constant region linked to a clotting factor and said second chain comprises at least a portion of an immunoglobulin constant region without a clotting factor linked to it.
  • Such chimeric proteins may be described using the formulas set forth in Table 1, where I, L, and F are as described above, and where (′) indicates a different molecule than without (′) and wherein (:) indicates a non-peptide bond TABLE 1 X-F:F-X X′-F:F-X X-L-F:F-X X-L-F:F-L-X X′-L-F:F-L-X X-L′-F:F-L-X X′-L′-F:F-L-X F:F-X F:F-L-X X-F:F X-L-F:F L-F:F-X X-F:F-L
  • Derivatives and analogs of the chimeric proteins of the invention, antibodies against the chimeric proteins of the invention and antibodies against binding partners of the chimeric proteins of the invention are all contemplated, and can be made by altering their amino acid sequences by substitutions, additions, and/or deletions/truncations or by introducing chemical modifications that result in functionally equivalent molecules. It will be understood by one of ordinary skill in the art that certain amino acids in a sequence of any protein may be substituted for other amino acids without adversely affecting the activity of the protein.
  • the derivative is functionally active, i.e., capable of exhibiting one or more activities associated with the chimeric proteins of the invention, e.g., clot formation, activation of a clotting factor.
  • Activity can be measured by assays known in the art, e.g., StaCLot FVIIa-rTF assay (Johannessen et al. 2000, Blood Coagulation and Fibrinolysis 11 :S1 59) prothrombin time (PT assay) or a APTT assay for factor IX
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 2).
  • various amino acids are commonly substituted with neutral amino acids, e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine (see, e.g., MacLennan et al. (1998) Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al. (1998) Adv. Biophys. 35:1-24).
  • the chimeric proteins of this invention include at least one clotting factor.
  • the clotting factor can include any molecule that has clotting activity or activates a molecule with clotting activity.
  • the clotting factor can be comprised of a polypeptide, a small organic molecule, or a small inorganic molecule.
  • the clotting factor can be a mutated clotting factor or an analog of a clotting factor so long as it maintains at least some clotting activity.
  • the clotting factor can be, as an example, but not as a limitation factor VII, including B domain deleted factor VIII, factor IX (U.S. Pat.
  • the clotting factor is factor VII or factor VIIa.
  • the clotting factor is a mutated factor VII or VIIa (see, e.g., Persson, et al. 2001, Proc. Natl. Acad. Sci. USA 98:13583; U.S. patent application Ser. No.10/109498).
  • the clotting factor can be a factor that participates in the extrinsic pathway.
  • the clotting factor can be a factor that participates in the intrinsic pathway.
  • the clotting factor can be a factor that participates in both the extrinsic and intrinsic pathway.
  • the clotting factor can be a human clotting factor or a non-human clotting factor, e.g., derived from a non-human primate, a pig, or any mammal.
  • the clotting factor can be chimeric clotting factor, e.g., the clotting factor can comprise a portion of a human clotting factor and a portion of a porcine, or any non-human clotting factor or a portion of a first non-human clotting factor and a portion of a second non-human clotting factor.
  • the clotting factor can be an activated clotting factor.
  • the clotting factor can be an inactive form of a clotting factor, e.g., a zymogen.
  • the inactive clotting factor can undergo activation subsequent to being linked to at least a portion of an immunoglobulin constant region.
  • the inactive clotting factor can be activated subsequent to administration to a subject.
  • the inactive clotting factor can be activated prior to administration.
  • the chimeric proteins of this invention include at least a portion of an immunoglobulin constant region.
  • Immunoglobulins are comprised of four protein chains that associate covalently—two heavy chains and two light chains. Each chain is further comprised of one variable region and one constant region.
  • the heavy chain constant region is comprised of 3 or 4 constant region domains (e.g., CH1, CH2, CH3, CH4).
  • Some isotypes can also include a hinge region.
  • the portion of an immunoglobulin constant region can be a portion of an immunoglobulin constant region obtained from any mammal.
  • the portion of an immunoglobulin constant region can include, but is not limited to, a portion of a human immunoglobulin constant region, a non-human primate immunoglobulin constant region, a bovine immunoglobulin constant region, a porcine immunoglobulin constant region, a murine immunoglobulin constant region, an ovine immunoglobulin constant region or a rat immunoglobulin constant region.
  • the portion of an immunoglobulin constant region can include the entire heavy chain constant region, or a fragment or analog thereof.
  • a heavy chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain, and/or a hinge region.
  • a heavy chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain, and/or a CH4 domain.
  • the immunoglobulin can be produced recombinantly or synthetically.
  • the immunoglobulin can be isolated from a cDNA library.
  • the immunoglobulin can be isolated from a phage library (see McCafferty et al. 1990, Nature 348:552).
  • the immunoglobulin can be obtained by gene shuffling of known sequences (Mark et al. 1992, Bio/Technol. 10:779).
  • the immunoglobulin can be isolated by in vivo recombination (Waterhouse et al. 1993, Nucl. Acid Res. 21:2265).
  • the immunoglobulin can be a humanized immunoglobulin (Jones et al. 1986, Nature 332:323).
  • the portion of an immunoglobulin constant region can include a portion of an IgG, an IgA, an IgM, an IgD, an IgE.
  • the immunoglobulin is an IgG.
  • the immunoglobulin is IgG1.
  • the immunoglobulin is IgG2.
  • the portion of an immunoglobulin constant region can include an Fc fragment.
  • An Fc fragment can be comprised of the CH2 and CH3 domains of an immunoglobulin and the hinge region of the immunoglobulin.
  • the Fc fragment can be the Fc fragment of an IgG1, an IgG2, an IgG3 or an IgG4.
  • the immunoglobulin is an Fc fragment of an IgG1.
  • the immunoglobulin is an Fc fragment of an IgG2.
  • the portion of an immunoglobulin constant region is comprised of the amino acid sequence of SEQ ID NO:2 ( FIG. 3B ) or an analog thereof.
  • the immunoglobulin is comprised of a protein, or fragment thereof, encoded by the nucleic acid sequence of SEQ ID NO:3 ( FIG. 3C ).
  • the portion of an immunoglobulin constant region can include an Fc variant.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (WO 97/34631).
  • Native refers to an Fc that has not been modified by a human.
  • WO 96/32478 describes exemplary Fc variants, as well as interaction with the salvage receptor.
  • the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc.
  • a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention.
  • Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • an immunoglobulin constant region is an neonatal Fc receptor (FcRn) binding partner.
  • FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner.
  • the FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377).
  • the FcRn receptor binds IgG (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos. 6,030,613 and 6,086,875) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.
  • IgG immunoglobulin classes such as IgA, IgM, IgD, and IgE
  • FcRn binding partners of the present invention encompass any molecule that can be specifically bound by the FcRn receptor including whole 1gG, the Fc fragment of 1gG, and other fragments that include the complete binding region of the FcRn receptor.
  • the region of the Fc portion of 1gG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379).
  • the major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain.
  • the FcRn binding partners include whole 1gG, the Fc fragment of 1gG, and other fragments of 1gG that include the complete binding region of FcRn.
  • the major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain.
  • References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.
  • the Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn.
  • modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn.
  • Fcy1 human IgG1 Fc
  • P238A S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K32
  • one embodiment incorporates N297A, removing a highly conserved N-glycosylation site.
  • the effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591).
  • At least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG1 “ELLG” to the corresponding sequence from IgG2 “PVA” (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII which mediate various effector functions will not bind to IgG1 when such mutations have been introduced (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613).
  • affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased “on” rate, a decreased “off” rate or both an increased “on” rate and a decreased “off” rate. Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).
  • the FcRn binding partner is a polypeptide including the sequence PKNSSMISNTP and optionally further including a sequence selected from HQSLGTQ, HQNLSDGK, HQNISDGK, or VISSHLGQ (U.S. Pat. No. 5,739,277).
  • FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer.
  • Linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a clotting factor thus provides a means of delivering the clotting factor orally or as an aerosol administered nasally, via an ocular route or via a pulmonary route.
  • portions of an immunoglobulin constant region for use in the chimeric protein of the invention can include mutants or analogs thereof, or can include chemically modified immunoglobulin constant regions or fragments thereof (e.g. pegylation) (see, e.g., Aslam and Dent 1998, Bioconjugation: Protein Coupling Techniques For the Biomedical Sciences Macmilan Reference , London).
  • a mutant can provide for enhanced binding of an FcRn binding partner for the FcRn.
  • peptide mimetics of at least a portion of an immunoglobulin constant region e.g., a peptide mimetic of an Fc fragment or a peptide mimetic of an FcRn binding partner.
  • the peptide mimetic is identified using phage display (See, e.g., McCafferty et al. 1990, Nature 348:552, Kang et al. 1991, Proc. Natl. Acad. Sci. USA 88:4363; EP 0 589 877 B1).
  • the chimeric protein of the invention can optionally comprise at least one linker molecule.
  • the linker is comprised of amino acids.
  • the linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids.
  • the linker can comprise the sequence G n .
  • the linker can comprise the sequence (GGS) n (SEQ ID NO.: 5). In each instance, n may be an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • linkers include, but are not limited to, GGG (SEQ ID NO.: 6), SGGSGGS(SEQ ID NO.: 7), GGSGGSGGSGGSGGG, (SEQ ID NO.: 8) and GGSGGSGGSGGSGGSGGS,. (SEQ ID NO.: 9)
  • the linker does not eliminate the clotting activity of the clotting factor.
  • the linker enhances the clotting activity of the clotting factor, e.g., by diminishing the effects of steric hindrance and making the clotting factor more accessible to its target binding site, e.g., another factor in the clotting cascade.
  • the invention relates to a nucleic acid construct comprising a nucleic acid sequence encoding the chimeric proteins of the invention, said nucleic acid sequence comprising a first nucleic acid sequence encoding, for example at least one clotting factor, operatively linked to a second nucleic acid sequence encoding at least a portion of an immunoglobulin constant region.
  • the nucleic acid sequence can also include additional sequences or elements known in the art (e.g. promoters, enhancers, poly A sequences, signal sequence).
  • the nucleic acid sequence can optionally include a sequence encoding a linker placed between the nucleic acid sequence encoding at least one clotting factor and the portion of an immunoglobulin.
  • the nucleic acid sequence can optionally include a linker sequence placed before or after the nucleic acid sequence encoding at least one clotting factor and the portion of an immunoglobulin.
  • the nucleic acid construct is comprised of DNA. In another embodiment, the nucleic acid construct is comprised of RNA.
  • the nucleic acid construct can be a vector, e.g., a viral vector or a plasmid.
  • viral vectors include, but are not limited to adeno virus vector, an adeno associated virus vector or a murine leukemia virus vector.
  • plasmids include but are not limited to, e.g., pUC and pGEX.
  • the nucleic acid construct comprises the nucleic acid sequence of FIG. 3D (SEQ ID NO:4).
  • a DNA sequence can vary from that shown in SEQ ID NOS:3 or 4 and still encode a polypeptide having the amino acid sequence of SEQ ID NOS:2 or 1 respectively.
  • Such variant DNA sequences can result from silent mutations (e.g. occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.
  • the invention thus provides isolated DNA sequences encoding polypeptides of the invention, selected from: (a) DNA comprising the nucleotide sequence of SEQ ID NO:3 or 4; (b) DNA encoding the polypeptide of SEQ ID NO:1 or 2; (c) DNA capable of hybridization to a DNA of (a) or (b) under conditions of moderate stringency and which encodes polypeptides of the invention; (d) DNA capable of hybridization to a DNA of (a) or (b) under conditions of high stringency and which encodes polypeptides of the invention, and (e) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), (b), (c), or (d) and which encode polypeptides of the invention.
  • polypeptides encoded by such DNA sequences are encompassed by the invention.
  • nucleic acid molecules of the invention also comprise nucleotide sequences that are at least 80% identical to a native sequence. Also contemplated are embodiments in which a nucleic acid molecule comprises a sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native sequence.
  • the percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. 1984, Nucl. Acids Res. 12:387, and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess 1986, Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., 1979, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Other programs used by one skilled in the art of sequence comparison may also be used.
  • Chimeric proteins comprising at least a portion of an immunoglobulin constant region and a clotting factor can be synthesized using techniques well known in the art.
  • the chimeric proteins of the invention can be synthesized recombinantly in cells (see, e.g., Sambrook et al. 1989, Molecular Cloning A Laboratory Manual , Cold Spring Harbor Laboratory, N.Y. and Ausubel et al. 1989, Current Protocols in Molecular Biology , Greene Publishing Associates and Wiley Interscience, N.Y.).
  • DNA sequences encoding immunoglobulins, or fragments thereof, or clotting factors, or fragments thereof, may be cloned from a variety of genomic or cDNA libraries known in the art.
  • the techniques for isolating such DNA sequences using probe-based methods are conventional techniques and are well known to those skilled in the art. Probes for isolating such DNA sequences may be based on published DNA sequences (see, for example, Hieter et al. 1980, Cell 22: 197-207).
  • the polymerase chain reaction (PCR) method disclosed by Mullis et al. (U.S. Pat. No.4,683,195) and Mullis (U.S. Pat. No. 4,683,202) may be used.
  • DNA sequences encoding immunoglobulins, or fragments thereof, or clotting factors can be obtained from vectors known in the art to contain immunoglobulins, or fragments thereof, or clotting factors.
  • a polynucleotide sequence encoding the chimeric protein is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • an appropriate expression vehicle i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • the nucleic acid encoding the chimeric protein is inserted into the vector in proper reading frame.
  • the nucleic acid can also encode for a propeptide clotting factor which is modified by the cell to yield the mature chimeric protein of the invention.
  • the propeptide clotting factor is recognized by a vitamin K-dependent ⁇ carboxylase which modifies the propeptide clotting factor to achieve full activity (e.g. factor VII, factor IX, factor X, prothrombin).
  • an endoprotease e.g., paired basic amino acid cleaving enzyme (PACE), or any PACE family member, such as PCSK1-9, including truncated versions thereof, or its yeast equivalent Kex2 from S.
  • PACE paired basic amino acid cleaving enzyme
  • Kex2 cerevisiae and truncated versions of Kex2 (see, e.g., U.S. Pat. Nos. 5,077,204; 5,162,220; 5,234,830; 5,885,821; 6,329,176 (Kex2 1-675)).
  • the expression vehicle is then transfected into a suitable target cell which will express the protein, e.g., a chimeric protein.
  • Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. 1978, Cell 14:725) and electroporation (Neumann et al. 1982, EMBO, J. 1:841).
  • a variety of host-expression vector systems may be utilized to express the chimeric proteins described herein in eukaryotic cells.
  • the eukaryotic cell is an animal cell, including mammalian cells (e.g. CHO, BHK, Cos, HeLa cells).
  • the DNA encoding the chimeric protein may also code for a signal sequence that will permit the chimeric protein to be secreted.
  • a signal sequence that will permit the chimeric protein to be secreted.
  • the signal sequence is cleaved by the cell to form the mature chimeric protein.
  • Various signal sequences are known in the art e.g., native factor VII signal sequence, native factor IX signal sequence and the mouse IgK light chain signal sequence.
  • the chimeric protein can be recovered by lysing the cells.
  • transgenic animals refers to non-human animals that have incorporated a foreign gene into their genome. Because this gene is present in germline tissues, it is passed from parent to offspring. Exogenous genes are introduced into single-celled embryos (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82:4438). Methods of producing transgenic animals are known in the art, including transgenics that produce immunoglobulin molecules (Wagner et al. 1981, Proc. Natl. Acad. Sci. USA 78:6376; McKnight et al.
  • the expression vectors can encode for tags that permit for easy purification or identification of the recombinantly produced protein.
  • tags include, but are not limited to, vector pUR278 (Ruther et al. 1983, EMBO J. 2:1791) in which the chimeric protein described herein coding sequence may be ligated into the vector in frame with the lac z coding region so that a hybrid protein is produced;
  • pGEX vectors may be used to express proteins with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the vectors include cleavage sites (e.g. PreCission ProteaseTM (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.
  • the polynucleotide can be designed to encode multiple units of the chimeric protein of the invention separated by enzymatic cleavage sites.
  • the resulting polypeptide can be cleaved (e.g. by treatment with the appropriate enzyme) in order to recover the polypeptide units.
  • This can increase the yield of polypeptides driven by a single promoter.
  • the translation of each polypeptide encoded by the mRNA is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES.
  • the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase yield of a polypeptide driven by a single promoter.
  • Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems this can include an antibiotic resistance gene such as ampicillin, blasticidin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • One amplifiable selectable marker is the dihydrofolate reductase gene (DHFR gene).
  • Another amplifiable marker is the DHFRr cDNA (Simonsen and Levinson 1983, Proc. Natl. Acad. Sci. (USA) 80:2495). Selectable markers are reviewed by Thilly ( Mammalian Cell Technology , Butterworth Publishers, Stoneham, Mass.) and the choice of selectable markers is well within the level of ordinary skill in the art.
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, U.S. Pat. No.4,713,339).
  • the expression elements of the expression systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used;
  • promoters such as the baculovirus polyhedron promoter may be used;
  • promoters derived from the genome of plant cells e.g.
  • heat shock promoters the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g. the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g.
  • the adenovirus late promoter may be used; when generating cell lines that contain multiple copies of expression product, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
  • the expression of sequences encoding linear or non-cyclized forms of the chimeric proteins of the invention may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al. 1987, EMBO J. 3:1671-1680; Broglie et al.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • a coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e. virus lacking the proteinaceous coat coded for by the polyhedron gene).
  • a number of viral based expression systems may be utilized.
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a polypeptide peptide in infected hosts (see, e.g., Logan & Shenk 1984, Proc.
  • the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:7415; Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:4927).
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a polypeptide in infected hosts (see, e.g., Logan & Shenk 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659).
  • the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al. 1984, J. Virol. 49:857-864; Panicali et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:4927).
  • the term “appropriate growth medium” means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the media contains vitamin K which is necessary for a cellular ⁇ carboxylase to confer activity upon a recombinantly produced clotting factor, e.g., factor VII.
  • the media can contain bovine calf serum or fetal calf serum.
  • the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient, which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.
  • Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g. MEM, DMEM). Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.
  • the recombinantly produced chimeric protein of the invention can be isolated from the culture media using procedures well-established in the art (e.g., affinity chromatography, size exclusion chromatography, ion exchange chromatography).
  • the chimeric protein of the invention can be isolated from the culture media by column chromatography, e.g., a protein A column, or by ion exchange chromatography.
  • column chromatography e.g., a protein A column, or by ion exchange chromatography.
  • the chromatographic separation can contribute to the activation of the chimeric protein of the invention, e.g., by converting factor VII to factor VIIa.
  • the culture medium from appropriately grown transformed or transfected host cells is separated from the cell material, and the presence of chimeric proteins is demonstrated.
  • One method of detecting the chimeric proteins is by the binding of the chimeric proteins or portions of the chimeric proteins to a specific antibody recognizing the chimeric protein of the invention (e.g., an anti-Fc antibody).
  • An anti-chimeric protein antibody may be a monoclonal or polyclonal antibody raised against the chimeric protein in question.
  • the chimeric protein can contain a portion of an immunoglobulin constant region.
  • Antibodies recognizing the constant region of many immunoglobulins are known in the art and are commercially available.
  • An antibody can be used to perform an ELISA or a western blot to detect the presence of the chimeric protein of the invention.
  • chimeric proteins of the invention have many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent.
  • the invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises at least a portion of an immunoglobulin constant region and at least one clotting factor.
  • the chimeric protein of the invention treats or prevents a hemostatic disorder by promoting the formation of a fibrin clot.
  • the chimeric protein of the invention can activate any member of a coagulation cascade.
  • the clotting factor can be a participant in the extrinsic pathway, the intrinsic pathway or both.
  • the clotting factor is factor VII or factor VIIa.
  • Factor VIIa can activate factor X which interacts with factor Va to cleave prothrombin to thrombin, which in turn cleaves fibrinogen to fibrin.
  • the chimeric protein of the invention can be used to treat any hemostatic disorder.
  • hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII.
  • the hemostatic disorder is an inherited disorder.
  • the subject has hemophilia A, and the chimeric protein comprises factor VIII or factor VIIIa.
  • the subject has hemophilia A and the chimeric protein comprises factor VII or factor VIIa.
  • the subject has hemophilia B and the chimeric protein comprises factor IX or factor IXa.
  • the subject has hemophilia B and the chimeric protein comprises factor VII or factor VIIa.
  • the subject has inhibitory antibodies to factor VIII or factor VIIIa and the chimeric protein comprises factor VII or factor VIIa.
  • the subject has inhibitory antibodies against factor IX or factor IXa and the chimeric protein comprises factor VII or factor VIIa.
  • the chimeric protein of the invention can be used to prophylactically treat a subject with a hemostatic disorder.
  • the chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder.
  • the hemostatic disorder is the result of a deficiency in a clotting factor, e.g., factor IX, factor VII, factor VII.
  • a clotting factor e.g., factor IX, factor VII, factor VII.
  • the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand's factor.
  • the hemostatic disorder can be an acquired disorder.
  • the acquired disorder can result from an underlying secondary disease or condition.
  • the unrelated condition can be, as an example, but not as a limitation, cancer, an auto-immmune disease, or pregnancy.
  • the acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).
  • the invention also relates to methods of treating a subject that does not have a hemostatic disorder or a secondary disease or condition resulting in acquisition of a hemostatic disorder.
  • the invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises at least a portion of an immunoglobulin constant region and at least one clotting factor.
  • the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery.
  • the chimeric protein of the invention can be administered prior to, during, or after surgery as a prophylactic.
  • the chimeric protein of the invention can be administered prior to, during, or after surgery to control an acute bleeding episode.
  • the surgery can include, but is not limited to liver transplantation, liver resection, or stem cell transplantation.
  • the chimeric protein of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder.
  • the acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding.
  • the chimeric protein of the invention can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route.
  • the chimeric protein can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the site of bleeding or implanted into bandage/dressing.
  • the dose of the chimeric protein of the invention will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 ⁇ g/kg body weight. In one embodiment, the dosing range is 0.1-1,000 ⁇ g/kg.
  • the protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e.g., STA-CLOT VIIa-rTF clotting assay. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a clotting factor, at least a portion of an immunoglobulin constant region and a pharmaceutically acceptable carrier or excipients.
  • suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin.
  • excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition can also contain pH buffering reagents, and wetting or emulsifying agents.
  • the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means.
  • the composition can also be prepared as a liquid for example a syrup or a suspension.
  • the liquid can include suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also include flavoring, coloring and sweetening agents.
  • the composition can be presented as a dry product for constitution with water or another suitable vehicle.
  • the composition may take the form of tablets or lozenges according to conventional protocols.
  • the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or nebulizer, with optionally a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous or intramuscular) by bolus injection.
  • parenteral administration i.e. intravenous or intramuscular
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative.
  • the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., pyrogen free water.
  • the pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the invention in another embodiment, relates to a method of treating a subject with a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region in combination with at least one other clotting factor or-agent that promotes hemostasis.
  • Said other clotting factor or agent that promotes hemostasis can be any therapeutic with demonstrated clotting activity.
  • the clotting factor or hemostatic agent can include factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue factor (rsTF) or activated forms of any of the preceding.
  • the clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamic acid.
  • the invention provides a kit for the diagnosis of a hemostatic disorder.
  • the kit can include a container and a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • the chimeric protein can be provided in an appropriate buffer or solvent.
  • the buffer can be an aqueous buffer, e.g., PBS or alternatively the chimeric protein can be lyophilized.
  • the kit can also provide instructions for detecting the presence of a clotting factor in a sample, e.g., contacting an aliquot of a sample with the chimeric protein of the invention and detecting the presence of a clot. Detection can include visible detection.
  • the kit can optionally provide an aliquot of blood lacking a known clotting factor.
  • the sequence for the FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)(SEQ ID NO: 23), a common affinity tag used to identify or purify proteins, was cloned into the pcDNA 3.1-Fc plasmid, which contains the mouse IgK signal sequence followed by the Fc fragment of human IgG1 (amino acids 221-447, EU numbering).
  • FlagFc-F1 5′- GCTGGCTAGCCACCATGGA -3′ (SEQ ID NO: 10)
  • FlagFc-R1 5′- CTTGTCATCGTCGTCCTTGTAGTCGTCA (SEQ ID NO: 11) CCAGTGGAACCTGGAAC -3′
  • FlagFc-F2 5′- GACTACAAGG ACGACGATGA (SEQ ID NO: 12) CAAGGACAAA ACTCACACAT GCCCACCGTG CCCAGCTCCG GAACTCC -3′
  • FlagFc-R2 5′- TAGTGGATCCTCATTTACCCG -3′ (SEQ ID NO: 13)
  • the pcDNA 3.1-Fc template was then added to two separate PCR reactions containing 50 pmol each of the primer pairs FlagFc-F1/R1 or FlagFc-F2/R2 in a 50 ⁇ l reaction using Pfu Ultra DNA polymerase (Stratagene, Calif.) according to manufacturer's standard protocol in a MJ Thermocycler using the following cycles: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 52° C. 30 seconds, 72° C. 45 seconds), followed by 72° C. for 10 minutes.
  • the products of these two reactions were then mixed in another PCR reaction (2 ⁇ l each) with 50 pmol of FlagFc-F1 and FlagFc-R2 primers in a 50 ⁇ l reaction using Pfu Ultra DNA polymerase (Stratagene, Calif.) according to manufacturer's standard protocol in a MJ Thermocycler using the following cycles: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 52° C. 30 seconds, 72° C. 45 seconds), followed by 72° C. for 10 minutes.
  • the resulting fragment was gel purified, digested and inserted into the pcDNA 3.1-Fc plasmid NheI-BamHI.
  • the resulting plasmid contains the mouse IgK signal sequence producing the FlagFc protein.
  • the coding sequence for human PACE (paired basic amino acid cleaving enzyme), an endoprotease, was obtained by RT-PCR.
  • the following primers were used: (SEQ ID NO: 15) PACE-F1: 5′- GGTAAGCTTGCCATGGAGCTGAGGCCCTGGTTGC - 3′ (SEQ ID NO: 16) PACE-R1: 5′- GTTTTCAATCTCTAGGACCCACTCGCC -3′ (SEQ ID NO: 17) PACE-F2: 5′- GCCAGGCCACATGACTACTCCGC -3′ (SEQ ID NO: 18) PACE-R2: 5′- GGTGAATTCTCACTCAGGCAGGTGTGAGGGCAGC - 3′
  • the PACE-F1 primer adds a HindIII site to the 5′ end of the PACE sequence beginning with 3 nucleotides before the start codon, while the PACE-R2 primer adds a stop codon after amino acid 715, which occurs at the end of the extracellular domain of PACE, as well as adding an EcoRI site to the 3′ end of the stop codon.
  • the PACE-R1 and -F2 primers anneal on the 3′ and 5′ sides of an internal BamHI site, respectively.
  • RT-PCR reactions Two RT-PCR reactions were then set up using 25 pmol each of the primer pairs of PACE-F1/R1 or PACE-F2/R2 with 20 ng of adult human liver RNA (Clontech; Palo Alto, Calif.) in a 50 ⁇ l RT-PCR reaction using the SuperScript.TM One-Step RT-PCR with PLATINUM® Taq system (Invitrogen, Carlsbad, Calif.) according to manufacturers protocol.
  • the reaction was carried out in a MJ Thermocycler using the following cycles: 50° C. 30 minutes; 94° C. 2 minutes; 30 cycles of (94° C. 30 seconds, 58° C. 30 seconds, 72° C. 2 minutes), followed by 72° C. 10 minutes.
  • the coding sequence for Factor VII was obtained by RT-PCR from human fetal liver RNA (Clontech, Palo Alto, Calif.).
  • the cloned region is comprised of the cDNA sequence from bp 36 to bp 1430 terminating just before the stop codon.
  • a SbfI site was introduced on the N-terminus.
  • a BspEI site was introduced on the C-terminus.
  • the construct was cloned by PCR using the primers: (SEQ ID NO: 19) Down- 5′ GCTACCTGCAGGCCACCATGGTCTCCCAGGCCCTCAGG stream: 3′ (SEQ ID NO: 20) Upstream: 5′ CAGTTCCGGAGCTGGGCACGGCGGGCACGTGTGAGTTT TGTCGGGAAAT GG 3′ and the following conditions: 95° C. for 5 minutes followed by 30 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 1 minute and 45 seconds, and a final extension cycle of 72° C. for 10 minutes.
  • the fragment was digested SbfI-BspE I and inserted into pED.dC-Fc a plasmid encoding for the Fc fragment of an IgG1.
  • CHO DG-44 cells expressing Factor VII-Fc were established. CHO DG-44 cells were grown at 37° C., 5% CO2, in MEM Alpha plus nucleoside and ribonucleosides and supplemented with 5% heat-inactivated fetal bovine serum until transfection.
  • DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%-60%. A total of 10 ⁇ g of DNA was used to transfect one 100 mm dish: either 9 ⁇ g pED.dC.FVII-Fc+1 ⁇ g of pcDNA6-PACE for the dimer transfection, or 7.5 ⁇ g of pED.dC.FVII-Fc+1.5 ⁇ g pcDNA3/Flag-Fc+1 ⁇ g of pcDNA6-PACE for the monomer transfection. The cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, Calif.).
  • the media was removed after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 ⁇ g/ml of Blasticidin (Invitrogen, Carlsbad, Calif.) for both transfections, while the monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, Calif.).
  • the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable cell lines were established. Protein expression was subsequently amplified by the addition 25 nM methotrexate.
  • Approximately 2 ⁇ 10 7 cells were used to inoculate 300 ml of growth medium in a 1700 cm 2 roller bottle (Corning, Corning, N.Y.) supplemented with 5 ⁇ g/L of vitamin K 3 (menadione sodium bisulfite) (Sigma, St Louis, Mo.).
  • the roller bottles were incubated in a 5% CO 2 at 37° C. for 72 hours.
  • the growth medium was then exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 ⁇ g/ml bovine insulin and 10 ⁇ g/ml Gentamicin) supplemented with 5 ⁇ g/L of vitamin K 3 .
  • the production medium (conditioned medium) was collected every day for 10 days and stored at 4° C.
  • Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Pooled media was first clarified using a Sartoclean glass fiber filter (3.0 ⁇ m+0.2 ⁇ m) (Sartorious Corp. Gottingen, Germany) followed by an Acropack 500 filter (0.8 ⁇ m+0.2 ⁇ m) (Pall Corp., East Hills, N.Y.). The clarified media was then concentrated approximately 20-fold using Pellicon Biomax tangential flow filtration cassettes (10 kDa MWCO) (Millipore Corp., Billerica, Mass.).
  • Fc chimeras were then captured from the concentrated media by passage over a Protein A Sepharose 4 Fast Flow Column (AP Biotech, Piscataway, N.J.).
  • a 5 ⁇ 5 cm (100 ml) column was loaded with ⁇ 5 mg Fc protein per ml column volume at a linear flow rate of 100 cm/hour to achieve a residence time of ⁇ 3 minutes.
  • the column was then washed with >5 column volumes of 1 ⁇ DPBS to remove non-specifically bound proteins.
  • the bound proteins were eluted with 100 mM Glycine pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1 part 1 M Tris-HCL, pH 8 to 10 parts elute fraction.
  • the monomer-dimer hybrid transfection protein sample was subject to further purification, as it contained a mixture of FVII-Fc:FVII-Fc homodimer, FVII-FC: FLAG-FC monomer-dimer hybrid, and FLAG-Fc: FLAG-Fc homodimers.
  • FVII-Fc FLAG-FC monomer-dimer hybrid
  • FLAG-Fc FLAG-Fc homodimers.
  • the Protein A Sepharose 4 Fast Flow pool was first dialyzed into 20 mM MES, 20 mM NaCl, pH 6.1 and was then passed over a Unosphere S cation-exchange column (BioRad Corp., Richmond, Calif.).
  • the dialyzed material was then loaded onto a 1.1 ⁇ 11 cm (9.9 ml) column at 150 cm/hour. During the wash and elution, the flow rate was increased to 500 cm/hour.
  • the column was washed sequentially with 8 column volumes of 20 mM MES, 20 mM NaCl, pH 6.1 and 8 column volumes of 20 mM MES, 40 mM NaCl, pH 6.1.
  • the bound protein was eluted with 20 mM MES, 750 mM NaCl, pH 6.1. Elution fractions containing the protein peak were pooled and sterile filtered through a 0.2 ⁇ m filter disc prior to storage at ⁇ 80° C.
  • An anti-FLAG MAB affinity column was used to separate chimeric Fc dimers with hFVII fused to both Fc molecules from those with one FLAG peptide and one hFVII fusion.
  • the Unosphere S Eluate pool was diluted 1:1 with 20 mM Tris, 50 mM NaCl, 5 mM CaCl 2 , pH 8 and loaded onto a 1.6 ⁇ 5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, Mo.) at a linear flow rate of 60 cm/hour. Loading was targeted to ⁇ 2.5 mg monomer-dimer hybrid/ml column volume.
  • the StaClot FVIIa-rTF assay kit was purchased from Diagnostica Stago (Parsippany, N.J.) and modified as described in Johannessen et al. 2000, Blood Coagulation and Fibrinolysis 11: S159. A standard curve was performed with the FVIIa World Health Organization standard 89/688.
  • the assay compared a homodimer comprised of two factor VII molecules and two Fc molecules with a monomer/dimer hybrid comprised of one factor VII molecule and two Fc molecules. Significant clotting activity was observed for both the monomer/dimer hybrid and the homodimer. The clotting activity of the monomer/dimer hybrid compared to the homodimer was almost four times as great ( FIG. 4 ).
  • 25 gram day 9 newborn rats were purchased from Charles River (Wilmington, Mass.) and allowed to acclimate for 24 hours.
  • the rats were dosed orally with FVIIa-Fc homodimer, or monomer/dimer hybrid(consisting of two Fc chains, one of which was linked to Fc-VII).
  • a volume of 200 ⁇ l of a FVIIa-Fc solution was used for a dose of 1 mg/kg.
  • the solution was comprised of a Tris-HCl buffer pH 7.4 with 5 mg/ml soybean trypsin inhibitor.
  • the rats were euthanized with CO 2 at several time points, and 200 ⁇ l of blood was drawn by cardiac puncture.
  • Plasma was obtained by the addition of a ⁇ fraction (1/10) ⁇ volume 3.8% sodium citrate solution and centrifuged at room temperature at a speed of 1268 ⁇ g.
  • the plasma samples were either used in the assays fresh or frozen at ⁇ 20° C.
  • the Asserachrom Factor VII:Ag ELISA was performed on the minipig samples.
  • a Factor VII:Ag Elisa assay was purchased from Diagnostica Stago (Parsippany, N.J.) and preformed as described in the kit manual with one change.
  • the standard for the standard curve was replaced with purified Factor VIIa-Fc.
  • the standard was run in the same percent normal animal plasma as being analyzed. Plasma levels of intravenously administered monomer/dimer hybrid are shown in FIG. 8A .
  • the half life was determined to be 9.4 hours.
  • a time course assay using monomer/dimer hybrid chimeric protein demonstrated sustained plasma levels of intravenously administered Factor VIIa-Fc over time with a T 1/2 of 22 hours ( FIG. 8A ).
  • Clotting activity using was measured by the StaClot FVIIa-rTF assay kit ( FIG. 8B ).
  • the T 1/2 for clotting was found to be 6.4 hours for one pig and 5.7 for the other pig.
  • the human Factor IX coding sequence was obtained by RT-PCR amplification from adult human liver RNA using the following primers: (SEQ ID NO: 21) natFIX- 5′-TTACTGCAGAAGGTTATGCAGCGCGTGAACATG- F: 3′ (SEQ ID NO: 22) F9-R: 5′-TTTTTCGAATTCAGTGAGCTTTGTTTTTTCCTTAATCC- 3′
  • the fragment was gel purified using Qiagen Gel Extraction Kit (Qiagen, Valencia, Calif.), and digested with PstI-EcoRI, gel purified, and cloned into the corresponding digest of the pED.dC.XFc plasmid.
  • the amino acid and DNA sequences of factor IX-Fc are shown in FIG. 9 .
  • CHO DG-44 cells expressing Factor IX-Fc were established. DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%-60%. A total of 10 ⁇ g of DNA was used to transfect one 100 mm dish: for the homodimer transfection, 8 ⁇ g of pED.dC.Factor IX-Fc+2 ⁇ g of pcDNA6-PACE was used; for the monomer-dimer hybrid transfection, 8 ⁇ g of pED.dC.Factor IX-Fc+1 ⁇ g of pcDNA3-FlagFc+1 ⁇ g pcDNA6-PACE was used.
  • the cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, Calif.). The media was removed from transfection after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 ⁇ g/ml of Blasticidin (Invitrogen, Carlsbad, Calif.) for both transfections, while the monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). After 3 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable cell lines were established. Protein expression was subsequently amplified by the addition 10 nM or 100 nM methotrexate for the homodimer or monomer-dimer hybrid, respectively.
  • Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 ⁇ m) filter from Pall Gelman Sciences (Ann Arbor, Mich.). All of the following steps were performed at 4° C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCl, and 137 mM NaCl), eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of 1 M Tris-HCl, pH 9.0. The protein was then dialyzed into PBS.
  • 1X PBS 10 mM phosphate, pH 7.4, 2.7 mM KCl, and 137 mM NaCl
  • the monomer-dimer hybrid transfection protein sample was subject to further purification, as it contained a mixture of FIX-Fc:FIX-Fc homodimer, FIX-Fc:Flag-Fc monomer-dimer hybrid, and Flag-Fc:Flag-Fc homodimer. Material was concentrated and applied to a 2.6 cm ⁇ 60 cm (318 ml) Superdex 200 Prep Grade column at a flow rate of 4 ml/minute (36 cm/hour) and then eluted with 3 column volumes of 1 ⁇ PBS. Fractions corresponding to two peaks on the UV detector were collected and analyzed by SDS-PAGE.
  • Fractions from the first peak contained either FIX-Fc:FIX-Fc homodimer or FIX-Fc:FlagFc monomer-dimer hybrid, while the second peak contained FlagFc:FlagFc homodimer. All fractions containing the monomer-dimer hybrid but no FlagFc homodimer were pooled and applied directly to a 1.6 ⁇ 5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, Mo.) at a linear flow rate of 60 cm/hour. After loading, the column was washed with 5 column volumes PBS. Monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0.
  • Elution fractions containing the protein peak were then neutralized by adding ⁇ fraction (1/10) ⁇ volume of 1 M Tris-HCl, and analyzed by reducing and nonreducing SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 1-5 mg/ml, and stored at ⁇ 80° C.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Diabetes (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Orthopedics, Nursing, And Contraception (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The invention relates to a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region. The invention relates to a method of treating a hemostatic disorder comprising administering a therapeutically effective amount of a chimeric protein wherein the chimeric protein comprises at least one clotting factor and at least a portion of an immunoglobulin constant region.

Description

  • This application claims priority to U.S. Provisional Appln. No. 60/468,837, filed on May 6, 2003, which is incorporated by reference.
    • FIELD OF THE INVENTION
  • The invention relates generally to the field of therapeutics for hemostatic disorders. More specifically, the invention relates to a chimeric protein for the treatment of a hemostatic disorder.
    • BACKGROUND OF THE INVENTION
  • Hemostatic disorders are characterized by uncontrolled bleeding resulting from the inability or reduced ability to form fibrin clots. Hemostatic disorders can result from a genetic defect or can be acquired as a result of an unrelated medical condition (e.g., cancer and related chemotherapy, autoimmune disease) (Kasper 2000, Hemophilia 6(Supp):13; Cohen et al. 1996, Bailiere's Clinical Hematology 9(2):331). Typically, hemostatic disorders result from the deficiency of a specific blood clotting factor. Classic examples of hemostatic disorders include hemophilia A, which results from a deficiency in factor VIII; hemophilia B (Christmas Disease), which results from a deficiency in factor IX; and von Willebrand's disease, which results in a defect in von Willebrand's factor. Von Willebrand factor circulates in association with factor VIII and stabilizes it. It mediates the adherence of platelets to each other and to injured blood vessel walls. Other, less common hemostatic disorders include factor XI deficiency (PTA deficiency), factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XII (Kasper 2000, Hemophilia 6(Supp): 13).
  • Clotting factors act in concert with one another in a coagulation cascade that ultimately results in the formation of a fibrin clot (FIG. 1). These factors can exist in a quiescent state as a proenzyme or zymogen or in an activated enzymatic state when stimulated to form a clot. Stimulation of these factors can occur by two distinct pathways, the intrinsic pathway and the extrinsic pathway. The intrinsic pathway refers to those reactions that lead to clot formation through utilization of factors present only in the plasma. In contrast, the extrinsic pathway refers to those reactions that lead to clot formation from release of membrane bound tissue factor upon vessel endothelium disruption.
  • Factor VII participates in both pathways cleaving factor X into the activated factor Xa in conjunction with tissue factor in the extrinsic pathway or interacting with factor IXa in the intrinsic pathway. Factor VII acts down stream and independently of factors VII and IX when acting through the extrinsic pathway, thus bypassing the need for these clotting factors. It is, therefore, an attractive therapeutic candidate for treating hemostatic disorders, especially Hemophilia A and B. (U.S. Pat. No. 6,310,183; WO 01/58935).
  • Factor VII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein with a molecular weight of 53 kDa (Broze et al. 1980, J. Biol. Chem. 255:1242). The factor VII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site, Arg152-IIe153, resulting in two chains, heavy (254 amino acids) and light (154 amino acids) linked by a single disulfide bond (Hagen et al. 1986, Proc. Natl. Acad. Sci. (USA) 83:2412). The activated form of factor VII binds to tissue factor, which then converts factor X to factor Xa. Factor Xa is required to convert prothrombin to thrombin, which converts fibrinogen to fibrin as a final stage in forming a fibrin clot.
  • Factor VII undergoes post-translational modifications, including vitamin K dependent carboxylation resulting in ten γ-carboxyglutamic acid residues in the N-terminal region of the molecule. Other post translational modifications include sugar moiety attachment at two naturally occurring N-linked glycosylation sites at position 145 and 322, respectively, and at two naturally occurring O-linked glycosylation sites at position 52 and 60, respectively (WO 01/58935).
  • Traditional therapy for hemostatic disorders calls for parenteral replacement of deficient clotting factors such as factor VII, factor VIII or factor IX (see, e.g., U.S. Pat. Nos. 6,310,183 and 4,784,950). Treatment often entails both administering clotting factors to treat acute bleeding episodes as well as administering clotting factors to prophylactically deter the occurrence of future bleeding episodes. Prophylaxis has been found to reduce the risk of developing joint problems and arthritis associated with frequent bleeding episodes (Petrini 2001, Hemophilia 7:99; Fischer et al. 2002, Blood 99(7):2337).
  • Traditional therapies, however, have many associated problems. Currently clotting factors must be administered parenterally in order to attain effective doses because to date non-invasive methods have not been successful in attaining therapeutic levels. Problems associated with parenteral administration include injection site occlusion, pain and infection. Use of an in-line catheter increases the risk of all of these events. Specific problems associated with the parenteral administration of clotting factors to infants and small children include central venous access. Additionally, parenteral administration of clotting factors runs the risk of precipitating a bleeding episode. These problems are particularly relevant when the patient is undergoing regular prophylactic administration of a clotting factor.
  • Clotting factors such as factor IX and factor VIII must be given frequently and in large doses resulting in the development of inhibitor antibodies against the clotting factor in a significant number of patients (see, e.g., Nilsson 1992, Transfusion Medicine Review 6(4):285; Cohen et al. 1996, Bailiere's Clinical Hematology 9(2):331).
  • One aspect of the invention provides a safer more effective treatment for hemostatic disorders. Another aspect of the invention provides for increased serum half life and increased bioavailability of therapeutics administered through non-invasive means for the treatment of hemostatic disorders thereby reducing the risk of incurring a bleeding episode, infection and injection site occlusion associated with parenteral administration. Another aspect of the invention provides therapy for hemostatic disorders with reduced risk, compared to current therapies, of developing inhibitor antibodies against the clotting factor. Yet another aspect of the invention provides for a prophylactic treatment of a hemostatic disorder.
  • The aspects of the invention provide for a chimeric protein comprised of at least one clotting factor and at least a portion of an immunoglobulin constant region, wherein the clotting factor is capable of promoting blood coagulation and/or fibrin clot formation.
  • Chimeric proteins comprising an Fc portion of an immunoglobulin are known (see, e.g., U.S. Pat. Nos. 6,030,613, 6,086,875, 6,485,726, and PCT Application No. US/02/21335) and while chimeric proteins comprised of mutant clotting factors without clotting activity, and immunoglobulins have been previously described (WO 01/02439), chimeric proteins comprising a clotting factor (i.e., having clotting activity) and at least a portion of an immunoglobulin constant region have not been described. Clotting factor as defined below and used herein refers to any molecule with clotting activity.
    • SUMMARY OF THE INVENTION
  • The invention relates to an improved chimeric protein for treating hemostatic disorders. The invention provides a chimeric protein to treat a hemostatic disorder that can be administered parenterally or non-invasively (e.g., via a pulmonary, nasal, or oral route). The invention thus relates to a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • The invention relates to a method of treating a hemostatic disorder comprising administering a therapeutically effective amount of a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • The invention relates to a method of making a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region; said method comprising transfecting a cell with a DNA construct comprising a first DNA sequence encoding at least one clotting factor operatively linked to a second DNA sequence encoding at least a portion of an immunoglobulin.; culturing said cell under conditions such that the chimeric protein is expressed; and isolating said chimeric protein from said cell.
  • The invention relates to a nucleic acid molecule comprising a sequence encoding at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • The invention relates to a nucleic acid construct comprising a DNA sequence encoding at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
  • It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a diagram showing the extrinsic and intrinsic pathway of the coagulation cascade.
  • FIG. 2 is a diagram of the inactive and active forms of factor VIIa-Fc.
  • FIG. 3A is the amino acid sequence of a chimeric protein comprising factor VIIa and an Fc fragment of an immunoglobulin (SEQ ID NO:1).
  • FIG. 3B is the amino acid sequence of an Fc fragment of an immunoglobulin (SEQ ID NO:2).
  • FIG. 3C is the nucleic acid sequence of an Fc fragment of an immunoglobulin (SEQ ID NO:3).
  • FIG. 3D is the nucleic acid sequence of a chimeric protein comprising factor VIIa and an Fc fragment of an immunoglobulin (SEQ ID NO:4).
  • FIG. 4 is a diagram of results from a STA-CLOT assay showing factor VIIa-Fc has significant clotting activity.
  • FIG. 5 is a diagram showing factor VIIa-Fc binds to soluble human Fc neonatal receptor (shFcRn).
  • FIG. 6 demonstrates plasma levels over time for orally administered factor VIIa-Fc in neonatal rats.
  • FIG. 7 compares oral uptake in neonatal rats of Factor VIIa-Fc monomer/dimer hybrids comprising two polypeptide chains, each chain comprising at least a portion of an immunoglobulin constant region and wherein factor VII is linked to only one of the two chains versus homodimers comprising two polypeptide chains wherein both chains comprise at least a portion of an immunoglobulin constant region and both chains also comprise factor VII.
  • FIG. 8A demonstrates plasma levels over time of factor VIIa-Fc administered intravenously to minipigs, wherein the factor VIIa-Fc is a monomer/dimer hybrid comprising two polypeptide chains, each chain comprising at least a portion of an immunoglobulin constant region and wherein factor VII is linked to only one of the two chains.
  • FIG. 8B demonstrates clotting activity over time of factor VIIa-Fc administered intravenously to minipigs wherein the factor VIIa-Fc is a monomer/dimer hybrid comprising two polypeptide chains, each chain comprising at least a portion of an immunoglobulin constant region and wherein factor VII is linked to only one of the two chains.
  • FIG. 9A is the amino acid sequence of the chimeric protein Factor IX-Fc. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell and the propeptide (bold) which is recognized by the vitamin K-dependent γ carboxylase which modifies the Factor IX to achieve full activity. The sequence is subsequently cleaved by PACE to yield Factor IX-Fc.
  • FIG. 9B is the nucleic acid sequence of the chimeric protein Factor IX-Fc. Included in the sequence is the signal peptide (underlined) and the propeptide (bold) which is recognized by the vitamin K-dependent γ carboxylase which modifies the Factor IX to achieve full activity. The translated sequence is subsequently cleaved by PACE to yield mature Factor IX-Fc.
    • SUMMARY OF THE SEQUENCES
  • Seq ID NO FIG. Description
    1 3A amino acid sequence of a chimeric protein
    comprising factor VIIA and an IgG FC fragment
    2 3B amino acid sequence of an FC fragment of IgG
    3 3C nucleic acid sequence corresponding to the
    amino acid sequence of SEQ ID NO: 2
    4 3D nucleic acid sequence corresponding to the
    amino acid sequence of SEQ ID NO: 1
    • DESCRIPTION OF THE EMBODIMENTS
  • A. Definitions
  • Affinity tag, as used herein, means a molecule attached to a second molecule of interest, capable of interacting with a specific binding partner for the purpose of isolating or identifying said second molecule of interest.
  • Analogs of, or proteins or peptides or substantially identical to the chimeric proteins of the invention, as used herein, means that a relevant amino acid sequence of a protein or a peptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a given sequence. By way of example, such sequences may be variants derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. Percent identity between two amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al. (1990) J. Mol. Biol., 215:403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48:444-453; the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4:11-17; or Tatusova et al. (1999) FEMS Microbiol. Lett., 174:247-250, etc. Such algorithms are incorporated into the BLASTN, BLASTP and “BLAST 2 Sequences” programs (see www.ncbi.nlm.nih.gov/BLAST). When utilizing such programs, the default parameters can be used. For example, for nucleotide sequences the following settings can be used for “BLAST 2 Sequences”: program BLASTN, reward for match 2, penalty for mismatch −2, open gap and extension gap penalties 5 and 2 respectively, gap x_dropoff 50, expect 10, word size 11, filter ON. For amino acid sequences the following settings can be used for “BLAST 2 Sequences”: program BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11 and 1 respectively, gap x_dropoff 50, expect 10, word size 3, filter ON.
  • Bioavailability, as used herein, means the extent and rate at which a substance is absorbed into a living system or is made available at the site of physiological activity.
  • A chimeric protein, as used herein, refers to any protein comprised of a first amino acid sequence derived from a first source, bonded, covalently or non-covalently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same. A first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein can include for example, a protein derived from at least 2 different biological sources. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog, as further described herein, of any of the above). A synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences). A chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source.
  • Clotting factor, as used herein, means any molecule, or analog thereof, naturally occurring or recombinantly produced which prevents or decreases the duration of a bleeding episode in a subject with a hemostatic disorder. In other words, it means any molecule having clotting activity.
  • Clotting activity, as used herein, means the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and/or reduces the severity, duration or frequency of hemorrhage or bleeding episode.
  • DNA construct, as used herein, means a DNA molecule, or a clone of such a molecule, either single- or double-stranded that has been modified through human intervention to contain segments of DNA combined in a manner that as a whole would not otherwise exist in nature. DNA constructs contain the information necessary to direct the expression of polypeptides of interest. DNA constructs can include promoters, enhancers and transcription terminators. DNA constructs containing the information necessary to direct the secretion of a polypeptide will also contain at least one secretory signal sequence.
  • A fragment, as used herein, refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, of at least 5 contiguous amino acid residues, of at least 10 contiguous amino acid residues, of at least 15 contiguous amino acid residues, of at least 20 contiguous amino acid residues, of at least 25 contiguous amino acid residues, of at least 40 contiguous amino acid residues, of at least 50 contiguous amino acid residues, of at least 100 contiguous amino acid residues, or of at least 200 contiguous amino acid residues or any deletion or truncation of a protein.
  • Hemostasis, as used herein, means the stoppage of bleeding or hemorrhage; or the stoppage of blood flow through a blood vessel or body part.
  • Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot.
  • Linked, as used herein, refers to a first nucleic acid sequence covalently joined to a second nucleic acid sequence. The first nucleic acid sequence can be directly joined or juxtaposed to the second nucleic acid sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. Linked as used herein can also refer to a first amino acid sequence covalently joined to a second amino acid sequence. The first amino acid sequence can be directly joined or juxtaposed to the second amino acid sequence or alternatively an intervening sequence can covalently join the first amino acid sequence to the second amino acid sequence. Linked can also refer to a first amino acid sequence non-covalently joined to a second amino acid sequence. Linked as used herein can also refer to a first amino acid sequence covalently joined to a nucleic acid sequence or a small organic or inorganic molecule.
  • Operatively linked, as used herein, means a first nucleic acid sequence linked to a second nucleic acid sequence such that both sequences are capable of being expressed as a biologically active polypeptide.
  • A small inorganic molecule, as used herein, means a molecule containing no carbon atoms and being no larger than 50 kD.
  • A small organic molecule, as used herein, means a molecule containing at least one carbon atom and being no larger than 50 kD.
  • High stringency, as used herein, includes conditions readily determined by the skilled artisan based on, for example, the length of the DNA. Generally, such conditions are defined as hybridization conditions as above, and with washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.
  • Moderate stringency, as used herein, include conditions that can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA. The basic conditions are set forth by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989), and include use of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6×SSC at 42° C. (or other similar hybridization solution, such as Stark's solution, in 50% formamide at 42° C.), and washing conditions of 60° C., 0.5×SSC, 0.1 % SDS.
  • Polypeptide, as used herein, refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term does not exclude post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, pegylation, addition of a lipid moiety, or the addition of any organic or inorganic molecule. Included within the definition, are for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids) and polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • Treat, treatment, treating, as used herein, means any of the following: the reduction in severity of a hemostatic disorder; the prophylaxis of one or more symptoms associated with a hemostatic disorder, e.g., a bleeding episode; the reduction in the duration of a disease course of a hemostatic disorder; the amelioration of one or more symptoms associated with a hemostatic disorder; the reduction in duration of a bleeding episode associated with a hemostatic disorder; the reduction in the titer of an inhibitor antibody against a clotting factor; the provision of beneficial effects to a subject with a hemostatic disorder, without necessarily curing the hemostatic disorder.
  • B. Improved Therapeutics for Hemostatic Disorders
  • The invention relates generally to improved therapeutics for hemostatic disorders. The invention thus relates to a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region.
  • The chimeric proteins of the invention have increased stability and other improved properties when compared to known therapeutic agents having clotting activity. They can also be administered parenterally or non-invasively. Accordingly, the invention provides for improved methods for administering therapeutic agents having clotting activity. While clotting factors presently are generally administered subcutaneously, intramuscularly or intravenously, the chimeric proteins of the invention can be administered using less invasive means such as oral administration, nasal administration, or pulmonary administration. In a specific embodiment, the chimeric proteins of the invention are useful in the prophylactic treatment of hemostatic disorders.
  • The invention also relates generally to improved methods of making therapeutic clotting factors. The invention relates to recombinant methods of producing therapeutic clotting factors with enhanced expression and improved yields. The invention thus relates to methods of making chimeric proteins comprising at least one clotting factor and at least a portion of an immunoglobulin constant region by transfecting a cell with a DNA construct, said construct comprising a DNA sequence encoding at least one clotting factor and a DNA sequence encoding at least a portion of an immunoglobulin constant region; culturing said cell under conditions such that the chimeric protein is expressed by said cell; and isolating said chimeric protein.
  • C. Chimeric Proteins
  • The invention relates generally to chimeric proteins comprising at least one clotting factor, at least a portion of an immunoglobulin constant region, and optionally a linker. The clotting factor can be linked covalently, or non-covalently with the portion of an immunoglobulin constant region. The portion of an immunoglobulin constant region will have both an N, or an amino terminus, and a C, or carboxy terminus. In one embodiment, the chimeric protein of the invention may have a clotting factor linked to the N terminus of the portion of an immunoglobulin constant region.
  • The chimeric protein can optionally comprise at least one linker, thus the clotting factor does not have to be directly linked to the portion of an immunoglobulin constant region. The linker can intervene in between the clotting factor and the portion of an immunoglobulin constant region. The linker can be linked to the N terminus of the portion of an immunoglobulin constant region, or the C terminus of a portion of an immunoglobulin constant region. The linker can be linked to the N terminus of the clotting factor.
  • The invention thus relates to a chimeric protein comprised of at least one clotting factor (X), optionally, a linker (L), and at least a portion of an immunoglobulin constant region (F). In one embodiment, the invention relates to a chimeric protein comprised of the formula
    X-La-F
    wherein X is linked at its C terminus to the N terminus of L, and L is linked at its C terminus to the N terminus of F and wherein a is any integer or zero. When a is zero X is directly linked at its C terminus to the N terminus of F. For example, but not as a limitation, a may be 0, 1, 2, 3, 4, 5, 10 or 20.
  • In one embodiment, the invention relates to a chimeric protein comprising the amino acid sequence of FIG. 3A (SEQ ID NO:1).
  • The chimeric protein of the invention includes monomers, dimers, as well as higher order multimers. In one embodiment, the chimeric protein is a monomer comprising one clotting factor and one portion of an immunoglobulin constant region. In another embodiment, the chimeric protein is a dimer comprising two clotting factors and two portions of an immunoglobulin. In one embodiment, the two clotting factors are the same. In one embodiment, the two clotting factors are different. In one embodiment, the two portions of an immunoglobulin are the same. In one embodiment, the two portions of an immunoglobulin are different. In another embodiment, the chimeric protein is a monomer/dimer hybrid wherein the chimeric protein has a dimeric aspect in that it is comprised of at least a portion of two immunoglobulin constant region polypeptides and a monomeric aspect in that it is comprised of only one clotting factor linked to one of the two immunoglobulin. The invention thus relates to a chimeric protein comprising a first chain and a second chain, wherein said first chain comprises at least a portion of an immunoglobulin constant region linked to a clotting factor and said second chain comprises at least a portion of an immunoglobulin constant region without a clotting factor linked to it.
  • Such chimeric proteins may be described using the formulas set forth in Table 1, where I, L, and F are as described above, and where (′) indicates a different molecule than without (′) and wherein (:) indicates a non-peptide bond
    TABLE 1
    X-F:F-X
    X′-F:F-X
    X-L-F:F-X
    X-L-F:F-L-X
    X′-L-F:F-L-X
    X-L′-F:F-L-X
    X′-L′-F:F-L-X
    F:F-X
    F:F-L-X
    X-F:F
    X-L-F:F
    L-F:F-X
    X-F:F-L
  • The skilled artisan will understand additional combinations are possible including the use of additional linkers.
  • 1. Chimeric Protein Variants
  • Derivatives and analogs of the chimeric proteins of the invention, antibodies against the chimeric proteins of the invention and antibodies against binding partners of the chimeric proteins of the invention are all contemplated, and can be made by altering their amino acid sequences by substitutions, additions, and/or deletions/truncations or by introducing chemical modifications that result in functionally equivalent molecules. It will be understood by one of ordinary skill in the art that certain amino acids in a sequence of any protein may be substituted for other amino acids without adversely affecting the activity of the protein.
  • Various changes may be made in the amino acid sequences of the chimeric proteins of the invention or DNA sequences encoding therefore without appreciable loss of their biological activity, function, or utility. Derivatives, analogs, or mutants resulting from such changes and the use of such derivatives is within the scope of the present invention. In a specific embodiment, the derivative is functionally active, i.e., capable of exhibiting one or more activities associated with the chimeric proteins of the invention, e.g., clot formation, activation of a clotting factor. Activity can be measured by assays known in the art, e.g., StaCLot FVIIa-rTF assay (Johannessen et al. 2000, Blood Coagulation and Fibrinolysis 11 :S1 59) prothrombin time (PT assay) or a APTT assay for factor IX
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 2). Furthermore, various amino acids are commonly substituted with neutral amino acids, e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine (see, e.g., MacLennan et al. (1998) Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al. (1998) Adv. Biophys. 35:1-24).
    TABLE 2
    Original Exemplary Typical
    Residues Substitutions Substitutions
    Ala (A) Val, Leu, Ile Val
    Arg (R) Lys, Gln, Asn Lys
    Asn (N) Gln Gln
    Asp (D) Glu Glu
    Cys (C) Ser, Ala Ser
    Gln (Q) Asn Asn
    Gly (G) Pro, Ala Ala
    His (H) Asn, Gln, Lys, Arg Arg
    Ile (I) Leu, Val, Met, Ala, Phe, Leu
    Norleucine
    Leu (L) Norleucine, Ile, Val, Met, Ile
    Ala, Phe
    Lys (K) Arg, 1,4-Diamino-butyric Arg
    Acid, Gln, Asn
    Met (M) Leu, Phe, Ile Leu
    Phe (F) Leu, Val, Ile, Ala, Tyr Leu
    Pro (P) Ala Gly
    Ser (S) Thr, Ala, Cys Thr
    Thr (T) Ser Ser
    Trp (W) Tyr, Phe Tyr
    Tyr (Y) Trp, Phe, Thr, Ser Phe
    Val (V) Ile, Met, Leu, Phe, Ala, Leu
    Norleucine

    2. Clotting Factors
  • The chimeric proteins of this invention include at least one clotting factor. The clotting factor can include any molecule that has clotting activity or activates a molecule with clotting activity. The clotting factor can be comprised of a polypeptide, a small organic molecule, or a small inorganic molecule. The clotting factor can be a mutated clotting factor or an analog of a clotting factor so long as it maintains at least some clotting activity. The clotting factor can be, as an example, but not as a limitation factor VII, including B domain deleted factor VIII, factor IX (U.S. Pat. No.4,994,371), factor XI, factor XII, fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII. In one embodiment, the clotting factor is factor VII or factor VIIa. In another embodiment, the clotting factor is a mutated factor VII or VIIa (see, e.g., Persson, et al. 2001, Proc. Natl. Acad. Sci. USA 98:13583; U.S. patent application Ser. No.10/109498). The clotting factor can be a factor that participates in the extrinsic pathway. The clotting factor can be a factor that participates in the intrinsic pathway. Alternatively, the clotting factor can be a factor that participates in both the extrinsic and intrinsic pathway.
  • The clotting factor can be a human clotting factor or a non-human clotting factor, e.g., derived from a non-human primate, a pig, or any mammal. The clotting factor can be chimeric clotting factor, e.g., the clotting factor can comprise a portion of a human clotting factor and a portion of a porcine, or any non-human clotting factor or a portion of a first non-human clotting factor and a portion of a second non-human clotting factor.
  • The clotting factor can be an activated clotting factor. Alternatively the clotting factor can be an inactive form of a clotting factor, e.g., a zymogen. The inactive clotting factor can undergo activation subsequent to being linked to at least a portion of an immunoglobulin constant region. The inactive clotting factor can be activated subsequent to administration to a subject. Alternatively, the inactive clotting factor can be activated prior to administration.
  • 3. Immunoglobulins
  • The chimeric proteins of this invention include at least a portion of an immunoglobulin constant region. Immunoglobulins are comprised of four protein chains that associate covalently—two heavy chains and two light chains. Each chain is further comprised of one variable region and one constant region. Depending upon the immunoglobulin isotype, the heavy chain constant region is comprised of 3 or 4 constant region domains (e.g., CH1, CH2, CH3, CH4). Some isotypes can also include a hinge region.
  • The portion of an immunoglobulin constant region can be a portion of an immunoglobulin constant region obtained from any mammal. The portion of an immunoglobulin constant region can include, but is not limited to, a portion of a human immunoglobulin constant region, a non-human primate immunoglobulin constant region, a bovine immunoglobulin constant region, a porcine immunoglobulin constant region, a murine immunoglobulin constant region, an ovine immunoglobulin constant region or a rat immunoglobulin constant region.
  • The portion of an immunoglobulin constant region can include the entire heavy chain constant region, or a fragment or analog thereof. A heavy chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain, and/or a hinge region. A heavy chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain, and/or a CH4 domain.
  • The immunoglobulin can be produced recombinantly or synthetically. The immunoglobulin can be isolated from a cDNA library. The immunoglobulin can be isolated from a phage library (see McCafferty et al. 1990, Nature 348:552). The immunoglobulin can be obtained by gene shuffling of known sequences (Mark et al. 1992, Bio/Technol. 10:779). The immunoglobulin can be isolated by in vivo recombination (Waterhouse et al. 1993, Nucl. Acid Res. 21:2265). The immunoglobulin can be a humanized immunoglobulin (Jones et al. 1986, Nature 332:323).
  • The portion of an immunoglobulin constant region can include a portion of an IgG, an IgA, an IgM, an IgD, an IgE. In one embodiment, the immunoglobulin is an IgG. In another embodiment, the immunoglobulin is IgG1. In yet another embodiment, the immunoglobulin is IgG2.
  • The portion of an immunoglobulin constant region can include an Fc fragment. An Fc fragment can be comprised of the CH2 and CH3 domains of an immunoglobulin and the hinge region of the immunoglobulin. The Fc fragment can be the Fc fragment of an IgG1, an IgG2, an IgG3 or an IgG4. In one embodiment, the immunoglobulin is an Fc fragment of an IgG1. In one embodiment, the immunoglobulin is an Fc fragment of an IgG2. In another embodiment, the portion of an immunoglobulin constant region is comprised of the amino acid sequence of SEQ ID NO:2 (FIG. 3B) or an analog thereof. In another embodiment, the immunoglobulin is comprised of a protein, or fragment thereof, encoded by the nucleic acid sequence of SEQ ID NO:3 (FIG. 3C).
  • The portion of an immunoglobulin constant region can include an Fc variant. Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (WO 97/34631). Native refers to an Fc that has not been modified by a human. WO 96/32478 describes exemplary Fc variants, as well as interaction with the salvage receptor. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
  • In another embodiment, the portion of an immunoglobulin constant region is an neonatal Fc receptor (FcRn) binding partner. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos. 6,030,613 and 6,086,875) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.
  • FcRn binding partners of the present invention encompass any molecule that can be specifically bound by the FcRn receptor including whole 1gG, the Fc fragment of 1gG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of 1gG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole 1gG, the Fc fragment of 1gG, and other fragments of 1gG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.
  • The Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example the following single amino acid residues in human IgG1 Fc (Fcy1) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wildtype proline substituted by alanine at position number 238. In addition to alanine other amino acids may be substituted for the wildtype amino acids at the positions specified above. Mutations may be introduced singly into Fc giving rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more FcRn binding partners.
  • Certain of the above mutations may confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG1 “ELLG” to the corresponding sequence from IgG2 “PVA” (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII which mediate various effector functions will not bind to IgG1 when such mutations have been introduced (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased “on” rate, a decreased “off” rate or both an increased “on” rate and a decreased “off” rate. Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).
  • In one embodiment, the FcRn binding partner is a polypeptide including the sequence PKNSSMISNTP and optionally further including a sequence selected from HQSLGTQ, HQNLSDGK, HQNISDGK, or VISSHLGQ (U.S. Pat. No. 5,739,277).
  • Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. Linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a clotting factor thus provides a means of delivering the clotting factor orally or as an aerosol administered nasally, via an ocular route or via a pulmonary route.
  • The skilled artisan will understand that portions of an immunoglobulin constant region for use in the chimeric protein of the invention can include mutants or analogs thereof, or can include chemically modified immunoglobulin constant regions or fragments thereof (e.g. pegylation) (see, e.g., Aslam and Dent 1998, Bioconjugation: Protein Coupling Techniques For the Biomedical Sciences Macmilan Reference, London). In one instance a mutant can provide for enhanced binding of an FcRn binding partner for the FcRn. Also contemplated for use in the chimeric protein of the invention are peptide mimetics of at least a portion of an immunoglobulin constant region, e.g., a peptide mimetic of an Fc fragment or a peptide mimetic of an FcRn binding partner. In one embodiment, the peptide mimetic is identified using phage display (See, e.g., McCafferty et al. 1990, Nature 348:552, Kang et al. 1991, Proc. Natl. Acad. Sci. USA 88:4363; EP 0 589 877 B1).
  • 4. Optional Linkers
  • The chimeric protein of the invention can optionally comprise at least one linker molecule. In one embodiment, the linker is comprised of amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids. The linker can comprise the sequence Gn. The linker can comprise the sequence (GGS)n (SEQ ID NO.: 5). In each instance, n may be an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of linkers include, but are not limited to, GGG (SEQ ID NO.: 6), SGGSGGS(SEQ ID NO.: 7),
    GGSGGSGGSGGSGGG, (SEQ ID NO.: 8)
    and
    GGSGGSGGSGGSGGSGGS,. (SEQ ID NO.: 9)
  • In one embodiment, the linker does not eliminate the clotting activity of the clotting factor. Optionally, the linker enhances the clotting activity of the clotting factor, e.g., by diminishing the effects of steric hindrance and making the clotting factor more accessible to its target binding site, e.g., another factor in the clotting cascade.
  • D. Nucleic Acid Constructs
  • The invention relates to a nucleic acid construct comprising a nucleic acid sequence encoding the chimeric proteins of the invention, said nucleic acid sequence comprising a first nucleic acid sequence encoding, for example at least one clotting factor, operatively linked to a second nucleic acid sequence encoding at least a portion of an immunoglobulin constant region. The nucleic acid sequence can also include additional sequences or elements known in the art (e.g. promoters, enhancers, poly A sequences, signal sequence). The nucleic acid sequence can optionally include a sequence encoding a linker placed between the nucleic acid sequence encoding at least one clotting factor and the portion of an immunoglobulin. The nucleic acid sequence can optionally include a linker sequence placed before or after the nucleic acid sequence encoding at least one clotting factor and the portion of an immunoglobulin.
  • In one embodiment, the nucleic acid construct is comprised of DNA. In another embodiment, the nucleic acid construct is comprised of RNA. The nucleic acid construct can be a vector, e.g., a viral vector or a plasmid. Examples of viral vectors include, but are not limited to adeno virus vector, an adeno associated virus vector or a murine leukemia virus vector. Examples of plasmids include but are not limited to, e.g., pUC and pGEX.
  • In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of FIG. 3D (SEQ ID NO:4).
  • Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NOS:3 or 4 and still encode a polypeptide having the amino acid sequence of SEQ ID NOS:2 or 1 respectively. Such variant DNA sequences can result from silent mutations (e.g. occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence. The invention thus provides isolated DNA sequences encoding polypeptides of the invention, selected from: (a) DNA comprising the nucleotide sequence of SEQ ID NO:3 or 4; (b) DNA encoding the polypeptide of SEQ ID NO:1 or 2; (c) DNA capable of hybridization to a DNA of (a) or (b) under conditions of moderate stringency and which encodes polypeptides of the invention; (d) DNA capable of hybridization to a DNA of (a) or (b) under conditions of high stringency and which encodes polypeptides of the invention, and (e) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), (b), (c), or (d) and which encode polypeptides of the invention. Of course, polypeptides encoded by such DNA sequences are encompassed by the invention.
  • In another embodiment, the nucleic acid molecules of the invention also comprise nucleotide sequences that are at least 80% identical to a native sequence. Also contemplated are embodiments in which a nucleic acid molecule comprises a sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native sequence. The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. 1984, Nucl. Acids Res. 12:387, and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess 1986, Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., 1979, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.
  • E. Synthesis of Chimeric Proteins
  • Chimeric proteins comprising at least a portion of an immunoglobulin constant region and a clotting factor can be synthesized using techniques well known in the art. For example the chimeric proteins of the invention can be synthesized recombinantly in cells (see, e.g., Sambrook et al. 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al. 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.).
  • DNA sequences encoding immunoglobulins, or fragments thereof, or clotting factors, or fragments thereof, may be cloned from a variety of genomic or cDNA libraries known in the art. The techniques for isolating such DNA sequences using probe-based methods are conventional techniques and are well known to those skilled in the art. Probes for isolating such DNA sequences may be based on published DNA sequences (see, for example, Hieter et al. 1980, Cell 22: 197-207). The polymerase chain reaction (PCR) method disclosed by Mullis et al. (U.S. Pat. No.4,683,195) and Mullis (U.S. Pat. No. 4,683,202) may be used. The choice of library and selection of probes for the isolation of such DNA sequences is within the level of ordinary skill in the art. Alternatively, DNA sequences encoding immunoglobulins, or fragments thereof, or clotting factors can be obtained from vectors known in the art to contain immunoglobulins, or fragments thereof, or clotting factors.
  • For recombinant production, a polynucleotide sequence encoding the chimeric protein is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The nucleic acid encoding the chimeric protein is inserted into the vector in proper reading frame.
  • In certain embodiments the nucleic acid can also encode for a propeptide clotting factor which is modified by the cell to yield the mature chimeric protein of the invention. In one specific embodiment the propeptide clotting factor is recognized by a vitamin K-dependent γ carboxylase which modifies the propeptide clotting factor to achieve full activity (e.g. factor VII, factor IX, factor X, prothrombin). To yield the mature chimeric protein of the invention, the propetide sequence is subsequently cleaved by an endoprotease, e.g., paired basic amino acid cleaving enzyme (PACE), or any PACE family member, such as PCSK1-9, including truncated versions thereof, or its yeast equivalent Kex2 from S. cerevisiae and truncated versions of Kex2 (see, e.g., U.S. Pat. Nos. 5,077,204; 5,162,220; 5,234,830; 5,885,821; 6,329,176 (Kex2 1-675)).
  • The expression vehicle is then transfected into a suitable target cell which will express the protein, e.g., a chimeric protein. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. 1978, Cell 14:725) and electroporation (Neumann et al. 1982, EMBO, J. 1:841). A variety of host-expression vector systems may be utilized to express the chimeric proteins described herein in eukaryotic cells. In one embodiment, the eukaryotic cell is an animal cell, including mammalian cells (e.g. CHO, BHK, Cos, HeLa cells). When the chimeric protein is expressed in a eukaryotic cell the DNA encoding the chimeric protein may also code for a signal sequence that will permit the chimeric protein to be secreted. One skilled in the art will understand that while the protein is translated the signal sequence is cleaved by the cell to form the mature chimeric protein. Various signal sequences are known in the art e.g., native factor VII signal sequence, native factor IX signal sequence and the mouse IgK light chain signal sequence. Alternatively, where a signal sequence is not included the chimeric protein can be recovered by lysing the cells.
  • The chimeric protein of the invention can be synthesized in a transgenic animal, such as a rodent, goat, sheep, pig, or cow. The term “transgenic animals” refers to non-human animals that have incorporated a foreign gene into their genome. Because this gene is present in germline tissues, it is passed from parent to offspring. Exogenous genes are introduced into single-celled embryos (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82:4438). Methods of producing transgenic animals are known in the art, including transgenics that produce immunoglobulin molecules (Wagner et al. 1981, Proc. Natl. Acad. Sci. USA 78:6376; McKnight et al. 1983, Cell 34:335; Brinster et al. 1983, Nature 306:332; Ritchie et al. 1984, Nature 312:517; Baldassarre et al. 2003, Theriogenology 59:831; Robl et al. 2003, Theriogenology 59:107; Malassagne et al. 2003, Xenotransplantation 10(3):267).
  • The expression vectors can encode for tags that permit for easy purification or identification of the recombinantly produced protein. Examples include, but are not limited to, vector pUR278 (Ruther et al. 1983, EMBO J. 2:1791) in which the chimeric protein described herein coding sequence may be ligated into the vector in frame with the lac z coding region so that a hybrid protein is produced; pGEX vectors may be used to express proteins with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (e.g. PreCission Protease™ (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.
  • To increase efficiency of production, the polynucleotide can be designed to encode multiple units of the chimeric protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g. by treatment with the appropriate enzyme) in order to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, the translation of each polypeptide encoded by the mRNA is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase yield of a polypeptide driven by a single promoter.
  • Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems this can include an antibiotic resistance gene such as ampicillin, blasticidin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase gene (DHFR gene). Another amplifiable marker is the DHFRr cDNA (Simonsen and Levinson 1983, Proc. Natl. Acad. Sci. (USA) 80:2495). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) and the choice of selectable markers is well within the level of ordinary skill in the art.
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, U.S. Pat. No.4,713,339).
  • The expression elements of the expression systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g. heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g. the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5 K promoter, the CMV promoter) may be used; when generating cell lines that contain multiple copies of expression product, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
  • In cases where plant expression vectors are used, the expression of sequences encoding linear or non-cyclized forms of the chimeric proteins of the invention may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al. 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al. 1984, EMBO J. 3:1671-1680; Broglie et al. 1984, Science 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al. 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, e.g., Weissbach & Weissbach 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
  • In one insect expression system that may be used to produce the chimeric proteins of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e. virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (see, e.g., Smith et al. 1983, J. Virol. 46:584; U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds 1989, Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.
  • In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a polypeptide peptide in infected hosts (see, e.g., Logan & Shenk 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:7415; Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:4927).
  • In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a polypeptide in infected hosts (see, e.g., Logan & Shenk 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al. 1984, J. Virol. 49:857-864; Panicali et al. 1982, Proc. Natl. Acad. Sci. (USA) 79:4927).
  • Host cells containing DNA constructs of the chimeric protein are grown in an appropriate growth medium. As used herein, the term “appropriate growth medium” means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors. In one embodiment, the media contains vitamin K which is necessary for a cellular γ carboxylase to confer activity upon a recombinantly produced clotting factor, e.g., factor VII. Optionally, the media can contain bovine calf serum or fetal calf serum. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient, which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g. MEM, DMEM). Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.
  • The recombinantly produced chimeric protein of the invention can be isolated from the culture media using procedures well-established in the art (e.g., affinity chromatography, size exclusion chromatography, ion exchange chromatography). The chimeric protein of the invention can be isolated from the culture media by column chromatography, e.g., a protein A column, or by ion exchange chromatography. When a protein A column or ion exchange column is used to separate the chimeric protein of the invention the chromatographic separation can contribute to the activation of the chimeric protein of the invention, e.g., by converting factor VII to factor VIIa. The culture medium from appropriately grown transformed or transfected host cells is separated from the cell material, and the presence of chimeric proteins is demonstrated. One method of detecting the chimeric proteins, for example, is by the binding of the chimeric proteins or portions of the chimeric proteins to a specific antibody recognizing the chimeric protein of the invention (e.g., an anti-Fc antibody). An anti-chimeric protein antibody may be a monoclonal or polyclonal antibody raised against the chimeric protein in question. For example, the chimeric protein can contain a portion of an immunoglobulin constant region. Antibodies recognizing the constant region of many immunoglobulins are known in the art and are commercially available. An antibody can be used to perform an ELISA or a western blot to detect the presence of the chimeric protein of the invention.
  • F. Methods of Using Chimeric Proteins
  • The chimeric proteins of the invention have many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent.
  • 1. Methods of Treating a Subject Having a Hemostatic Disorder
  • The invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises at least a portion of an immunoglobulin constant region and at least one clotting factor.
  • The chimeric protein of the invention treats or prevents a hemostatic disorder by promoting the formation of a fibrin clot. The chimeric protein of the invention can activate any member of a coagulation cascade. The clotting factor can be a participant in the extrinsic pathway, the intrinsic pathway or both. In one embodiment, the clotting factor is factor VII or factor VIIa. Factor VIIa can activate factor X which interacts with factor Va to cleave prothrombin to thrombin, which in turn cleaves fibrinogen to fibrin.
  • a. Conditions That May Be Treated
  • The chimeric protein of the invention can be used to treat any hemostatic disorder. The hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII.
  • In one embodiment, the hemostatic disorder is an inherited disorder. In one embodiment, the subject has hemophilia A, and the chimeric protein comprises factor VIII or factor VIIIa. In another embodiment, the subject has hemophilia A and the chimeric protein comprises factor VII or factor VIIa. In another embodiment, the subject has hemophilia B and the chimeric protein comprises factor IX or factor IXa. In another embodiment, the subject has hemophilia B and the chimeric protein comprises factor VII or factor VIIa. In another embodiment, the subject has inhibitory antibodies to factor VIII or factor VIIIa and the chimeric protein comprises factor VII or factor VIIa. In yet another embodiment, the subject has inhibitory antibodies against factor IX or factor IXa and the chimeric protein comprises factor VII or factor VIIa.
  • The chimeric protein of the invention can be used to prophylactically treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder.
  • In one embodiment, the hemostatic disorder is the result of a deficiency in a clotting factor, e.g., factor IX, factor VII, factor VII. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand's factor.
  • In another embodiment, the hemostatic disorder can be an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an auto-immmune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).
  • 2. Methods of Treating a Subject In Need of a General Hemostatic Agent
  • The invention also relates to methods of treating a subject that does not have a hemostatic disorder or a secondary disease or condition resulting in acquisition of a hemostatic disorder. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises at least a portion of an immunoglobulin constant region and at least one clotting factor.
  • a. Conditions That May Be Treated
  • In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to, during, or after surgery as a prophylactic. The chimeric protein of the invention can be administered prior to, during, or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to liver transplantation, liver resection, or stem cell transplantation.
  • The chimeric protein of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding.
  • b. Treatment Modalities
  • The chimeric protein of the invention can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route. The chimeric protein can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the site of bleeding or implanted into bandage/dressing.
  • The dose of the chimeric protein of the invention will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 μg/kg body weight. In one embodiment, the dosing range is 0.1-1,000 μg/kg. The protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e.g., STA-CLOT VIIa-rTF clotting assay. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).
  • The invention also relates to a pharmaceutical composition comprising a clotting factor, at least a portion of an immunoglobulin constant region and a pharmaceutically acceptable carrier or excipients. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin. Examples of excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.
  • For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can include suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.
  • For buccal administration, the composition may take the form of tablets or lozenges according to conventional protocols.
  • For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or nebulizer, with optionally a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • The pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous or intramuscular) by bolus injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., pyrogen free water.
  • The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • C. Combination Therapy
  • In another embodiment, the invention relates to a method of treating a subject with a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region in combination with at least one other clotting factor or-agent that promotes hemostasis. Said other clotting factor or agent that promotes hemostasis can be any therapeutic with demonstrated clotting activity. As an example, but not as a limitation, the clotting factor or hemostatic agent can include factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue factor (rsTF) or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamic acid.
  • G. Kits
  • The invention provides a kit for the diagnosis of a hemostatic disorder. The kit can include a container and a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region. The chimeric protein can be provided in an appropriate buffer or solvent. The buffer can be an aqueous buffer, e.g., PBS or alternatively the chimeric protein can be lyophilized. The kit can also provide instructions for detecting the presence of a clotting factor in a sample, e.g., contacting an aliquot of a sample with the chimeric protein of the invention and detecting the presence of a clot. Detection can include visible detection. The kit can optionally provide an aliquot of blood lacking a known clotting factor.
    • EXAMPLES Example 1 Cloning of pCDNA 3.1-Flag-Fc
  • The sequence for the FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)(SEQ ID NO: 23), a common affinity tag used to identify or purify proteins, was cloned into the pcDNA 3.1-Fc plasmid, which contains the mouse IgK signal sequence followed by the Fc fragment of human IgG1 (amino acids 221-447, EU numbering). The construct was created by overlapping PCR using the following primers:
    FlagFc-F1:
    5′- GCTGGCTAGCCACCATGGA -3′ (SEQ ID NO: 10)
    FlagFc-R1:
    5′- CTTGTCATCGTCGTCCTTGTAGTCGTCA (SEQ ID NO: 11)
    CCAGTGGAACCTGGAAC -3′
    FlagFc-F2:
    5′- GACTACAAGG ACGACGATGA (SEQ ID NO: 12)
    CAAGGACAAA
    ACTCACACAT GCCCACCGTG CCCAGCTCCG
    GAACTCC -3′
    FlagFc-R2:
    5′- TAGTGGATCCTCATTTACCCG -3′ (SEQ ID NO: 13)
  • The pcDNA 3.1-Fc template was then added to two separate PCR reactions containing 50 pmol each of the primer pairs FlagFc-F1/R1 or FlagFc-F2/R2 in a 50 μl reaction using Pfu Ultra DNA polymerase (Stratagene, Calif.) according to manufacturer's standard protocol in a MJ Thermocycler using the following cycles: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 52° C. 30 seconds, 72° C. 45 seconds), followed by 72° C. for 10 minutes. The products of these two reactions were then mixed in another PCR reaction (2 μl each) with 50 pmol of FlagFc-F1 and FlagFc-R2 primers in a 50 μl reaction using Pfu Ultra DNA polymerase (Stratagene, Calif.) according to manufacturer's standard protocol in a MJ Thermocycler using the following cycles: 95° C. 2 minutes; 30 cycles of (95° C. 30 seconds, 52° C. 30 seconds, 72° C. 45 seconds), followed by 72° C. for 10 minutes. The resulting fragment was gel purified, digested and inserted into the pcDNA 3.1-Fc plasmid NheI-BamHI. The resulting plasmid contains the mouse IgK signal sequence producing the FlagFc protein.
    • Example 2 Cloning of PACE Construct
  • The coding sequence for human PACE (paired basic amino acid cleaving enzyme), an endoprotease, was obtained by RT-PCR. The following primers were used:
    (SEQ ID NO: 15)
    PACE-F1: 5′- GGTAAGCTTGCCATGGAGCTGAGGCCCTGGTTGC -
    3′
    (SEQ ID NO: 16)
    PACE-R1: 5′- GTTTTCAATCTCTAGGACCCACTCGCC -3′
    (SEQ ID NO: 17)
    PACE-F2: 5′- GCCAGGCCACATGACTACTCCGC -3′
    (SEQ ID NO: 18)
    PACE-R2: 5′- GGTGAATTCTCACTCAGGCAGGTGTGAGGGCAGC -
    3′
  • The PACE-F1 primer adds a HindIII site to the 5′ end of the PACE sequence beginning with 3 nucleotides before the start codon, while the PACE-R2 primer adds a stop codon after amino acid 715, which occurs at the end of the extracellular domain of PACE, as well as adding an EcoRI site to the 3′ end of the stop codon. The PACE-R1 and -F2 primers anneal on the 3′ and 5′ sides of an internal BamHI site, respectively. Two RT-PCR reactions were then set up using 25 pmol each of the primer pairs of PACE-F1/R1 or PACE-F2/R2 with 20 ng of adult human liver RNA (Clontech; Palo Alto, Calif.) in a 50 μl RT-PCR reaction using the SuperScript.™ One-Step RT-PCR with PLATINUM® Taq system (Invitrogen, Carlsbad, Calif.) according to manufacturers protocol. The reaction was carried out in a MJ Thermocycler using the following cycles: 50° C. 30 minutes; 94° C. 2 minutes; 30 cycles of (94° C. 30 seconds, 58° C. 30 seconds, 72° C. 2 minutes), followed by 72° C. 10 minutes. These fragments were each ligated into the vector pGEM T-Easy (Promega, Madison, Wis.) and sequenced fully. The F2-R2 fragment was then subcloned into pcDNA6 V5/His (Invitrogen, Carlsbad, Calif.) using the BamHI/EcoRI sites, and then the F1-R1 fragment was cloned into this construct using the HindIII/BamHI sites. The final plasmid, pcDNA6-PACE, produces a soluble form of PACE (amino acids 1-715), as the transmembrane region has been deleted. The sequence of PACE in pcDNA6-PACE is essentially as described in Harrison et al. 1998, Seminars in Hematology 35:4.
    • Example 3 Cloning of Fc-Factor VII construct
  • The coding sequence for Factor VII, was obtained by RT-PCR from human fetal liver RNA (Clontech, Palo Alto, Calif.). The cloned region is comprised of the cDNA sequence from bp 36 to bp 1430 terminating just before the stop codon. A SbfI site was introduced on the N-terminus. A BspEI site was introduced on the C-terminus. The construct was cloned by PCR using the primers:
    (SEQ ID NO: 19)
    Down- 5′ GCTACCTGCAGGCCACCATGGTCTCCCAGGCCCTCAGG
    stream: 3′
    (SEQ ID NO: 20)
    Upstream: 5′ CAGTTCCGGAGCTGGGCACGGCGGGCACGTGTGAGTTT
    TGTCGGGAAAT GG
    3′

    and the following conditions: 95° C. for 5 minutes followed by 30 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 1 minute and 45 seconds, and a final extension cycle of 72° C. for 10 minutes.
  • The fragment was digested SbfI-BspE I and inserted into pED.dC-Fc a plasmid encoding for the Fc fragment of an IgG1.
    • Example 4 Factor VII-Fc monomer-dimer Hybrid Expression and Purification
  • CHO DG-44 cells expressing Factor VII-Fc were established. CHO DG-44 cells were grown at 37° C., 5% CO2, in MEM Alpha plus nucleoside and ribonucleosides and supplemented with 5% heat-inactivated fetal bovine serum until transfection.
  • DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%-60%. A total of 10 μg of DNA was used to transfect one 100 mm dish: either 9 μg pED.dC.FVII-Fc+1 μg of pcDNA6-PACE for the dimer transfection, or 7.5 μg of pED.dC.FVII-Fc+1.5 μg pcDNA3/Flag-Fc+1 μg of pcDNA6-PACE for the monomer transfection. The cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, Calif.). The media was removed after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 μg/ml of Blasticidin (Invitrogen, Carlsbad, Calif.) for both transfections, while the monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). After 10 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable cell lines were established. Protein expression was subsequently amplified by the addition 25 nM methotrexate.
  • Approximately 2×107 cells were used to inoculate 300 ml of growth medium in a 1700 cm2 roller bottle (Corning, Corning, N.Y.) supplemented with 5 μg/L of vitamin K3 (menadione sodium bisulfite) (Sigma, St Louis, Mo.). The roller bottles were incubated in a 5% CO2 at 37° C. for 72 hours. The growth medium was then exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 μg/ml bovine insulin and 10 μg/ml Gentamicin) supplemented with 5 μg/L of vitamin K3. The production medium (conditioned medium) was collected every day for 10 days and stored at 4° C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Pooled media was first clarified using a Sartoclean glass fiber filter (3.0 μm+0.2 μm) (Sartorious Corp. Gottingen, Germany) followed by an Acropack 500 filter (0.8 μm+0.2 μm) (Pall Corp., East Hills, N.Y.). The clarified media was then concentrated approximately 20-fold using Pellicon Biomax tangential flow filtration cassettes (10 kDa MWCO) (Millipore Corp., Billerica, Mass.).
  • Fc chimeras were then captured from the concentrated media by passage over a Protein A Sepharose 4 Fast Flow Column (AP Biotech, Piscataway, N.J.). A 5×5 cm (100 ml) column was loaded with ≦5 mg Fc protein per ml column volume at a linear flow rate of 100 cm/hour to achieve a residence time of ≧3 minutes. The column was then washed with >5 column volumes of 1× DPBS to remove non-specifically bound proteins. The bound proteins were eluted with 100 mM Glycine pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1 part 1 M Tris-HCL, pH 8 to 10 parts elute fraction. Passage of the chimeric protein over the Protein A column converted inactive factor VII to activated factor VIIa (FIG. 2). Further activation could be achieved by passing the protein over an anion exchange column (Peders et al. 1989, Biochemistry 28:9331-36).
  • The monomer-dimer hybrid transfection protein sample was subject to further purification, as it contained a mixture of FVII-Fc:FVII-Fc homodimer, FVII-FC: FLAG-FC monomer-dimer hybrid, and FLAG-Fc: FLAG-Fc homodimers. To remove FLAG-Fc homodimers from the preparation, the Protein A Sepharose 4 Fast Flow pool was first dialyzed into 20 mM MES, 20 mM NaCl, pH 6.1 and was then passed over a Unosphere S cation-exchange column (BioRad Corp., Richmond, Calif.). Under the operating conditions for the column, the FLAG-Fc monomer-dimer hybrid has a net neutral charge (FLAG-Fc theoretical pl=6.19) and flows through the column while the hFVII-Fc constructs are positively charged, and thus bind to the column and elute at higher ionic strength. The dialyzed material was then loaded onto a 1.1×11 cm (9.9 ml) column at 150 cm/hour. During the wash and elution, the flow rate was increased to 500 cm/hour. The column was washed sequentially with 8 column volumes of 20 mM MES, 20 mM NaCl, pH 6.1 and 8 column volumes of 20 mM MES, 40 mM NaCl, pH 6.1. The bound protein was eluted with 20 mM MES, 750 mM NaCl, pH 6.1. Elution fractions containing the protein peak were pooled and sterile filtered through a 0.2 μm filter disc prior to storage at −80° C.
  • An anti-FLAG MAB affinity column was used to separate chimeric Fc dimers with hFVII fused to both Fc molecules from those with one FLAG peptide and one hFVII fusion. The Unosphere S Eluate pool was diluted 1:1 with 20 mM Tris, 50 mM NaCl, 5 mM CaCl2, pH 8 and loaded onto a 1.6×5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, Mo.) at a linear flow rate of 60 cm/hour. Loading was targeted to <2.5 mg monomer-dimer hybrid/ml column volume. After loading the column was washed with 5 column volumes 20 mM Tris, 50 mM NaCl, 5 mM CaCl2, pH 8.0. monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1 part 1 M Tris-HCl, pH 8 to 10 parts eluate fraction. Pools were stored at −80° C.
    • Example 5 Clotting Activity Analysis of Factor VII-Fc Homodimer and Monomer/Dimer Hybrids
  • The StaClot FVIIa-rTF assay kit was purchased from Diagnostica Stago (Parsippany, N.J.) and modified as described in Johannessen et al. 2000, Blood Coagulation and Fibrinolysis 11: S159. A standard curve was performed with the FVIIa World Health Organization standard 89/688. The assay compared a homodimer comprised of two factor VII molecules and two Fc molecules with a monomer/dimer hybrid comprised of one factor VII molecule and two Fc molecules. Significant clotting activity was observed for both the monomer/dimer hybrid and the homodimer. The clotting activity of the monomer/dimer hybrid compared to the homodimer was almost four times as great (FIG. 4).
    • Example 6 Binding of Factor VII-Fc to shFcRn
  • Binding of factor VII-Fc to soluble human neonatal Fc receptor (shFcRn) was analyzed using a Biacore 3000 instrument (Biacore, Uppsala, Sweden). A CM5 chip (Biacore, Uppsala, Sweden) was coated with 3500 resonance units (RU) of shFcRn using amine chemistry. Factor VII-Fc was diluted, 10 fold or 100 fold, in 50 mM PO4, pH 6.0,100 mM NaCl, 0.01% Tween-20 and injected (in duplicate) over the surface for 10 minutes at 10 μL/minute. The samples were also injected over a mock-coated surface which served as a reference control. The chip was regenerated with 100 mM PO4 pH 8.0. The response (in RU), recorded 30 seconds before the injection was stopped, indicated specific binding (FIG. 5).
    • Example 7 Factor VII-Fc Oral Uptake in Neonatal Rats
  • 25 gram day 9 newborn rats were purchased from Charles River (Wilmington, Mass.) and allowed to acclimate for 24 hours. The rats were dosed orally with FVIIa-Fc homodimer, or monomer/dimer hybrid(consisting of two Fc chains, one of which was linked to Fc-VII). A volume of 200 μl of a FVIIa-Fc solution was used for a dose of 1 mg/kg. The solution was comprised of a Tris-HCl buffer pH 7.4 with 5 mg/ml soybean trypsin inhibitor. The rats were euthanized with CO2 at several time points, and 200 μl of blood was drawn by cardiac puncture. Plasma was obtained by the addition of a {fraction (1/10)} volume 3.8% sodium citrate solution and centrifuged at room temperature at a speed of 1268×g. The plasma samples were either used in the assays fresh or frozen at −20° C.
  • Samples were then analyzed by ELISA. The Asserachrom Factor VII:Ag ELISA was performed on the neonatal rat plasma samples. A Factor VII:Ag ELISA assay was purchased from Diagnostica Stago (Parsippany, N.J.) and performed as described in the kit manual with one change. The standard for the standard curve was replaced with purified Factor VIIa-Fc. For in vivo experiments, the standard was run in the same percent normal animal plasma as being analyzed. The results showed oral administration of both the monomer/dimer hybrids and the dimers was successful (FIG. 7). A time course assay using monomer/dimer hybrids demonstrated sustained plasma levels of orally administered Factor VIIa-Fc over time with a T1/2 of 18 hours (FIG. 6).
    • Example 8 Intravenous Dosing of Factor VII-Fc in Minipigs
  • Adult Gottingen minipigs (6 kg) were purchased from Marshall Farms and allowed to acclimate for two weeks. The pigs were anethestized with 12 mg/kg Telazol and 8 mg/kg Xylaxine and dosed intravenously through the jugular vein with 3 ml of a 0.5 mg/kg Factor VIIa-Fc solution in a Tris-HCl buffer pH 7.4. Three mis of blood was collected in citrated vacutainer tubes (BD Sciences, Franklin Lakes, N.J.) at various time points via venous puncture. Plasma was obtained by centrifuging samples at room temperature at a speed of 1268×g. The plasma samples were frozen at −20° C. and subsequently analyzed by ELISA.
  • The Asserachrom Factor VII:Ag ELISA was performed on the minipig samples. A Factor VII:Ag Elisa assay was purchased from Diagnostica Stago (Parsippany, N.J.) and preformed as described in the kit manual with one change. The standard for the standard curve was replaced with purified Factor VIIa-Fc. For in vivo experiments, the standard was run in the same percent normal animal plasma as being analyzed. Plasma levels of intravenously administered monomer/dimer hybrid are shown in FIG. 8A. The half life was determined to be 9.4 hours. A time course assay using monomer/dimer hybrid chimeric protein demonstrated sustained plasma levels of intravenously administered Factor VIIa-Fc over time with a T1/2 of 22 hours (FIG. 8A).
  • Clotting activity using was measured by the StaClot FVIIa-rTF assay kit (FIG. 8B). The T1/2 for clotting was found to be 6.4 hours for one pig and 5.7 for the other pig.
    • Example 9 Cloning of Fc-Factor IX construct
  • The human Factor IX coding sequence, including the prepropeptide sequence, was obtained by RT-PCR amplification from adult human liver RNA using the following primers:
    (SEQ ID NO: 21)
    natFIX- 5′-TTACTGCAGAAGGTTATGCAGCGCGTGAACATG-
    F: 3′
    (SEQ ID NO: 22)
    F9-R: 5′-TTTTTCGAATTCAGTGAGCTTTGTTTTTTCCTTAATCC-
    3′
  • 20 ng of adult human liver RNA (Clontech, Palo Alto, Calif.) and 25 pmol each primer were added to a RT-PCR reaction using the SuperScript.™ One-Step RT-PCR with PLATINUM® Taq system (Invitrogen, Carlsbad, Calif.) according to manufacturers protocol. Reaction was carried out in a MJ Thermocycler using the following cycles: 50° C. 30 minutes; 94° C. 2 minutes; 35 cycles of (94° C. 30 seconds, 58° C. 30 seconds, 72° C. 1 minute), and a final 72° C. 10 minutes. The fragment was gel purified using Qiagen Gel Extraction Kit (Qiagen, Valencia, Calif.), and digested with PstI-EcoRI, gel purified, and cloned into the corresponding digest of the pED.dC.XFc plasmid. The amino acid and DNA sequences of factor IX-Fc are shown in FIG. 9.
    • Example 10 Factor IX-Fc homodimer and monomer-dimer Hybrid Expression and Purification
  • CHO DG-44 cells expressing Factor IX-Fc were established. DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%-60%. A total of 10 μg of DNA was used to transfect one 100 mm dish: for the homodimer transfection, 8 μg of pED.dC.Factor IX-Fc+2 μg of pcDNA6-PACE was used; for the monomer-dimer hybrid transfection, 8 μg of pED.dC.Factor IX-Fc+1 μg of pcDNA3-FlagFc+1 μg pcDNA6-PACE was used. The cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, Calif.). The media was removed from transfection after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 μg/ml of Blasticidin (Invitrogen, Carlsbad, Calif.) for both transfections, while the monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). After 3 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable cell lines were established. Protein expression was subsequently amplified by the addition 10 nM or 100 nM methotrexate for the homodimer or monomer-dimer hybrid, respectively.
  • For both cell lines, approximately 2×107 cells were used to inoculate 300 ml of growth medium in a 1700 cm2 roller bottle (Corning, Corning, N.Y.), supplemented with 5 μg/L of vitamin K3 (menadione sodium bisulfite) (Sigma, St. Louis, Mo.). The roller bottles were incubated in a 5% CO2 at 37° C. for approximately 72 hours. The growth medium was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 μg/ml bovine insulin and 10 μg/ml Gentamicin), supplemented with 5 μg/L of vitamin K3. The production medium (conditioned medium) was collected everyday for 10 days and stored at 4° C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 μm) filter from Pall Gelman Sciences (Ann Arbor, Mich.). All of the following steps were performed at 4° C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCl, and 137 mM NaCl), eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of 1 M Tris-HCl, pH 9.0. The protein was then dialyzed into PBS.
  • The monomer-dimer hybrid transfection protein sample was subject to further purification, as it contained a mixture of FIX-Fc:FIX-Fc homodimer, FIX-Fc:Flag-Fc monomer-dimer hybrid, and Flag-Fc:Flag-Fc homodimer. Material was concentrated and applied to a 2.6 cm×60 cm (318 ml) Superdex 200 Prep Grade column at a flow rate of 4 ml/minute (36 cm/hour) and then eluted with 3 column volumes of 1× PBS. Fractions corresponding to two peaks on the UV detector were collected and analyzed by SDS-PAGE. Fractions from the first peak contained either FIX-Fc:FIX-Fc homodimer or FIX-Fc:FlagFc monomer-dimer hybrid, while the second peak contained FlagFc:FlagFc homodimer. All fractions containing the monomer-dimer hybrid but no FlagFc homodimer were pooled and applied directly to a 1.6×5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, Mo.) at a linear flow rate of 60 cm/hour. After loading, the column was washed with 5 column volumes PBS. Monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were then neutralized by adding {fraction (1/10)} volume of 1 M Tris-HCl, and analyzed by reducing and nonreducing SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 1-5 mg/ml, and stored at −80° C.
  • All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supercede and/or take precedence over any such contradictory material.
  • All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
  • Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (58)

1. A chimeric protein comprising at least a portion of an immunoglobulin constant region, at least one clotting factor, and optionally at least one linker.
2. The chimeric protein of claim 1, wherein said immunoglobulin is an IgG.
3. The chimeric protein of claim 2, wherein the portion of an immunoglobulin constant region is an Fc fragment.
4. The chimeric protein of claim 2, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.
5. The chimeric protein of claim 2, wherein the Fc fragment comprises an amino acid sequence having at least 80% identity with the sequence set forth in SEQ ID NO:3.
6. The chimeric protein of claim 1, 2, 3, or 4, wherein the clotting factor is factor VII or VIIa.
7. The chimeric protein of claim 6, wherein the clotting factor is an analog of factor VII or factor VIIa.
8. The chimeric protein of claim 1, 2, 3, or 4, wherein the clotting factor is factor VIII, or factor VIIIa, factor V, factor Va, factor IX, factor X, factor XI, factor XII, factor XIII or von Willebrand's factor.
9. A pharmaceutical composition comprising the chimeric protein of claim 6, and pharmaceutically acceptable excipient.
10. The chimeric protein of claim 1, 2, 3, or 4, wherein said chimeric protein is a dimer.
11. The chimeric protein of claim 10, wherein the dimer is a homodimer.
12. The chimeric protein of claim 10, wherein the chimeric protein is a dimer comprising a first chain and a second chain, wherein said first chain comprises an Fc fragment of an immunoglobulin and Factor VII or Factor VIIa and said second chain comprises an Fc fragment of an immunoglobulin without factor VII or factor VIIa.
13. The chimeric protein of claim 1, 2, 3, or 4, comprising the formula

X-L-F
wherein F is an Fc fragment of an immunoglobulin and L is a linker or a direct bond, and X is factor VII, or factor VIIa.
14. The chimeric protein of claim 13, wherein F comprises an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO:2.
15. The chimeric protein of claim 13, wherein L is a linker comprising about 1 to about 20 amino acids.
16. The chimeric protein of claim 13, wherein L is a linker comprising about 1 to about 10 amino acids.
17. The chimeric protein of claim 13, wherein L is a linker comprising about 1 to about 5 amino acids.
18. The chimeric protein of claim 13, wherein L is a linker comprising the sequence -(Gly)n- (SEQ ID NO: 30), wherein n is an integer of about 1 to about 10.
19. The chimeric protein of claim 13, wherein L is a linker comprising the sequence -(GGS)n- (SEQ ID NO: 5) or (GGGGS)n (SEQ ID NO: 31), wherein n is an integer of about 1 to about 10.
20. The chimeric protein of claim 1, 2, 3, or 4, wherein the protein comprises SEQ ID NO:1.
21. The chimeric protein of claim 1, 2, 3, or 4, wherein the chimeric protein promotes hemostasis.
22. A method of treating a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein having clotting activity to a subject, wherein said chimeric protein comprises at least a portion of an immunoglobulin and a clotting factor, and allowing the chimeric protein to treat the hemostatic disorder.
23. The method of claim 22, wherein the hemostatic disorder is hemophilia A or B.
24. The method of claim 22, wherein the portion of an immunoglobulin constant region is an Fc fragment.
25. The method of claim 22, wherein the clotting factor is factor VII or factor VIIa.
26. The method of claim 24, wherein said portion of an immunoglobulin constant region is an FcRn binding partner.
27. The method of claim 22, 24, or 26, wherein the chimeric protein is administered intravenously, subcutaneously, intra-muscularly, or via a mucosal surface.
28. The method of claim 27, wherein the chimeric protein is administered orally, nasally, rectally, vaginally, or via buccal administration.
29. The method of claim 27, wherein the chimeric protein is administered via a pulmonary route.
30. The method of claim 22, wherein the chimeric protein is administered non-parenterally.
31. The method of claim 22, wherein the chimeric protein is administered at a dose of 0.1-1000 μg/kg.
32. The method of claim 22, wherein the chimeric protein is administered daily.
33. A method of making a chimeric protein having clotting activity comprising at least a portion of an immunoglobulin constant region and a clotting factor, said method comprising:
a) transfecting a cell with a DNA construct comprising a first DNA sequence encoding at least a portion of an immunoglobulin constant region operatively linked to a second DNA sequence encoding a clotting factor and optionally a third DNA sequence encoding an endoprotease;
b) culturing said cell under conditions such that the chimeric protein is expressed; and
c) isolating said chimeric protein from said cell.
34. The method of claim 33, wherein the culture conditions include vitamin K.
35. The method of claim 34, wherein the endoprotease is PACE.
36. The method of claim 33, wherein the cell is a eukaryotic cell.
37. The method of claim 36, wherein the cell is a CHO cell or a BHK cell.
38. The method of claim 33 further comprising transfecting a cell with a second DNA construct comprising a DNA sequence encoding at least a portion of an immunoglobulin constant region without said second sequence encoding a clotting factor.
39. The method of claim 33 or 38, wherein said portion of an immunoglobulin constant region is an Fc fragment.
40. The method of claim 33 or 38, wherein said portion of an immunoglobulin constant region is an IgG Fc fragment.
41. The method of claim 33 or 38, wherein said portion of an immunoglobulin constant region is FcRn binding partner.
42. The method of claim 33 or 38, wherein the clotting factor is factor VII or factor VIIa.
43. A nucleic acid molecule comprising a nucleic acid sequence encoding at least one clotting factor, and at least a portion of an immunoglobulin constant region.
44. A vector comprising the nucleic acid molecule of claim 43.
45. The nucleic acid molecule of claim 43, wherein said portion of an immunoglobulin constant region is an Fc fragment.
46. The nucleic acid molecule of claim 43, wherein said portion of an immunoglobulin constant region is an FcRn binding partner.
47. The vector of claim 43, wherein said portion of an immunoglobulin constant region is a portion of an IgG.
48. The vector of claim 43, wherein the clotting factor is factor VII or factor VIIa.
49. A host cell comprising the vector of claim 44.
50. The host cell of claim 49, wherein said host cell is a CHO cell or a BHK cell.
51. The chimeric protein of claim 1, 2, 3, or 4, comprising a protein encoded by the DNA sequence of SEQ ID NO:1.
52. The chimeric protein of claim 1, 2, 3, or 4, comprising the amino acid sequence of SEQ ID NO:2.
53. The vector of claim 48, comprising the DNA sequence of SEQ ID NO:2.
54. The vector of claim 48, comprising the DNA sequence of SEQ ID NO:3.
55. A method of treating a hemostatic disorder comprising administering to a subject a therapeutically effective amount of a first agent to treat a hemostatic disorder, wherein said first agent comprises at least one chimeric protein having clotting activity, and wherein said chimeric protein comprises at least a portion of an immunoglobulin and a clotting factor, in combination with a second agent, wherein said second agent is any agent known to have clotting factor activity and allowing the first and second agent to treat the hemostatic disorder.
56. The method of claim 55 wherein said second agent is factor VIII, factor IX, factor XI, factor XII, fibrinogen, prothrombin, factor V, factor VII, factor X, factor XIII or von Willebrand factor.
57. The chimeric protein of claim 10, wherein the chimeric protein is a dimer comprising a first chain and a second chain, wherein said first chain comprises an Fc fragment of an immunoglobulin and Factor VIII and said second chain comprises an Fc fragment of an immunoglobulin without factor VIII.
58. The chimeric protein of claim 10, wherein the chimeric protein is a dimer comprising a first chain and a second chain, wherein said first chain comprises an Fc fragment of an immunoglobulin and Factor IX and said second chain comprises an Fc fragment of an immunoglobulin without factor IX.
US10/841,819 2003-05-06 2004-05-06 Clotting factor-Fc chimeric proteins to treat hemophilia Abandoned US20050147618A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US10/841,819 US20050147618A1 (en) 2003-05-06 2004-05-06 Clotting factor-Fc chimeric proteins to treat hemophilia
US12/949,564 US8449884B2 (en) 2003-05-06 2010-11-18 Clotting factor-fc chimeric proteins to treat hemophilia
US13/792,889 US8815250B2 (en) 2003-05-06 2013-03-11 Clotting factor-Fc chimeric proteins to treat hemophilia
US14/334,396 US20150044207A1 (en) 2003-05-06 2014-07-17 Clotting Factor-Fc Chimeric Proteins to Treat Hemophilia
US15/866,203 US20180237762A1 (en) 2003-05-06 2018-01-09 Clotting factor-fc chimeric proteins to treat hemophilia
US17/239,208 US20210348150A1 (en) 2003-05-06 2021-04-23 Clotting factor-fc chimeric proteins to treat hemophilia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46883703P 2003-05-06 2003-05-06
US10/841,819 US20050147618A1 (en) 2003-05-06 2004-05-06 Clotting factor-Fc chimeric proteins to treat hemophilia

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/949,564 Division US8449884B2 (en) 2003-05-06 2010-11-18 Clotting factor-fc chimeric proteins to treat hemophilia

Publications (1)

Publication Number Publication Date
US20050147618A1 true US20050147618A1 (en) 2005-07-07

Family

ID=33452235

Family Applications (6)

Application Number Title Priority Date Filing Date
US10/841,819 Abandoned US20050147618A1 (en) 2003-05-06 2004-05-06 Clotting factor-Fc chimeric proteins to treat hemophilia
US12/949,564 Expired - Lifetime US8449884B2 (en) 2003-05-06 2010-11-18 Clotting factor-fc chimeric proteins to treat hemophilia
US13/792,889 Expired - Lifetime US8815250B2 (en) 2003-05-06 2013-03-11 Clotting factor-Fc chimeric proteins to treat hemophilia
US14/334,396 Abandoned US20150044207A1 (en) 2003-05-06 2014-07-17 Clotting Factor-Fc Chimeric Proteins to Treat Hemophilia
US15/866,203 Abandoned US20180237762A1 (en) 2003-05-06 2018-01-09 Clotting factor-fc chimeric proteins to treat hemophilia
US17/239,208 Abandoned US20210348150A1 (en) 2003-05-06 2021-04-23 Clotting factor-fc chimeric proteins to treat hemophilia

Family Applications After (5)

Application Number Title Priority Date Filing Date
US12/949,564 Expired - Lifetime US8449884B2 (en) 2003-05-06 2010-11-18 Clotting factor-fc chimeric proteins to treat hemophilia
US13/792,889 Expired - Lifetime US8815250B2 (en) 2003-05-06 2013-03-11 Clotting factor-Fc chimeric proteins to treat hemophilia
US14/334,396 Abandoned US20150044207A1 (en) 2003-05-06 2014-07-17 Clotting Factor-Fc Chimeric Proteins to Treat Hemophilia
US15/866,203 Abandoned US20180237762A1 (en) 2003-05-06 2018-01-09 Clotting factor-fc chimeric proteins to treat hemophilia
US17/239,208 Abandoned US20210348150A1 (en) 2003-05-06 2021-04-23 Clotting factor-fc chimeric proteins to treat hemophilia

Country Status (19)

Country Link
US (6) US20050147618A1 (en)
EP (5) EP2298347B1 (en)
AT (2) ATE497783T1 (en)
AU (2) AU2004239244C1 (en)
BE (1) BE2016C058I2 (en)
CA (1) CA2522859C (en)
CY (4) CY1109640T1 (en)
DE (2) DE602004031390D1 (en)
DK (3) DK2077121T3 (en)
ES (3) ES2558102T3 (en)
FR (1) FR16C0041I2 (en)
HK (2) HK1134432A1 (en)
HU (2) HUE026384T2 (en)
LU (1) LU93265I2 (en)
NL (1) NL300835I2 (en)
PL (3) PL2077121T3 (en)
PT (3) PT1624891E (en)
SI (3) SI2077121T1 (en)
WO (1) WO2004101740A2 (en)

Cited By (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050261229A1 (en) * 1999-07-21 2005-11-24 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US20070172928A1 (en) * 2003-05-06 2007-07-26 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
WO2010111414A1 (en) * 2009-03-24 2010-09-30 Bayer Healthcare Llc Factor viii variants and methods of use
US20110182896A1 (en) * 2003-05-06 2011-07-28 Syntonix Pharmaceuticals, Inc. CLOTTING FACTOR-Fc CHIMERIC PROTEINS TO TREAT HEMOPHILIA
WO2011060242A3 (en) * 2009-11-13 2011-10-13 Talecris Biotherapeutics, Inc. Von willebrand factor (vwf)-containing preparations, and methods, kits, and uses related thereto
WO2012061654A1 (en) 2010-11-03 2012-05-10 Catalyst Biosciences, Inc. Modified factor ix polypeptides and uses thereof
US20130108629A1 (en) * 2009-12-06 2013-05-02 Biogen Idec Hemophilia Inc. Factor VIII-Fc Chimeric and Hybrid Polypeptides, and Methods of Use Thereof
CN103180439A (en) * 2010-07-09 2013-06-26 比奥根艾迪克依蒙菲利亚公司 Chimeric clotting factors
US20130296534A1 (en) * 2010-10-20 2013-11-07 Sk Chemicals Co., Ltd. Fusion protein having factor ix activity
US20140370035A1 (en) * 2012-01-12 2014-12-18 Biogen Idec Ma Inc. Methods of reducing immunogenicity against factor viii in individuals undergoing factor viii therapy
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US8980610B2 (en) 2008-11-20 2015-03-17 Biogen Idec Ma Inc. Arginine inactivation of viruses
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9062115B2 (en) 2010-09-22 2015-06-23 Novo Nordisk A/S Therapeutic factor VIII antibodies
US20150184142A1 (en) * 2012-06-08 2015-07-02 Biogen Idec Ma Inc. Procoagulant compounds
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US20150315262A1 (en) * 2012-12-10 2015-11-05 Vib Vzw Novel interleukin-33 inhibitors
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US20150353911A1 (en) * 2012-06-08 2015-12-10 Biogen Idec Ma Inc. Chimeric clotting factors
US9238878B2 (en) 2009-02-17 2016-01-19 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9540438B2 (en) 2011-01-14 2017-01-10 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and methods of use thereof
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
EP1984503B2 (en) † 2006-02-06 2018-08-15 CSL Behring GmbH Modified coagulation factor viia with extended half-life
US10138291B2 (en) 2012-07-11 2018-11-27 Bioverativ Therapeutics Inc. Factor VIII complex with XTEN and von Willebrand Factor protein, and uses thereof
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US10370430B2 (en) 2012-02-15 2019-08-06 Bioverativ Therapeutics Inc. Recombinant factor VIII proteins
US10421798B2 (en) 2012-02-15 2019-09-24 Bioverativ Therapeutics Inc. Factor VIII compositions and methods of making and using same
US10656167B2 (en) 2011-07-25 2020-05-19 Bioverativ Therapeutics Inc. Assays to monitor bleeding disorders
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10896749B2 (en) 2017-01-27 2021-01-19 Shire Human Genetic Therapies, Inc. Drug monitoring tool
US11081211B2 (en) 2013-06-20 2021-08-03 Baxalta Incorporated Method and apparatus for providing a pharmacokinetic drug dosing regimen
WO2021154414A2 (en) 2020-01-29 2021-08-05 Catalyst Biosciences, Inc. Gene therapy for hemophilia b with a chimeric aav capsid vector encoding modified factor ix polypeptides
US11135275B2 (en) * 2011-03-02 2021-10-05 Novo Nordisk Healthcare Ag Coagulation factor-targeting to TREM-like transcript 1 (TLT-1) on activated platelets
US11192936B2 (en) 2014-01-10 2021-12-07 Bioverativ Therapeutics Inc. Factor VIII chimeric proteins and uses thereof
US11208632B2 (en) 2016-04-26 2021-12-28 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
US11220554B2 (en) 2018-09-07 2022-01-11 Novo Nordisk A/S Procoagulant antibodies
US11370827B2 (en) 2012-01-12 2022-06-28 Bioverativ Therapeutics Inc. Chimeric factor VIII polypeptides and uses thereof
US11491212B1 (en) 2017-09-27 2022-11-08 Catalyst Biosciences, Inc. Subcutaneous administration of modified factor IX polypeptides and treatment of hemophilia B
US11642398B2 (en) 2013-03-15 2023-05-09 Bioverativ Therapeutics Inc. Factor IX polypeptide formulations
US12030925B2 (en) 2018-05-18 2024-07-09 Bioverativ Therapeutics Inc. Methods of treating hemophilia A
US12084512B2 (en) 2017-02-01 2024-09-10 Novo Nordisk A/S Procoagulant antibodies

Families Citing this family (95)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7214660B2 (en) 2001-10-10 2007-05-08 Neose Technologies, Inc. Erythropoietin: remodeling and glycoconjugation of erythropoietin
WO2004099231A2 (en) 2003-04-09 2004-11-18 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
US7173003B2 (en) 2001-10-10 2007-02-06 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
US7348004B2 (en) * 2003-05-06 2008-03-25 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
WO2005012484A2 (en) 2003-07-25 2005-02-10 Neose Technologies, Inc. Antibody-toxin conjugates
US8007805B2 (en) 2003-08-08 2011-08-30 Paladin Labs, Inc. Chimeric antigens for breaking host tolerance to foreign antigens
US8110665B2 (en) 2003-11-13 2012-02-07 Hanmi Holdings Co., Ltd. Pharmaceutical composition comprising an immunoglobulin FC region as a carrier
US7736653B2 (en) 2003-11-13 2010-06-15 Hanmi Pharm. Co., Ltd Pharmaceutical composition comprising an immunoglobulin Fc region as a carrier
US20080305992A1 (en) 2003-11-24 2008-12-11 Neose Technologies, Inc. Glycopegylated erythropoietin
WO2006010143A2 (en) 2004-07-13 2006-01-26 Neose Technologies, Inc. Branched peg remodeling and glycosylation of glucagon-like peptide-1 [glp-1]
US20080176790A1 (en) 2004-10-29 2008-07-24 Defrees Shawn Remodeling and Glycopegylation of Fibroblast Growth Factor (Fgf)
MX2007008229A (en) 2005-01-10 2007-09-11 Neose Technologies Inc Glycopegylated granulocyte colony stimulating factor.
CN104109209B (en) 2005-01-24 2016-06-08 得克萨斯大学体系董事会 The Fc fusion constructs being combined with phosphatidylserine and therepic use thereof
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
AU2006239117A1 (en) 2005-04-28 2006-11-02 Novo Nordisk Health Care Ag A closed container comprising an activated factor VII polypeptide, processes for the preparation of the same, and a kit and a method for use of the kit
JP5335422B2 (en) 2005-06-17 2013-11-06 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Selective reduction and derivatization of engineered proteins containing at least one unnatural cysteine
US20070105755A1 (en) 2005-10-26 2007-05-10 Neose Technologies, Inc. One pot desialylation and glycopegylation of therapeutic peptides
WO2007056191A2 (en) 2005-11-03 2007-05-18 Neose Technologies, Inc. Nucleotide sugar purification using membranes
EP2650305B1 (en) * 2006-03-24 2024-05-08 Bioverativ Therapeutics Inc. PC5 as a factor IX propeptide processing enzyme
RU2008145084A (en) 2006-05-24 2010-06-27 Ново Нордиск Хелс Кеа Аг (Ch) ANALOGUES OF FACTOR IX HAVING A LONG-TERM HALF-TIME IN VIVO
EP1867660A1 (en) * 2006-06-14 2007-12-19 CSL Behring GmbH Proteolytically cleavable fusion protein comprising a blood coagulation factor
ES2889920T3 (en) 2006-06-14 2022-01-14 Csl Behring Gmbh Proteolytically Cleavable Fusion Proteins with High Molar Specific Activity
US7939632B2 (en) 2006-06-14 2011-05-10 Csl Behring Gmbh Proteolytically cleavable fusion proteins with high molar specific activity
EP2049144B8 (en) 2006-07-21 2015-02-18 ratiopharm GmbH Glycosylation of peptides via o-linked glycosylation sequences
EP2054521A4 (en) 2006-10-03 2012-12-19 Novo Nordisk As Methods for the purification of polypeptide conjugates
EP3231440A1 (en) 2006-12-22 2017-10-18 CSL Behring GmbH Modified coagulation factors with prolonged in vivo half-life
EP1935430A1 (en) * 2006-12-22 2008-06-25 CSL Behring GmbH Modified coagulation factors with prolonged in vivo half-life
WO2008098720A1 (en) 2007-02-12 2008-08-21 Csl Behring Gmbh Therapeutic application of kazal-type serine protease inhibitors
JP2010523582A (en) 2007-04-03 2010-07-15 バイオジェネリクス アクチェンゲゼルシャフト Treatment method using glycoPEGylated G-CSF
MX2009013259A (en) 2007-06-12 2010-01-25 Novo Nordisk As Improved process for the production of nucleotide sugars.
CN103497247A (en) 2008-02-27 2014-01-08 诺沃—诺迪斯克有限公司 Conjugated factor VIII molecules
EP2291523B1 (en) * 2008-06-24 2014-12-17 CSL Behring GmbH Factor viii, von willebrand factor or complexes thereof with prolonged in vivo half-life
EP2371857A1 (en) 2010-04-01 2011-10-05 CSL Behring GmbH Factor XII inhibitors for treating interstitial lung disease
EP3508573A1 (en) 2010-07-09 2019-07-10 Bioverativ Therapeutics Inc. Systems for factor viii processing and methods thereof
CN103140237A (en) 2010-07-09 2013-06-05 比奥根艾迪克依蒙菲利亚公司 Factor ix polypeptides and methods of use thereof
CA2829037C (en) 2011-03-09 2022-05-17 Csl Behring Gmbh Factor xii inhibitors for the administration with medical procedures comprising contact with artificial surfaces
EP2497489A1 (en) 2011-03-09 2012-09-12 CSL Behring GmbH Factor XII inhibitors for the treatment of silent brain ischemia and ischemia of other organs
PT3513804T (en) 2011-07-08 2022-06-02 Bioverativ Therapeutics Inc Factor viii chimeric and hybrid polypeptides, and methods of use thereof
EP2734552A1 (en) 2011-07-22 2014-05-28 CSL Behring GmbH Inhibitory anti -factor xii/xiia monoclonal antibodies and their uses
WO2013057219A1 (en) 2011-10-18 2013-04-25 Csl Behring Gmbh Method for improving the stability of purified factor viii after reconstitution
JP6029674B2 (en) 2011-10-18 2016-11-24 ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー Use of a combination of sulfated glycosaminoglycan and hyaluronidase to improve the bioavailability of factor VIII
WO2013057167A1 (en) 2011-10-18 2013-04-25 Csl Behring Gmbh Use of sulfated glycosaminoglycans for improving the bioavailability of factor viii
EP2623110A1 (en) 2012-01-31 2013-08-07 CSL Behring GmbH Factor XII inhibitors for the treatment of neurological inflammatory disorders
EP2814502B1 (en) 2012-02-15 2017-09-13 CSL Behring GmbH Von willebrand factor variants having improved factor viii binding affinity
US10023628B2 (en) 2012-07-06 2018-07-17 Bioverativ Therapeutics Inc. Cell line expressing single chain factor VIII polypeptides and uses thereof
WO2014018777A2 (en) 2012-07-25 2014-01-30 Biogen Idec Ma Inc. Blood factor monitoring assay and uses thereof
EP3542861A1 (en) 2012-09-25 2019-09-25 Bioverativ Therapeutics Inc. Methods of using fix polypeptides
US10391152B2 (en) 2012-10-18 2019-08-27 Bioverativ Therapeutics Inc. Methods of using a fixed dose of a clotting factor
EP3446700A1 (en) 2012-10-30 2019-02-27 Bioverativ Therapeutics Inc. Methods of using fviii polypeptide
CN105188750A (en) 2013-03-08 2015-12-23 德国杰特贝林生物制品有限公司 Treatment and prevention of remote ischemia-reperfusion injury
RS60003B1 (en) 2013-03-15 2020-04-30 Bioverativ Therapeutics Inc Factor viii polypeptide formulations
DK2796145T3 (en) 2013-04-22 2018-01-29 Csl Ltd A covalent complex of von Willebrand factor and factor VIII linked by a disulfide bridge
KR20160026905A (en) 2013-06-28 2016-03-09 체에스엘 베링 게엠베하 Combination therapy using a factor xii inhibitor and a c1-inhibitor
US10611794B2 (en) 2013-09-25 2020-04-07 Bioverativ Therapeutics Inc. On-column viral inactivation methods
WO2015085276A1 (en) 2013-12-06 2015-06-11 Biogen Idec Ma Inc. Population pharmacokinetics tools and uses thereof
WO2015148444A1 (en) 2014-03-24 2015-10-01 Biogen Ma Inc. Lyophilized factor ix formulations
WO2015193457A1 (en) 2014-06-18 2015-12-23 Csl Behring Gmbh Therapy using a factor xii inhibitor in a neurotraumatic disorder
DK3164150T3 (en) 2014-07-02 2021-02-08 CSL Behring Lengnau AG MODIFIED BY WILLEBRAND FACTOR
CN104292341B (en) * 2014-10-11 2018-08-10 上海兴迪金生物技术有限公司 A kind of eight factor fusion protein of blood coagulation and its preparation method and application
ES2824151T3 (en) 2014-12-19 2021-05-11 Alkermes Inc Single-chain Fc fusion proteins
KR20170125941A (en) 2015-03-06 2017-11-15 체에스엘 베링 리컴비넌트 퍼실리티 아게 Modified von Willebrand factor with improved half-life
DK3298036T3 (en) 2015-05-22 2022-06-07 CSL Behring Lengnau AG Methods for preparing modified von Willebrand factor
CA2986626A1 (en) 2015-05-22 2016-12-01 Csl Behring Recombinant Facility Ag Truncated von willebrand factor polypeptides for treating hemophilia
EP3184149A1 (en) 2015-12-23 2017-06-28 Julius-Maximilians-Universität Würzburg Soluble glycoprotein v for treating thrombotic diseases
AU2017205776B2 (en) 2016-01-07 2020-09-10 CSL Behring Lengnau AG Mutated von Willebrand factor
RU2018128582A (en) 2016-01-07 2020-02-11 Цсл Беринг Ленгнау Аг MUTED CROPPED VILLEBRAND BACKGROUND FACTOR
AU2017247004B2 (en) 2016-04-06 2022-07-07 Csl Limited Method of treating atherosclerosis
AU2017291321B2 (en) 2016-06-22 2020-06-18 Alkermes, Inc. Compositions and methods for modulating IL-10 immunostimulatory and anti-inflammatory properties
EP3502143A4 (en) 2016-08-19 2020-07-15 Ampsource Biopharma Shanghai Inc. Linker peptide for constructing fusion protein
CN106317226B (en) 2016-08-19 2017-09-05 安源医药科技（上海）有限公司 Linker peptide for construction of fusion proteins
CN106279437B (en) 2016-08-19 2017-10-31 安源医药科技（上海）有限公司 Hyperglycosylated human coagulation factor VIII fusion proteins and preparation method thereof and purposes
EP3538133B1 (en) 2016-11-11 2021-02-17 CSL Behring Lengnau AG Truncated von willebrand factor polypeptides for treating hemophilia
CA3043397A1 (en) 2016-11-11 2018-05-17 CSL Behring Lengnau AG Truncated von willebrand factor polypeptides for extravascular administration in the treatment or prophylaxis of a blood coagulation disorder
EP3548066A1 (en) 2016-12-02 2019-10-09 Bioverativ Therapeutics Inc. Methods of treating hemophilic arthropathy using chimeric clotting factors
JP2019536794A (en) 2016-12-02 2019-12-19 バイオベラティブ セラピューティクス インコーポレイテッド Methods for inducing immune tolerance to coagulation factors
US11872194B2 (en) 2017-03-14 2024-01-16 Ohio State Innovation Foundation Methods and compositions related to a tissue factor-targeting IGG3 immunoconjugates
PL3641800T3 (en) 2017-06-22 2024-03-18 CSL Behring Lengnau AG Modulation of fviii immunogenicity by truncated vwf
AU2018338608A1 (en) 2017-09-27 2020-04-09 Sigilon Therapeutics, Inc. Methods, compositions, and implantable elements comprising active cells
MX2020009975A (en) 2018-03-28 2020-10-12 Bristol Myers Squibb Co Interleukin-2/interleukin-2 receptor alpha fusion proteins and methods of use.
IL312315A (en) 2018-04-04 2024-06-01 Sigilon Therapeutics Inc Implantable particles and related methods
US20210145889A1 (en) 2018-04-04 2021-05-20 Sigilon Therapeutics, Inc. Methods, compositions, and implantable elements comprising stem cells
UY38389A (en) 2018-09-27 2020-04-30 Sigilon Therapeutics Inc IMPLANTABLE DEVICES FOR CELLULAR THERAPY AND RELATED METHODS
US20220211808A1 (en) 2019-05-17 2022-07-07 Universitaet Zuerich Haptoglobin for use in treating an adverse secondary neurological outcome following a haemorrhagic stroke
AU2020300820A1 (en) 2019-07-04 2022-03-03 CSL Behring Lengnau AG A truncated von willebrand factor (vWF) for increasing the in vitro stability of coagulation factor VIII
WO2021094344A1 (en) 2019-11-11 2021-05-20 CSL Behring Lengnau AG Polypeptides for inducing tolerance to factor viii
BR112022009938A2 (en) 2019-11-25 2022-09-13 Alkermes Inc SUBSTITUTED MACROCYCLIC COMPOUNDS AND RELATED TREATMENT METHODS
KR20220139915A (en) 2020-02-06 2022-10-17 브리스톨-마이어스 스큅 컴퍼니 IL-10 and its uses
US11981718B2 (en) 2020-05-27 2024-05-14 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation
EP4236962A1 (en) 2020-10-29 2023-09-06 Bristol-Myers Squibb Company Fusion proteins for the treatment of disease
EP4247416A1 (en) 2020-11-20 2023-09-27 CSL Behring GmbH Method for treating antibody-mediated rejection
EP4284407A1 (en) 2021-02-01 2023-12-06 CSL Behring AG Method of treating or preventing an adverse secondary neurological outcome following a haemorrhagic stroke
KR20240005075A (en) 2021-05-07 2024-01-11 체에스엘 베링 아게 Expression system for producing recombinant haptoglobin (HP) beta chain
TW202423475A (en) 2022-09-02 2024-06-16 瑞士商Ｃｓｌ貝林股份有限公司 Haptoglobin for use in treating or preventing exaggerated erectile response or erectile dysfunction
WO2024081310A1 (en) 2022-10-11 2024-04-18 Sigilon Therapeutics, Inc. Engineered cells and implantable elements for treatment of disease
WO2024081309A1 (en) 2022-10-11 2024-04-18 Sigilon Therapeutics, Inc. Engineered cells and implantable elements for treatment of disease

Citations (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941763A (en) * 1975-03-28 1976-03-02 American Home Products Corporation PGlu-D-Met-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 and intermediates
US4695623A (en) * 1982-05-06 1987-09-22 Amgen Consensus human leukocyte interferon
US4784950A (en) * 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
US4831119A (en) * 1984-11-05 1989-05-16 Ole Nordfang Preparation for the treatment of hemophilia A inhibitor patients and a process for producing such a preparation
US5077204A (en) * 1984-06-21 1991-12-31 Chiron Corporation Yeast endopeptidase for basic amino-acid site cleavage, preparation and use
US5093246A (en) * 1986-12-03 1992-03-03 University Patents, Inc. Rna ribozyme polymerases, dephosphorylases, restriction endoribo-nucleases and methods
US5116964A (en) * 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US5162220A (en) * 1988-02-03 1992-11-10 Suntory Limited Kex2 endoprotease without a c-terminal hydrophobic region
US5175096A (en) * 1989-05-11 1992-12-29 Alfa-Laval Agri International Aktiebolag Dna encoding a fibronectin binding protein as well as its preparation
US5180583A (en) * 1985-11-26 1993-01-19 Hedner Ulla K E Method for the treatment of bleeding disorders
US5189015A (en) * 1984-05-30 1993-02-23 Alfa-Laval Agri International Ab Method for prophylactic treatment of the colonization of a Staphylococcus aureus bacterial strain by bacterial cell surface protein with fibronectin and fibrinogen binding ability
US5234830A (en) * 1988-02-03 1993-08-10 Suntory Limited DNA encoding a KEX2 endoprotease without a C-terminal hydrophobic region
US5258498A (en) * 1987-05-21 1993-11-02 Creative Biomolecules, Inc. Polypeptide linkers for production of biosynthetic proteins
US5428130A (en) * 1989-02-23 1995-06-27 Genentech, Inc. Hybrid immunoglobulins
US5464933A (en) * 1993-06-07 1995-11-07 Duke University Synthetic peptide inhibitors of HIV transmission
US5480981A (en) * 1992-05-26 1996-01-02 Immunex Corporation CD30 ligand
US5579277A (en) * 1995-05-01 1996-11-26 Apple Computer, Inc. System and method for interleaving memory banks
US5585089A (en) * 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5591573A (en) * 1995-04-10 1997-01-07 Alpha Therapeutic Corporation Method and system for testing blood samples
US5605689A (en) * 1991-05-31 1997-02-25 Genentech, Inc. Treatment of HIV-associated immune thrombocytopenic purpura
US5624821A (en) * 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5648240A (en) * 1994-05-24 1997-07-15 Texas A&M University MHC II analog from Staphylococcus aureus
US5723125A (en) * 1995-12-28 1998-03-03 Tanox Biosystems, Inc. Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide
US5739277A (en) * 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US5807706A (en) * 1995-03-01 1998-09-15 Genentech, Inc. Method for making heteromultimeric polypeptides
US5808029A (en) * 1989-09-12 1998-09-15 Hoffmann-La Roche Inc. DNA encoding a human TNF binding protein
US5869046A (en) * 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5885821A (en) * 1996-03-04 1999-03-23 Suntory Limited Process for production of secretory Kex2 derivatives
US5910573A (en) * 1992-01-23 1999-06-08 Merck Patent Gesellschaft Mit Beschrankter Haftung Monomeric and dimeric antibody-fragment fusion proteins
US6013263A (en) * 1993-06-07 2000-01-11 Trimeris, Inc. Measles virus peptides with antifusogenic and antiviral activities
US6015881A (en) * 1998-03-23 2000-01-18 Trimeris, Inc. Methods and compositions for peptide synthesis
US6017536A (en) * 1993-06-07 2000-01-25 Trimeris, Inc. Simian immunodeficiency virus peptides with antifusogenic and antiviral activities
US6030613A (en) * 1995-01-17 2000-02-29 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6060613A (en) * 1997-02-05 2000-05-09 Ajinomoto Co., Inc. Process for producing N-long-chain acyl acidic amino acids or salts thereof
US6086875A (en) * 1995-01-17 2000-07-11 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of immunogens
US6093799A (en) * 1998-03-16 2000-07-25 Pharmacopeia, Inc. Universal linker for combinatorial synthesis
US6184344B1 (en) * 1995-05-04 2001-02-06 The Scripps Research Institute Synthesis of proteins by native chemical ligation
US6280994B1 (en) * 1998-11-25 2001-08-28 Zymogenetics, Inc. Zace 1: a human metalloenzyme
US6310183B1 (en) * 1997-09-10 2001-10-30 Novo Nordisk A/S Coagulation factor VIIa composition
US6310180B1 (en) * 1993-06-21 2001-10-30 Vanderbilt University Method for synthesis of proteins
US6323323B1 (en) * 1988-01-22 2001-11-27 Zymogenetics, Inc. Ligand-binding, dimerized polypeptide fusions
US6326468B1 (en) * 1997-06-13 2001-12-04 Gryphon Sciences Solid phase native chemical ligation of unprotected or n-terminal cysteine protected peptides in aqueous solution
US6329176B1 (en) * 1998-11-06 2001-12-11 Novo Nordisk A/S Method for the production of factor VII
US20020081664A1 (en) * 1999-05-19 2002-06-27 Kin-Ming Lo Expression and export of interferon-alpha proteins as Fc fusion proteins
US20020106374A1 (en) * 2000-09-15 2002-08-08 Olson William C. Compositions and methods for inhibition of HIV-1 infection
US6469136B1 (en) * 1999-07-07 2002-10-22 Trimeris, Inc. Methods and composition for peptide synthesis
US6475491B1 (en) * 1995-06-07 2002-11-05 Trimeris, Inc. Treatment of HIV and other viral infections using combinatorial therapy
US6485726B1 (en) * 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6518013B1 (en) * 1993-06-07 2003-02-11 Trimeris, Inc. Methods for the inhibition of epstein-barr virus transmission employing anti-viral peptides capable of abrogating viral fusion and transmission
US20030180287A1 (en) * 2002-02-27 2003-09-25 Immunex Corporation Polypeptide formulation
US6660843B1 (en) * 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US20030235536A1 (en) * 2002-03-15 2003-12-25 The Brigham And Women's Hospital, Inc. Central airway administration for systemic delivery of therapeutics
US20040077022A1 (en) * 1998-10-23 2004-04-22 Ulrich Feige Modified peptides as therapeutic agents
US20040110929A1 (en) * 2002-07-12 2004-06-10 Bjorn Soren E. TF binding compound
US20050027109A1 (en) * 2003-05-06 2005-02-03 Mezo Adam R. Methods for chemically synthesizing immunoglobulin chimeric proteins
US20050037941A1 (en) * 2001-10-24 2005-02-17 Benchmark Research And Technology, Inc. Contaminant-tolerant foaming additive
US20050037947A1 (en) * 2003-05-06 2005-02-17 Bitonti Alan J. Inhibition of drug binding to serum albumin
US7084109B2 (en) * 1999-07-02 2006-08-01 Genentech, Inc. FVIIa antagonists
US7348004B2 (en) * 2003-05-06 2008-03-25 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US7385032B2 (en) * 1999-12-30 2008-06-10 Apotech Research And Development Ltd. Bimer or an oligomer of a dimer, trimer, quadromer or pentamer of recombinant fusion proteins

Family Cites Families (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4215051A (en) 1979-08-29 1980-07-29 Standard Oil Company (Indiana) Formation, purification and recovery of phthalic anhydride
US4456591A (en) * 1981-06-25 1984-06-26 Baxter Travenol Laboratories, Inc. Therapeutic method for activating factor VII
US4713339A (en) 1983-01-19 1987-12-15 Genentech, Inc. Polycistronic expression vector construction
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4902505A (en) 1986-07-30 1990-02-20 Alkermes Chimeric peptides for neuropeptide delivery through the blood-brain barrier
US5116742A (en) 1986-12-03 1992-05-26 University Patents, Inc. RNA ribozyme restriction endoribonucleases and methods
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
US4994371A (en) 1987-08-28 1991-02-19 Davie Earl W DNA preparation of Christmas factor and use of DNA sequences
ZA89430B (en) 1988-01-22 1989-10-25 Gen Hospital Corp Cloned genes encoding ig-cd4 fusion proteins and the use thereof
FR2641468A1 (en) 1989-01-11 1990-07-13 Merieux Inst Chimeric antibodies to human interleukin-2 receptor, their preparation and pharmaceutical compositions containing them
GB9009106D0 (en) 1990-04-23 1990-06-20 3I Res Expl Ltd Processes and intermediates for synthetic antibody derivatives
DK0464533T3 (en) * 1990-06-28 1999-04-26 Gen Hospital Corp Fusion proteins with immunoglobulin moieties, their preparation and use
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
EP0563214A4 (en) 1990-12-04 1994-08-17 Wistar Inst Bifunctional antibodies and method of preparing same
US5374617A (en) * 1992-05-13 1994-12-20 Oklahoma Medical Research Foundation Treatment of bleeding with modified tissue factor in combination with FVIIa
AU2795892A (en) * 1991-10-03 1993-05-03 Center For Blood Research, The Cd36 immunoadhesins and their use in selectively killing (plasmodium falciparum) infected erythrocytes
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
WO1994005328A1 (en) * 1992-08-28 1994-03-17 The Scripps Research Institute Inhibition of tumor metastasis via neutralization of tissue factor function
GB2270845B (en) 1992-09-24 1996-07-10 Smiths Ind Med Syst Inc Suction catheter assemblies
US5563045A (en) * 1992-11-13 1996-10-08 Genetics Institute, Inc. Chimeric procoagulant proteins
US5621039A (en) 1993-06-08 1997-04-15 Hallahan; Terrence W. Factor IX- polymeric conjugates
AU3559695A (en) 1994-09-30 1996-04-26 Inex Pharmaceuticals Corp. Glycosylated protein-liposome conjugates and methods for their preparation
JP4026848B2 (en) 1995-01-17 2007-12-26 ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド Receptor-specific transepithelial transport of immunogens
US6036482A (en) † 1995-02-10 2000-03-14 Tokyo Electron Limited Heat treatment method
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
JP4046354B2 (en) 1996-03-18 2008-02-13 ボード オブ リージェンツ，ザ ユニバーシティ オブ テキサス システム Immunoglobulin-like domain with increased half-life
BR9806793A (en) 1997-01-22 2000-05-16 Univ Texas Tissue factor processes and compositions for coagulation and treatment of tumors.
CA2225189C (en) * 1997-03-06 2010-05-25 Queen's University At Kingston Canine factor viii gene, protein and methods of use
HUP0100813A3 (en) 1998-02-25 2003-08-28 Lexigen Pharmaceuticals Corp L Enhancing the circulating half-life of antibody-based fusion proteins
AU4090299A (en) 1998-05-20 1999-12-06 Sdg, Inc. Liposomal delivery complex
DK1100830T3 (en) 1998-07-28 2004-01-19 Micromet Ag Straight Mini Antibodies
US20020142374A1 (en) 1998-08-17 2002-10-03 Michael Gallo Generation of modified molecules with increased serum half-lives
EP1117693B1 (en) 1998-09-30 2006-05-17 New England Biolabs, Inc. Intein mediated peptide ligation
CA2270600A1 (en) 1999-05-03 2000-11-03 Infectio Recherche Inc. Method and formulations for the treatment of diseases, particularly those caused by human immunodeficiency virus and leishmania
JP2003504315A (en) 1999-07-01 2003-02-04 エール ユニバーシティ Angiogenesis target immune complex
IL147270A0 (en) * 1999-07-02 2002-08-14 Genentech Inc Fusion peptides comprising a peptide ligand domain and a multimerization domain
ATE407697T1 (en) † 1999-07-13 2008-09-15 Bolder Biotechnology Inc ERYTHROPOIETIN IMMUNOLOBULIN FUSION PROTEINS
AU780693B2 (en) 1999-11-05 2005-04-14 Curis, Inc. Hedgehog fusion proteins and uses
WO2001036489A2 (en) 1999-11-12 2001-05-25 Merck Patent Gmbh Erythropoietin forms with improved properties
WO2001036637A1 (en) 1999-11-17 2001-05-25 Immunex Corporation Receptor activator of nf-kappa b
ATE428445T1 (en) 2000-02-11 2009-05-15 Bayer Healthcare Llc CLOTTING FACTOR VII OR VIIA CONJUGATES
AU4314801A (en) 2000-02-11 2001-08-20 Lexigen Pharm Corp Enhancing the circulating half-life of antibody-based fusion proteins
US6756480B2 (en) 2000-04-27 2004-06-29 Amgen Inc. Modulators of receptors for parathyroid hormone and parathyroid hormone-related protein
US20020090646A1 (en) † 2000-05-03 2002-07-11 Amgen Inc. Calcitonin-related molecules
AU2001274904B2 (en) 2000-05-26 2006-08-31 Ortho-Mcneil Pharmaceutical, Inc. Neuroprotective peptides
WO2002018417A1 (en) 2000-09-01 2002-03-07 Gryphon Therapeutics, Inc. Nucleophile-stable thioester generating compounds, methods of production and use
EP1339427A4 (en) 2000-11-01 2004-09-15 Elusys Therapeutics Inc Method of producing biospecific molecules by protein trans-splicing
WO2002089828A2 (en) 2001-05-04 2002-11-14 Institute Of Molecular And Cell Biology Interferons in the treatment of ischemia
US7419949B2 (en) * 2001-07-16 2008-09-02 Novo Noridsk Healthcare A/G Single-dose administration of factor VIIa
MXPA05000063A (en) 2002-07-01 2005-04-08 Kenneth S Warren Inst Inc Recombinant tissue protective cytokines and encoding nucleic acids thereof for protection, restoration, and enhancement of responsive cells, tissues, and organs.
WO2004004798A2 (en) 2002-07-03 2004-01-15 The Brigham And Women's Hospital, Inc. Central airway administration for systemic delivery of therapeutics
EP1523504A2 (en) * 2002-07-12 2005-04-20 Novo Nordisk A/S A tissue factor binding immunoconjugate comprising factor viia
US20050281829A1 (en) * 2003-05-06 2005-12-22 Hehir Cristina A T Fc chimeric proteins with anti-HIV drugs
ES2558102T3 (en) 2003-05-06 2016-02-02 Biogen Hemophilia Inc. Chimeric coagulation factor proteins for the treatment of a hemostatic disorder
US7736653B2 (en) 2003-11-13 2010-06-15 Hanmi Pharm. Co., Ltd Pharmaceutical composition comprising an immunoglobulin Fc region as a carrier
US8110665B2 (en) 2003-11-13 2012-02-07 Hanmi Holdings Co., Ltd. Pharmaceutical composition comprising an immunoglobulin FC region as a carrier
US8263084B2 (en) 2003-11-13 2012-09-11 Hanmi Science Co., Ltd Pharmaceutical composition for treating obesity-related disease comprising insulinotropic peptide conjugate
CA2546580A1 (en) * 2003-11-18 2005-06-09 Iconic Therapeutics, Inc. Homogeneous preparations of chimeric proteins
KR100754667B1 (en) 2005-04-08 2007-09-03 한미약품 주식회사 Immunoglobulin Fc fragment modified by non-peptide polymer and pharmaceutical composition comprising the same
PT3513804T (en) * 2011-07-08 2022-06-02 Bioverativ Therapeutics Inc Factor viii chimeric and hybrid polypeptides, and methods of use thereof
US10504058B2 (en) 2015-04-28 2019-12-10 Sato Holdings Kabushiki Kaisha Storage cabinet

Patent Citations (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941763A (en) * 1975-03-28 1976-03-02 American Home Products Corporation PGlu-D-Met-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 and intermediates
US4695623A (en) * 1982-05-06 1987-09-22 Amgen Consensus human leukocyte interferon
US4897471A (en) * 1982-05-06 1990-01-30 Amgen Consensus human leukocyte interferon
US5189015A (en) * 1984-05-30 1993-02-23 Alfa-Laval Agri International Ab Method for prophylactic treatment of the colonization of a Staphylococcus aureus bacterial strain by bacterial cell surface protein with fibronectin and fibrinogen binding ability
US5077204A (en) * 1984-06-21 1991-12-31 Chiron Corporation Yeast endopeptidase for basic amino-acid site cleavage, preparation and use
US4831119A (en) * 1984-11-05 1989-05-16 Ole Nordfang Preparation for the treatment of hemophilia A inhibitor patients and a process for producing such a preparation
US4784950A (en) * 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
US5180583A (en) * 1985-11-26 1993-01-19 Hedner Ulla K E Method for the treatment of bleeding disorders
US5093246A (en) * 1986-12-03 1992-03-03 University Patents, Inc. Rna ribozyme polymerases, dephosphorylases, restriction endoribo-nucleases and methods
US5624821A (en) * 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5258498A (en) * 1987-05-21 1993-11-02 Creative Biomolecules, Inc. Polypeptide linkers for production of biosynthetic proteins
US5482858A (en) * 1987-05-21 1996-01-09 Creative Biomolecules, Inc. Polypeptide linkers for production of biosynthetic proteins
US6323323B1 (en) * 1988-01-22 2001-11-27 Zymogenetics, Inc. Ligand-binding, dimerized polypeptide fusions
US5162220A (en) * 1988-02-03 1992-11-10 Suntory Limited Kex2 endoprotease without a c-terminal hydrophobic region
US5234830A (en) * 1988-02-03 1993-08-10 Suntory Limited DNA encoding a KEX2 endoprotease without a C-terminal hydrophobic region
US5585089A (en) * 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5116964A (en) * 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US5428130A (en) * 1989-02-23 1995-06-27 Genentech, Inc. Hybrid immunoglobulins
US5175096A (en) * 1989-05-11 1992-12-29 Alfa-Laval Agri International Aktiebolag Dna encoding a fibronectin binding protein as well as its preparation
US5808029A (en) * 1989-09-12 1998-09-15 Hoffmann-La Roche Inc. DNA encoding a human TNF binding protein
US5605689A (en) * 1991-05-31 1997-02-25 Genentech, Inc. Treatment of HIV-associated immune thrombocytopenic purpura
US5910573A (en) * 1992-01-23 1999-06-08 Merck Patent Gesellschaft Mit Beschrankter Haftung Monomeric and dimeric antibody-fragment fusion proteins
US5480981A (en) * 1992-05-26 1996-01-02 Immunex Corporation CD30 ligand
US6228983B1 (en) * 1993-06-07 2001-05-08 Trimeris, Inc. Human respiratory syncytial virus peptides with antifusogenic and antiviral activities
US6068973A (en) * 1993-06-07 2000-05-30 Trimeris, Inc. Methods for inhibition of membrane fusion-associated events, including influenza virus
US6518013B1 (en) * 1993-06-07 2003-02-11 Trimeris, Inc. Methods for the inhibition of epstein-barr virus transmission employing anti-viral peptides capable of abrogating viral fusion and transmission
US6060065A (en) * 1993-06-07 2000-05-09 Trimeris, Inc. Compositions for inhibition of membrane fusion-associated events, including influenza virus transmission
US6017536A (en) * 1993-06-07 2000-01-25 Trimeris, Inc. Simian immunodeficiency virus peptides with antifusogenic and antiviral activities
US5464933A (en) * 1993-06-07 1995-11-07 Duke University Synthetic peptide inhibitors of HIV transmission
US6013263A (en) * 1993-06-07 2000-01-11 Trimeris, Inc. Measles virus peptides with antifusogenic and antiviral activities
US6310180B1 (en) * 1993-06-21 2001-10-30 Vanderbilt University Method for synthesis of proteins
US5648240A (en) * 1994-05-24 1997-07-15 Texas A&M University MHC II analog from Staphylococcus aureus
US6030613A (en) * 1995-01-17 2000-02-29 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6485726B1 (en) * 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US6086875A (en) * 1995-01-17 2000-07-11 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of immunogens
US5807706A (en) * 1995-03-01 1998-09-15 Genentech, Inc. Method for making heteromultimeric polypeptides
US5591573A (en) * 1995-04-10 1997-01-07 Alpha Therapeutic Corporation Method and system for testing blood samples
US5739277A (en) * 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US5869046A (en) * 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5579277A (en) * 1995-05-01 1996-11-26 Apple Computer, Inc. System and method for interleaving memory banks
US6184344B1 (en) * 1995-05-04 2001-02-06 The Scripps Research Institute Synthesis of proteins by native chemical ligation
US6475491B1 (en) * 1995-06-07 2002-11-05 Trimeris, Inc. Treatment of HIV and other viral infections using combinatorial therapy
US5723125A (en) * 1995-12-28 1998-03-03 Tanox Biosystems, Inc. Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide
US5885821A (en) * 1996-03-04 1999-03-23 Suntory Limited Process for production of secretory Kex2 derivatives
US6060613A (en) * 1997-02-05 2000-05-09 Ajinomoto Co., Inc. Process for producing N-long-chain acyl acidic amino acids or salts thereof
US6326468B1 (en) * 1997-06-13 2001-12-04 Gryphon Sciences Solid phase native chemical ligation of unprotected or n-terminal cysteine protected peptides in aqueous solution
US6310183B1 (en) * 1997-09-10 2001-10-30 Novo Nordisk A/S Coagulation factor VIIa composition
US6093799A (en) * 1998-03-16 2000-07-25 Pharmacopeia, Inc. Universal linker for combinatorial synthesis
US6281331B1 (en) * 1998-03-23 2001-08-28 Trimeris, Inc. Methods and compositions for peptide synthesis
US6015881A (en) * 1998-03-23 2000-01-18 Trimeris, Inc. Methods and compositions for peptide synthesis
US6660843B1 (en) * 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US20040077022A1 (en) * 1998-10-23 2004-04-22 Ulrich Feige Modified peptides as therapeutic agents
US6329176B1 (en) * 1998-11-06 2001-12-11 Novo Nordisk A/S Method for the production of factor VII
US6280994B1 (en) * 1998-11-25 2001-08-28 Zymogenetics, Inc. Zace 1: a human metalloenzyme
US20020081664A1 (en) * 1999-05-19 2002-06-27 Kin-Ming Lo Expression and export of interferon-alpha proteins as Fc fusion proteins
US7084109B2 (en) * 1999-07-02 2006-08-01 Genentech, Inc. FVIIa antagonists
US6469136B1 (en) * 1999-07-07 2002-10-22 Trimeris, Inc. Methods and composition for peptide synthesis
US7385032B2 (en) * 1999-12-30 2008-06-10 Apotech Research And Development Ltd. Bimer or an oligomer of a dimer, trimer, quadromer or pentamer of recombinant fusion proteins
US20020106374A1 (en) * 2000-09-15 2002-08-08 Olson William C. Compositions and methods for inhibition of HIV-1 infection
US20050037941A1 (en) * 2001-10-24 2005-02-17 Benchmark Research And Technology, Inc. Contaminant-tolerant foaming additive
US20030180287A1 (en) * 2002-02-27 2003-09-25 Immunex Corporation Polypeptide formulation
US20030235536A1 (en) * 2002-03-15 2003-12-25 The Brigham And Women's Hospital, Inc. Central airway administration for systemic delivery of therapeutics
US20040110929A1 (en) * 2002-07-12 2004-06-10 Bjorn Soren E. TF binding compound
US20050027109A1 (en) * 2003-05-06 2005-02-03 Mezo Adam R. Methods for chemically synthesizing immunoglobulin chimeric proteins
US20070172928A1 (en) * 2003-05-06 2007-07-26 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US7348004B2 (en) * 2003-05-06 2008-03-25 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US7381408B2 (en) * 2003-05-06 2008-06-03 Syntonix Pharmaceuticals, Inc. Methods for chemically synthesizing immunoglobulin chimeric proteins
US20050037947A1 (en) * 2003-05-06 2005-02-17 Bitonti Alan J. Inhibition of drug binding to serum albumin
US7404956B2 (en) * 2003-05-06 2008-07-29 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US20080249288A1 (en) * 2003-05-06 2008-10-09 Syntonix Pharmaceuticals, Inc. Methods for Chemically Synthesizing Immunoglobulin Chimeric Proteins

Cited By (135)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7955590B2 (en) 1999-07-21 2011-06-07 Merck Patent Gmbh Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US20050261229A1 (en) * 1999-07-21 2005-11-24 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US8043608B2 (en) 1999-07-21 2011-10-25 Merck Patent Gmbh Methods of using Fc-cytokine fusion proteins
US20100068175A1 (en) * 1999-07-21 2010-03-18 Gillies Stephen D Methods of using Fc-Cytokine fusion proteins
US9725496B1 (en) 2003-05-06 2017-08-08 Bioverative Therapeutics Inc. Immunoglobulin chimeric monomer-dimer hybrids
US20070172928A1 (en) * 2003-05-06 2007-07-26 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US7862820B2 (en) 2003-05-06 2011-01-04 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US11401322B2 (en) 2003-05-06 2022-08-02 Bioverativ Therapeutics Inc. Immunoglobulin chimeric monomer-dimer hybrids
US20110182896A1 (en) * 2003-05-06 2011-07-28 Syntonix Pharmaceuticals, Inc. CLOTTING FACTOR-Fc CHIMERIC PROTEINS TO TREAT HEMOPHILIA
US20110182919A1 (en) * 2003-05-06 2011-07-28 Peters Robert T Immunoglobulin Chimeric Monomer-Dimer Hybrids
US8932830B2 (en) 2003-05-06 2015-01-13 Biogen Idec Hemophilia, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US20080249288A1 (en) * 2003-05-06 2008-10-09 Syntonix Pharmaceuticals, Inc. Methods for Chemically Synthesizing Immunoglobulin Chimeric Proteins
US8815250B2 (en) 2003-05-06 2014-08-26 Biogen Idec Hemophilia Inc. Clotting factor-Fc chimeric proteins to treat hemophilia
US11168125B2 (en) 2003-05-06 2021-11-09 Bioverativ Therapeutics Inc. Immunoglobulin chimeric monomer-dimer hybrids
US9636416B2 (en) 2003-05-06 2017-05-02 Bioverativ Therapeutics Inc. Immunoglobulin chimeric monomer-dimer hybrids
US8449884B2 (en) 2003-05-06 2013-05-28 Syntonix Pharmaceuticals, Inc. Clotting factor-fc chimeric proteins to treat hemophilia
US8329182B2 (en) 2003-05-06 2012-12-11 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
US7820162B2 (en) 2003-05-06 2010-10-26 Syntonix Pharmaceuticals, Inc. Methods for chemically synthesizing immunoglobulin chimeric proteins
EP2233576B1 (en) * 2006-02-06 2020-04-08 CSL Behring GmbH Modified coagulation factor VIIa with extended half-life
EP1984503B2 (en) † 2006-02-06 2018-08-15 CSL Behring GmbH Modified coagulation factor viia with extended half-life
US8980610B2 (en) 2008-11-20 2015-03-17 Biogen Idec Ma Inc. Arginine inactivation of viruses
US9238878B2 (en) 2009-02-17 2016-01-19 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
US9879249B2 (en) 2009-02-17 2018-01-30 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
CN102427823A (en) * 2009-03-24 2012-04-25 拜耳医药保健有限公司 Factor VIII variants and methods of use
WO2010111414A1 (en) * 2009-03-24 2010-09-30 Bayer Healthcare Llc Factor viii variants and methods of use
RU2579977C2 (en) * 2009-11-13 2016-04-10 Грифольс Терапьютикс Инк. PREPARATIONS CONTAINING VON WILLEBRAND FACTOR (vWF) AND METHODS, KITS AND APPLICATIONS RELATED TO THEM
US8759293B2 (en) * 2009-11-13 2014-06-24 Grifols Therapeutics Inc. von Willebrand factor (vWF)-containing preparations, and methods, kits, and uses related thereto
WO2011060242A3 (en) * 2009-11-13 2011-10-13 Talecris Biotherapeutics, Inc. Von willebrand factor (vwf)-containing preparations, and methods, kits, and uses related thereto
US20120289468A1 (en) * 2009-11-13 2012-11-15 Grifols Therapeutics Inc. Von willebrand factor (vwf)-containing preparations, and methods, kits, and uses related thereto
CN102648212A (en) * 2009-11-13 2012-08-22 基立福疗法公司 Von willebrand factor (vWF)-containing preparations, and methods, kits, and uses related thereto
AU2010319425B2 (en) * 2009-11-13 2015-07-02 Grifols Therapeutics Inc. Von Willebrand factor (vWF)-containing preparations, and methods, kits, and uses related thereto
US9050318B2 (en) * 2009-12-06 2015-06-09 Biogen Idec Hemophilia Inc. Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof
US20130108629A1 (en) * 2009-12-06 2013-05-02 Biogen Idec Hemophilia Inc. Factor VIII-Fc Chimeric and Hybrid Polypeptides, and Methods of Use Thereof
AU2016213822B2 (en) * 2009-12-06 2017-08-31 Bioverativ Therapeutics Inc. Factor VIII-Fc Chimeric And Hybrid Polypeptides, And Methods Of Use Thereof
AU2010325787B2 (en) * 2009-12-06 2016-05-12 Bioverativ Therapeutics Inc. Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof
US9241978B2 (en) 2009-12-06 2016-01-26 Biogen Hemophilia Inc. Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof
US11266720B2 (en) 2009-12-06 2022-03-08 Bioverativ Therapeutics Inc. Factor VIII-FC chimeric and hybrid polypeptides, and methods of use thereof
CN103180439A (en) * 2010-07-09 2013-06-26 比奥根艾迪克依蒙菲利亚公司 Chimeric clotting factors
US10968442B2 (en) 2010-07-09 2021-04-06 Bioverativ Therapeutics Inc. Chimeric clotting factors
US20130216513A1 (en) * 2010-07-09 2013-08-22 Biogen Idec Hemophilia Inc. Chimeric Clotting Factors
US10927362B2 (en) * 2010-07-09 2021-02-23 Bioverativ Therapeutics Inc. Processable single chain molecules and polypeptides made using same
US9856468B2 (en) * 2010-07-09 2018-01-02 Bioverativ Therapeutics Inc. Processable single chain molecules and polypeptides made using same
US20130202596A1 (en) * 2010-07-09 2013-08-08 Biogen Idec Hemophilia Inc. Processable Single Chain Molecules and Polypeptides Made Using Same
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9062115B2 (en) 2010-09-22 2015-06-23 Novo Nordisk A/S Therapeutic factor VIII antibodies
US9657295B2 (en) 2010-10-01 2017-05-23 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US10064959B2 (en) 2010-10-01 2018-09-04 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9701965B2 (en) 2010-10-01 2017-07-11 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US20130296534A1 (en) * 2010-10-20 2013-11-07 Sk Chemicals Co., Ltd. Fusion protein having factor ix activity
US9617328B2 (en) * 2010-10-20 2017-04-11 Sk Chemicals Co., Ltd. Fusion protein having factor IX activity
EP3663399A1 (en) 2010-11-03 2020-06-10 Catalyst Biosciences, Inc. Modified factor ix polypeptides and uses thereof
US8778870B2 (en) 2010-11-03 2014-07-15 Catalyst Biosciences, Inc. Modified factor IX polypeptides and uses thereof
US10982203B2 (en) 2010-11-03 2021-04-20 Catalyst Biosciences, Inc. Modified factor IX polypeptides and uses thereof
US9328339B2 (en) 2010-11-03 2016-05-03 Catalyst Biosciences, Inc. Modified factor IX polypeptides and uses thereof
WO2012061654A1 (en) 2010-11-03 2012-05-10 Catalyst Biosciences, Inc. Modified factor ix polypeptides and uses thereof
US9540438B2 (en) 2011-01-14 2017-01-10 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and methods of use thereof
US10183998B2 (en) 2011-01-14 2019-01-22 Redwood Bioscience, Inc. Aldehyde-tagged immunoglobulin polypeptides and methods of use thereof
US11135275B2 (en) * 2011-03-02 2021-10-05 Novo Nordisk Healthcare Ag Coagulation factor-targeting to TREM-like transcript 1 (TLT-1) on activated platelets
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9950068B2 (en) 2011-03-31 2018-04-24 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US10656167B2 (en) 2011-07-25 2020-05-19 Bioverativ Therapeutics Inc. Assays to monitor bleeding disorders
US11747351B2 (en) 2011-07-25 2023-09-05 Bioverativ Therapeutics Inc. Assays to monitor bleeding disorders
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10751386B2 (en) 2011-09-12 2020-08-25 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US20140370035A1 (en) * 2012-01-12 2014-12-18 Biogen Idec Ma Inc. Methods of reducing immunogenicity against factor viii in individuals undergoing factor viii therapy
US11370827B2 (en) 2012-01-12 2022-06-28 Bioverativ Therapeutics Inc. Chimeric factor VIII polypeptides and uses thereof
US11286528B2 (en) 2012-01-12 2022-03-29 Bioverativ Therapeutics Inc. Methods of reducing immunogenicity against factor VIII in individuals undergoing factor VIII therapy
US10221455B2 (en) * 2012-01-12 2019-03-05 Bioverativ Therapeutics Inc. Methods of reducing immunogenicity against factor VIII in individuals undergoing factor VIII therapy
US10421798B2 (en) 2012-02-15 2019-09-24 Bioverativ Therapeutics Inc. Factor VIII compositions and methods of making and using same
US10370430B2 (en) 2012-02-15 2019-08-06 Bioverativ Therapeutics Inc. Recombinant factor VIII proteins
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US9782462B2 (en) 2012-04-02 2017-10-10 Modernatx, Inc. Modified polynucleotides for the production of proteins associated with human disease
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9220792B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides encoding aquaporin-5
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9221891B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. In vivo production of proteins
US9216205B2 (en) 2012-04-02 2015-12-22 Moderna Therapeutics, Inc. Modified polynucleotides encoding granulysin
US9089604B2 (en) 2012-04-02 2015-07-28 Moderna Therapeutics, Inc. Modified polynucleotides for treating galactosylceramidase protein deficiency
US9233141B2 (en) 2012-04-02 2016-01-12 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9192651B2 (en) 2012-04-02 2015-11-24 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
US9255129B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9220755B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9301993B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides encoding apoptosis inducing factor 1
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9061059B2 (en) 2012-04-02 2015-06-23 Moderna Therapeutics, Inc. Modified polynucleotides for treating protein deficiency
US9050297B2 (en) 2012-04-02 2015-06-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9254311B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9149506B2 (en) 2012-04-02 2015-10-06 Moderna Therapeutics, Inc. Modified polynucleotides encoding septin-4
US9114113B2 (en) 2012-04-02 2015-08-25 Moderna Therapeutics, Inc. Modified polynucleotides encoding citeD4
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9587003B2 (en) 2012-04-02 2017-03-07 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US11261437B2 (en) 2012-06-08 2022-03-01 Bioverativ Therapeutics Inc. Procoagulant compounds
US10287564B2 (en) * 2012-06-08 2019-05-14 Bioverativ Therapeutics Inc. Procoagulant compounds
US10202595B2 (en) * 2012-06-08 2019-02-12 Bioverativ Therapeutics Inc. Chimeric clotting factors
US20150353911A1 (en) * 2012-06-08 2015-12-10 Biogen Idec Ma Inc. Chimeric clotting factors
US20150184142A1 (en) * 2012-06-08 2015-07-02 Biogen Idec Ma Inc. Procoagulant compounds
US11168316B2 (en) 2012-06-08 2021-11-09 Bioverativ Therapeutics, Inc. Chimeric clotting factors
US10138291B2 (en) 2012-07-11 2018-11-27 Bioverativ Therapeutics Inc. Factor VIII complex with XTEN and von Willebrand Factor protein, and uses thereof
US11091534B2 (en) 2012-07-11 2021-08-17 Bioverativ Therapeutics Inc. Factor VIII complex with XTEN and von Willebrand Factor protein, and uses thereof
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US20150315262A1 (en) * 2012-12-10 2015-11-05 Vib Vzw Novel interleukin-33 inhibitors
US10703799B2 (en) * 2012-12-10 2020-07-07 Vib Vzw IL-33R and IL-1RAcP fusion proteins
US11642398B2 (en) 2013-03-15 2023-05-09 Bioverativ Therapeutics Inc. Factor IX polypeptide formulations
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US11749394B2 (en) 2013-06-20 2023-09-05 Takeda Pharmaceutical Company Limited Method and apparatus for providing a pharmacokinetic drug dosing regimen
US11081211B2 (en) 2013-06-20 2021-08-03 Baxalta Incorporated Method and apparatus for providing a pharmacokinetic drug dosing regimen
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US20220275057A1 (en) * 2014-01-10 2022-09-01 Bioverativ Therapeutics Inc. Factor viii chimeric proteins and uses thereof
US11192936B2 (en) 2014-01-10 2021-12-07 Bioverativ Therapeutics Inc. Factor VIII chimeric proteins and uses thereof
US11670409B2 (en) 2016-04-15 2023-06-06 Takeda Pharmaceutical Company Limited Method and apparatus for providing a pharmacokinetic drug dosing regiment
US11208632B2 (en) 2016-04-26 2021-12-28 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
US11788066B2 (en) 2016-04-26 2023-10-17 R.P. Scherer Technologies, Llc Antibody conjugates and methods of making and using the same
US10896749B2 (en) 2017-01-27 2021-01-19 Shire Human Genetic Therapies, Inc. Drug monitoring tool
US11783931B2 (en) 2017-01-27 2023-10-10 Takeda Pharmaceutical Company Limited Drug monitoring tool
US12084512B2 (en) 2017-02-01 2024-09-10 Novo Nordisk A/S Procoagulant antibodies
US11491212B1 (en) 2017-09-27 2022-11-08 Catalyst Biosciences, Inc. Subcutaneous administration of modified factor IX polypeptides and treatment of hemophilia B
US12030925B2 (en) 2018-05-18 2024-07-09 Bioverativ Therapeutics Inc. Methods of treating hemophilia A
US11220554B2 (en) 2018-09-07 2022-01-11 Novo Nordisk A/S Procoagulant antibodies
WO2021154414A2 (en) 2020-01-29 2021-08-05 Catalyst Biosciences, Inc. Gene therapy for hemophilia b with a chimeric aav capsid vector encoding modified factor ix polypeptides

Also Published As

Publication number Publication date
AU2010201711B2 (en) 2012-04-05
EP1624891B1 (en) 2009-08-26
US8449884B2 (en) 2013-05-28
CY1117090T1 (en) 2017-03-15
PT1624891E (en) 2010-01-05
WO2004101740A3 (en) 2005-10-27
ES2558102T3 (en) 2016-02-02
FR16C0041I1 (en) 2016-09-12
DE602004022800D1 (en) 2009-10-08
EP2298347A1 (en) 2011-03-23
CY2016039I1 (en) 2017-03-15
PL2077121T3 (en) 2011-07-29
EP2077121B1 (en) 2011-02-09
WO2004101740A2 (en) 2004-11-25
EP3002012A1 (en) 2016-04-06
SI1624891T1 (en) 2010-01-29
US20210348150A1 (en) 2021-11-11
AU2010201711A1 (en) 2010-05-20
CY2016039I2 (en) 2017-03-15
US8815250B2 (en) 2014-08-26
ES2333598T3 (en) 2010-02-24
DK2298347T3 (en) 2016-01-11
EP1624891A2 (en) 2006-02-15
PL1624891T5 (en) 2014-02-28
ES2361036T3 (en) 2011-06-13
EP2298347B1 (en) 2015-09-30
AU2004239244B2 (en) 2010-01-28
HK1153947A1 (en) 2012-04-20
AU2004239244C1 (en) 2015-04-23
PL1624891T3 (en) 2010-02-26
HK1134432A1 (en) 2010-04-30
US20180237762A1 (en) 2018-08-23
SI1624891T2 (en) 2013-09-30
FR16C0041I2 (en) 2019-07-05
US20150044207A1 (en) 2015-02-12
CY1111725T1 (en) 2015-10-07
AU2004239244A1 (en) 2004-11-25
US20130273047A1 (en) 2013-10-17
EP2077121A1 (en) 2009-07-08
CA2522859A1 (en) 2004-11-25
BE2016C058I2 (en) 2023-08-09
DK1624891T4 (en) 2013-07-15
ATE497783T1 (en) 2011-02-15
AU2010201711C1 (en) 2012-11-08
US20110182896A1 (en) 2011-07-28
DK2077121T3 (en) 2011-06-06
SI2077121T1 (en) 2011-07-29
PT2077121E (en) 2011-05-23
CY1109640T1 (en) 2014-08-13
NL300835I2 (en) 2017-03-16
HUE026384T2 (en) 2016-06-28
DK1624891T3 (en) 2010-01-04
PT2298347E (en) 2016-01-29
SI2298347T1 (en) 2016-03-31
LU93265I2 (en) 2016-12-19
HUS1600041I1 (en) 2016-11-28
PL2298347T3 (en) 2016-03-31
DE602004031390D1 (en) 2011-03-24
EP3552627A1 (en) 2019-10-16
EP3002012A9 (en) 2016-11-16
EP1624891A4 (en) 2007-06-06
ES2333598T5 (en) 2013-09-04
EP1624891B2 (en) 2013-04-10
CA2522859C (en) 2017-10-24
ATE440619T1 (en) 2009-09-15

Similar Documents

Publication Publication Date Title
US20210348150A1 (en) Clotting factor-fc chimeric proteins to treat hemophilia
US20230023927A1 (en) Immunoglobulin chimeric monomer-dimer hybrids
US7348004B2 (en) Immunoglobulin chimeric monomer-dimer hybrids
AU2012203896B2 (en) Clotting Factor-Fc Chimeric Proteins to Treat Hemophilia
AU2012200470B2 (en) Immunoglobulin Chimeric Monomer-Dimer Hybrids
AU2016244273A1 (en) Immunoglobulin Chimeric Monomer-Dimer Hybrids

Legal Events

Date Code Title Description
AS Assignment

Owner name: SYNTONIX PHARMACEUTICALS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RIVERA, DANIEL S.;PETERS, ROBERT T.;BITONTI, ALAN J.;REEL/FRAME:018501/0640;SIGNING DATES FROM 20041006 TO 20041108

AS Assignment

Owner name: BIOGEN IDEC INC., MASSACHUSETTS

Free format text: SECURITY AGREEMENT;ASSIGNOR:SYNTONIX PHARMACEUTICALS, INC.;REEL/FRAME:018764/0586

Effective date: 20061221

AS Assignment

Owner name: SYNTONIX PHARMACEUTICALS, INC., MASSACHUSETTS

Free format text: RELEASE & REASSIGNMENT;ASSIGNOR:BIOGEN IDEC INC.;REEL/FRAME:018964/0431

Effective date: 20070221

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: BIOGEN IDEC HEMOPHILIA INC., MASSACHUSETTS

Free format text: CHANGE OF NAME;ASSIGNOR:SYNTONIX PHARMACEUTICALS, INC.;REEL/FRAME:032753/0837

Effective date: 20100519

AS Assignment

Owner name: BIOVERATIV THERAPEUTICS INC., MASSACHUSETTS

Free format text: CHANGE OF NAME;ASSIGNOR:BIOGEN HEMOPHILIA INC.;REEL/FRAME:041735/0662

Effective date: 20160901