WO2004019872A2 - Delivrance orale de proteines hybrides modifiees de la transferrine - Google Patents

Delivrance orale de proteines hybrides modifiees de la transferrine Download PDF

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Publication number
WO2004019872A2
WO2004019872A2 PCT/US2003/026778 US0326778W WO2004019872A2 WO 2004019872 A2 WO2004019872 A2 WO 2004019872A2 US 0326778 W US0326778 W US 0326778W WO 2004019872 A2 WO2004019872 A2 WO 2004019872A2
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Prior art keywords
pharmaceutical composition
protein
peptide
glp
transfenin
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PCT/US2003/026778
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English (en)
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WO2004019872A3 (fr
Inventor
Christopher P. Prior
Homayoun Sadeghi
Andrew Turner
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Biorexis Pharmaceutical Corporation
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Priority claimed from US10/378,094 external-priority patent/US7176278B2/en
Application filed by Biorexis Pharmaceutical Corporation filed Critical Biorexis Pharmaceutical Corporation
Priority to AU2003270009A priority Critical patent/AU2003270009A1/en
Priority to US10/515,232 priority patent/US8129504B2/en
Publication of WO2004019872A2 publication Critical patent/WO2004019872A2/fr
Publication of WO2004019872A3 publication Critical patent/WO2004019872A3/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70582—CD71
    • 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
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the present invention relates to orally administerable therapeutic proteins or peptides with extended seram stability or seram half-life, particularly to therapeutic proteins or peptides fused to or inserted in a transfenin molecule modified to reduce or inhibit glycosylation.
  • Therapeutic proteins or peptides in their native state or when recombinantly produced are typically labile molecules exhibiting short periods of seram stability or short in vivo circulatory half-lives. In addition, these molecules are often extremely labile when formulated, particularly when formulated in aqueous solutions for diagnostic and therapeutic pu ⁇ oses.
  • Polyethylene glycol is a substance that can be attached to a protein, resulting in longer-acting, sustained activity of the protein. If the activity of a protein is prolonged by the attachment to PEG, the frequency that the protein needs to be administered may be decreased. PEG attachment, however, often decreases or destroys the protein's therapeutic activity. While in some instance PEG attachment can reduce immunogenicity of the protein, in other instances it may increase immunogenicity.
  • Therapeutic proteins or peptides have also been stabilized by fusion to a protein capable of extending the in vivo circulatory half-life of the therapeutic protein. For instance, therapeutic proteins fused to albumin or to antibody fragments may exhibit extended in vivo circulatory half-life when compared to the therapeutic protein in the unfused state. See U.S. Patents 5,876,969 and 5,766,883.
  • glycosylated human transfenin has also been used to make fusions with therapeutic proteins to target delivery to the interior of cells or to carry agents across the blood-brain barrier.
  • These fusion proteins comprising glycosylated human Tf have been used to target nerve growth factor (NGF) or ciliary neurotrophic factor (CNTF) across the blood-brain banier by fusing full-length Tf to the agent.
  • NGF nerve growth factor
  • CNTF ciliary neurotrophic factor
  • the Tf portion of the molecule is glycosylated and binds to two atoms of iron, which is required for Tf binding to its receptor on a cell and, according to the inventors of these patents, to target delivery of the NGF or CNTF moiety across the blood-brain banier.
  • Transfenin fusion proteins have also been produced by inserting an HIV-1 protease target sequence into surface exposed loops of glycosylated transfenin to investigate the ability to produce another form of Tf fusion for targeted delivery to the inside of a cell via the Tf receptor (Ali et al. (1999) J. Biol. Chem. 274(34):24066-24073).
  • Seram transfenin is a monomeric glycoprotein with a molecular weight of 80,000 daltons that binds iron in the circulation and transports it to various tissues via the transfenin receptor (TfR) (Aisen et al. (1980) Ann. Rev. Biochem. 49: 357-393; MacGillivray et al. (1981) J. Biol. Chem. 258: 3543-3553, U.S. Patent 5,026,651). Tf is one of the most common seram molecules, comprising up to about 5-10% of total seram proteins.
  • Carbohydrate deficient transfenin occurs in elevated levels in the blood of alcoholic individuals and exhibits a longer half life (approximately 14-17 days) than that of glycosylated transfenin (approximately 7-10 days).
  • Carbohydrate deficient transfenin occurs in elevated levels in the blood of alcoholic individuals and exhibits a longer half life (approximately 14-17 days) than that of glycosylated transfenin (approximately 7-10 days).
  • Tf has been well characterized and the mechanism of receptor binding, iron binding and release and carbonate ion binding have been elucidated (U.S. Patents 5,026,651, 5,986,067 and MacGillivray et al. (1983) J. Biol. Chem. 258(6):3543- 3546).
  • Transfenin and antibodies that bind the transfenin receptor have also been used to deliver or carry toxic agents to tumor cells as cancer therapy (Baselga and Mendelsohn, 1994), and transferrin has been used as a non- viral gene therapy vector to deliver DNA to cells (Frank et al, 1994; Wagner et al, 1992).
  • Transfenin fusion proteins have not, however, been modified or engineered to extend the in vivo circulatory half-life of a therapeutic protein nor peptide or to increase bioavailability by reducing or inhibiting glycosylation of the Tf moiety nor to reduce or prevent iron and/or Tf receptor binding.
  • the present invention includes orally administerable modified Tf fusion proteins comprising at least one therapeutic protein, polypeptide or peptide entity, wherein the Tf portion is engineered to extend the seram half- life or bioavailability of the molecule.
  • the invention also includes pharmaceutical formulations and compositions formulated for oral administration comprising the fusion proteins, methods of extending the seram stability, seram half-life and bioavailability of a therapeutic protein by fusion to modified transferrin, nucleic acid molecules encoding the modified Tf fusion proteins, and the like.
  • the present invention relates to methods of treating a patient with a modified Tf fusion protein by oral administration. Further, the present invention relates to methods of treating a patient with a modified Tf fusion protein by intranasal administration. Additionally, the present invention relates to methods of treating a patient with a modified Tf fusion protein by pulmonary administration.
  • Figure 1 shows an alignment of the N and C Domains of Human (Hu) transfenin (Tf) (SEQ ID NO: 3) with similarities and identities highlighted.
  • Figure 2A-2B shows an alignment of transfenin sequences from different species
  • Figure 3 shows the location of a number of Tf surface exposed insertion sites for therapeutic proteins, polypeptides or peptides.
  • a therapeutic protein e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof
  • a therapeutic protein can be stabilized to extend the in vivo circulatory half-life and/or retain the therapeutic protein's activity for extended periods of time in vivo by genetically fusing or chemically conjugating the therapeutic protein, polypeptide or peptide to all or a portion of modified transfenin sufficient to extend its half life in seram.
  • the modified transfenin fusion proteins include a transfenin protein or domain covalently linked to a therapeutic protein or peptide, wherein the transfenin portion is modified to contain one or more amino acid substitutions, insertions or deletions compared to a wild- type transfenin sequence.
  • Tf fusion proteins are engineered to reduce or prevent glycosylation within the Tf or a Tf domain.
  • the Tf protein or Tf domain(s) is modified to exhibit reduced or no binding to iron or carbonate ion, or to have a reduced affinity or not bind to a Tf receptor (TfR) .
  • the present invention therefore includes transfenin fusion proteins, therapeutic compositions comprising the fusion proteins, and methods of treating, preventing, or ameliorating diseases or disorders by administering the fusion proteins.
  • a transfenin fusion protein of the invention includes at least a fragment or variant of a therapeutic protein and at least a fragment or variant of modified transfenin, which are associated with one another, preferably by genetic fusion (z ' .e., the transfenin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of modified transfenin) or chemical conjugation to one another.
  • the therapeutic protein and transfenin protein once part of the transfenin fusion protein, may be refened to as a "portion", "region” or “moiety” of the transfenin fusion protein (e.g., a "therapeutic protein portion' or a "transfenin protein portion”).
  • the invention provides a transfenin fusion protein comprising, or alternatively consisting of, a therapeutic protein and a modified seram transfenin protein. In other embodiments, the invention provides a transfenin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a therapeutic protein and a modified transfenin protein. In other embodiments, the invention provides a transfenin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a therapeutic protein and modified transfenin protein. In further embodiments, the invention provides a transfenin fusion protein comprising a therapeutic protein, and a biologically active and/or therapeutically active fragment of modified transfenin. In another embodiment, the therapeutic protein portion of the transfenin fusion protein is the active form of the therapeutic protein.
  • an "amino acid conesponding to" or an "equivalent amino acid” in a transfenin sequence is identified by alignment to maximize the identity or similarity between a first transfenin sequence and at least a second transfenin sequence.
  • the number used to identify an equivalent amino acid in a second transfenin sequence is based on the number used to identify the conesponding amino acid in the first transfenin sequence.
  • these phrases may be used to describe the amino acid residues in human transfenin compared to certain residues in rabbit seram transfenin.
  • biological activity refers to a function or set of activities performed by a therapeutic molecule, protein or peptide in a biological context (i.e., in an organism or an in vitro facsimile thereof).
  • Biological activities may include but are not limited to the functions of the therapeutic molecule portion of the claimed fusion proteins, such as, but not limited to, the induction of extracellular matrix secretion from responsive cell lines, the induction of hormone secretion, the induction of chemotaxis, the induction of mitogenesis, the induction of differentiation, or the inhibition of cell division of responsive cells.
  • a fusion protein or peptide of the invention is considered to be biologically active if it exhibits one or more biological activities of its therapeutic protein's native counte ⁇ art.
  • binders are agents used to impart cohesive qualities to the powdered material. Binders, or “granulators” as they are sometimes known, impart a cohesiveness to the tablet formulation, which insures the tablet remaining intact after compression, as well as improving the free- flowing qualities by the formulation of granules of desired hardness and size.
  • binders include starch; gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, Veegum, microcrystalline cellulose, microcrystalline dextrose, amylose, and larch arabogalactan, and the like.
  • sugars such as sucrose, glucose, dextrose, molasses, and lactose
  • natural and synthetic gums such as acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, Veegum, microcrystalline cellulose, microcrystalline dextrose, amylose
  • canier refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered.
  • Such pharmaceutical caniers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • coloring agents are agents that give tablets a more pleasing appearance, and in addition help the manufacturer to control the product during its preparation and help the user to identify the product.
  • Any of the approved certified water- soluble FD&C dyes, mixtures thereof, or their conesponding lakes may be used to color tablets.
  • a color lake is the combination by adso ⁇ tion of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye.
  • diluents are inert substances added to increase the bulk of the formulation to make the tablet a practical size for compression. Commonly used diluents include calcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar, silica, and the like.
  • disintegrators or “disintegrants” are substances that facilitate the breakup or disintegration of tablets after administration.
  • Materials serving as disintegrants have been chemically classified as starches, clays, celluloses, algins, or gums.
  • Other disintegrators include Veegum HV, methylcellulose, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, alginic acid, guar gum, citras pulp, cross- linked polyvinylpyrrolidone, carboxymethylcellulose, and the like.
  • the term "dispersibility” or “dispersible” means a dry powder having a moisture content of less than about 10% by weight (%w) water, usually below about 5%w and preferably less than about 3%w; a particle size of about 1.0-5.0 ⁇ m mass median diameter (MMD), usually 1.0-4.0 ⁇ m MMD, and preferably 1.0-3.0 ⁇ m MMD; a delivered dose of about >30%, usually >40%, preferably >50%, and most prefened >60%; and an aerosol particle size distribution of 1.0-5.0 ⁇ m mass median aerodynamic diameter (MMAD), usually 1.5-4.5 ⁇ m MMAD, and preferably 1.5-4.0 ⁇ m MMAD.
  • MMD mass median diameter
  • MMAD aerodynamic diameter
  • dry means that the composition has a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol. This moisture content is generally below about 10% by weight (%w) water, usually below about 5%ow and preferably less than about 3%w.
  • effective amount means an amount of a drug or pharmacologically active agent that is sufficient to provide the desired local or systemic effect and perfonnance at a reasonable benefit/risk ratio attending any medical treatment.
  • flavoring agents vary considerably in their chemical structure, ranging from simple esters, alcohols, and aldehydes to carbohydrates and complex volatile oils. Synthetic flavors of almost any desired type are now available.
  • the tenns "fragment of a Tf protein” or “Tf protein,” or “portion of a Tf protein” refer to an amino acid sequence comprising at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a naturally occuning Tf protein or mutant thereof.
  • the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • a heterologous polynucleotide or a “heterologous nucleic acid” or a “heterologous gene” or a “heterologous sequence” or an “exogenous DNA segment” refers to a polynucleotide, nucleic acid or DNA segment that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but has been modified.
  • the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found.
  • a signal sequence native to a yeast cell but attached to a human Tf sequence is heterologous.
  • an "isolated" nucleic acid sequence refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electrophoresis.
  • an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced.
  • the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and inco ⁇ oration of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated.
  • the nucleic acid sequence may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combinations thereof.
  • two or more DNA coding sequences are said to be “joined” or “fused” when, as a result of in-frame fusions between the DNA coding sequences, the DNA coding sequences are translated into a fusion polypeptide.
  • fusion in reference to Tf fusions includes, but is not limited to, attachment of at least one therapeutic protein, polypeptide or peptide to the N -tenninal end of Tf, attachment to the C-terminal end of Tf, and/or insertion between any two amino acids within Tf.
  • lubricants are materials that perform a number of functions in tablet manufacture, such as improving the rate of flow of the tablet granulation, preventing adhesion of the tablet material to the surface of the dies and punches, reducing inte ⁇ article friction, and facilitating the ejection of the tablets from the die cavity.
  • Commonly used lubricants include talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oils. Typical amounts of lubricants range from about 0.1% by weight to about 5% by weight.
  • Modified transfenin refers to a transfenin molecule that exhibits at least one modification of its amino acid sequence, compared to wild-type transfenin.
  • Modified transfenin fusion protein refers to a protein formed by the fusion of at least one molecule of modified transfenin (or a fragment or variant thereof) to at least one molecule of a therapeutic protein (or fragment or variant thereof).
  • nucleic acid or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded fonn. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occuning nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • DNA segment is refened to as "operably linked" when it is placed into a functional relationship with another DNA segment.
  • DNA for a signal sequence is operably linked to DNA encoding a fusion protein of the invention if it is expressed as a preprotein that participates in the secretion of the fusion protein; a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
  • DNA sequences that are operably linked are contiguous, and in the case of a signal sequence or fusion protein both contiguous and in reading phase.
  • enhancers need not be contiguous with the coding sequences whose transcription they control. Linking, in this context, is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
  • pharmaceutically acceptable refers to materials and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized phannacopeia for use in animals, and more particularly in humans.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect. This amount is specific for each drag and its ultimate approved dosage level.
  • the term "powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli.
  • the powder is said to be “respirable.”
  • the average particle size is less than about 10 microns ( ⁇ m) in diameter with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 ⁇ m and most preferably less than about 5.0 ⁇ m.
  • the particle size distribution is between about 0.1 ⁇ m and about 5 ⁇ m in diameter, particularly about 0.3 ⁇ m to about 5 ⁇ m.
  • promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
  • recombinant refers to a cell, tissue or organism that has undergone transformation with a new combination of genes or DNA.
  • subject can be a human, a mammal, or an animal. The subject being treated is a patient in need of treatment.
  • a targeting entity, protein, polypeptide or peptide refers to a molecule that binds specifically to a particular cell type [normal (e.g., lymphocytes) or abnormal e.g., (cancer cell)] and therefore may be used to target a Tf fusion protein or compound (drag, or cytotoxic agent) to that cell type specifically.
  • tablettes are solid pharmaceutical dosage forms containing drag substances with or without suitable diluents and prepared either by compression or molding methods well known in the art. Tablets have been in widespread use since the latter part of the 19 th century and their popularity continues. Tablets remain popular as a dosage form because of the advantages afforded both to the manufacturer (e.g., simplicity and economy of preparation, stability, and convenience in packaging, shipping, and dispensing) and the patient (e.g., accuracy of dosage, compactness, portability, blandness of taste, and ease of administration). Although tablets are most frequently discoid in shape, they may also be round, oval, oblong, cylindrical, or triangular.
  • tablets may differ greatly in size and weight depending on the amount of drag substance present and the intended method of administration. They are divided into two general classes, (1) compressed tablets, and (2) molded tablets or tablet triturates. In addition to the active or therapeutic ingredient or ingredients, tablets contain a number or inert materials or additives.
  • a first group of such additives includes those materials that help to impart satisfactory compression characteristics to the formulation, including diluents, binders, and lubricants.
  • a second group of such additives helps to give additional desirable physical characteristics to the finished tablet, such as disintegrators, colors, flavors, and sweetening agents.
  • the term "therapeutically effective amount” refers to that amount of the transfenin fusion protein comprising a therapeutic molecule which, when administered to a subject in need thereof, is sufficient to effect treatment.
  • the amount of transfenin fusion protein which constitutes a “therapeutically effective amount” will vary depending on the therapeutic protein used, the severity of the condition or disease, and the age and body weight of the subject to be treated, but can be determined routinely by one or ordinary skill in the art having regard to his/her own knowledge and to this disclosure.
  • therapeutic protein refers to proteins, polypeptides, peptides or fragments or variants thereof, having one or more therapeutic and/or biological activities.
  • Therapeutic proteins encompassed by the invention include but are not limited to proteins, polypeptides, peptides, antibodies, and biologies.
  • the terms peptides, proteins, and polypeptides are used interchangeably herein.
  • the term "therapeutic protein” may refer to the endogenous or naturally occuning conelate of a therapeutic protein.
  • a polypeptide displaying a "therapeutic activity” or a protein that is “therapeutically active” is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a therapeutic protein such as one or more of the therapeutic proteins described herein or otherwise known in the art.
  • a "therapeutic protein” is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder.
  • a disease, condition or disorder may be in humans or in a non- human animal, e.g., veterinary use.
  • transformation refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
  • gene transformation refers to the transfer and inco ⁇ oration of DNA, especially recombinant DNA, into a cell.
  • transformationant refers to a cell, tissue or organism that has undergone transformation.
  • transgene refers to a nucleic acid that is inserted into an organism, host cell or vector in a manner that ensures its function.
  • transgenic refers to cells, cell cultures, organisms, bacteria, fungi, animals, plants, and progeny of any of the preceding, which have received a foreign or modified gene and in particular a gene encoding a modified Tf fusion protein by one of the various methods of transformation, wherein the foreign or modified gene is from the same or different species than the species of the organism receiving the foreign or modified gene.
  • variants refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
  • variant refers to a therapeutic protein portion of a transfenin fusion protein of the invention, differing in sequence from a native therapeutic protein but retaining at least one functional and/or therapeutic property thereof as described elsewhere herein or otherwise known in the art.
  • vector refers broadly to any plasmid, phagemid or virus encoding an exogenous nucleic acid.
  • the term is also be construed to include non-plasmid, non-phagemid and non- viral compounds which facilitate the transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like.
  • the vector may be a viral vector that is suitable as a delivery vehicle for delivery of the nucleic acid, or mutant thereof, to a cell, or the vector may be a non- viral vector which is suitable for the same pu ⁇ ose. Examples of viral and non- viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746).
  • viral vectors include, but are not limited to, a recombinant vaccinia virus, a recombinant adenoviras, a recombinant retroviras, a recombinant adeno-associated viras, a recombinant avian pox viras, and the like (Cranage et al, 1986, EMBO J. 5:3057-3063; International Patent Application No. WO94/17810, published August 18, 1994; International Patent Application No. WO94/23744, published October 27, 1994).
  • non- viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.
  • wild type refers to a polynucleotide or polypeptide sequence that is naturally occuning.
  • Tf wild-type human Tf
  • N about 330 amino acids
  • C about 340 amino acids
  • GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847 and S95936 www.ncbi.nlm.nih.gov/
  • the two domains have diverged over time but retain a large degree of identity/similarity (Fig. 1).
  • TfR Tf receptor
  • TfR Tf receptor
  • endocytosis then occurs whereby the TfR/Tf complex is transported to the endosome, at which point the localized drop in pH results in release of bound iron and the recycling of the TfR/Tf complex to the cell surface and release of Tf (known as apoTf in its un-iron bound form).
  • Receptor binding is through the C domain of Tf.
  • the two glycosylation sites in the C domain do not appear to be involved in receptor binding as unglycosylated iron bound Tf does bind the receptor.
  • Each Tf molecule can carry two iron ions (Fe 3+ ). These are complexed in the space between the NI and N2, CI and C2 sub domains resulting in a conformational change in the molecule. Tf crosses the blood brain banier (BBB) via the Tf receptor.
  • BBB blood brain banier
  • the iron binding sites comprise at least amino acids Asp 63 (Asp 82 of SEQ ID NO: 2 which includes the native Tf signal sequence), Asp 392 (Asp 411 of SEQ ID NO: 2), Tyr 95 (Tyr 114 of SEQ ID NO: 2), Tyr 426 (Tyr 445 of SEQ ID NO: 2), Tyr 188 (Tyr 207 of SEQ ID NO: 2), Tyr 514 or 517 (Tyr 533 or Tyr 536 SEQ ID NO: 2), His 249 (His 268 of SEQ ID NO: 2), and His 585 (His 604 of SEQ ID NO: 2) of SEQ ID NO: 3.
  • the hinge regions comprise at least N domain amino acid residues 94-96, 245- 247 and/or 316-318 as well as C domain amino acid residues 425-427, 581-582 and/or 652-658 of SEQ ID NO: 3.
  • the carbonate binding sites comprise at least amino acids Thr 120 (Thr 139 of SEQ ID NO: 2), Thr 452 (Thr 471 of SEQ ID NO: 2), Arg 124 (Arg 143 of SEQ ID NO: 2), Arg 456 (Arg 475 of SEQ ID NO: 2), Ala 126 (Ala 145 of SEQ ID NO: 2), Ala 458 (Ala 477 of SEQ ID NO: 2), Gly 127 (Gly 146 of SEQ ID NO: 2), and Gly 459 (Gly 478 of SEQ ID NO: 2) of SEQ ID NO: 3.
  • the modified transfenin fusion protein includes a modified human transfenin, although any animal Tf molecule may be used to produce the fusion proteins of the invention, including human Tf variants, cow, pig, sheep, dog, rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog, hornworm, monkey, as well as other bovine, canine and avian species. All of these Tf sequences are readily available in GenBank and other public databases.
  • the human Tf nucleotide sequence is available (see SEQ ID NOS 1, 2 and 3 and the accession numbers described above and available at www.ncbi.nlm.nih.gov/) and can be used to make genetic fusions between Tf or a domain of Tf and the therapeutic molecule of choice. Fusions may also be made from related molecules such as lacto transfenin (lactofenin) GenBank Ace: NM__002343) or melanotransfenin (GenBank Ace. NM_013900, murine melanotransfenin).
  • Melanotransfenin is a glycosylated protein found at high levels in malignant melanoma cells and was originally named human melanoma antigen p97 (Brown et al, 1982, Nature, 296: 171-173). It possesses high sequence homology with human seram transfenin, human lactofenin, and chicken transfenin (Brown et al, 1982, Nature, 296: 171-173; Rose et al, Proc. Natl. Acad. Sci. USA, 1986, 83: 1261-1265). However, unlike these receptors, no cellular receptor has been identified for melanotransfenin.
  • Lactofenin (Lf), a natural defense iron-binding protein, has been found to possess antibacterial, antimycotic, antiviral, antineoplastic and anti-inflammatory activity.
  • the protein is present in exocrine secretions that are commonly exposed to normal flora: milk, tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus and seminal fluid.
  • Lf is a major constituent of the secondary specific granules of circulating polymo ⁇ honuclear neutrophils (PMNs). The apoprotein is released on degranulation of the PMNs in septic areas.
  • Lf A principal function of Lf is that of scavenging free iron in fluids and inflamed areas so as to suppress free radical-mediated damage and decrease the availability of the metal to invading microbial and neoplastic cells.
  • the transfenin portion of the transfenin fusion protein of the invention includes a transfenin splice variant.
  • a transfenin splice variant can be a splice variant of human transfenin.
  • the human transfenin splice variant can be that of Genbank Accession AAA61140.
  • the transfenin portion of the transfenin fusion protein of the invention includes a lactofenin splice variant.
  • a human seram lactofenin splice variant can be a novel splice variant of a neutrophil lactofenin.
  • the neutrophil lactofenin splice variant can be that of Genbank Accession AAA59479.
  • the neutrophil lactofenin splice variant can comprise the following amino acid sequence EDCIALKGEADA (SEQ ID NO: 8), which includes the novel region of splice-variance.
  • the transfenin portion of the transfenin fusion protein of the invention includes a melanotransfenin variant.
  • Modified Tf fusions may be made with any Tf protein, fragment, domain, or engineered domain.
  • fusion proteins may be produced using the full-length Tf sequence, with or without the native Tf signal sequence.
  • Tf fusion proteins may also be made using a single Tf domain, such as an individual N or C domain or a modified form of Tf comprising 2N or 2C domains (see U.S. Provisional Application 60/406,977, filed August 30, 2002, which is herein inco ⁇ orated by reference in its entirety).
  • fusions of a therapeutic protein to a single C domain may be produced, wherein the C domain is altered to reduce, inhibit or prevent glycosylation.
  • the use of a single N domain is advantageous as the Tf glycosylation sites reside in the C domain and the N domain, on its own.
  • a prefened embodiment is the Tf fusion protein having a single N domain which is expressed at a high level.
  • a C tenninal domain or lobe modified to function as an N-like domain is modified to exhibit glycosylation patterns or iron binding properties substantially like that of a native or wild-type N domain or lobe.
  • the C domain or lobe is modified so that it is not glycosylated and does not bind iron by substitution of the relevant C domain regions or amino acids to those present in the conesponding regions or sites of a native or wild-type N domain.
  • a Tf moiety comprising "two N domains or lobes" includes a Tf molecule that is modified to replace the native C domain or lobe with a native or wild-type N domain or lobe or a modified N domain or lobe or contains a C domain that has been modified to function substantially like a wild-type or modified N domain.
  • the transfenin portion of the transfenin fusion protein includes at least two N terminal lobes of transfenin. In further embodiments, the transfenin portion of the transfenin fusion protein includes at least two N tenninal lobes of transfenin derived from human seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes, comprises, or consists of at least two C terminal lobes of transfenin. In further embodiments, the transfenin portion of the transfenin fusion protein includes at least two C terminal lobes of transfenin derived from human seram transfenin.
  • the C tenninal lobe mutant further includes a mutation of at least one of Asn413 and Asn611 of SEQ ID NO: 3 which does not allow glycosylation.
  • the Tf or Tf portion will be of sufficient length to increase the in vivo circulatory half-life, serum stability, in vitro solution stability or bioavailability of the therapeutic protein compared to the in vivo circulatory half-life, seram stability, in vitro solution stability or bioavailability of the therapeutic protein in an unfused state. Such an increase in stability, serum half-life or bioavailability may be about a 30%, 50%, 70%, 80%, 90% or more increase over the unfused therapeutic protein.
  • the modified transfenin fusion proteins exhibit a seram half-life of about 10-20 or more days, about 12-18 days or about 14-17 days.
  • the two N-linked glycosylation sites, amino acid residues conesponding to N413 and N611 of SEQ ID NO:3 may be mutated for expression in a yeast system to prevent glycosylation or hypermannosylationn and extend the seram half-life of the fusion protein and/or therapeutic protein ( to produce asialo-, or in some instances, monosialo-Tf or disialo-Tf).
  • mutations may be to the adjacent residues within the N-X-S/T glycosylation site to prevent or substantially reduce glycosylation. See U.S.
  • the transfenin fusion protein includes a modified transfenin molecule wherein the transfenin exhibits reduced glycosylation, including but not limited to asialo- monosialo- and disialo- forms of Tf.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant that is mutated to prevent glycosylation.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant that is fully glycosylated.
  • the transfenin portion of the transfenin fusion protein includes a recombinant human seram transfenin mutant that is mutated to prevent glycosylation, wherein at least one of Asn413 and Asn611 of SEQ ID NO:3 are mutated to an amino acid which does not allow glycosylation.
  • the transfenin portion of the transferrin fusion protein includes a recombinant human seram transfenin mutant that is mutated to prevent or substantially reduce glycosylation, wherein mutations may be to the adjacent residues within the N-X- S/T glycosylation site.
  • glycosylation may be reduced or prevented by mutating the serine or threonine residue. Further, changing the X to proline is known to inhibit glycosylation.
  • the iron binding is retained and the iron binding ability of Tf may be used to deliver a therapeutic protein or peptide(s) to the inside of a cell, across an epithelial or endothelial cell membrane and/or across the BBB.
  • Tf iron binding ability
  • These embodiments that bind iron and/or the Tf receptor will often be engineered to reduce or prevent glycosylation to extend the seram half-life of the therapeutic protein.
  • the N domain alone will not bind to TfR when loaded with iron, and the iron bound C domain will bind TfR but not with the same affinity as the whole molecule.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant does not retain the ability to bind metal ions.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant has a weaker binding avidity for metal ions than wild-type seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant has a stronger binding avidity for metal ions than wild-type seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant does not retain the ability to bind to the transfenin receptor.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant has a weaker binding avidity for the transfenin receptor than wild-type seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant has a stronger binding avidity for the transfenin receptor than wild-type seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant does not retain the ability to bind to carbonate ions.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant has a weaker binding avidity for carbonate ions than wild-type seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant transfenin mutant having a mutation wherein the mutant has a stronger binding avidity for carbonate ions than wild-type seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant human seram transfenin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO:3, wherein the mutant retains the ability to bind metal ions.
  • a recombinant human seram transfenin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a reduced ability to bind metal ions.
  • a recombinant human serum transfenin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO:3, wherein the mutant does not retain the ability to bind metal ions.
  • the transfenin portion of the transfenin fusion protein includes a recombinant human serum transfenin mutant having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the mutant has a stronger binding avidity for metal ions than wild-type human seram transfenin (see U.S. Patent 5,986,067, which is herein inco ⁇ orated by reference in its entirety).
  • the transfenin portion of the transfenin fusion protein includes a recombinant human serum transfenin mutant having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the mutant has a weaker binding avidity for metal ions than wild-type human seram transfenin.
  • the transfenin portion of the transfenin fusion protein includes a recombinant human seram transfenin mutant having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the mutant does not bind metal ions.
  • any available technique may be used to make the fusion proteins of the invention, including but not limited to molecular techniques commonly available, for instance, those disclosed in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989.
  • the encoded amino acid changes are preferably of a minor nature, that is, conservative amino acid substitutions, although other, non-conservative, substitutions are contemplated as well, particularly when producing a modified transfenin portion of a Tf fusion protein, e.g., a modified Tf fusion protein exhibiting reduced glycosylation, reduced iron binding and the like.
  • amino acid substitutions small deletions or insertions, typically of one to about 30 amino acids; insertions between transfenin domains; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, or small linker peptides of less than 50, 40, 30, 20 or 10 residues between transfenin domains or linking a transfenin protein and a therapeutic protein or peptide; or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative amino acid substitutions are substitutions made within the same group such as within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).
  • basic amino acids such as arginine, lysine, histidine
  • acidic amino acids such as glutamic acid and aspartic acid
  • polar amino acids such as glutamine and asparagine
  • hydrophobic amino acids such as leucine, isoleucine, valine
  • aromatic amino acids such as phenylalanine, tryptophan, tyrosine
  • small amino acids such as gly
  • Non-conservative substitutions encompass substitutions of amino acids in one group by amino acids in another group.
  • a non-conservative substitution would include the substitution of a polar amino acid for a hydrophobic amino acid.
  • Non-conservative substitutions, deletions and insertions are particularly useful to produce TF fusion proteins of the invention that exhibit no or reduced binding of iron, no or reduced binding of the fusion protein to the Tf receptor.
  • Iron binding and/or receptor binding may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues conesponding to one or more of Tf N domain residues Asp63, Tyr95, Tyrl88, His249 and/or C domain residues Asp 392, Tyr 426, Tyr 514 and/or His 585 of SEQ ID NO: 3. Iron binding may also be affected by mutation to amino acids Lys206, His207 or Arg632 of SEQ ID NO: 3.
  • Carbonate binding may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues conesponding to one or more of Tf N domain residues Thrl20, Argl24, Alal26, Gly 127 and/or C domain residues Thr 452, Arg 456, Ala 458 and/or Gly 459 of SEQ ID NO: 3.
  • a reduction or disruption of carbonate binding may adversely affect iron and/or receptor binding.
  • Binding to the Tf receptor may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues conesponding to one or more of Tf N domain residues described above for iron binding.
  • glycosylation may be reduced or prevented by mutation, including deletion, substitution or insertion into, amino acid residues conesponding to one or more of Tf C domain residues around the N-X-S/T sites conesponding to C domain residues N413 and/or N611 (See U.S. Patent No. 5,986,067).
  • the N413 and/or N611 may be mutated to Glu residues.
  • the Tf fusion proteins of the invention are not modified to prevent glycosylation, iron binding, carbonate binding and/or receptor binding, glycosylation, iron and/or carbonate ions may be stripped from or cleaved off of the fusion protein.
  • available deglycosylases may be used to cleave glycosylation residues from the fusion protein, in particular the sugar residues attached to the Tf portion, yeast deficient in glycosylation enzymes may be used to prevent glycosylation and/or recombinant cells may be grown in the presence of an agent that prevents glycosylation, e.g., tunicamycin.
  • an agent that prevents glycosylation e.g., tunicamycin.
  • the carbohydrates on the fusion protein may also be reduced or completely removed enzymatically by treating the fusion protein with deglycosylases.
  • Deglycosylases are well known in the art. Examples of deglycosylases include but are not limited to galactosidase, PNGase A, PNGase F, glucosidase, mannosidase, fucosidase, and Endo H deglycosylase.
  • the Tf portion of the fusion protein be fully glycosylated Additional mutations may be made with Tf to alter the three dimensional structure of
  • Tf such as modifications to the hinge region to prevent the conformational change needed for iron biding and Tf receptor recognition.
  • mutations may be made in or around N domain amino acid residues 94-96, 245-247 and/or 316-318 as well as C domain amino acid residues 425-427, 581-582 and/or 652-658.
  • mutations may be made in or around the flanking regions of these sites to alter Tf structure and function.
  • the transfenin fusion protein can function as a canier protein to extend the half life or bioavailability of the therapeutic protein as well as, in some instances, delivering the therapeutic protein inside a cell and/or across the blood brain banier.
  • the transfenin fusion protein includes a modified transfenin molecule wherein the transfenin does not retain the ability to cross the blood brain banier.
  • the transfenin fusion protein includes a modified transfenin molecule wherein the transfenin molecule retains the ability to bind to the transfenin receptor and transport the therapeutic peptide inside cells.
  • the transfenin fusion protein includes a modified transfenin molecule wherein the transfenin molecule does not retain the ability to bind to the transfenin receptor and transport the therapeutic peptide inside cells.
  • the transfenin fusion protein includes a modified transfenin molecule wherein the transfenin molecule retains the ability to bind to the transfenin receptor and transport the therapeutic peptide inside cells and retains the ability to cross the blood brain banier.
  • the transfenin fusion protein includes a modified transfenin molecule wherein the transfenin molecule retains the ability to cross the blood brain banier, but does not retain the ability to bind to the transfenin receptor and transport the therapeutic peptide inside cells.
  • the fusion proteins of the invention may contain one or more copies of the therapeutic protein or polypeptide attached to the N-terminus and/or the C-terminus of the Tf protein.
  • the therapeutic protein or polypeptide is attached to both the N- and C-terminus of the Tf protein and the fusion protein may contain one or more equivalents of the therapeutic protein or polypeptide on either or both ends of Tf.
  • the therapeutic protein or polypeptide is inserted into known domains of the Tf protein, for instance, into one or more of the loops of Tf (see Ali et al. (1999) J. Biol. Chem. 274(34):24066-24073).
  • the therapeutic protein or therapeutic peptide is inserted between the N and C domains of Tf.
  • the transfenin fusion protein of the invention may have one modified transfenin-derived region and one therapeutic protein-derived region. Multiple regions of each protein, however, may be used to make a transferrin fusion protein of the invention. Similarly, more than one therapeutic protein may be used to make a transferrin fusion protein of the invention, thereby producing a multi-functional modified Tf fusion protein.
  • the present invention provides transfenin fusion protein containing a therapeutic protein or polypeptide or portion thereof fused to a transfenin molecule or portion thereof. In one embodiment, the transfenin fusion protein of the invention contains a therapeutic protein or polypeptide fused to the N terminus of a transfenin molecule.
  • the transfenin fusion protein of the invention contains a therapeutic protein fused to the C terminus of a transfenin molecule.
  • the present invention also provides transfenin fusion protein containing a therapeutic protein or polypeptide or protion thereof fused to a modified transfenin morlecule or portion thererof.
  • the transferrin fusion protein of the inventions contains a therapeutic protein fused to both the N-terminus and the C-terminus of modified transfenin.
  • the therapeutic proteins fused at the N- and C- termini are the same therapeutic proteins.
  • the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins.
  • the therapeutic proteins fused to the N- and C- tennini are different therapeutic proteins which may be used to treat or prevent the same disease, disorder, or condition.
  • the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins which may be used to treat or prevent diseases or disorders which are known in the art to commonly occur in patients simultaneously.
  • transfenin fusion protein of the inventions of the invention may also be produced by inserting the therapeutic protein or peptide of interest (e.g., a therapeutic protein or peptide as disclosed herein, or, for instance, a single chain antibody that binds a therapeutic protein or a fragment or variant thereof) into an internal region of the modified transfenin.
  • the therapeutic protein or peptide of interest e.g., a therapeutic protein or peptide as disclosed herein, or, for instance, a single chain antibody that binds a therapeutic protein or a fragment or variant thereof
  • Internal regions of modified transfenin include, but are not limited to, the iron binding sites, the hinge regions, the bicarbonate binding sites, or the receptor binding domain.
  • modified transfenin molecule Within the protein sequence of the modified transfenin molecule a number of loops or turns exist, which are stabilized by disulfide bonds. These loops are useful for the insertion, or internal fusion, of therapeutically active peptides, particularly those requiring a secondary structure to be functional, or therapeutic proteins to generate a modified transfenin molecule with specific biological activity.
  • insertions may be made within any of the surface exposed loop regions, in addition to other areas of Tf. For instance, insertions may be made within the loops comprising Tf amino acids 32-33, 74-75, 256-257, 279-280 and 288-289 (Ali et al, supra) (See Figure 3). As previously described, insertions may also be made within other regions of Tf such as the sites for iron and bicarbonate binding, hinge regions, and the receptor binding domain as described in more detail below.
  • the loops in the Tf protein sequence that are amenable to modification/replacement for the insertion of proteins or peptides may also be used for the development of a screenable library of random peptide inserts. Any procedures may be used to produce nucleic acid inserts for the generation of peptide libraries, including available phage and bacterial display systems, prior to cloning into a Tf domain and/or fusion to the ends of Tf. In other embodiments, the library is made directly in or on the ends of a Tf peptide as described below.
  • the N-terminus of Tf is free and points away from the body of the molecule. Fusions of proteins or peptides on the N-terminus may therefore be a prefened embodiment. Such fusions may include a linker region, such as but not limited to a poly-glycine stretch, to separate the therapeutic protein or peptide from Tf. Attention to the junction between the leader sequence, the choice of leader sequence, and the structure of the mRNA by codon manipulation/optimization (no major stem loops to inhibit ribosome progress) will increase secretion and can be readily accomplished using standard recombinant protein techniques. The C-terminus of Tf appears to be more buried and secured by a disulfide bond 6 amino acids from the C-terminus.
  • the C-terminal amino acid is a proline which, depending on the way that it is orientated, will either point a fusion away or into the body of the molecule.
  • a linker or spacer moiety at the C-terminus may be used in some embodiments of the invention.
  • the proline at the N- and/or the C- termini may be changed out.
  • the C-terminal disulfide bond may be eliminated to untether the C- terminus.
  • small molecule therapeutics may be complexed with iron and loaded on a modified Tf protein fusion for delivery to the inside of cells and across the BBB.
  • a targeting peptide or, for example, a single chain antibody (SCA) can be used to target the payload to a particular cell type, e.g., a cancer cell.
  • Nucleic acid molecules are also provided by the present invention. These encode a modified Tf fusion protein comprising a transfenin protein or a portion of a transfenin protein covalently linked or joined to a therapeutic protein. As discussed in more detail below, any therapeutic protein may be used.
  • the fusion protein may further comprise a linker region, for instance a linker less than about 50, 40, 30, 20, or 10 amino acid residues. The linker can be covalently linked to and between the transfenin protein or portion thereof and the therapeutic protein. Nucleic acid molecules of the invention may be purified or not.
  • Host cells and vectors for replicating the nucleic acid molecules and for expressing the encoded fusion proteins are also provided. Any vectors or host cells may be used, whether prokaryotic or eukaryotic, but eukaryotic expression systems, in particular yeast expression systems, may be prefened. Many vectors and host cells are known in the art for such pu ⁇ oses. It is well within the skill of the art to select an appropriate set for the desired application. DNA sequences encoding transfenin, portions of transfenin and therapeutic proteins of interest 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 or protein sequences (see, for example, Baldwin, G.S. (1993) Comparison of Transfenin Sequences from Different
  • PCR polymerase chain reaction
  • similarity between two polynucleotides or polypeptides is determined by comparing the nucleotide or amino acid sequence and its conserved nucleotide or amino acid substitutes of one polynucleotide or polypeptide to the sequence of a second polynucleotide or polypeptide.
  • identity also known in the art is “identity” which means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the identity of the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • identity and similarity are well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J. Applied Math. 48:1073 (1988).
  • Prefened methods to determine identity are designed to give the largest match between the two sequences tested. Methods to determine identity and similarity are codified in computer programs. Prefened computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, et al, Nucl. Acid Res. 12(1):387 (1984)), BLASTP, BLASTN, FASTA
  • the degree of similarity or identity refened to above is determined as the degree of identity between the two sequences, often indicating a derivation of the first sequence from the second.
  • the degree of identity between two nucleic acid sequences may be detennined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch J. Mol. Biol. 48:443-453 (1970)).
  • GAP is used with the following settings: GJAP creation penalty of 5.0 and GAP extension penalty of 0.3.
  • the degeneracy of the genetic code permits variations of the nucleotide sequence of a transfenin protein and/or therapeutic protein of interest, while still producing a polypeptide having the identical amino acid sequence as the polypeptide encoded by the native DNA sequence.
  • the procedure known as "codon optimization" (described in U.S. Patent 5,547,871 which is inco ⁇ orated herein by reference in its entirety) provides one with a means of designing such an altered DNA sequence.
  • the design of codon optimized genes should take into account a variety of factors, including the frequency of codon usage in an organism, nearest neighbor frequencies, RNA stability, the potential for secondary structure formation, the route of synthesis and the intended future DNA manipulations of that gene.
  • the prefened codon usage frequencies for a synthetic gene should reflect the codon usages of nuclear genes derived from the exact (or as closely related as possible) genome of the cell/organism that is intended to be used for recombinant protein expression, particularly that of yeast species.
  • the human Tf sequence is codon optimized, before or after modification as herein described for yeast expression as may be the therapeutic protein nucleotide sequence(s).
  • Expression units for use in the present invention will generally comprise the following elements, operably linked in a 5' to 3' orientation: a transcriptional promoter, a secretory signal sequence, a DNA sequence encoding a modified Tf fusion protein comprising transfenin protein or a portion of a transfenin protein joined to a DNA sequence encoding a therapeutic protein or peptide of interest and a transcriptional terminator.
  • a transcriptional promoter a secretory signal sequence
  • a DNA sequence encoding a modified Tf fusion protein comprising transfenin protein or a portion of a transfenin protein joined to a DNA sequence encoding a therapeutic protein or peptide of interest and a transcriptional terminator.
  • any anangement of the therapeutic protein or peptide fused to or within the Tf portion may be used in the vectors of the invention.
  • suitable promoters, signal sequences and terminators will be determined by the selected host cell and will be evident to one skilled in the art and are discussed more specifically below.
  • yeast plasmid vectors also include pRS403-406, pRS413-416 and the Pichia vectors available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids ⁇ RS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and inco ⁇ orate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • PlasmidspRS413-41.6 are Yeast Centromere plasmids (YCps).
  • Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transfonnants to be selected.
  • selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al. ibid.), URA3 (Botstein et al, Gene 8: 17, 1979), HIS3 (Struhl et al, ibid.) or POT1 (Kawasaki and Bell, EP 171,142).
  • Other suitable selectable markers include the CAT gene, which confers chloramphenicol resistance on yeast cells.
  • promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al, J Biol. Chem. 225: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Young et al, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, (eds.), p. 355, Plenum, NY., 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983).
  • prefened promoters are the TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311) and the ADH2-4 C (see U.S. Patent 6,291,212 promoter (Russell et al., Nature 304: 652-654, 1983).
  • the expression units may also include a transcriptional terminator.
  • a prefened transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, ibid.).
  • prefened vectors and prefened components such as promoters and tenninators of a yeast expression system are disclosed in European Patents EP 0258067, EP 0286424, EP0317254, EP 0387319, EP 0386222, EP 0424117, EP 0431880, and EP 1002095; European Patent Publications EP 0828759, EP 0764209, EP 0749478, and EP 0889949; PCT Publication WO 00/44772 and WO 94/04687; and U.S.
  • modified fusion proteins of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus.
  • useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the adh3 promoter (McKnight et al, EMBO J. 4: 2093-2099, 1985) and the tpiA promoter.
  • a suitable terminator is the adh3 terminator (McKnight et al, ibid.).
  • the expression units utilizing such components may be cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus, for example.
  • Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of the modified Tf fusion protein.
  • Prefened promoters include viral promoters and cellular promoters.
  • Prefened viral promoters include the major late promoter from adenoviras 2 (Kaufman and Sha ⁇ , Mol. Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subramani et al, Mol. Cell. Biol. 1: 854-864, 1981).
  • Prefened cellular promoters include the mouse metallothionein 1 promoter (Palmiter et al, Science 222: 809-814, 1983) and a mouse VK (see U.S.
  • Patent 6,291,212 promoter (Grant et al, Nuc. Acids Res. 15: 5496, 1987).
  • a particularly prefened promoter is a mouse V H (see U.S. Patent 6,291,212) promoter (Loh et al, ibid.).
  • Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the transfenin fusion protein. Prefened RNA splice sites may be obtained from adenovirus and/or i munoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest.
  • Polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sha ⁇ , ibid.), the polyadenylation signal from the adenoviras 5 E1B region and the human growth hormone gene terminator (DeNoto et al, Nucl. Acid Res. 9: 3719-3730, 1981).
  • a particularly prefened polyadenylation signal is the V H (see U.S. Patent 6,291,212) gene terminator (Loh et al, ibid.).
  • the expression vectors may include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites.
  • Prefened vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse ⁇ (see U.S. Patent 6,291,212) enhancer (Gillies, Cell 33: 717-728, 1983).
  • Expression vectors may also include sequences encoding the adenoviras VA RNAs.
  • Cloned DNA sequences comprising modified Tf fusion proteins of the invention may be introduced into cultured mammalian cells by, for example, calcium phosphate- mediated transfection (Wigler et al, Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973.)
  • Other techniques for introducing cloned DNA sequences into mammalian cells such as electroporation (Neumann et al, EMBO J. 1: 841-845, 1982), or lipofection may also be used.
  • a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
  • Prefened selectable markers for use in cultured mammalian cells include genes that confer resistance to drags, such as neomycin, hygromycin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • a prefened amplifiable selectable marker is the DHFR gene.
  • a particularly prefened amplifiable marker is the DHFR r (see U.S. Patent 6,291,212) cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983).
  • 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.
  • the present invention also includes a cell, preferably a yeast cell transformed to express a modified transfenin fusion protein of the invention.
  • a cell preferably a yeast cell transformed to express a modified transfenin fusion protein of the invention.
  • the present invention also includes a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away.
  • Host cells for use in practicing the present invention include eukaryotic cells, and in some cases prokaryotic cells, capable of being transformed or transfected with exogenous DNA and grown in culture, such as cultured mammalian, insect, fungal, plant and bacterial cells.
  • Fungal cells including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp.) may be used as host cells within the present invention.
  • yeasts e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp.
  • yeasts contemplated to be useful in the practice, of the present invention as hosts for expressing the, transfenin fusion protein of the inventions are Pichia (some species of which were formerly classified as Hansenula), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia,
  • Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.
  • Kluyveromyces spp. are K. fragilis, K. lactis and K. marxianus.
  • a suitable Torulaspora species is T delbrueckii.
  • Pichia spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P. pastoris.
  • Pichia pastoris is the methylotrophic Pichia pastoris (Steinlein et al. (1995) Protein Express. Purifi 6:619- 624).
  • Pichia pastoris has been developed to be an outstanding host for the production of foreign proteins since its alcohol oxidase promoter was isolated and cloned; its transformation was first reported in 1985.
  • P. pastoris can utilize methanol as a carbon source in the absence of glucose.
  • the P. pastoris expression system can use the methanol- induced alcohol oxidase (AOX1) promoter, which controls the gene that codes for the expression of alcohol oxidase, the enzyme which catalyzes the first step in the metabolism of methanol.
  • AOX1 methanol- induced alcohol oxidase
  • This promoter has been characterized and inco ⁇ orated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded conectly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E. coli expression systems. A number of proteins have been produced using this system, including tetanus toxin fragment, Bordatella pertussis pertactin, human seram albumin and lysozyme.
  • yeast Saccharomyces cerevisiae are another prefened host.
  • a yeast cell or more specifically, a Saccharomyces cerevisiae host cell that contains a genetic deficiency in a gene required for asparagine-linked glycosylation of glycoproteins is used.
  • S. cerevisiae host cells having such defects may be prepared using standard techniques of mutation and selection, although many available yeast strains have been modified to prevent or reduce glycosylation or hypermannosylation. Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) have described the isolation of mannoprotein biosynthesis mutants that are defective in genes which affect asparagine-linked glycosylation.
  • Gentzsch and Tanner (Glycobiology 7:481-486, 1997) have described a family of at least six genes (PMT1-6) encoding enzymes responsible for the first step in O- glycosylation of proteins in yeast. Mutants defective in one or more of these genes show reduced O-linked glycosylation and/or altered specificity of O-glycosylation. To optimize production of the heterologous proteins, it is also prefened that the host strain canies a mutation, such as the S. cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in reduced proteolytic activity. Host strains containing mutations in other protease encoding regions are particularly useful to produce large quantities of the Tf fusion proteins of the invention.
  • Host cells containing DNA constracts of the present invention are grown in an appropriate growth medium.
  • 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 growth medium will generally select for cells containing the DNA constract by, for example, drag selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA constract or co-transfected with the DNA constract.
  • Yeast cells for example, are preferably grown in a chemically defined medium, comprising a carbon source, e.g.
  • sucrose, a non-amino acid nitrogen source, inorganic salts, vitamins and essential amino acid supplements The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 5.5-6.5.
  • Methods for maintaining a stable pH include buffering and constant pH control.
  • Prefened buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.).
  • Yeast cells having a defect in a gene required for asparagine-linked glycosylation are preferably grown in a medium containing an osmotic stabilizer.
  • a prefened osmotic stabilizer is sorbitol supplemented into the medium at a concentration between 0.1 M and 1.5 M., preferably at 0.5 M or 1.0 M.
  • Cultured mammalian cells are generally grown in commercially available seram- containing or seram-free media. Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.
  • Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest.
  • Drag selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drag concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.
  • Baculoviras/insect cell expression systems may also be used to produce the modified Tf fusion proteins of the invention.
  • the BacPAKTM Baculoviras Expression System (BD Biosciences (Clontech) expresses recombinant proteins at high levels in insect host cells.
  • the target gene is inserted into a transfer vector, which is cotransfected into insect host cells with the linearized BacPAK ⁇ viral DNA.
  • the BacP AK6 DNA is missing an essential portion of the baculoviras genome. When the DNA recombines with the vector, the essential element is restored and the target gene is transfened to the baculoviras genome.
  • recombination Following recombination, a few viral plaques are picked and purified, and the recombinant phenotype is verified. The newly isolated recombinant viras can then be amplified and used to infect insect cell cultures to produce large amounts of the desired protein.
  • Tf fusion proteins of the present invention may also be produced using transgenic plants and animals.
  • sheep and goats can make the therapeutic protein in their milk.
  • tobacco plants can include the protein in their leaves.
  • Both transgenic plant and animal production of proteins comprises adding a new gene coding the fusion protein into the genome of the organism. Not only can the transgenic organism produce a new protein, but it can also pass this ability onto its offspring.
  • secretory signal sequence or “signal sequence” or “secretion leader sequence” are used interchangeably and are described, for example in U.S. Pat. 6,291,212 and U.S. Pat 5,547,871, both of which are herein inco ⁇ orated by reference in their entirety.
  • Secretory signal sequences or signal sequences or secretion leader sequences encode secretory peptides.
  • a secretory peptide is an amino acid sequence that acts to direct the secretion of a mature polypeptide or protein from a cell.
  • Secretory peptides are generally characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins.
  • Secretory peptides may contain processing sites that allow cleavage of the signal peptide from the mature protein as it passes through the secretory pathway. Processing sites may be encoded within the signal peptide or may be added to the signal peptide by, for example, in vitro mutagenesis. Secretory peptides may be used to direct the secretion of modified Tf fusion proteins of the invention.
  • One such secretory peptide that may be used in combination with other secretory peptides is the alpha mating factor leader sequence.
  • Secretory signal sequences or signal sequences or secretion leader sequences are required for a complex series of post- translational processing steps which result in secretion of a protein. If an intact signal sequence is present, the protein being expressed enters the lumen of the rough endoplasmic reticulum and is then transported through the Golgi apparatus to secretory vesicles and is finally transported out of the cell. Generally, the signal sequence immediately follows the initiation codon and encodes a signal peptide at the amino-terminal end of the protein to be secreted. In most cases, the signal sequence is cleaved off by a specific protease, called a signal peptidase. Prefened signal sequences improve the processing and export efficiency of recombinant protein expression using viral, mammalian or yeast expression vectors. In some cases, the native Tf signal sequence may be used to express and secrete fusion proteins of the invention.
  • the Tf moiety and therapeutic protein moiety(s) of the modified transfenin fusion proteins of the invention can be fused directly or using a linker peptide of various lengths to provide greater physical separation and allow more spatial mobility between the fused proteins and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor.
  • the linker peptide may consist of amino acids that are flexible or more rigid.
  • a linker such as but not limited to a poly-glycine stretch.
  • the linker can be less than about 50, 40, 30, 20, or 10 amino acid residues.
  • the linker can be covalently linked to and between the transfenin protein or portion thereof and the therapeutic protein.
  • Detection of Tf Fusion Proteins may include Western transfer, protein blot or colony filter as well as activity based assays that detect the fused therapeutic protein.
  • a Western transfer filter may be prepared using the method described by Towbin et al. (Proc. Natl. Acad. Sci. USA 16: 4350-4354, 1979). Briefly, samples are elecfrophoresed in a sodium dodecylsulfate polyacrylamide gel. The proteins in the gel are electrophoretically transfened to nitrocellulose paper.
  • Protein blot filters may be prepared by filtering supernatant samples or concentrates through nitrocellulose filters using, for example, a Minifold (Schleicher & Schuell, Keene, N.H.). Colony filters may be prepared by growing colonies on a nitrocellulose filter that has been laid across an appropriate growth medium. In this method, a solid medium is prefened. The cells are allowed to grow on the filters for at least 12 hours. The cells are removed from the filters by washing with an appropriate buffer that does not remove the proteins bound to the filters.
  • a prefened buffer comprises 25 mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.
  • Fusion proteins of the invention may also be detected by assaying for the activity of the therapeutic protein moiety.
  • assays are readily available, including but not limited to, those assays described in Table 1.
  • transfenin fusion proteins of the invention may be assayed for functional activity (e.g., biological activity or therapeutic activity) using the assay referenced in the "Exemplary Activity Assay" column of Table 1.
  • functional activity e.g., biological activity or therapeutic activity
  • one of skill in the art may routinely assay fragments of a therapeutic protein conesponding to a therapeutic protein portion of a fusion protein of the invention, for activity using assays referenced in its conesponding row of Table 1.
  • one of skill in the art may routinely assay fragments of a modified transfenin protein for activity using assays known in the art.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immuno
  • antibody binding is detected by detecting a label on the. primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • binding partner e.g., a receptor or a ligand
  • binding to that binding partner by a transfenin fusion protein containing that therapeutic protein as the therapeutic protein portion of the fusion can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. Other methods will be known to the skilled artisan and are within the scope of the invention.
  • Secreted, biologically active, modified transfenin fusion proteins may be isolated from the medium of host cells grown under conditions that allow the secretion of the biologically active fusion proteins.
  • the cell material is removed from the culture medium, and the biologically active fusion proteins are isolated using isolation techniques known in the art. Suitable isolation techniques include precipitation and fractionation by a variety of chromatographic methods, including gel filtration, ion exchange chromatography and affinity chromatography.
  • a particularly prefened purification method is affinity chromatography on an iron binding or metal chelating column or an immunoaffinity chromatography using an antibody directed against the transfenin or therapeutic protein or peptide portion of the polypeptide fusion.
  • the antibody is preferably immobilized or attached to a solid support or substrate.
  • a particularly prefened substrate is CNBr-activated Sepharose (Pharmacia LKB
  • the medium is combined with the antibody/substrate under conditions that will allow binding to occur.
  • the complex may be washed to remove unbound material, and the transfenin fusion protein is released or eluted through the use of conditions unfavorable to complex formation.
  • Particularly useful methods of elution include changes in pH, wherein the immobilized antibody has a high affinity for the ligand at a first pH and a reduced affinity at a second (higher or lower) pH; changes in concentration of certain chaotropic agents; or through the use of detergents.
  • Transfenin fusion proteins of the present invention may also be labeled with a radioisotope or other imaging agent and used for in vivo diagnostic pu ⁇ oses.
  • Prefened radioisotope imaging agents include iodine-125 and technetium-99, with technetium-99 being particularly prefened.
  • Methods for producing protein-isotope conjugates are well known in the art, and are described by, for example, Eckelman et al. (U.S. Pat. No. 4,652,440), Parker et al. (WO 87/05030) and Wilber et al. (EP 203,764).
  • the transfenin fusion proteins may be bound to spin label enhancers and used for magnetic resonance (MR) imaging.
  • MR magnetic resonance
  • Suitable spin label enhancers include stable, sterically hindered, free radical compounds such as nitroxides.
  • Methods for labeling ligands for MR imaging are disclosed by, for example, Coffman et al. (U.S. Pat. No. 4,656,026).
  • the labeled transfenin fusion proteins are combined with a pharmaceutically acceptable canier or diluent, such as sterile saline or sterile water.
  • Administration is preferably by bolus injection, preferably intravenously.
  • the present invention further provides methods for producing a modified fusion protein of the invention using nucleic acid molecules herein described.
  • the production of a recombinant form of a protein typically involves the following steps.
  • a nucleic acid molecule is first obtained that encodes a transfenin fusion protein of the invention.
  • the nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
  • the expression unit is used to transfonn a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
  • the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • Each of the foregoing steps can be accomplished in a variety of ways.
  • the constraction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transfonnation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier and are otherwise known to persons skilled in the art.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce a desired recombinant protein.
  • any expression system may be used, including yeast, bacterial, animal, plant, eukaryotic and prokaryotic systems.
  • yeast, mammalian cell culture and transgenic animal or plant production systems are prefened.
  • yeast systems that have been modified to reduce native yeast glycosylation, hyper-glycosylation or proteolytic activity may be used.
  • a therapeutic molecule may be used as the fusion partner to Tf according to the methods and compositions of the present invention.
  • a therapeutic molecule is typically a protein or peptide capable of exerting a beneficial biological effect in vitro or in vivo and includes proteins or peptides that exert a beneficial effect in relation to nonnal homeostasis, physiology or a disease state.
  • Therapeutic molecules do not include, fusion partners commonly used as markers or protein purification aids, such as bacterial galactosidases (see for example, U.S. Patent 5, 986, 067 and Aldred et al. (1984) Biochem. Biophys. Res. Commun. 122: 960-965).
  • a beneficial effect as related to a disease state includes any effect that is advantageous to the treated subject, including disease prevention, disease stabilization, the lessening or alleviation of disease symptoms or a modulation, alleviation or cure of the underlying defect to produce an effect beneficial to the treated subject.
  • a modified transfenin fusion protein of the invention includes at least a fragment or variant of a therapeutic protein and at least a fragment or variant of modified seram transfenin, which are associated with one another, preferably by genetic fusion.
  • the transfenin fusion protein includes a modified transfenin molecule linked to a neuropharmaceutical agent.
  • the modified transfenin fusion protein includes transfenin at the carboxyl terminus linked to a neuropharmaceutical agent at the amino tenninus. In an alternate embodiment, the modified transfenin fusion protein includes transfenin at the amino tenninus linked to a neuropharmaceutical agent at the carboxy terminus. In specific embodiments, the neuropharmaceutical agent is either nerve growth factor or ciliary neurotrophic factor.
  • a modified transfenin fusion protein of the invention may contain at least a fragment or variant of a therapeutic protein, and/or at least a fragment or variant of an antibody.
  • the transfenin fusion proteins can contain peptide fragments or peptide variants of proteins or antibodies wherein the variant or fragment retains at least one biological or therapeutic activity.
  • the transfenin fusion proteins can contain therapeutic proteins that can be peptide fragments or peptide variants at least about 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least about 40, at least about 50, at least about 55, at least about 60 or at least about 70 or more amino acids in length fused to the N and/or C termini, inserted within, or inserted into a loop of a modified transfenin.
  • the modified transfenin fusion molecules contain a therapeutic protein portion that can be fragments of a therapeutic protein that include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence.
  • the modified transfenin fusion molecules contain a therapeutic protein portion that can be fragments of a therapeutic protein that include the full length protein as well as polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence.
  • the modified transfenin fusion molecules contain a therapeutic protein portion that can have one or more amino acids deleted from both the amino and the carboxy tennini.
  • the modified transferrin fusion molecules contain a therapeutic protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference therapeutic protein set forth herein, or fragments thereof.
  • the transfenin fusion molecules contain a therapeutic protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to reference polypeptides having the amino acid sequence of N- and C-terminal deletions as described above.
  • the modified transfenin fusion molecules contain the therapeutic protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the native or wild-type amino acid sequence of a therapeutic protein. Fragments, of these polypeptides are also provided.
  • the therapeutic proteins conesponding to a therapeutic protein portion of a modified transfenin fusion protein of the invention can be modified by the attachment of one or more oligosaccharide groups.
  • the modification refened to as glycosylation can significantly affect the physical properties of proteins and can be important in protein stability, secretion, and localization. Glycosylation occurs at specific locations along the polypeptide backbone.
  • glycosylation characterized by O-linked oligosaccharides, which are attached to serine or threonine residues; and glycosylation characterized by N-linked oligosaccharides, which are attached to asparagine residues in an Asn-X-Ser/Thr sequence, where X can be an amino acid except proline.
  • Variables such as protein structure and cell type influence the number and nature of the carbohydrate units within the chains at different glycosylation sites.
  • Glycosylation isomers are also common at the same site within a given cell type. For example, several types of human interferon are glycosylated.
  • Therapeutic proteins conesponding to a therapeutic protein portion of a transfenin fusion protein of the invention may be modified so that glycosylation at one or more sites is altered as a result of manipulation(s) of their nucleic acid sequence by the host cell in which they are expressed, or due to other conditions of their expression.
  • glycosylation isomers may be produced by abolishing or introducing glycosylation sites, e.g., by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or unglycosylated recombinant proteins may be produced by expressing the proteins in host cells that will not glycosylate them, e.g. in glycosylation-deficient yeast.
  • transfenin fusion proteins of the invention are capable of a therapeutic activity and/or biologic activity, conesponding to the therapeutic activity and/or biologic activity of the therapeutic protein listed in the conesponding row of Table 1 and elsewhere in this application.
  • the therapeutically active protein portions of the transfenin fusion proteins of the invention are fragments or variants of the reference sequences cited herein.
  • the present invention is further directed to modified Tf fusion proteins comprising fragments of the therapeutic proteins herein described. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the therapeutic protein portion, other therapeutic activities and/or functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of polypeptides with N-tenninal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained with less than the majority of the residues of the complete polypeptide removed from the N-terminus.
  • other therapeutic activities and/or functional activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • deletion of one or more amino acids from the N- terminus or C-terminus of a therapeutic protein results in modification or loss of one or more biological functions of the protein
  • other functional activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • therapeutic activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • therapeutic activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • therapeutic activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • the ability of polypeptides with C-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus.
  • Peptide fragments of the therapeutic proteins can be fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a therapeutic activity and/or functional activity (e.g. biological activity) of the polypeptide sequence of the therapeutic protein of which the amino acid sequence is a fragment.
  • the peptide fragments of the therapeutic protein may comprise only the N- and C- termini of the protein, i.e., the central portion of the therapeutic protein has been deleted.
  • the peptide fragments may comprise non-adjacent and/or adjacent portions of the central part of the therapeutic protein.
  • polypeptide fragments are biologically active fragments.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a therapeutic protein used in the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • variants of proteins are overall very similar, and, in many regions, identical to the amino acid sequence of the therapeutic protein conesponding to a therapeutic protein portion of a transfenin fusion protein of the invention. Nucleic acids encoding these variants are also encompassed by the invention.
  • a polypeptide-having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.
  • These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence, or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least about 80%, 85%, 90%, 95%o, 96%o, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a transfenin fusion protein of the invention or a fragment thereof (such, as the therapeutic protein portion of the transfenin fusion protein or the transfenin portion of the transfenin fusion protein), can be determined conventionally using known computer programs.
  • a prefened method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also refened to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brafiag et al. (Comp. App. Biosci 245 (1990)).
  • polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both.
  • Polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide may be used to produce modified Tf fusion proteins.
  • Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code can be utilized.
  • polypeptide variants in which less than about 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination can also be utilized.
  • Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those prefened by a host, such as, yeast or E. coli as described above).
  • the therapeutic protein moiety has conservative substitutions compared to the wild-type sequence.
  • conservative substitutions is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and T ⁇ , and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of a therapeutic protein described herein and/or serum transfenin, and/ modified transfenin protein of the invention, wherein the fragments or variants have 1- 5, 5-10, 5-25, 5-50, 10-50 or 50-150 amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence.
  • the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the invention.
  • the modified fusion proteins of the present invention can be composed of amino- acids joined to each other by peptide bonds or modified peptide bonds and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxy termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond fonnation, demethylation, formation of covalent cross-links, formation of cysteine, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Therapeutic molecules that may be fused to or inserted into Tf include, but are not limited to, hormones, matrix proteins, immunosuppressants, bronchodilators, cardiovascular agents, enzymes, CNS agents, neurotransmitters, receptor proteins or peptides, growth hormones, growth factors, antiviral peptides, fusogenic inhibitor peptides, cytokines, lymphokines, monokines, interleukins, colony stimulating factors, differentiation factors, angiogenic factors, receptor ligands, cancer-associated proteins, antineoplastics, viral peptides, antibiotic peptides, blood proteins, antagonist proteins, transcription factors, antiangiogenic factors, antagonist proteins or peptides, receptor antagonists, antibodies, single chain antibodies and cell adhesion molecules.
  • Different therapeutic molecules may be combined into a single fusion protein to produce a bi or multi-functional therapeutic molecule.
  • Different molecules may also be used in combination to produce a fusion protein with a therapeutic entity and a targeting entity.
  • Cytokines are soluble proteins released by cells of the immune system, which act nonenzymatically through specific receptors to regulate immune responses. Cytokines resemble hormones in that they act at low concentrations bound with high affinity to a specific receptor.
  • the term "cytokine” is used herein to describe naturally occuning or recombinant proteins, analogs thereof, and fragments thereof which elicit a specific biological response in a cell which has a receptor for that cytokine. Cytokines preferably include interleukins such as interleukin-2 (IL-2) (GenBank Ace. No. S77834), IL-3 (GenBank Ace. No. M14743), IL-4 (GenBank Ace. No. M23442), IL-5 (GenBank Ace. No.
  • IL-6 GenBank Ace. No. M14584
  • IL-7 GenBank Ace. No. NM_O0088O
  • IL-10 GenBank Ace. No. NM_000572
  • IL-12 GenBank Ace. No.AF180562 and GenBank Ace. No. AF180563
  • IL-13 GenBank Ace. No. U10307
  • IL-14 GenBank Ace. No. XM_170924
  • IL-15 GenBank Ace. No. X91233
  • IL-16 GenBank Ace. No. NM_004513
  • IL- 17 GenBank Ace. No. NM_002190
  • IL- 18 GenBank Ace. No.
  • hematopoietic factors such as granulocyte-macrophage colony stimulating factor (GM-CSF) (GenBank Ace. No. X03021), granulocyte colony stimulating factor (G- CSF) (GenBank Ace. No. X03656), platelet activating factor (GenBank Ace. No. NM_000437) and erythropoeitin (GenBank Ace. No. X02158), tumor necrosis factors (TNF) such as TNF ⁇ (GenBank Ace. No. X02910), lymphokines such as lymphotoxin- ⁇ (GenBank Ace. No. X02911), lymphotoxin- ⁇ (GenBank Ace. No.
  • TNF tumor necrosis factors
  • lymphokines such as lymphotoxin- ⁇ (GenBank Ace. No. X02911), lymphotoxin- ⁇ (GenBank Ace. No.
  • leukoregulin a factor that influences the rate of production of leukoregulin
  • macrophage migration inhibitory factor GeneBank Ace. No. M25639
  • neuroleukin GeneBank Ace. No. K03515)
  • regulators of metabolic processes such as leptin (GenBank Acc. No. U43415)
  • interferons such as interferon ⁇ (IFN ⁇ ) (GenBank Ace. No. M54886), IFN ⁇ (GenBank Ace. No. V00534), IFN ⁇ (GenBank Ace. No. J00219), IFNo (GenBank Ace. No. NM_002177), thrombospondin 1 (THBS1) (GenBank Ace. No. NM_003246), THBS2 (GenBank Ace. No.
  • the modified transfenin- cytokine fusion protein of the present invention displays cytokine biological activity.
  • Hormone is used herein to describe any one of a number of biologically active substances that are produced by certain cells or tissues and that cause specific biological changes or activities to occur in another cell or tissue located elsewhere in the body. Hormones preferably include GLP-1 of glucagon preproprotein (GenBank Ace. No. NM_002045), proinsulin (GenBank Ace. No. V00565), insulin (GenBank Ace. No. NM_000207), growth hormone 1 (GenBank Ace. No. V00520), growth hormone 2 (GenBank Ace. No. F006060), growth hormone release factor (GenBank Ace. No. NM_021081), insulin-like growth factor I (GenBank Ace. No. M27544), insulin-like growth factor II (GenBank Ace. No.
  • IGFBP-1 insulin-like growth factor binding protein 1
  • IGFBP-2 Insulation-like growth factor binding protein 2
  • IGFBP-3 Insulation-like growth factor binding protein 3
  • IGFBP-4 Insulation-like growth factor binding protein 4
  • IGFBP-5 GeneBank Ace. No. M65062
  • IGFBP-6 GeneBank Ace. No. NM_002178
  • IGFBP-7 GeneBank Ace. No. NM_001553
  • chorionic gonadofropin ⁇ chain GenBank Ace. No. NM_033142
  • chorionic gonadofropin ⁇ chain GenBank Ace. No. NM_033142
  • NM_000735 luteinizing hormone ⁇ (GenBank Ace. No. X00264), follicle-stimulating hormone ⁇ (GenBank Ace. No. NM_000510), thyroid-stimulating hormone ⁇ (GenBank Ace. No. NM_000549), prolactin (GenBank Ace. No. NM_000948), pro-opiomelanocortin (GenBank Ace. No. V01510), corticotropin (ACTH), ⁇ -lipotropin, ⁇ -melanocyte stimulating hormone ( ⁇ -MSH), ⁇ -lipotropin, ⁇ -MSH, ⁇ -endo ⁇ hin, and corticotropin-like intermediate lobe peptide (CLIP).
  • ACTH corticotropin
  • ⁇ -MSH ⁇ -melanocyte stimulating hormone
  • ⁇ -lipotropin ⁇ -MSH
  • ⁇ -endo ⁇ hin corticotropin-like intermediate lobe peptide
  • growth factor is used herein to describe any protein or peptide that binds to a receptor to stimulate cell proliferation.
  • Growth factors preferably include platelet- derived growth factor- ⁇ (PDGF- ⁇ ) (GenBank Ace. No. X03795), PDGF- ⁇ (GenBank Ace. No. X02811), steroid hormones, epidermal growth factor (EGF) (GenBank Ace. No. NM_001963), fibroblast growth factors such as fibroblast growth factor 1 (FGF1) (GenBank Ace. No. NM_000800), FGF2 (GenBank Ace. No. NM_002006), FGF3 (GenBank Ace. No. NM_005247), FGF4 (GenBank Ace. No. NM_002007), FGF5 (GenBank Ace. No. M37825), FGF6 (GenBank Ace. No. X57075), FGF7 (GenBank Ace.
  • PDGF- ⁇ platelet- derived growth factor- ⁇
  • PDGF- ⁇ GeneBank Ace. No
  • NM_004112 FGF12 (GenBank Ace. No. NM_021032), FGF13 (GenBank Ace. No. NM_004114), FGF14 (GenBank Ace. No. NM_004115), FGF16 (GenBank Ace. No.
  • FGF17 GenBank Ace. No. NM_003867
  • FGF18 GenBank Ace. No.
  • FGF 19 GenBank Ace. No. NM_005117
  • FGF20 GenBank Ace. No.
  • FGF21 GenBank Ace. No. NM_019113
  • FGF22 GenBank Ace. No.
  • NM_020637 and FGF23 (GenBank Ace. No. NM_020638), angiogenin (GenBank Ace. No. Ml 1567), brain-derived neurotrophic factor (GenBank Ace. No. M61176), ciliary neurotrophic growth factor (GenBank Ace. No. X60542), transforming growth factor- ⁇
  • TGF- ⁇ (GenBank Ace. No. X70340), TGF- ⁇ (GenBank Ace. No. X02812), nerve growth factor- ⁇ (NGF- ⁇ ) (GenBank Ace. No. NM_010915), NGF- ⁇ (GenBank Ace. No. X52599), tissue inhibitor of metalloproteinase 1 (TIMP1) (GenBank Ace. No. NM_003254), TIMP2 (GenBank Ace. No. NM_003255), TIMP3 (GenBank Ace. No. U02571), TIMP4 (GenBank
  • matrix protein is used herein to describe proteins or peptides that are normally found in the extracellular matrix. These proteins may be functionally important for strength, filtration, or adhesion.
  • Matrix proteins preferably include collagens such as collagen I (GenBank Ace. No. Z74615), collagen II (GenBank Ace. No. X16711), collagen
  • L34155 L34155
  • LAMA4 GenBank Ace. No. NM_002290
  • LAMB1 GenBank Ace. No.
  • LAMB3 GenBank Ace. No. L25541
  • LAMC1 GenBank Ace. No.
  • NM_002293 nidogen (GenBank Ace. No. NM_002508), ⁇ -tectorin (GenBank Ace. No. NM_005422), ⁇ -tectorin (GenBank Ace. No. NM_058222), and fibronectin (GenBank Ace.
  • blood proteins are traditionally defined as those sourced from plasma, many now commonly produced by recombinant means, and include, but are not limited to native serum proteins, derivatives, fragments and mutants or variants thereof, blood clotting factors, derivatives, mutants, variants and fragments (including factors VII, VIII, IX, X), protease inhibitors (antithrombin 3, alpha- 1 antitrypsin), urokinase-type plasminogen activator, immunoglobulins, von Willebrand factor and von Willebrand mutants, fibronectin, fibrinogen, thrombin and hemoglobin.
  • Enzyme is used herein to describe any protein or proteinaceous substance which catalyzes a specific reaction without itself being permanently altered or destroyed. Enzymes preferably include coagulation factors such as F2 (GenBank Ace. No. XM_170688), F7 (GenBank Ace. No. XM_027508), F8 (GenBank Ace. No. XM_013124), F9 (GenBank Ace. No. NM_000133), F 10 (GenBank Ace. No. AF503510) and others, matrix metalloproteinases such as matrix metalloproteinase I (GenBank Ace. No. MMP1) (GenBank Ace. No. NM_002421), MMP2 (GenBank Ace. No.
  • coagulation factors such as F2 (GenBank Ace. No. XM_170688), F7 (GenBank Ace. No. XM_027508), F8 (GenBank Ace. No. XM_013124), F9 (GenBank Ace. No. NM_000133
  • MMP3 GenBank Ace. No. NM_002422
  • MMP7 GeneBank Ace. No. NM_002423
  • MMP8 GeneBank Ace. No. NM_002424
  • MMP9 GeneBank Ace. No. NM_004994
  • MMP10 GeneBank Ace. No. NM_002425
  • MMP12 GeneBank Ace. No. NM_002426
  • MMP13 GeneBank Ace. No. X75308
  • MMP20 GenBank Ace. No. NM_004771
  • adenosine deaminase GenBank Ace. No. NM_000022
  • mitogen activated protein kinases such as MAPK3 (GenBank Ace. No.
  • MAP2K2 (GenBank Ace. No. NM_030662), MAP2K1 (GenBank Ace. No. NM_002755), MAP2K4 (GenBank Ace. No. NM_003010), MAP2K7 (AF013588), and MAPK12 (NM_002969), kinases such as JNKKl (GenBank Ace. No. U17743), JNKK2 (GenBank Ace. No. AF014401), JAK1 (M64174), JAK2 (NM_004972), and JAK3 (NM_000215), and phosphatases such as PPM1 A (GenBank Ace. No. NM_021003) and PPM1D (GenBank Ace. No. NM_003620).
  • Transcription factors is used herein to describe any protein or peptide involved in the transcription of protein-coding genes. Transcription factors may include Spl, Sp2 (GenBank Ace. No. NM_003110), Sp3 (GenBank Ace. No. AY070137), Sp4 (GenBank Ace. No. NM_003112) NFYB (GenBank Ace. No. NM_006166), Hap2 (GenBank Ace. No. M59079), GATA-1 (GenBank Ace. No. NM_002049), GATA-2 (GenBank Ace. No. NM_002050), GATA-3 (GenBank Ace. No. X55122), GATA-4 (GenBank Ace. No.
  • NM_005144 mothers against decapentaplegic proteins such as MADH1 (GenBank Ace. No. NM_005900), MADH2 (GenBank Ace. No. NM_005901), MADH3 (GenBank Ace. No. NM_005902), MADH4 (GenBank Ace. No. NM_005359), MADH5 (GenBank Ace. No. AF009678), MADH6 (GenBank Ace. No. NM_005585), MADH7 (GenBank Ace. No. NM_005904), MADH9 (GenBank Ace. No. NM_005905), and signal transducer and activator of transcription proteins such as STAT1 (GenBank Ace. No. XM_010893), STAT2 (GenBank Ace.
  • the therapeutic molecule is a non- human or non-mammalian protein.
  • HIV gpl20 HIV Tat, surface proteins of other virases such as hepatitis, he ⁇ es, influenza, adenoviras and RSV, other HIV components, parasitic surface proteins such as malarial antigens, and bacterial surface proteins are prefened.
  • non-human proteins may be used, for example, as antigens, or because they have useful activities.
  • the therapeutic molecule may be streptokinase, staphylokinase, asparaginase, or other proteins with useful enzymatic activities.
  • the therapeutic molecule is a ligand-binding protein with biological activity.
  • ligand-binding proteins may, for example, (1) block receptor- ligand interactions at the cell surface; or (2) neutralize the biological activity of a molecule in the fluid phase of the blood, thereby preventing it from reaching its cellular target.
  • the modified transfenin fusion proteins include a modified transfenin molecule fused to a ligand-binding domain of a receptor selected from the group consisting of, but not limited to, a low density lipoprotein (LDL) receptor, an acetylated LDL receptor, a tumor necrosis factor ⁇ receptor, a transforming growth factor ⁇ receptor, a cytokine receptor, an immunoglobulin Fc receptor, a hormone receptor, a glucose receptor, a glycolipid receptor, and a glycosaminoglycan receptor.
  • LDL low density lipoprotein
  • ligand- binding proteins include CD2 (M14362), CD3G (NM_000073), CD3D (NM_000732), CD3E (NM_000733), CD3Z (J04132), CD28 (NM_006139), CD4 (GenBank Ace. No. NM_000616), CD1A (GenBank Ace. No. M28825), CD1B (GenBank Ace. No. NM_001764), CD1C (GenBank Ace. No. NM_001765), CD1D (GenBank Ace. No. NM_001766), CD80 (GenBank Ace. No. NM_005191), GNB3 (GenBank Ace. No. AF501884), CTLA-4 (GenBank Ace. No.
  • NM_005214 intercellular adhesion molecules such as ICAM-1 (NM_000201), ICAM-2 (NM_000873), and ICAM-3 (NM_002162), tumor necrosis factor receptors such as TNFRSF1A (GenBank Ace. No. X55313), TNFR1SFB (GenBank Ace. No. NM_001066), TNFRSF9 (GenBank Ace. No. NM_001561), TNFRSF10B (GenBank Ace. No. NM_003842), TNFRSF1 IB (GenBank Ace. No. NM_002546), and TNFRSF13B (GenBank Ace. No. NM_006573), and interleukin receptors such as IL2RA (GenBank Ace.
  • the Tf-ligand-binding protein fusion of the present invention displays the biological activity of the ligand-binding protein.
  • cancer-associated proteins is used herein to describe proteins or polypeptides whose expression is associated with cancer or the maintenance of controlled cell growth, such as proteins encoded by rumor suppressor genes or oncogenes.
  • Cancer- associated proteins may include pl6 (GenBank Ace. No. AH005371), p53 (GenBank Ace. No. NM_000546), p63 (GenBank Ace. No. NM_003722), p73 (GenBank Ace. No. NM_005427), BRCAl (GenBank Ace. No. U14680), BRCA2 (GenBank Ace. No. NM_000059), CTBP interacting protein (GenBank Ace. No. U72066), DMBT1 (GenBank Ace. No.
  • NM_004406 HRAS (GenBank Ace. No. NM D05343), NCYM (GenBank Ace. No. NM_006316), FGR (GenBank Ace. No. NM_005248), myb (GenBank Ace. No. AF104863), rafl (GenBank Ace. No. NM_002880), erbB2 (GenBank Ace. No. NM_004448), VAV (GenBank Ace. No. X16316), c-fos (V GenBank Ace. No. 01512), c- fes (GenBank Ace. No. X52192), c-jun (GenBank Ace. No. NM_002228), MAS1 (GenBank Ace. No.
  • NM_005228 erbA (GenBank Ace. No. X04707), c-src tyrosine kinase (GenBank Ace. No. XM_044659), c-abl (GenBank Ace. No. M14752), N-ras (GenBank Ace. No. X02751), K- ras (GenBank Ace. No. M54968), jun-B (GenBank Ace. No. M29039), c-myc (GenBank Ace. No. AH001511), RBI (GenBank Ace. No. M28419), DCC (GenBank Ace. No. X76132), APC (GenBank Ace. No. NM_000038), NFl (GenBank Ace. No. M89914), NF2 (GenBank Ace. No. Y18000), and bcl-2 (GenBank Ace. No. M13994).
  • “Fusogenic inhibitor peptides” is used herein to describe peptides that show antiviral activity, anti-membrane fusion capability, and/or an ability to modulate intracellular processes, for instance, those involving coiled-coil peptide structures.
  • Antiviral activity includes, but is not limited to, the inhibition of HIV- 1, HIV-2, RSV, SIV, EBV, measles viras, influenza virus, or CMV transmission to uninfected cells. Additionally, the antifusogenic capability, antiviral activity or intracellular modulatory activity of the peptides merely requires the presence of the peptides and specifically does not require the stimulation of a host immune response directed against such peptides.
  • Antifusogenic refers to a peptide 's ability to inhibit or reduce the level of membrane fusion events between two or more moieties relative to the level of membrane fusion which occurs between said moieties in the absence of the peptide.
  • the moieties may be, for example, cell membranes or viral structures, such as viral envelopes or pili.
  • the term "antiviral peptide”, as used herein, refers to the peptide 's ability to inhibit viral infection of cells or some viral activity required for productive viral infection and/or viral pathogenesis, via, for example, cell-cell fusion or free virus infection. Such infection may involve membrane fusion, as occurs in the case of enveloped virases, or some other fusion event involving a viral structure and a cellular structure. Fusogenic inhibitor peptides and antiviral peptides often have amino acid sequences that are derived from greater than one viral protein (e.g., an HIV-1, HIV-2, RSV, and SIV-derived polypeptide).
  • fusogenic inhibitor peptides and antiviral peptides can be found in WO 94/2820, WO 96/19495, WO 96/40191, WO 01/64013 and US patents 6,333,395, 6,258,782, 6,228,983, 6,133,418, 6,093,794, 6,068,973, 6,060,065, 6,054,265, 6,020,459, 6,017,536, 6,013,263, 5,464,933, 5,346,989, 5,603,933, 5,656,480, 5,759,517, 6,245,737; 6,326,004, and 6,348,568; all of which are herein inco ⁇ orated by reference.
  • antifusogenic peptides are selected from the group consisting of HIV T-20 (FWNWLSAWKDLELLEQENKEQQNQSEEILSHILSTY, SEQ ID NO: 4), HIV T-1249, RSV T786 (VYPSDEYDASISQVNEEINQALAYIRKADELLENV, SEQ ID NO: 5), RSV T1584 (AVSKVLHLEGEVNKIKSALLSTNKAWSLSNGVSVLTSKVLDLKNYIDKQL, SEQ ID NO: 6) and RSV TI 12 (VFPSDEFDASISQVNEKINQSLAFIRESDELLHNV, SEQ ID NO: 7).
  • peptides examples include fragments of therapeutic proteins as described herein, in particular, fragments of human proteins that retain at least one activity of the parent molecule.
  • Peptides that may be used to produce modified Tf fusion proteins of the invention also include mimetic peptides and peptides that exhibit a biological activity of a therapeutic protein but differ in sequence or three-dimensional stracture from a full-length therapeutic protein.
  • peptides include erythropoeitin mimetic peptides disclosed by Johnson et al. (2000) Nephrol Dial. Transplant 15(9): 1274-7, Kuai et al. (2000) J. Pept. Res. 56(2):59-62, Barbone et al.
  • Therapeutic molecules also include allergenic proteins and digested fragments thereof. These include pollen allergens from ragweed, rye, June grass, orchard grass, sweet vernal grass, red top grass, timothy grass, yellow dock, wheat, corn, sagebrash, blue grass, California annual grass, pigweed, Bermuda grass, Russian thistle, mountain cedar, oak, box elder, sycamore, maple, elm, etc., dust mites, bee venom, food allergens, animal dander, and other insect venoms.
  • pollen allergens from ragweed, rye, June grass, orchard grass, sweet vernal grass, red top grass, timothy grass, yellow dock, wheat, corn, sagebrash, blue grass, California annual grass, pigweed, Bermuda grass, Russian thistle, mountain cedar, oak, box elder, sycamore, maple, elm, etc., dust mites, bee venom, food allergen
  • microbial vaccines which include viral, bacterial and protozoal vaccines and their various components such as surface antigens.
  • vaccines which contain glycoproteins, proteins or peptides derived from these proteins.
  • Such vaccines are prepared from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Salmonella spp., Shigella spp., Escherichia coli, Klebsiella spp., Proteus spp., Vibrio cholerae, Campylobacter pylori, Pseudomonas aeruginosa, Haemophilus influenzae, Bordetella pertussis, Mycobacterium tuberculosis, Legionella pneumophila, Treponema pallidum, chlamydia, tetanus toxoid, diphtheria toxoid, influenza viruses, adenoviruses, paramyxovirases (m
  • Prefened fusion molecules may contain anti-HIV viral peptides, anti-RSV peptides, human growth hormone, ⁇ and/or ⁇ interferons, erythropoietin (EPO), EPO like peptides, granulocyte-colony stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GMCSF), insulin, insulin-like growth factor (IGF), thrombopoeitin, peptides conesponding to the CDR of an antibody, Islet ⁇ eogenesis Associated Protein (I ⁇ GJAP), calcitonin, angiostatin, endostatin, interleukin-2, growth hormone releasing factor, human parathyroid hormone, anti-tumor necrosis factor (T ⁇ F) peptides, interleukin-1 (IL-1) receptor and/or single chain antibodies.
  • EPO erythropoietin
  • EPO EPO like peptides
  • GCSF gran
  • Fusion proteins of the invention may also be prepared to include peptides or polypeptides derived from peptide libraries to screen for molecules with new or novel functions.
  • peptide libraries may include those commercially or publicly available, e.g., American Peptide Co. Inc., Cell Sciences Inc., Invitrogen Co ⁇ oration, Phoenix Pharmaceuticals Inc., United States Biological, as well as those produced by available technologies, e.g., bacteriophage and bacterial display libraries made using standard procedures.
  • Tf fusion proteins may be prepared by using therapeutic protein moieties as known in the art and exemplified by the peptides and proteins cunently approved by the Food and Drug Administration at
  • Table 1 (adapted from PCT International Publication No. WO 01/79444) provides a non-exhaustive list of therapeutic proteins that conespond to a therapeutic protein portion of a modified transfenin fusion protein of the invention.
  • the "Therapeutic Protein X" column discloses therapeutic protein molecules followed by parentheses containing scientific and brand names that comprise or alternatively consist of that therapeutic protein molecule or a fragment or variant thereof.
  • “Therapeutic protein X" as used herein may refer either to an individual therapeutic protein molecule (as defined by the amino acid sequence obtainable from the CAS and Genbank accession numbers), or to the entire group of therapeutic proteins associated with a given therapeutic protein molecule disclosed in this column.
  • the 'Exemplary Identifier' column provides Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or Genbank Accession Numbers (e.g. , Locus ID, NP - XXXXX (Reference Sequence Protein), and XP-XXXXX (Model Protein) identifiers available through the National Center for Biotechnology Information (NCBI) webpage (www.ncbi.nlm.nih.gov) that conespond to entries in the CAS Registry or Genbank database which contain an amino acid sequence of the protein molecule or of a fragment or variant of the therapeutic protein molecule.
  • GenSeq Accession numbers and/or journal publication citations are given to identify the exemplary amino acid sequence for some polypeptides.
  • the summary pages associated with each of these CAS and Genbank and GenSeq Accession Numbers as well as the cited journal publications are available (e.g., PubMed ID number (PMID)) and are herein inco ⁇ orated by reference in their entirety.
  • the PCT/Patent Reference column provides U. S. Patent numbers, or PCT International Publication Numbers conesponding to patents and/or published patent- applications that describe the therapeutic protein molecule all of which are herein inco ⁇ orated by reference in their entirety.
  • the Biological Activity column describes biological activities associated with the therapeutic protein molecule.
  • the Exemplary Activity Assay column provides references that describe assays which may be used to test the therapeutic and/or biological activity of a therapeutic protein or a transfenin fusion protein of the invention comprising a therapeutic protein X portion. These references are also herein inco ⁇ orated by reference in their entirety. "The Prefened Indication Y" column describes disease, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by therapeutic protein X or a transfenin fusion protein of the invention comprising a therapeutic protein X portion.
  • cytokines including ⁇ -IFN
  • ⁇ -IFN have relatively short circulation half-lives since they are produced in vivo to act locally and transiently.
  • the present invention provides ⁇ -IFN/transferrin fusion proteins with increased half- lives and pharmaceutical compositions comprising such fusion proteins with increased stability. Such fusion proteins can be administered to patients at lower doses, thus reducing the toxic side effects associated with ⁇ -IFN.
  • the present invention contemplates the use of the ⁇ -IFN/transferrin fusion proteins to treat various diseases and conditions associated with ⁇ -IFN, such as but not limited to multiple sclerosis, cancer including brain tumors and skin cancer, and viral infections such as hepatitis B and C.
  • the ⁇ -IFN/transferrin fusion proteins are used to treat subjects suffering from multiple sclerosis.
  • formulations for oral administration may also be produced.
  • ⁇ -interferon is a glycoprotein with an apparent molecular weight (MW) of 23 kilodaltons.
  • the gene encoding ⁇ -IFN is located on chromosome 9. Its amino acid sequence containing 166 residues was determined by K. Hosoi et al. (J. Interferon Res., 8, pp 375-384 (1988)), and its glucoside sequence was reported by Y. Kagawa et al. (J. Biol. Chem., 263, pp 17508-17515 (1988)).
  • ⁇ -IFN is secreted by fibroblasts in response to a viral or bacterial infection, or exposure to foreign cells, macromolecules, or RNA.
  • ⁇ -IFN inhibits the proliferation of infected cells and stimulates the immune system.
  • the specific antiviral activity of homogeneous Hu- ⁇ -IFN is considered to be between 3 x 10 8 and 1 x 10 9 iu/mg (international units per milligram of total protein) inclusive (see U.S. Pat. No. 4,289,689 and EP-A-94 672).
  • "Interferon-beta" (IFN- ⁇ ) or "beta-interferon” ( ⁇ -IFN) includes native and recombinant Type I interferons exhibiting the same or similar pharmaceutical characteristics as the Type I interferons commonly known as IFN- ⁇ -la and IFN- ⁇ -lb.
  • any ⁇ -IFN sequence may be used to prepare Tf fusion proteins of the present invention.
  • U.S. Patent 4,738,931 discloses the human ⁇ -IFN gene derived from human chromosomal DNA.
  • a 1.8 kb EcoRI fragment, containing the nucleic acid encoding the human ⁇ -IFN, introduced into Escherichia coli has been deposited with the American Type Culture Collection in U.S.A. as Escherichia coli CI4 under accession number ATCC 31905.
  • the GenBank accession number for the amino acid sequence of Human ⁇ -IFN amino acid sequence is AAA72588.
  • the ⁇ -IFN could also be a mutein as described in U.S. Pat. No.
  • ⁇ -IFN is regarded as an active principle not only in the treatment and prophylaxis of viral diseases such as herpes, influenza etc, but also in the treatment of tumoral conditions such as encephaloma and leukemia.
  • ⁇ -IFN is used to treat multiple sclerosis, brain tumor, skin cancer and hepatitis B and C.
  • ⁇ -IFN fusion proteins of the present invention may be used to treat any of these diseases.
  • Human ⁇ -IFN is also effective in treating coronary restenosis in humans by selectively inhibiting the proliferation of coronary smooth muscle cell at the site of vascular injury following a surgical procedure while having no inhibitory effect on the normal proliferation of coronary endothelial cells following the procedure.
  • U.S. Patent 5,681,558 discloses a method of treating restenosis comprising administering ⁇ -IFN to the patient. Accordingly, ⁇ -IFN fusion proteins of the present invention may be used to treat restenosis.
  • ⁇ -IFN has an erythropoietic effect on the growth of progenitor cells from individuals suffering from several diseases with a very low production of red blood cells.
  • ⁇ -IFN increases burst formation as well as promotes a more rapid maturation toward normoblasts and even late reticulocytes.
  • U.S. Patent 5,104,653 discloses a method for the stimulation of erythropoiesis in a patient suffering from a disorder characterized by lack of maturation of progenitor blood cells to red blood cells comprising administering to said patient an erythropoietic effective amount of human ⁇ -IFN. Therefore, ⁇ -IFN fusion proteins of the present invention may be used to stimulate erythropoiesis.
  • ⁇ -IFN acting via STAT1 and STAT2 is known to upregulate and downregulate a wide variety of genes, most of which are involved in the antiviral immune response.
  • ⁇ -IFN Interferon ⁇ -IFN exerts its biological effects by binding to specific receptors on the surface of human cells. This binding initiates a complex cascade of intracellular events that leads to the expression of numerous interferon-induced gene products and markers, for example, 2', 5'-oligoadenylate synthetase, b 2 -microglobulin, and neopterin.
  • (2'-5')-Oligoadenylate synthetase and dsRNA dependent protein kinase are the two best-known IFN- ⁇ -induced proteins (Biron, 1998, supra).
  • (2'-5')-oligoadenylate synthetase polymerizes ATP in a unique 2'-5' fashion (Janeway et al, Immunobiology: The Immune System in Health and Disease, 4th Edition, New York, Elsevier Science/Garland Publishing pp 385-386(1999)); the resultant oligomers activate RNase L, which cleaves mRNA (Biron, 1998, supra).
  • dsRNA dependent protein kinase phosphorylates and inactivates elF2, a transcriptional initiator.
  • Both (2'-5')-oligoadenylate synthetase and dsRNA dependent protein kinase act only in the presence of dsRNA, i.e. in virally infected cells. The net result of the action of these two proteins is to inhibit protein translation, which will retard viral replication (Biron, 1998, supra).
  • ⁇ -IFN dependent upregulation of TAP transporter associated with antigen processing
  • Lmp2 Lmp7 serves to increase presentation of viral peptides by MHC class I molecules in order to facilitate CD8 T cell recognition and destruction of infected cells.
  • TAP is the molecule responsible for loading peptide fragments onto MHC class I molecules in the ER; the Lmp proteins are components of the proteasome which cleave proteins specifically for MHC class I presentation (Janeway et al, 1999, supra).
  • ⁇ -IFN is known to both activate and induce some proliferation in natural killer (NK) cells (Janeway et al, 1999, supra).
  • NK natural killer
  • interferons themselves are not mitogens.
  • the proliferation of NK cells is probably caused by an intermediary cytokine which is induced by IFN- ⁇ (Biron, 1998, supra).
  • NK cells can kill cells which exhibit atypical patterns of MHC class I expression; such cells are generally virally infected (Janeway et al, 1999, supra).
  • T cells die by apoptosis as the immune system returns to a homeostatic balance, some T cells must avoid apoptosis and enter a G 0 /G ⁇ memory state to preserve immunological memory. These memory T cells are rescued from apoptosis by interacting with stromal cells, which secrete ⁇ -IFN and some IFN- ⁇ (Pilling et al, European Journal of Immunology 29:1041-1050 (1999)). T cell apoptosis may be induced by either cytokine deprivation or ligation of Fas on the cell surface, but ⁇ -IFN is able to block both apoptotic pathways.
  • ⁇ -IFN increased transcription of well over 100 proteins in human fibrosarcoma cells. Induced proteins ranged in function from cytochromes and cell scaffolding proteins to immunologically active proteins such as Complement components and dsRNA adenosine deaminase. These results indicate that ⁇ -IFN has truly pleiotropic effects, many of which are not fully understood. Much clinical research on ⁇ -IFN is currently focused on its use as a treatment for multiple sclerosis (MS).
  • MS multiple sclerosis
  • MS is an autoimmune disease in which T cells mount an immune response against self myelin antigens in the glial cells of the central nervous system (Goodkin, 1999. Multiple sclerosis: Treatment options for patients with relapsing-remitting and secondary progressive multiple sclerosis. ⁇ http://www.msnews.org/goodkinl 99,htm>).
  • the FDA approved subcutaneous injections of IFN- ⁇ lb for treatment of MS Revelle M., 1993, FDA licenses interferon beta- lb. r ⁇ http://www.fda.gov/bbs/topics/NEWS/NEW00424.html> ⁇
  • ⁇ -IFN lb is a non- glycosylated form of IFN- ⁇ produced in E.
  • ⁇ -IFN lb Adverse experiences associated with ⁇ -IFN lb therapy include: injection site reactions (inflammation, pain, hypersensitivity and necrosis), and a flu-like symptom complex (fever, chills, anxiety and confusion). These adverse side effects may be, in fact, reduced or alleviated by fusing ⁇ -IFN lb to transferrin as described above.
  • ⁇ -IFN la an eukaryotic, glycosylated form
  • Interferon beta-la is a 166 amino acid glycoprotein with a predicted molecular weight of approximately 22,500 daltons. It is produced by mammalian cells (Chinese Hamster Ovary cells) into which the human IFN- ⁇ gene has been introduced.
  • the amino acid sequence of ⁇ -IFN la is identical to that of natural human ⁇ -IFN and may be used to make Tf fusion proteins of the present invention.
  • ⁇ -IFN/transferrin fusion proteins treatment may also ameliorate autoimmune attacks by restoring suppressor T cell function; cotreatment with all-tr ⁇ Hs-retinoic acid seems to increase this restorative action for unknown reasons (Qu et al, 1998. All-trans retinoic acid potentiates the ability of interferon beta- lb. ⁇ http .'//members .tripod. com/ ⁇ Th Juland/ra- betalb.html>).
  • ⁇ -IFN may also inhibit the induction of inducible nitric oxide synthase (INOS) expression by IL-1 and IFN- ⁇ .
  • IOS inducible nitric oxide synthase
  • Beta inteferon prevents nitric oxide/peroxynitrate from damaging the central nervous system. ( ⁇ htt ⁇ .-//members .tripod. comATh Juland/nitric-oxide beta.html ⁇ .
  • the present invention includes the use of ⁇ -IFN analogs that are therapeutically effective for treating various diseases associated with ⁇ -IFN for generating ⁇ -IFN/transferrin fusion proteins.
  • the present invention includes the use of the ⁇ -IFN/transferrin fusion protein in the methods described above to inhibit or stimulate various cellular processes and for the treatment and prevention of the various disease and conditions described above.
  • the ⁇ -IFN/transferrin ifusion protein may be used to treat multiple sclerosis, herpes, influenza, brain tumor, and skin cancer.
  • the ⁇ -IFN/transferrin fusion protein of the present invention can be formulated into pharmaceutical compositions by well known methods. See, e.g., Remington's Pharmaceutical Sciences by E. W. Martin, hereby incorporated by reference, describes suitable formulations.
  • the pharmaceutical composition of the ⁇ -IFN/transferrin fusion protein of the present invention may be formulated in a variety of forms, including liquid, gel, lyophilized, or any other suitable form. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art
  • the ⁇ -IFN/transferrin fusion protein can be administered in pure form or in an appropriate pharmaceutical composition. Administration can be carried out via any of the accepted modes. Thus, administration can be, for example, orally, nasally, parenterally, topically, transdermally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • the compositions will include a conventional pharmaceutical carrier or excipient and the ⁇ -IFN/transferrin fusion protein as the active agent, and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.
  • the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the ⁇ -IFN /transferrin fusion protein, and 99% to 1% by weight of a suitable pharmaceutical excipient.
  • the composition could be about 5% to 75% by weight of the ⁇ -IFN/transferrin fusion protein with the rest being suitable pharmaceutical excipients.
  • the route of administration could be parenterally, using a convenient daily dosage regimen which can be adjusted according to the degree of severity of the disease, preferably multiple sclerosis, to be treated.
  • a pharmaceutically acceptable composition containing the ⁇ -IFN/transferrin fusion protein may be formed by the methods disclosed in U.S. Pat. Nos. 4,462,940, 4,588,585 and 4,992,271.
  • the ⁇ -IFN/transferrin fusion protein pharmaceutical compositions may be administered orally, intravenously, intramuscularly, intraperitoneally, intradermally or subcutaneously or in any other acceptable manner.
  • the preferred mode of administration will depend upon the particular indication being treated and will be apparent to one of sl ll in the art.
  • U.S. Patent 6,333,032 describes effective methods of using ⁇ -IFN to treat diseases in warm-blooded vertebrates, such as multiple sclerosis.
  • Treatment of multiple sclerosis comprises administering ⁇ -IFN at a dosage of 0.01 to about 5 IU/lb per day in a dosage form adapted to promote contact of said dosage of interferon with the oral and pharyngeal mucosa of said animal.
  • the dosage of interferon could be from 0.1 to about 4.0 IU/lb per day, or from 0.5 to about 1.5 IU/lb of body weight per day.
  • the present invention contemplates administering the ⁇ -IFN in a dosage form adapted to assure maximum contact of the interferon in said dosage form with the oral and pharyngeal mucosa of the human or animal undergoing treatment.
  • Contact of interferon with the mucosa can be enhanced by maximizing residence time of the treatment solution in the oral or pharyngeal cavity.
  • best results seem to be achieved in human patients when the patient is requested to hold said solution of interferon in the mouth for a period of time.
  • Contact of interferon with the oral and pharyngeal mucosa and thereafter with the lymphatic system of the treated human or animal is unquestionably the most efficient method administering immunotherapeutic amounts of interferon.
  • the present invention contemplates the use of the ⁇ -IFN/transferrin protein for the manufacture of a medicament which is useful for the treatment of diseases associated with ⁇ -IFN.
  • the diseases contemplated by the present invention include but are not limited to those described above.
  • Glucagon-Like Peptide-1 (GLP-1) is a gastrointestinal hormone that regulates insulin secretion belonging to the so-called enteroinsular axis.
  • the enteroinsular axis designates a group of hormones, released from the gastrointestinal mucosa in response to the presence and absorption of nutrients in the gut, which promote an early and potentiated release of insulin.
  • the incretin effect which is the enhancing effect on insulin secretion is probably essential for a normal glucose tolerance.
  • GLP-1 is a physiologically important insulinotropic hormone because it is responsible for the incretin effect.
  • GLP-1 is a product of proglucagon (Bell, et al, Nature, 1983, 304: 368-371). It is synthesized in intestinal endocrine cells in two principal major molecular forms, as GLP- l(7-36)amide and GLP-l(7-37). The peptide was first identified following the cloning of cDNAs and genes for proglucagon in the early 1980s. Initial studies done on the full length peptides GLP-l(l-37) and GLP-l(l-36 amide ) concluded that the larger GLP-1 molecules are devoid of biological activity. In 1987, three independent research groups demonstrated that removal of the first six amino acids resulted in a GLP-1 molecule with enhanced biological activity.
  • GLP-1 The amino acid sequence of GLP-1 is disclosed by Schmidt et al. (1985 Diabetologia 28 704-707).
  • Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized in the L-cells in the distal ileum, in the pancreas, and in the brain. Processing of preproglucagon to GLP-1 (7-36 amide ), GLP-1 (7-37) and GLP-2 occurs mainly in the L-cells.
  • the amino acid sequence of GLP-l(7-36 am ⁇ de ) and GLP-l(7-37) is (SEQ ID NO: 6): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys-Glu-Phe-Ile-Ala-Trp-Leu-Nal-Lys-Gly-Arg-X wherein X is NH 2 for GLP-l(7-36 amide ) and X is Gly for GLP-l(7-37).
  • GLP-1 like molecules possesses anti-diabetic activity in human subjects suffering from Type II (non-insulin-dependent diabetes mellitus (NIDDM)) and, in some cases, even Type I diabetes.
  • Treatment with GLP-1 elicits activity, such as increased insulin secretion and biosynthesis, reduced glucagon secretion, delayed gastric emptying, only at elevated glucose levels, and thus provides a potentially much safer therapy than insulin or sulfonylureas.
  • Post-prandial and glucose levels in patients can be moved toward normal levels with proper GLP-1 therapy.
  • GLP-1 -like molecules possess the ability to preserve and even restore pancreatic beta cell function in Type-II patients.
  • GLP-1 sequence may be used to make Tf fusion proteins of the present invention, including GLP-l(7-35), GLP-l(7-36), and GLP-l(7-37).
  • GLP-1 also has powerful actions on the gastrointestinal tract. Infused in physiological amounts, GLP-1 potently inhibits pentagastrin-induced as well as meal-induced gastric acid secretion (Schjoldager et al, Dig. Dis. Sci. 1989, 35:703-708; Wettergren et al, Dig Dis Sci 1993; 38:665-673). It also inhibits gastric emptying rate and pancreatic enzyme secretion (Wettergren et al, Dig Dis Sci 1993; 38:665-673).
  • GLP-1 seems to have an effect on food intake. Intraventricular administration of GLP-1 profoundly inhibits food intake in rats (Schick et al. in Ditschuneit et al. (eds.), Obesity in Europe, John Libbey & Company ltd, 1994; pp. 363-367; Turton et al, Nature 1996, 379: 69-72). This effect seems to be highly specific.
  • N-terminally extended GLP-1 (PG 72-107) amide is inactive and appropriate doses of the GLP-1 antagonist, exendin 9-39, abolish the effects of GLP-1 (Tang-Christensen et al, Am. J. Physiol., 1996, 271(4 Pt 2):R848-56).
  • GLP-1 secreted from the intestinal L-cells may also act as a satiety signal.
  • GLP's insulinotropic effects and the effects of GLP-1 on the gastrointestinal tract are preserved (Willms et al, Diabetologia 1994; 37, suppl.1: Al 18), which may help curtail meal-induced glucose excursions, but, more importantly, may also influence food intake.
  • Administered intravenously, continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to dramatically improve glycaemic control in NIDDM patients without significant side effects (Larsen et al, Diabetes 1996; 45, suppl. 2: 233 A.).
  • GLP-1 /transferrin fusion proteins comprising at least one analog of GLP-1 and fragments thereof are useful in the treatment of Type 1 and Type 2 diabetes and obesity and may be formulated for oral administration as described below.
  • GLP-1 molecule means GLP-1, a GLP-1 analog, or GLP- 1 derivative.
  • GLP-1 analog is defined as a molecule having one or more amino acid substitutions, deletions, inversions, or additions compared with GLP-1.
  • Many GLP-1 analogs are known in the art and include, for example, GLP-1 (7-34), GLP- 1(7-35), GLP-l(7-36), Val 8 -GLP-l(7-37), Gly 8 -GLP-l(7-37), Ser 8 -GLP-l(7-37), Gln 9 - GLPl(7-37), D-Gln 9 -GLP-l(7-37), Thr ,6 -Lys 18 -GLP- 1(7-37), and Lys 18 -GLP-l(7-37).
  • analogs include dipeptidyl-peptidase resistant versions of GLP-1, wherein the N- terminal end of the peptide is protected.
  • Such analogs include, but are not limited to GLP-1 with additional amino acids, such as histidine residue added to the N-terminal end or substituted into the N-terminal amino acids (amino acid 7 or 8 in GLP-1 (7-36) or GLP-1 (7- 37).
  • the N-terminal end may comprise the residues His-His-Ala, Gly-His- Ala, His-Gly-Glu, His-Ser-Glu, His-Ala-Glu, His-Gly-Glu, His-Ser-Glu, His-His-Ala-Glu, His-His-Gly-Glu, His-His-Ser-Glu, Gly-His-Ala-Glu, Gly-His-Gly-Glu, Gly-His-Ser-Glu, His-X-Ala-Glu, His-X-Gly-Glu, His-X-Ser-Glu, wherein X is any amino acid.
  • Patent 5,118,666 discloses examples of GLP-1 analogs such as GLP-l(7-34) and GLP-l(7-35).
  • GLP-1 derivative is defined as a molecule having the amino acid sequence of GLP-1 or a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, ⁇ -carbon atoms, terminal amino group, or terminal carboxylic acid group.
  • a chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.
  • GLP-1 related compound refers to any compound falling within the GLP-1, GLP-1 analog, or GLP-1 derivative definition.
  • WO 91/11457 discloses analogs of the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 which can also be useful as GLP-1 moieties.
  • EP 0708179- A2 (Eli Lilly & Co.) discloses GLP-1 analogs and derivatives that include an N-terminal imidazole group and optionally an unbranched C 6 -Cio acyl group in attached to the lysine residue in position 34.
  • EP 0699686- A2 (Eli Lilly & Co.) discloses certain N-terminal truncated fragments of GLP-1 that are reported to be biologically active.
  • U.S. Patent 5,545,618 discloses GLP-1 molecules consisting essentially of GLP-1(7- 34), GLPl(7-35), GLP-l(7-36), or GLP-l(7-37), or the amide forms thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of: (a) substitution of glycine,serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or a D-
  • U.S. Pat. No. 5,118,666 discloses a GLP-1 molecule having insulinotropic activity.
  • Such molecule is selected from the group consisting of a peptide having the amino acid sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Nal-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Nal-Lys (SEQ ID NO: 7) or His-Ala-Glu-Gly-Thr-Phe- Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-T -Leu- Val-Lys-Gly (SEQ ID NO: 8); and a derivative of said peptide and wherein said peptide is selected from
  • U.S. Patent 6,277,819 teaches a method of reducing mortality and morbidity after myocardial infarction comprising administering GLP-1, GLP-1 analogs, and GLP-1 derivatives to the patient.
  • the GLP-1 analog being represented by the following structural formula (SEQ ID NO: 9): R ⁇ -X ⁇ -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-X 2 - Gly-Gln-Ala-Ala-Lys- X 3 -Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R 2 and pharmaceutically- acceptable salts thereof, wherein: Ri is selected from the group consisting of L-histidine, D- histidine, desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine, homohistidine, alpha- fluoromethyl-histidine
  • Patent 6,429,197 teaches that GLP-1 treatment after acute stroke or hemorrhage, preferably intravenous administration, can be an ideal treatment because it provides a means for optimizing insulin secretion, increasing brain anabolism, enhancing insulin effectiveness by suppressing glucagon, and maintaining euglycemia or mild hypoglycemia with no risk of severe hypoglycemia or other adverse side effects.
  • the present invention provides a method of treating the ischemic or reperfused brain with GLP- 1 or its biologically active analogues after acute stroke or hemorrhage to optimize insulin secretion, to enhance insulin effectiveness by suppressing glucagon antagonism, and to maintain euglycemia or mild hypoglycemia with no risk of severe hypoglycemia.
  • U.S. Patent 6,277,819 provides a method of reducing mortality and morbidity after myocardial infarction, comprising administering to a patient in need thereof, a compound selected from the group consisting of GLP-1, GLP-1 analogs, GLP-1 derivatives and pharmaceutically-acceptable salts thereof, at a dose effective to normalize blood glucose.
  • U.S. Patent 6,191,102 discloses a method of reducing body weight in a subject in need of body weight reduction by administering to the subject a composition comprising a glucagon-like peptide-1 (GLP-1), a glucagon-like peptide analog (GLP-1 analog), a glucagon-like peptide derivative (GLP-1 derivative) or a pharmaceutically acceptable salt thereof in a dose sufficient to cause reduction in body weight for a period of time effective to produce weight loss, said time being at least 4 weeks.
  • GLP-1 glucagon-like peptide-1
  • GLP-1 analog glucagon-like peptide analog
  • GLP-1 derivative a glucagon-like peptide derivative
  • GLP-1 is fully active after subcutaneous administration (Ritzel et al, Diabetologia 1995; 38: 720-725), but is rapidly degraded mainly due to degradation by dipeptidyl peptidase IV-like enzymes (Deacon et al, J Clin Endocrinol Metab 1995, 80: 952-957; Deacon et ⁇ /.,1995, Diabetes 44: 1126-1131).
  • GLP-1 and many of its analogues have a short plasma half-life in humans (Orskov et al, Diabetes 1993; 42:658- 661).
  • transferrin fusion proteins comprising GLP-1 or analogues thereof which have a protracted profile of action relative to GLP-l(7-37). It is a further object of the invention to provide derivatives of GLP-1 and analogues thereof which have a lower clearance than GLP- 1(7-37). Moreover, it is an object of the invention to provide pharmaceutical compositions comprising GLP- 1 /transferrin fusion proteins or GLP-1 analog/transferrin fusion proteins with improved stability. Additionally, the present invention includes the use of GLP-1 /transferrin fusion proteins or GLP-1 analog/transferrin fusion proteins to treat diseases associated with GLP-1 such as but not limited to those described above.
  • the pharmaceutical compositions comprising the GLP-1 peptide/transferrin fusion proteins and GLP-1 analog/transferrin fusion proteins may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences, 1985.
  • the composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution.
  • the compositions may be sterilized by conventional sterilization techniques which are well known in the art.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the GLP-1 /transferrin fusion proteins and GLP-1 analog/transferrin fusion proteins of the present invention may also be adapted for oral, nasal, transdermal, pulmonal or rectal administration.
  • the pharmaceutically acceptable carrier or diluent employed in the composition may be any conventional solid carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • composition of the invention in the form of a sustained release formulation.
  • the composition may be formulated as microcapsules or microparticles containing the GLP-1/transferrin fusion protein or GLP-1 analog/transferrin fusion protein encapsulated by or dispersed in a suitable pharmaceutically acceptable biodegradable polymer such as polylactic acid, polyglycolic acid or a lactic acid/glycolic acid copolymer.
  • the preparation may contain GLP-1 /transferrin fusion proteins or GLP-1 analog/transferrin fusion proteins dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application.
  • the carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
  • solubilizing agents e.g. propylene glycol
  • surfactants e.g. propylene glycol
  • absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin
  • preservatives such as parabenes.
  • the compounds of the present invention are dispensed in unit dosage form comprising 0.5-500 mg of the fusion protein together with a pharmaceutically acceptable carrier per unit dosage.
  • the present invention contemplates the use of the GLP-1 /transferrin and GLP-1 analog/transferrin fusion proteins for the manufacture of a medicinal product which can be used in the treatment of diseases associated with elevated glucose level, such as but not to limited to those described above.
  • the present invention contemplates the use of GLP-1 /transferrin fusion protein for the treatment of diabetes including type II diabetes, obesity, severe burns, and heart failure, including congestive heart failure and acute coronary syndrome.
  • the N-terminus of GLP-1 is normally amidated. In yeast, amidation does not occur.
  • an extra amino acid is added on the N-terminus of GLP-1.
  • the addition of an amino acid to the N-terminus of GLP-1 may prevent dipeptidyl peptidase from cleaving at the second amino acid of GLP-1 due to steric hindrance. Therefore, GLP-1 will remain functionally active. Any one of the 20 amino acids may be added to the N- terminus of GLP-1. In some instances, an uncharged or positively charged amino acid may be used and preferably, a smaller amino acid such as Glycine is added. The GLP-1 with the extra amino acid is then fused to transferrin.
  • the GLP-1 with the added amino acid will be fused at the N-terminus of the GLP-1 /transferrin fusion protein.
  • a His residue is added at the N-terminus of GLP-1 or is inserted after the His residue at the N-terminus of GLP-1, so that the N-terminus of GLP-1 begins with His-His.
  • the second residue from the N-terminus in the GLP-l(7-36) or GLP- 1(7-37) peptide is substituted with another amino acid.
  • the Ala residue at the second residue from the N-terminus in the GLP- 1(7-36) or GLP- 1(7-37) peptide may be substituted with Ser, Gly, Nai, or another amino acid.
  • GLP-1 activates and regulates important endocrine hormone systems in the body and plays a critical management role in the metabolism of glucose. Unlike all other diabetic treatments on the market GLP-1 has the potential to be restorative by acting as a growth factor for B-cells thus improving the ability of the pancreas to secrete insulin and also, to make the existing insulin levels act more efficiently by improving sensitivity and better stabilizing glucose levels. This reduces the burden on daily monitoring of glucose levels and potentially offers a delay in the serious long term side effects caused by fluctuations in blood glucose due to diabetes. Furthermore, GLP-1 can reduce appetite and reduce weight. Obesity is an inherent consequence of poor control of glucose metabolism and this only serves to aggravate the diabetic condition.
  • GLP-1 fusion proteins retain the activity of GLP-1 but have the long half-life (14-17 days), solubility, and biodistribution properties of transferrin (mTf) and they can be administered orally. These properties could provide for a low cost, small volume, monthly s.c. (subcutaneous) injection and this type of product is absolutely needed for long term chronic use.
  • Insulin Human insulin contains two peptide chains, known as the A and B chains, which are
  • proinsulin a single chain peptide composed of the B and A chains linked to a connecting peptide of approximately 31 amino acids, known as the C- peptide, by adjacent pairs of basic residues.
  • the arrangement of these three peptides in the proinsulin molecule, beginning with the amino-terminal end, is as follows: B chain-Arg- Arg-C-peptide-Lys-Arg-A chain.
  • Preproinsulin is synthesized in pancreatic beta cells located within the islets of Langerhans, which are dispersed throughout the pancreas. Removal of the signal peptide occurs in the rough endoplasmic reticulum, and the resulting proinsulin is then transported to the Golgi apparatus for packaging into secretion granules. The folded proinsulin is stabilized by disulfide bonds. During processing of the secretion granules, the folded proinsulin molecule is cleaved by specific proteases at the paired basic residues to liberate insulin and the C-peptide. Diabetes mellitus is a disease that affects approximately 17 million people in the
  • Type 2 diabetes formerly called adult-onset diabetes, which begins as insulin resistance (failure of the body's cells to use insulin properly) as progresses to an inability of the pancreas to produce insulin.
  • Type 1 diabetes formerly called juvenile diabetes or insulin-dependent diabetes, accounts for the remaining 5-10% of diabetes cases.
  • insulin contains only 51 amino acid residues, it is readily made by recombinant techniques, and a large number of insulin analogues and variants have been prepared. Any of these analogues or variants can be used to make mTf fusion proteins of the invention.
  • Diabetics typically require insulin replacement therapy, which involves one or more doses of the drug per day by subcutaneous injection. Treatment by injection, however, is both psychologically and physically painful, as well as demanding of technical expertise, and many diabetics require assistance in administering injections. Oral formulations of insulin have not been successful, however, because the peptide is rapidly degraded in the acidic environment of the GI tract, particularly in the stomach. Nevertheless, alternatives to injection, such as oral, nasal and topical formulations have been attempted. U.S.
  • Patent 5,824,638, Burnside et al describes oral emulsion preparations in which insulin is dissolved in a hydrophilic phase, such as water, saline or a water-miscible alcohol, and dispersed with a surfactant in a hydrophobic phase, such as a long chain fatty acid or fatty acid ester. Although an emulsion keeps insulin dispersed, it cannot protect the peptide from the harsh conditions of the stomach.
  • Nasal preparations, which deliver insulin in an aerosol to the lungs are disclosed in U.S. Patent 6,427,681, Gonda et al, while topical preparations are disclosed in U.S. Patent 6,399,566, Dardai et al.
  • Modified insulins for injection containing amino acid substitutions or glycosylated residues, to enhance activity, inhibit degradation or inhibit peptide aggregation have also been developed (see U.S. Patent 4,478,746, Kim et al, glycosylated insulin derivatives; U.S. Patent 4,992,418, Katsoyanis et al, Aspio-containing insulin (B chain) for increased activity; U.S.
  • Patent 5,716,927 Balschmidt et al, Lys or Arg at position 28 in the B chain, or A18, A21 or B3 modified from Asn, or other modifications at the C-terminal end of the B chain, to prevent aggregation and reduced activity) Additional amino acid substitutions that confer a longer active phase, because they can be acylated, are disclosed in U.S. Patent 5,750,497, Havelund et al. A21 B3 and B30 can be replaced by any amino acid except Lys, Arg or Cys. BI may be deleted, and B30 may be replaced by a lipophilic chain of 10-24 carbon atoms.
  • Fusion proteins for improved recombinant production of insulin are described in U.S. Patent 6,534,288, Habermann et al. These peptides contain a fusion portion at the amino terminal end of the B chain, followed by amino acids RDVP-Y n -A chain, where Y is a peptide 2-50 amino acids in length, terminating with a basic amino acid.
  • the present invention includes fusion proteins comprising transferrin and an insulin protein or peptide. In one embodiment, the fusion proteins are formulated for oral delivery.
  • the present invention therefore, also includes methods of orally administering insulin fusion proteins of the invention to a patient in need thereof, in particular, a diabetic patient.
  • the present invention includes transferrin fusion protein comprising single chain insulin analog (Lee et al, 2000, Nature, 408: 483).
  • the insulin in the transferrin fusion protein may contain a protease cleavage site specific to the gastrointestinal (GI) tract, or a specific part of the gastrointestinal tract, such that the site would be recognized by one or more enzymes in the GI tract.
  • the proinsulin could be activated in this manner.
  • the cleavage site could reside in the peptide linking the A and B chain.
  • EPO Mimetic Peptide (EMP) Erythropoietin (EPO) is a glycoprotein hormone that is synthesized in the kidneys of mammals for stimulating mitotic cell division and differentiation of erythrocyte precursor cells. Accordingly, EPO acts to stimulate and regulate the production of erythrocytes. Because of its role in red blood cell formation, EPO is useful in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. Studies have shown the efficacy of EPO therapy in a variety of disease states, disorders, and states of hematologic irregularity, for example, beta-thalassemia (Nedovato et al. (1984) Acta. Haematol.
  • cystic fibrosis (Nichinsky et al. (1984) J. Pediatric 105:15-21); pregnancy and menstrual disorders (Cotes et al. (1983) Brit. J. Ostet. Gyneacol. 90:304-311); early anemia of prematurity (Haga et al. (1983) Acta Pediatr. Scand. 72:827- 831); spinal cord injury (Claus-Walker et al. (1984) Arch. Phys. Med. Rehabil. 65:370- 374); space flight (Dunn et al. (1984) Eur. J. Appl. Physiol. 52:178-182); acute blood loss (Miller et al.
  • EPO has been used for the treatment of the anemia of renal failure, anemia of chronic disease associated with rheumatoid arthritis, inflammatory bowel disease, AIDS, and cancer, as well as for the treatment of anemia in hematopoietic malignancies, post-bone marrow transplantation, and autologous blood donation.
  • EPO-receptor belongs to the class of growth-factor-type receptors which are activated by a ligand-induced protein dimerization.
  • Other hormones and cytokines such as human growth hormone (hGH), granulocyte colony stimulating factor (G-CSF), epidermal growth factor (EGF) and insulin can cross-link two receptors resulting in juxtaposition of two cytoplasmic tails.
  • hGH human growth hormone
  • G-CSF granulocyte colony stimulating factor
  • EGF epidermal growth factor
  • insulin can cross-link two receptors resulting in juxtaposition of two cytoplasmic tails.
  • Many of these dimerization-activated receptors have protein kinase domains within the cytoplasmic tails that phosphorylate the neighboring tail upon dimerization. While some cytoplasmic tails lack intrinsic kinase activity, these function by association with protein kinases.
  • the EPO receptor is of the latter type. In each case, phosphorylation results in the activation of
  • EPO erythropoietin receptor
  • EPO EPO mimetic peptides
  • EMP1 can act as an EPO mimetic.
  • EMP1 competes with EPO in receptor binding assays to cause cellular proliferation of cell lines engineered to be responsive to EPO (Wrighton et al, 1996, Science, 273:458-463). Both EPO and EMP1 induce a similar cascade of phosphorylation events and cell cycle progression in EPO responsive cells (Wrighton et al, 1996, Science, 273:458-463). Further, EMP1 demonstrates significant erythropoietic effects in mice as monitored by two different in vivo assays of nascent red blood cell production (Wrighton et al, 1996, Science, 273:458-463).
  • the present invention provides EMP1 /transferrin fusion proteins with increased half-life and pharmaceutical compositions comprising such fusion proteins.
  • Any EMP1 sequence may be used to make EMP1 /transferrin fusion proteins, including EMP1 sequences wherein one or more C residues is deleted or replaced. These sequences can then be inserted into a mTf loop to provide three dimensional structure to the EMP1 region of the fusion protein.
  • the present invention contemplates the use of the fusion protein to treat various diseases and conditions associated with EPO such as but not limited to those described above.
  • the pharmaceutical compositions comprising the EMP1 /transferrin fusion protein and may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences, 1985.
  • the composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution.
  • a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution.
  • These pharmaceutical compositions may contain buffers, salts and other excipients to stabilize the composition or assist in the delivery of the transferrin fusion proteins.
  • the present invention provides a method for treating disorders associated with EPO.
  • the method is accomplished by administering a EMP1 /transferrin fusion protein provided herein for a time and under conditions sufficient to alleviate the symptoms of the disorder, i.e. sufficient to effect dimerization or biological activation of EPO receptors.
  • EPO such methodology is useful in the treatment of end-stage renal failure/dialysis; anemia, especially associated with AIDS or chronic inflammatory diseases such as rheumatoid arthritis and chronic bowel inflammation; auto-immune disease; and for boosting the red blood cell count of patient when necessary, e.g. prior to surgery or as pretreatment to transfusion.
  • EMP1 /transferrin fusion protein of the present invention which behave as EPO agonists can be used to activate megakaryocytes.
  • EPO has been shown to have a mitogenic and chemotactic effect on vascular endothelial cells as well as an effect on central cholinergic neurons (Amagnostou et al. (1990) Proc. Natl. Acad. Sci. USA 87:597805982; Konishi et al. (1993) Brain Res.
  • the compounds of this invention can also be used to treat a variety of vascular disorders, such as promoting wound healing, growth of collateral coronary blood vessels (such as those that may occur after myocardial infarction), trauma, and post vascular graft treatment, and a variety of neurological disorders, generally characterized by low absolute levels of acetyl choline or low relative levels of acetyl choline as compared to other neuroactive substances e.g., neurotransmitters.
  • vascular disorders such as promoting wound healing, growth of collateral coronary blood vessels (such as those that may occur after myocardial infarction), trauma, and post vascular graft treatment
  • neurological disorders generally characterized by low absolute levels of acetyl choline or low relative levels of acetyl choline as compared to other neuroactive substances e.g., neurotransmitters.
  • the present invention includes pharmaceutical compositions comprising, as an active ingredient, the EMP1 /transferrin fusion protein of the present invention in association with a pharmaceutical carrier or diluent.
  • the EMP1 /transferrin fusion protein of this invention can be administered by oral, parenteral (intramuscular, intraperitoneal, intravenous (IN) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation) or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration in dosage forms appropriate for each route of administration.
  • Solid dosage forms for oral administration include capsules, tablets, pill, powders, and granules.
  • the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
  • Such dosage forms can also comprise, as it normal practice, additional substances other than inert diluents, e.g., lubricating, agents such as magnesium stearate.
  • the dosage forms may also comprise buffering, agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
  • Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
  • compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.
  • Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
  • the dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient shall be such that a suitable dosage form is obtained.
  • the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Generally dosage levels of between 0.001 to 10 mg/kg of body weight daily are administered to mammals.
  • the present invention also contemplates the use of the transferrin fusion protein comprising EMP 1 or analogs thereof for the manufacture of a medicinal product which can be used in the treatment of diseases associated with low or defective red blood cell production. Examples of such diseases are not limited to those described above.
  • T-20 and T-1249 HIN infection is pandemic and HIN associated diseases represent a major world health problem.
  • considerable effort is being put into the successful design of effective therapeutics, currently no curative anti-retro viral drugs against AIDS exist.
  • several stages of the HIN life cycle have been considered as targets for therapeutic intervention (Mitsuya, H. et al, 1991, FASEB J. 5:2369-2381).
  • virally encoded reverse transcriptase has been one focus of drug development.
  • a number of reverse-transcriptase-targeted drugs including 2',3'-dideoxynucleoside analogs such as AZT, ddl, ddc, and d4T have been developed which have been shown to been active against HIN (Mitsuya, H. et al, 1991, Science 249:1533-1544). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIN mutants (Lander, B. et al, 1989, Science 243 : 1731 - 1734). In addition, the drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver function abnormalities.
  • Entry inhibitors are distinct from the existing classes of drugs that fight HIN. Other drugs work inside the infected cell. Nucleoside reverse transcriptase inhibitors such as AZT and abacavir and non-nucleoside reverse transcriptase inhibitors like nevirapine and efavirenz all act by shutting down the reverse transcriptase enzyme that HIN uses to replicate itself once it is inside the cell. Protease inhibitors shut down the viral protease enzyme HIN uses to package itself up for export. By contrast, entry inhibitors are drugs that interfere with the processes involved in the virus' initial assault on the cell's outer membrane.
  • T-20 is the most studied of all the entry inhibitors and is the first member of the fusion inhibitor class. Unlike existing AIDS drugs that work inside the cell and target viral enzymes involved in the replication of the virus, T-20 inhibits fusion of HIN with host cells before the virus enters the cell and begins its replication process. T-20 binds to one of the two helical domains of gp41. Gp41 is a spring-loaded HIN-1 protein that is activated when CD4 binds to HIN gp-120. The fusion action of gp41 is inhibited if its two helical domains cannot fold together. T-20 binds to gp41, effectively keeping the protein from functioning. It has been shown in early, single-arm clinical studies to be about as potent as a protease inhibitor by itself-giving greater than 10 fold reductions in viral load-and to be safe in combination with other antiretrovirals.
  • T-20 (pentafuside, DP-178) as a 36 amino acid synthetic peptide. Since this drug is a peptide, it cannot be given orally because it is readily broken down by the digestive system.
  • T-20 When administered by subcutaneous injection, T-20 achieves sufficient levels in the blood to have anti-HIN activity. It is administered by subcutaneous injection twice daily. However, patients develop skin reactions at the injection site. The most frequently reported treatment related adverse events were mild to moderate local injection site reactions. These consist of mild pain, temporary swelling and redness at the site of injection.
  • U.S. Patent 6,479,055 discloses peptide analogs of the DP-178 (peptides corresponding to amino acid residues 638 to 673 of transmembrane protein gp41 of HIN- 1 L A I5 which exhibit anti-membrane fusion capability, antiviral activity, such as the ability to inhibit HIN transmission to uninfected CD-4 + cells, or an ability to modulate intracellular processes involving coiled-coil peptide structures. Further, the patent relates to the use of DP-178 and DP-178 portions and/or analogs as antifusogenic or antiviral compounds or as inhibitors of intracellular events involving coiled-coil peptide structures. Further, the patent teaches the use of the peptides as diagnostic agents. For example, a DPI 78 peptide may be used as an HIN subtype-specific diagnostic.
  • T-1249 is a sister compound of T-20. Like T-20, T-1249 targets the HIN glycoprotein known as gp41 which HIN uses to bind onto CD4 cells. T-1249 has shown potent anti-HIN effects in animal and laboratory studies. Preliminary safety, dosing and efficacy studies in humans have provided support for ongoing research. T-1249 is currently administered by subcutaneous (under the skin) injection once or twice daily. The first safety study of T-1249 conducted in humans found two serious adverse events: hypersensitivity reaction (oral ulcers, maculopapular rash, fever) and severe neutropenia. Forty percent of recipients developed injection site reactions but these were deemed to be mild. Dizziness, diarrhea, headache and fever have also been reported by recipients. No dose-limiting toxicity was identified and experiments with higher doses are likely. T-1249 has completed phase I/II safety and dosing studies. Initial results indicated that higher doses produced an average viral load drop of 1.3 log.
  • the present invention provides transferrin fusion proteins comprising T-20, T-1249, or analogs thereof with increased half-life and pharmaceutical compositions comprising such fusion proteins.
  • the present invention also provides pharmaceutical compositions comprising these transferrin fusion proteins for therapeutic purposes.
  • the present invention contemplates the use of such fusion proteins as inhibitors of human and non-human retroviral, especially HIN, transmission to uninfected cells.
  • the human retroviruses whose transmission may be inhibited by the peptides of the invention include, but are not limited to all strains of HIN- 1 and HIN-2 and the human T-lymphocyte viruses (HTLN-I, II, III).
  • the non-human retroviruses whose transmission may be inhibited by the peptides of the invention include, but are not limited to bovine leukosis virus, feline sarcoma and leukemia viruses, simian sarcoma and leukemia viruses, and sheep progress pneumonia viruses.
  • the transferrin fusion protein of the present invention comprising T-20, T-1249 or analogs thereof may be used as a therapeutic in the treatment of AIDS.
  • These transferrin fusion proteins may be administered using techniques well known to those in the art.
  • the pharmaceutical compositions comprising these transferrin fusion proteins are formulated and administered systemically.
  • Suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as inrrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. Most preferably, administration is intravenous.
  • the transferrin fusion proteins comprising T- 20, T1249, or analogs thereof may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the transferrin fusion protein comprising T-20, T1249, or analogs thereof may be used as a prophylactic measure in previously uninfected individuals after acute exposure to an HIN virus.
  • prophylactic use of the peptides may include, but are not limited to, prevention of virus transmission from mother to infant and other settings where the likelihood of HIN transmission exists, such as, for example, accidents in health care settings wherein workers are exposed to HIN-containing blood products.
  • the transferrin fusion proteins of the present invention comprising T-20, T-1249, or analogs thereof in such cases may serve the role of a prophylactic vaccine, wherein the host raises antibodies against the fusion proteins of the invention, which then serve to neutralize HIN viruses by, for example, inhibiting further HIN infection.
  • Administration of the transferrin fusion proteins of the invention as a prophylactic vaccine would comprise administering to a host a concentration of transferrin fusion protein effective in raising an immune response which is sufficient to neutralize HIN, by, for example, inhibiting HIN ability to infect cells.
  • concentration will depend upon the specific peptide in the transferrin fusion protein to be administered, but may be determined by using standard techniques for assaying the development of an immune response which are well known to those of ordinary skill in the art.
  • the transferrin fusion proteins to be used as vaccines are usually administered intramuscularly.
  • Effective dosages of the transferrin fusion proteins comprising T-20, T-1249, or analogs thereof to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity. Given the data presented below in Section 6, DP-178, for example, may prove efficacious in vivo at doses required achieve circulating levels of 10 ng per ml of peptide.
  • the present invention contemplates the use of the transferrin fusion proteins comprising T-20, T-1249, or analogs thereof for the manufacture of a medicinal product for the treatment of diseases associated with the transmission of a virus.
  • BBB Blood Brain Barrier
  • the modified transferrin fusion proteins may be used as a carrier to deliver a molecule or small molecule therapeutic complexed to the ferric ion of transferrin to the inside of a cell or across the blood brain barrier or other barriers including across the cell membrane of any cell type that naturally or engineered to express a Tf receptor.
  • the Tf fusion protein will typically be engineered or modified to inhibit, prevent or remove glycosylation to extend the serum half-life of the fusion protein and/or therapeutic protein portion.
  • the addition of a targeting peptide is specifically contemplated to further target the Tf fusion protein to a particular cell type, e.g., a cancer cell.
  • the iron-containing, anti-anemic drug, ferric-sorbitol-citrate complex is loaded onto a modified Tf fusion protein of the invention.
  • Ferric-sorbitol-citrate (FSC) has been shown to inhibit proliferation of various murine cancer cells in vitro and cause tumor regression in vivo, while not having any effect on proliferation of non- malignant cells (Poljak-Blazi et al. (June 2000) Cancer Biotherapy and Radiopharmaceuticals (United States), 15/3:285-293).
  • the antineoplastic drug Adriamycin® (doxorubicin) and/or the chemotherapeutic drug bleomycin, both of which are known to form complexes with ferric ion, is loaded onto a Tf fusion protein of the invention.
  • a salt of a drug for instance, a citrate or carbonate salt, may be prepared and complexed with the ferric iron that is then bound to Tf.
  • transferrin modified to carry at least one anti-tumor agent may provide a means of increasing agent exposure or load to the tumor cells.
  • the modified fusion proteins of the invention may be administered to a patient in need thereof using standard administration protocols.
  • the modified Tf fusion proteins of the present invention can be provided alone, or in combination, or in sequential combination with other agents that modulate a particular pathological process.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same or near the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal and buccal routes.
  • an agent may be administered locally to a site of injury via microinfusion.
  • administration may be noninvasive by either the oral, inhalation, nasal, or pulmonary route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • administration or delivery orally may be a preferred embodiment for certain classes of fusion proteins or to treat certain conditions.
  • the present invention further provides compositions containing one or more fusion proteins of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise about 1 pg/kg to about 100 mg/kg body weight. The preferred dosages for systemic administration comprise about 100 ng/kg to about 100 mg/kg body weight. The preferred dosages for direct administration to a site via microinfusion comprise about 1 ng/kg to about 1 mg/kg body weight. When administered via direct injection or microinfusion, modified fusion proteins of the invention may be engineered to exhibit reduced or no binding of iron to prevent, in part, localized iron toxicity.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and dextran.
  • the suspension may also contain stabilizers.
  • Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the agents of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds of this invention may be co-administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, ex vivo or in vitro.
  • Modified fusion proteins of the present invention may be used in the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders relating to diseases and disorders of the endocrine system, the nervous system, the immune system, respiratory system, cardiovascular system, reproductive system, digestive system, diseases and/or disorders relating to cell proliferation, and/or diseases or disorders relating to the blood.
  • modified Tf fusion proteins may be used in the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders relating to diseases and disorders known to be associated with or treatable by therapeutic protein moieties as known in the art and exemplified by PCT Patent Publication Nos. WO 01/79258, WO 01/77137, WO 01/79442, WO 01/79443, WO 01/79444 and WO 01/79480, all of which are herein incorporated by reference in their entirety.
  • the present invention encompasses a method of treating a disease or disorder listed in the "Preferred Indication Y" column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired a modified transferrin fusion protein of the invention that comprises a therapeutic protein portion corresponding to a therapeutic protein disclosed in the "Therapeutic Protein X" column of Table 1 in an amount effective to treat, prevent or ameliorate the disease or disorder.
  • a transferrin fusion protein of the present invention may be used to diagnose and/or prognose diseases and/or disorders.
  • Modified transferrin fusion proteins of the invention and polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention can be used as a marker or detector of a particular immune system disease or disorder.
  • fusion proteins of the invention and/or polynucleotides encoding modified transferrin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, diagnosing, and/or prognosing autoimmune disorders.
  • Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention, that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
  • Modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells.
  • Transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia.
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
  • Allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosing and using modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention.
  • these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to modulate IgE concentrations in vitro or in vivo.
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may inhibit the activation, proliferation, and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions.
  • Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury), neurodegenerative disorders (e.g., Parkinson's disease and Alzußmer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myo
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochiftis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrin
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease
  • GNHD Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GNHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GNHD.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as an adjuvant to enhance anti-viral immune responses.
  • Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant include virus and virus associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of AIDS, meningitis, Dengue, EBN, and hepatitis (e.g., hepatitis B).
  • compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIN/ AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
  • a virus, disease, or symptom selected from the group consisting of: HIN/ AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses.
  • Anti-bacterial or anti- fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a bacterium or fungus disease, or symptom selected from the group consisting of tetanus, Diphtheria, botulism, meningitis type B, and candidiasis.
  • compositions of the invention are used as an adjuvant to enhance an immune response to a bacterium or fungus, disease, or symptom selected from the group consisting of Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella par atyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Candida.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as an adjuvant to enhance anti-parasitic immune responses.
  • j iti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite.
  • the compositions of the invention are used as an, adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities, of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used in one or more of the applications described herein, as they may apply to veterinary medicine.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as a means of blocking various aspects of immune responses to foreign agents or self.
  • diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury, and diseases/disorders associated with pathogens.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
  • modified transferrin fusion proteins or polynucleotides encoding transferrin fusion proteins of the invention are used as an inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
  • modified transferrin fusion proteins of the invention, and/or polynucleotides encoding transferrin fusion proteins of the invention are used as a therapy for chronic hypergammaglobulinen evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monocional gammopathies, and plasmacytomas.
  • MGUS monoclonal gammopathy of undetermined significance
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD 8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion protein of the invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
  • modified transferrin fusion proteins of the invention and/or-polynucleotides encoding transferrin fusion proteins of the invention may be employed to treat adult respiratory distress syndrome (ARDS).
  • ARDS adult respiratory distress syndrome
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to stimulate the regeneration of mucosal surfaces.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction.
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, Common Nariable Immunodeficiency (CNID), other primary immune deficiencies, HIN disease, Chronic Lymphocytic Leukemia (CLL), recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carn
  • fusion proteins of the invention include, but are not limited to, HIN infection, HTLN-BLN infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms.
  • cancers or neoplasms that may be prevented, diagnosed, or treated by fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBN transformed diseases, and/or diseases and disorders described in the section entitled "Hyperproliferative Disorders" elsewhere herein.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
  • compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity.
  • hemostatic or thrombolytic activity fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
  • blood coagulation diseases, disorders, and/or conditions e.g., afibrinogenemia, factor deficiencies, hemophilia
  • blood platelet diseases, disorders, and/or conditions e.g. thrombocytopenia
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to prevent diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina.
  • the transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention maybe used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atria fibrillation, for reducing the risk of embolism associated with mechanical heart valves and/or mitral valves disease.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canals, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed.
  • the modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to modulate hematopoietic activity (the formation of blood cells).
  • the transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets.
  • the ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis, and/or treatment of anemias and leukopenias described below.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention maybe used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets.
  • the ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis, and/or treatment of leukocytoses, such as, for example eosinophilia.
  • leukocytoses such as, for example eosinophilia.
  • the modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to prevent, treat, or diagnose blood dyscrasia.
  • Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis).
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias.
  • Anemias that may be treated prevented or diagnosed by the transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary sideroblastic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B 12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmunune hemolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria).
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleen.
  • the transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemia arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfa drugs.
  • modified fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention maybe useful in treating, preventing, and/or diagnosing anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease). Additionally, the transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in diagnosing, preventing, and/or prognosing in treating thalassemias, including, but not limited to, major and minor forms of alpha- thalassemia and beta-thalassemia.
  • the modified fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Non Willebrand' s disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolyticuremic syndrome, hemophilias such as hemophilia A or Factor Nil deficiency and Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Webe syndrome, allergic
  • fusion proteins of the invention, and/or polynucleotides encoding transferrin fusion proteins of the invention can be used to treat or detect hyperprohferative disorders, including neoplasms.
  • Transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may inhibit the proliferation of the disorder through direct or indirect interactions.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may cause proliferation of other cells which can inhibit the hyperproliferative disorder.
  • hyperproliferative disorders can be treated.
  • This immune response may be increased by either enhancing an existing immune response, of by initiating a new immune response.
  • decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
  • hyperproliferative disorders that can be treated or detected by modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to neoplasms located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
  • hyperproliferative disorders can also be treated or detected by modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention.
  • hyperproliferative disorders include, but are not limited to Acute Childhood Lymphoblastic Leukemia; Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS- Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer,
  • Endometrial Cancer Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung
  • Rhabdomyosarcoma Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilm's Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
  • Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth is consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins. and Angell, 1976, Basic Pathology, 2d Ed. W. B. Saunders Co., Philadelphia, pp. 68-79).
  • Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to, angiofoUicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cemenrum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, foca epithelial hyperplasia
  • modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention conjugated to a toxin or a radio-active isotope, as described herein may be used to treat cancers and neoplasms, including, but not limited to, those described herein.
  • transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein may be used to treat acute myelogenous leukemia.
  • modified fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis.
  • diseases associated with increased cell survival or the inhibition of apoptosis include cancers (such as follicular- lymphomas, carcinomas with p53 mutations, and hormone- dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
  • Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, and/or treated by modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention, include but are not limited to, progression and or metastases of malignancies and related disorders such as leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, mylomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not
  • AIDS dementia
  • neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebral degeneration and brain tumor or prion associated disease
  • autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis
  • myelodysplastic syndromes such as aplastic anemia
  • graft Y host disease ischemic injury (such as that caused by myocardial infarction, stroke and repercussion injury
  • Another preferred embodiment utilizes polynucleotides encoding modified transferrin fusion proteins of the invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
  • the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide encoding modified fransferrin fusion protein of the present invention, wherein said polynucleotide represses said expression.
  • Another embodiment of the present invention provides a method of treating cell proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells.
  • the polynucleotides of the present invention may be delivered directly to cell proliferative disorderly disease sites in internal organs, body cavities, and the like by use of imaging devices used to guide an injecting needle directly to the disease site.
  • the polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
  • cell proliferative disease any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
  • any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotides of the present invention simultaneously to the same site.
  • biologically inhibiting is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells.
  • Modified transferrin fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering these transferrin fusion proteins and/or polynucleotides, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha, integrins, (See, e.g., Curr. Top. Mirobiol. Immunol. 1998; 231:1 41, which is hereby incorporated by reference). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
  • the invention provides a method of delivering compositions containing the fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention to targeted cells expressing a polypeptide bound by, that binds to, or associates with a modified transferrin fusion protein of the invention.
  • Transferrin fusion proteins of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
  • Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, post infectious glomerulonephritis, rapidly progressive glomerulonephritis, nephritic syndrome, membranous glomerulonephritis, familial nephritic syndrome, membrane proliferative glomerulonephritis and mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulo-interstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis(PSGN), pyelonephritis, lupus nephritis, chronic ne
  • compositions of the invention can be used to diagnose, prognose, prevent, and/or treat metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephritic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus
  • compositions of the invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or lecrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal Fsegmental glomerulo sclerosis (FSGS), narcotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's rumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernafremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
  • compositions of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gel foam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Compositions of the invention may be administered as part of a Therapeutic, described in more detail below.
  • Modified fransferrin fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
  • Cardiovascular disorders includes, but is not limited to, cardiovascular abnormalities, such as arterio arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
  • cardiovascular abnormalities such as arterio arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
  • Congenital heart defects include, but are not limited to, aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septald defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
  • Cardiovascular disorders also include, but are not limited to, heart disease, such arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiactamponade, endocarditis (including bacteria), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy left ventricular hypertrophy, right ventricularhypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including consfrictive and tuberculous), pricumopericardium, post pericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
  • heart disease such arrhythmias, carcinoid heart disease, high
  • Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre- excitation syndrome, Wolff-Parkinson- White syndrome, sick sinus syndrome, itachycardias, and ventricular fibrillation.
  • Tachycardias include paroxysmal tachycardia, suprayentriculai tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachyeardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoattial nodalreentry * tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
  • Heart valve diseases include, but are not limited to, aortic valve insufficiency aortic valve stenosis, heart murmurs, aortic valve prolapse, neutral valve prolapse, fricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, fricuspid atresia, fricuspid valve insufficiency, and fricuspid valve stenosis.
  • Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
  • Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunning.
  • coronary disease such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunning.
  • Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomiatosis, Hippel-Lindau Disease, Klippel Trenaunay Weber Syndrome, Sturge Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arthritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arthritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno- occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein oc
  • Cerebrovascular disorders include, but are not limited to, cardio artery diseases but includes respiratory disorders.
  • Transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders of the respiratory system.
  • Diseases and disorders of the respiratory system include, but are not limited to, nasalvestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma
  • Mycoplasma pneumoniae pneumonia e.g., Hemophilus influenza pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)
  • viral pneumonia e.g., influenza, chickenpox (varicella).
  • Additional diseases and disorders of the respiratory system include, but are not limited to bronchiohtis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubellapanencephalitis, German measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergiUosis, caused by Aspergillus spp.) candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypicalpneumonias (e.g., Mycoplasma and
  • pulmonary fibrosis e.g., usual interstitial pneumonia
  • idiopathic pulmonary fibrosis desquamative interstitial pneumonia
  • lymphoid interstitial pneumonia histiocytosis (e.g., Letterer-Siwe disease, Hand-Sch ⁇ ller- Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary, alveolar proteinosis)
  • Acute respiratory distress syndrome also called, e.g., adult respiratory distress syndrome
  • bronchitis e.g., viral, bacterial
  • bronchiectasis atelectasis
  • lung abscess caused
  • Cancers which may be treated with modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemia.
  • solid tumors including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometri
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
  • Modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be useful, in treating other disorders, besides cancers, which involve angiogenesis.
  • disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenicgranulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygiaab normal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scar
  • disorders which can be treated with modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma; and vascular adhesions.
  • disorders and/or states which can be treated, prevented, diagnosed, and/or prognosed with the modified fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemia, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocularangiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, refinoblastoma, and uvietis, delayed wound healing, endometriosis, vasculogenesis,
  • an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after” method.
  • Modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
  • Modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be utilized in a wide variety of surgical procedures.
  • cancers such as follicular lymphomas, carcinomas with mutations, and hormone- dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis
  • modified fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
  • Additional diseases or conditions associated with increased cell survival that could be treated or detected by modified fusion proteins of the invention and/or polynucleotides encoding, fransferrin fusion proteins of the invention include, but are not limited to, progression, and/or metastases of malignances and related disorders such as leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycytemia vera
  • AIDS Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prion associated disease
  • autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cimhosis, Behcet's disease, Crohn's' disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis
  • Myelodysplastic syndromes such as aplastic anemia
  • graft v aplastic anemia
  • ischemic injury such as that caused, by myocardial. infarction, stroke and reperfusion injury
  • liver injury e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer
  • toxin-induced liver disease such as that caused by alcohol
  • septic shock cachexia and anorexia.
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be, used to treat or prevent the onset of diabetes mellitus.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease.
  • fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system.
  • Nervous system disorders that can be treated with the compositions of the invention (e.g., fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention), limited to nervous systems include, but are not limited injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
  • Nervous system lesions which may be freated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from nervous system tissue; (4) infectious lesions in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zo
  • the modified fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention are used to protect neural cells from the damaging effects of hypoxia.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia.
  • motor neuron disorders that may be freated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motor sensory Neuropathy (Charcot-Marie-Tooth Disease).
  • disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulb
  • modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may play a role in neuronal survival; synapse formation; conductance; neural differentiation, etc.
  • compositions of the invention may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders.
  • the compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioral disorders.
  • Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • neurologic diseases which can be treated or detected with modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, brain diseases, such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxi
  • Additional neurologic diseases which can be treated or detected with modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include cerebro vascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidermal hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular
  • Additional neurologic diseases which can be treated or detected with modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt- Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi- infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemicencephalitis, Japanese Encephalitis, St.
  • dementia such as AIDS Dementia Complex
  • presenile dementia such as Alzheimer's Disease and Creutzfeldt- Jakob Syndrome
  • senile dementia such as Alzheimer's Disease and progressive supranuclear palsy
  • vascular dementia such as multi- infarct dementia
  • encephalitis which include encephalitis periaxialis
  • viral encephalitis such as epidemicencephalitis, Japanese Encephalitis, St.
  • epilepsy such as generalized epilepsy, which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic- clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-fraumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.
  • Additional neurologic diseases which can be freated or detected with modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include hydrocephalus such as Dandy- Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebripseudo tumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranialtuberculoma and Zellweger Syndrome, central nervous system infections such as AIDS, Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhabaic Encephalomyelitis, Visna, and cerebral malaria.
  • hydrocephalus such as Dand
  • Additional neurologic diseases which can be treated or detected with modified fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention include meningitis such as arachnoiditis, aseptic meningitis such as viral meningitis which includes lymphocytic chronic meningitis, Bacterial meningitis which includes Haemophilus Meningitis, Listeria Meningitis, Meningococcal Meningitis such as Waterhouse-Fridericlisen Syndrome, Pneumococcal Meningitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningitis, subdural effusion, meningencephalitis, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and post poliomyelitis syndrome, prion diseases (such as Creutzfeldt- ak
  • Additional neurologic diseases which can be treated or detected with modified fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention include central nervous system neoplasms such as brain neoplasms that include cerebellarneoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroids plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningealneoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sculleries which include sadrenoleukodysfrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic ence
  • Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle, (e.g., dysmenorrhea and endometriosis).
  • pregnancy and labor e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor
  • disorders and/or diseases of the menstrual cycle e.g., dysmenorrhea and endometriosis
  • Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Gushing' s Syndrome, hyperaldosterlonism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease
  • endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer.
  • Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neotestis.
  • endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
  • the modified transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used for the diagnosis, freatment, or prevention of diseases and/or disorders of the reproductive system.
  • Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders will result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
  • Reproductive system disorders and/or diseases include diseases and/or disorders, of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and.
  • anorchia typically results from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis
  • testiculartorsiori vasitis nodosa
  • germ cell tumors e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas
  • stromal tumors e. g., Leydig cell tumors
  • hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production e. g. immotile cilia syndrome, spermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).
  • Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, postatodystonia, prostatosis, granulomatotis prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
  • compositions of the invention may be useful in the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphmosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, frichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminarum, condyloma latum, and pearly penile papules, urethral abnormalities, such as hypospadias, epispadias, and phimosis, premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, criant condyloma of Buscke-Lowenstein, and varrucous
  • the fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chioride diarrhea, and polycystic kidney disease.
  • disorders and/or diseases of the male reproductive system include, for example, Klinefelters syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
  • polynucleotides, modified fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used in the diagnosis freatment and/or prevention of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, Candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvartrauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condylomaacuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paaet's disease, lichensclerosus, lichen planus, vulvodynia, toxic shock syndrome, vag
  • disorders and/or diseases of the uterus include dysmenorrhea, refroverted uterus, endomefriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushiner's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas.
  • the fransferrin fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention may be useful as a marker or detector of, as well as, in the diagnosis, freatment, and/or prevention of congenital uterine abnormalities, such as bicomuate uterus, septate uterus, simple unicomuate uterus, unicomuate uterus with a noncavitary rudimentary horn, unicorriuate uterus with a non- communicating cavitary rudimentary horn, unicomuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.
  • congenital uterine abnormalities such as bicomuate uterus, septate uterus, simple unicomuate uterus, unicomuate uterus with a noncavitary rudimentary horn, unicorriu
  • Ovarian diseases and/or disorders include an ovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian over production of androgens, right ovarian vein syndrome, in amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endomefriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
  • ovarian cancer including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endomefriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and O
  • Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, and cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
  • Modified fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be freated.
  • the immune response may be increased by either enhancing an existing immune response, or by fusion proteins of the invention and/or initiating a new immune response.
  • polynucleotides encoding fransferrin fusion proteins of the invention may also directly inhibit infectious agent, without necessarily eliciting an immune response.
  • viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by transferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention.
  • viruses include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arteriyirus, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae Hepatitis, Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza),
  • bacterial and fungal agents that can cause disease or symptoms that can be treated or detected by fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but not limited to, the following Gram-negative and Gram-positive bacteria, bacterial families, and fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorfer ⁇ ), Brucella, Candidia, Campylobacter, Chlamydia, Clostridiuffi (e.g., Clostridium botulinum, Clostridium pulpe, Clostridium perfringens, Clostridiumtetani),
  • Enterobacter e.g. Enterotoxigenic E. coli and Enterohemorrhagic E. coli
  • Enterobacter e.g. Enterobacter aerogenes
  • Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.
  • Legionella pneumophila Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseriagonorrhea, Neisseria meningitidis), Pasteurellaceae, Proteus, Pseudomonas (e.g., Pseudomionas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema.
  • Listeria e.g., Listeria monocytogenes
  • Mycoplasma Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis)
  • Vibrio e.g., Vibrio cholerae
  • spp. Leptospiraspp., Borrielia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas.
  • Staphylococcus e.g., Staphylococcus aureus
  • Meningiococcus Meningiococcus
  • Pneumococcus and Streptococcus e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci
  • Ureaplasmas Ureaplasmas.
  • parasitic agents causing disease or that can be treated, prevented, and/or diagnosed by fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium vivax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale).
  • Modified fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention can be used to differentiate, proliferate, and attract cells, pleading to the regeneration of tissues.
  • the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
  • Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue.
  • organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vasculature including vascular and lymphatics
  • nervous hematopoietic
  • hematopoietic skeletal
  • skeletal bone, cartilage, tendon, and ligament
  • Modified fransferrin fusion proteins of the invention and/or polynucleotides encoding transferrin fusion proteins of the invention may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowel lymphoma), and ulcers, such as peptic ulcers.
  • cancers e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowel lymphoma)
  • ulcers such as peptic ulcers.
  • Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and structuring, Mallory- Weiss lesions, lipomas, epidennal cancers, adeoncarcinomas, gastric retention disorders, gasfroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Menetrier's), and peritoneal diseases (e.g., chylo perioneum, hemoperitoneum, mesenteric cyst, mesentericlymphadenitis, mesenteric vascular occlusion, panniculiti, neoplasms, peritonitis,
  • Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndrome's, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp. and T. solium
  • Liver diseases and/or disorders include infrahepatic cholestasis (Alagille syndrome, biliary liver cirrhosis), fatty, liver (alcoholic fatty liver, Reye's syndrome), hepatic veiri, thrombosis, hepatolenfricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal, syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver, enlargement, ascites, hepatitis (alcoholic hepatitis
  • Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucacronoma, cysticcitmeoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cysticfibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
  • pancreatic diseases e.g., cysticfibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency).
  • Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholeeystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
  • Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplastris, colon cancer, adenomatous colon polyps (e.g., villous adenoma), coloncarcinoma, colorectal cancer, colonic diverticulitis, colonic diverticulosis, megacolon, Hirschsprung disease, toxic inegacolon, sigmoid diseases proctocolitis, sigmoinneoplasmsj, constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasins, duodenal obstruction, duodenal ulcer, duodenitis), enteritis
  • anorectal disorders e.g., fissures,
  • enterolitis HIV enteropathy
  • leal diseases leal neoplasins, ileitis
  • immunoproliferative small intestinal disease inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, proamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neolasms, duodenalpneoplasms, leal neoplasms, intestinal polyps, jejunal neoplasins, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo obstruction cecal volvulus, intussusception), intestinal perforation, intestinal polyps (col
  • biliary tract diseases such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastricfistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasins, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasins, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic eystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., gastroschisis, fistula (e.
  • neoplasms perforation (e.g., Boerhaave syndrome, Mallory- Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic
  • Modified fransferrin fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention may have chemotaxis activity.
  • a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T- cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • Modified transferrin fusion proteins of the invention and/or polynucleotides encoding fransferrin fusion proteins of the invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body.
  • Tf fusion proteins including but not limited to modified Tf fusion proteins, may be formulated for oral delivery.
  • certain fusion proteins of the invention that are used to treat certain classes of diseases or medical conditions may be particularly amenable for oral formulation and delivery.
  • Such classes of diseases or conditions include, but are not limited to, acute, chronic and recurrent diseases.
  • Chronic or recurrent diseases include, but are not limited to, viral disease or infections, cancer, a metabolic diseases, obesity, autoimmune diseases, inflammatory diseases, allergy, graft-vs.- host disease, systemic microbial infection, anemia, cardiovascular disease, psychosis, genetic diseases, neurodegenerative diseases, disorders of hematopoietic cells, diseases of the endocrine system or reproductive systems, gastrointestinal diseases.
  • these classes of disease include diabetes, multiple sclerosis, asthma, HCV or HIV infections, hypertension, hypercholesterolemia, arterial scherosis, arthritis, and Alzheimer's disease.
  • oral formulations of Tf fusion proteins of the invention and methods of administration are particularly useful because they allow long-term patient care and therapy via home oral administration without reliance on injectable treatment or drug protocols.
  • Tf fusion proteins of the invention take advantage of, in part, transferrin receptor mediated transcytosis across the gastrointestinal (GI) epithelium.
  • the Tf receptor is found at a very high density in the human GI epithelium, transferrin is highly resistant to tryptic and chymotryptic digestion and Tf chemical conjugates have been used to successfully deliver proteins and peptides across the GI epithelium (Xia et al, (2000) J. Pharmacol. Experiment. Therap., 295:594- 600; Xia et al. (2001) Pharmaceutical Res., 18(2):191-195; and Shah et al. (1996) J.
  • Tf fusion proteins of the invention Once transported across the GI epithelium, Tf fusion proteins of the invention exhibit extended half-life in serum, that is, the therapeutic protein or peptide(s) attached or inserted into Tf exhibit an extended serum half-life compared to the protein or peptide in its non-fused state. Oral formulations of Tf fusion proteins of the invention may be prepared so that they are suitable for transport to the GI epithelium and protection of the Tf fusion protein component and other active components in the stomach.
  • Such formulations may include carrier and dispersant components and may be in any suitable form, including aerosols (for oral or pulmonary delivery), syrups, elixirs, tablets, including chewable tablets, hard or soft capsules, troches, lozenges, aqueous or oily suspensions, emulsions, cachets or pellets granulates, and dispersible powders.
  • Tf fusion protein formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages.
  • Solid dosage forms for oral administration are preferably tablets, capsules, or the like.
  • the amount of Tf fusion protein may be increased over that theoretically required or other known measures such as coating or encapsulation may be taken to protect the polypeptides from enzymatic action in the stomach.
  • peptide and protein drugs have been administered by injection because of the poor bioavailability when administered non-parenterally, and in particular orally. These drugs are prone to chemical and conformational instability and are often degraded by the acidic conditions in the stomach, as well as by enzymes in the stomach and gastrointestinal tract.
  • certain technologies for oral delivery have been developed, such as encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation in microparticles, insertion into liposomes in emulsions, and conjugation to other molecules.
  • nanoparticles examples include mucoadhesive nanoparticles coated with chitosan and Carbopol (Takeuchi et al, Adv. Drug Deliv. Rev. 47(l):39-54, 2001) and nanoparticles containing charged combination polyesters, poly(2-sulfobutyl- vinyl alcohol) and poly(D,L- lactic-co-glycolic acid) (Jung et al, Eur. J. Pharm. Biopharm. 50(1): 147-160, 2000).
  • Nanoparticles containing surface polymers with poly-N-isopropylacrylamide regions and cationic poly-vinylamine groups showed improved absorption of salmon calcitonin when administered orally to rats.
  • Drug delivery particles composed of alginate and pectin, strengthened with polylysine, are relatively acid and base resistant and can be used as a carrier for drugs.
  • lipoamino acid groups and liposaccharide groups conjugated to the N- and C-termini of peptides such as synthetic somatostatin, creating an amphipathic surfactant were shown to produce a composition that retained biological activity (Toth et al, J. Med. Chem. 42(19):4010-4013, 1999).
  • Examples of other peptide delivery technologies include carbopol-coated mucoadhesive emulsions containing the peptide of interest and either nitroso-N-acetyl-D,L- penicillamine and carbolpol or taurocholate and carbopol. These were shown to be effective when orally administered to rats to reduce serum calcium concentrations (Ogiso et al, Biol. Pharm. Bull. 24(6):656-661, 2001).
  • Phosphatidylethanol derived from phosphatidylcholine, was used to prepare liposomes containing phosphatidylethanol as a carrier of insulin. These liposomes, when administered orally to rats, were shown to be active (Kisel et al, Int. J. Pharm. 216(1-2):105-114, 2001).
  • Insulin has also been formulated in poly(vinyl alcohol)-gel spheres containing insulin and a protease inhibitor, such as aprotinin or bacitracin.
  • a protease inhibitor such as aprotinin or bacitracin.
  • the glucose-lowering properties of these gel spheres have been demonstrated in rats, where insulin is released largely in the lower intestine (Kimura et al, Biol. Pharm. Bull. 19(6): 897-900, 1996.
  • Oral delivery of insulin has also been studied using nanoparticles made of poly(alkyl cyanoacrylate) that were dispersed with a surfactant in an oily phase (Damge et al, J. Pharm. Sci. 86(12): 1403-1409, 1997) and using calcium alginate beads coated with chitosan (Onal et al, Artif. Cells Blood Substit. Immobil. Biotechnol. 30(3):229-237, 2002).
  • N- and C-termini of a peptide are linked to polyethylene glycol and then to allyl chains to form conjugates with improved resistance to enzymatic degradation and improved diffusion through the GI wall (www.nobexcorp.com).
  • BioPORTER® is a cationic lipid mixture, which interacts non-covalently with peptides to create a protective coating or layer.
  • the peptide-lipid complex can fuse to the plasma membrane of cells, and the peptides are internalized into the cells (www.genetherapysystems.com).
  • cochleate-shaped particles have been developed as a pharmaceutical vehicle.
  • a peptide is added to a suspension of liposomes containing mainly negatively charged lipids.
  • the addition of calcium causes the collapse and fusion of the liposomes into large sheets composed of lipid bilayers, which then spontaneously roll up or stack into cochleates (U.S. Patent 5,840,707; http://www.biodeliverysciences.com).
  • compositions comprising Tf fusion protein intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents in order to provide a pharmaceutically elegant and palatable preparation.
  • Tf fusion protein is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract.
  • the tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations.
  • compositions comprising Tf fusion protein may be prepared as described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein incorporated by reference.
  • many of the oral formulations of the invention may contain inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. Such formulations, or enteric coatings, are well known in the art.
  • tablets containing Tf fusion protein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for manufacture of tablets may be used.
  • excipients may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid, or talc.
  • the tablets may be uncoated or they may be coated with known techniques to delay disintegration and abso ⁇ tion in the gastrointestinal track and thereby provide a sustained action over a longer period of time.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin or as soft gelatin capsules wherein the active ingredient is mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate, or kaolin
  • an aqueous or an oil medium for example, arachis oil, peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions may contain Tf fusion protein in the admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum fragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecylethyloxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example poly
  • the aqueous suspensions may also contain one or more preservatives for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oil suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient and admixture with dispersing or wetting agent, suspending agent and one or more preservatives.
  • Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutical compositions containing Tf fusion protein may also be in the form of oil-in- water emulsions.
  • the oil phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil for example, gum acacia or gum fragacanth, naturally- occurring phosphotides, for example soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of the same partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs containing Tf fusion protein may be formulated with sweetening agents, for example, glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparations may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvate, for example as a solution in 1, 3-butanediol.
  • a non-toxic parenterally-acceptable diluent or solvate for example as a solution in 1, 3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this period any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions may also be formulated for oral delivery using polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase and Morrel, Oral Delivery of Microencapsulated Proteins, in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).
  • the proportion of pharmaceutically active Tf fusion protein to carrier and/or other substances may vary from about 0.5 to about 100 wt. % (weight percent).
  • the pharmaceutical formulation will generally contain from about 5 to about 100% by weight of the active material.
  • the formulation will generally have from about 0.5 to about 50 wt. % of the active material.
  • Tf fusion protein formulations employed in the invention provide an effective amount of Tf fusion protein upon administration to an individual.
  • an "effective amount" of Tf fusion is an amount that is effective to ameliorate a symptom of a disease.
  • the Tf fusion protein composition of the present invention may be, though not necessarily, administered daily, in an effective amount to ameliorate a symptom.
  • the total daily dosage will be at least about 50 mg, preferably at least about 100 mg, and more preferably at least about 200 mg, and preferably not more than 500 mg per day, administered orally, e.g., in 4 capsules or tablets, each containing 50 mg Tf fusion protein.
  • Capsules or tablets for oral delivery can conveniently contain up to a full daily oral dose, e.g., 200 mg or more.
  • oral pharmaceutical compositions comprising Tf fusion protein are formulated in buffered liquid form which is then encapsulated into soft or hard-coated gelatin capsules which are then coated with an appropriate enteric coating.
  • the location of release may be anywhere in the GI system, including the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
  • oral compositions of the invention are formulated to slowly release the active ingredients, including the Tf fusion proteins of the invention, in the GI system using known delayed release formulations.
  • Tf fusion proteins of the invention for oral delivery are capable of binding the Tf receptor found in the GI epithelium.
  • Tf fusion proteins of the invention are typically produced with iron and in some instances carbonate, bound to the Tf moiety.
  • Processes and methods to load the Tf moiety of the fusion protein compositions of the invention with iron and carbonate are known in the art
  • the Tf moiety of the Tf fusion protein may be modified to increase the affinity or avidity of the Tf moiety to iron.
  • mutagenesis can be used to produce mutant fransferrin moieties that bind iron more avidly than natural transferrin.
  • the amino acids which are ligands for metal ion chelation include, but are not limited to N lobe amino acids Asp63, Tyr 95, Tyrl 88, Lys206, His207 and His249; and C lobe amino acids As ⁇ 392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3 (the number beside the amino acid indicates the position of the amino acid residue in the primary amino acid sequence where the valine of the mature protein is designated position 1). See U.S. Patent 5,986,067, which is herein inco ⁇ orated be reference.
  • the Lys206 and His207 residues within the N lobe are replaced with Gin and Glu, respectively.
  • the Tf fusion protein is engineered to contain a cleavage site between the therapeutic protein or peptide and the Tf moiety. Such cleavable sites or linkers are known in the art.
  • compositions of the invention and methods of the invention may include the addition of a transcytosis enhancer to facilitate transfer of the fusion protein across the GI epithelium.
  • a transcytosis enhancer to facilitate transfer of the fusion protein across the GI epithelium.
  • Such enhancers are known in the art. See Xia et al, (2000) J. Pharmacol. Experiment. Therap., 295:594-600; and Xia et al. (2001) Pharmaceutical Res., 18(2):191-195.
  • oral pharmaceutical formulations include Tf fusion proteins comprising a modified Tf moiety exhibiting reduced or no glycosylation fused at the N terminal end to an insulin or GLP-1 protein or peptide as described above.
  • Such pharmaceutical compositions may be used to treat glucose imbalance disorders such as diabetes by oral administration of the pharmaceutical composition comprising an effective dose of fusion protein.
  • the effective dose of fusion protein may be measured in a numbers of ways, including dosages calculated to alleviate symptoms associated with a specific disease state in a patient, such as the symptoms of diabetes.
  • dosages are calculated to comprise an effective amount of fusion protein to induce a detectable change in blood glucose levels in the patient.
  • detectable changes in blood glucose may include a decrease in blood glucose levels of between about 1% and 90%, or between about 5% and about 80%. These decreases in blood glucose levels will be dependent on the disease condition being treated and pharmaceutical compositions or methods of administration may be modified to achieve the desired result for each patient.
  • the pharmaceutical compositions are formulated and methods of administration modified to detect an increase in the activity level of the therapeutic protein or peptide in the patient, for instance, detectable increases in the activities of insulin or GLP-1.
  • Such formulations and methods may deliver between about 1 pg to about 100 mg /kg body weight of fusion protein, about 100 ng to about 100 ⁇ g/kg body weight of fusion protein, about 100 ⁇ g/ to about 100 mg/kg body weight of fusion protein, about 1 ⁇ g to about 1 g of fusion protein, about 10 ⁇ g to about 100 mg of fusion protein or about 10 mg to about 50 mg of fusion protein.
  • Formulations may also be calculated using a unit measurement of therapeutic protein activity, such as about 5 to about 500 units of human insulin or about 10 to about 100 units of human insulin.
  • the measurements by weight or activity can be calculated using known standards for each therapeutic protein or peptide fused to Tf.
  • the invention also includes methods of orally administering the pharmaceutical compositions of the invention.
  • Such methods may include, but are not limited to, steps of orally administering the compositions by the patient or a caregiver.
  • Such administration steps may include administration on intervals such as once or twice per day depending on the Tf fusion protein, disease or patient condition or individual patient.
  • Such methods also include the administration of various dosages of the individual Tf fusion protein.
  • the initial dosage of a pharmaceutical composition may be at a higher level to induce a desired effect, such as reduction in blood glucose levels. Subsequent dosages may then be decreased once a desired effect is achieved.
  • These changes or modifications to administration protocols may be done by the attending physician or health care worker.
  • the changes in the administration protocol may be done by the individual patient, such as when a patient is monitoring blood glucose levels and administering a mTf- insulin or mTf-GLP-1 oral composition of the invention.
  • the invention also includes methods of producing oral compositions or medicant compositions of the invention comprising formulating a Tf fusion protein of the invention into an orally administerable form. In other instances, the invention includes methods of producing compositions or medicant compositions of the invention comprising formulating a Tf fusion protein of the invention into a form suitable for oral administration.
  • the present invention includes pulmonary delivery of the Tf fusion protein formulations.
  • Pulmonary delivery is particularly promising for the delivery of macromolecules which are difficult to deliver by other routes of administration.
  • Such pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs, since drugs delivered to the lung are readily absorbed through the alveolar region directly into the blood circulation.
  • the present invention provides compositions suitable for forming a drug dispersion for oral inhalation (pulmonary delivery) to treat various conditions or diseases.
  • the Tf fusion protein formulation could be delivered by different approaches such as liquid nebulizers, aerosol-based metered dose inhalers (MDFs), and dry powder dispersion devices.
  • MDFs aerosol-based metered dose inhalers
  • dry powder dispersion devices In formulating compositions for pulmonary delivery, pharmaceutically acceptable carriers including surface active agents or surfactants and bulk carriers are commonly added to provide stability, dispersibility, consistency, and/or bulking characteristics to enliance uniform pulmonary delivery of the composition to the subject.
  • Useful surface active agents or surfactants promotes abso ⁇ tion of polypeptide through mucosal membrane or lining.
  • Useful surface active agents or surfactants include fatty acids and salts thereof, bile salts, phospholipid, or an alkyl saccharide. Examples of fatty acids and salts thereof include sodium, potassium and lysine salts of caprylate (C 8 ), caprate (C I Q), laurate ( 2 ) and myristate (C ⁇ 4 ).
  • bile salts include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, and ursodeoxycholic acid.
  • Examples of phospholipids include single-chain phospholipids, such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine; or double-chain phospholipids, such as diacylphosphatidylcholines, diacylphosphatidylglycerols, diacylphosphatidylethanolamines, diacylphosphatidylinositols and diacylphosphatidylserines.
  • alkyl saccharides include alkyl glucosides or alkyl maltosides, such as decyl glucoside and dodecyl maltoside.
  • compositions that are useful as carriers include stabilizers such as human serum albumin (HSA); bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amo ⁇ hous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • carbohydrates for use as bulking agents include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, frehalose, and the like; cyclodexfrins, such as 2-hydroxypropyl-.beta.-cyclodexfrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like.
  • polypeptides for use as bulking agents include aspartame.
  • Amino acids include alanine and glycine, with glycine being preferred.
  • Additives which are minor components of the composition, may be included for conformational stability during spray drying and for improving dispersibility of the powder.
  • additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.
  • Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • the Tf fusion compositions for pulmonary delivery may be packaged as unit doses where a therapeutically effective amount of the composition is present in a unit dose receptacle, such as a blister pack, gelatin capsule, or the like.
  • a unit dose receptacle such as a blister pack, gelatin capsule, or the like.
  • the manufacture of blister packs or gelatin capsules is typically carried out by methods that are generally well known in the packaging art.
  • U.S. Patent 6,524,557 discloses a pharmaceutical aerosol formulation comprising (a) a HFA propellant; (b) a pharmaceutically active polypeptide dispersible in the propellant; and (c) a surfactant which is a C 8 -C ⁇ 6 fatty acid or salt thereof, a bile salt, a phospholipid, or an alkyl saccharide, which surfactant enhances the systemic abso ⁇ tion of the polypeptide in the lower respiratory fract.
  • the invention also provides methods of manufacturing such formulations and the use of such formulations in treating patients.
  • One approach for the pulmonary delivery of dry powder drugs utilizes a hand-held device with a hand pump for providing a source of pressurized gas.
  • the pressurized gas is abruptly released through a powder dispersion device, such as a venturi nozzle, and the dispersed powder made available for patient inhalation.
  • the present invention provides formulating Tf fusion protein for oral inhalation.
  • the formulation comprises Tf fusion protein and suitable pharmaceutical excipients for pulmonary delivery.
  • the present invention also provides administering the Tf fusion protein composition via oral inhalation to subjects in need thereof.
  • transgenic non-human animals that contain a modified transferrin fusion construct with increased serum half-life increased serum stability or increased bioavailability of the instant invention is contemplated in one embodiment of the present invention.
  • lactoferrin may be used as the Tf portion of the fusion protein so that the fusion protein is produced and secreted in milk.
  • mice such as domesticated mammals including cows, pigs, goats, horses, cattle, and sheep
  • animals which express large quantities of exogenous proteins in an easily harvested form (e.g., expression into the milk or blood)
  • animals with increased weight gain, feed efficiency, carcass composition, milk production or content, disease resistance and resistance to infection by specific microorganisms and the production of animals having enhanced growth rates or reproductive performance.
  • Animals which contain exogenous DNA sequences in their genome are referred to as transgenic animals.
  • the most widely used method for the production of transgenic animals is the microinjection of DNA into the pronuclei of fertilized embryos (Wall et al, J. Cell.
  • PCT International Application WO 90/08832 [1990]; and Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995].
  • PCT International Application WO 90/08832 describes the injection of wild-type feline leukemia virus B into the perivitelline space of sheep embryos at the 2 to 8 cell stage. Fetuses derived from injected embryos were shown to contain multiple sites of integration.
  • U.S. Patent 6,291,740 (issued September 18, 2001) describes the production of transgenic animals by the introduction of exogenous DNA into pre-maturation oocytes and mature, unfertilized oocytes (i.e., pre- fertilization oocytes) using retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]).
  • retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]).
  • MMV murine leukemia virus
  • U.S. Patent 6,281,408 (issued August 28, 2001) describes methods for producing transgenic animals using embryonic stem cells. Briefly, the embryonic stem cells are used in a mixed cell co-culture with a morula to generate transgenic animals. Foreign genetic material is introduced into the embryonic stem cells prior to co-culturing by, for example, electroporation, microinjection or retroviral delivery. ES cells transfected in this manner are selected for integrations of the gene via a selection marker such as neomycin.
  • a selection marker such as neomycin.
  • Patent 6,271,436 (issued August 7, 2001) describes the production of transgenic animals using methods including isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals.
  • the efficiency at which transgenic animals are generated is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.
  • modified transferrin fusion constructs for gene therapy wherein a modified transferrin protein or transferrin domain is joined to a therapeutic protein or peptide is contemplated in one embodiment of this invention.
  • modified fransferrin fusion constructs with increased serum half-life or serum stability of the instant invention are ideally suited to gene therapy treatments.
  • gene therapy via injection of an adenovirus vector containing a gene encoding a soluble fusion protein consisting of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglubulin GI was recently shown in Ijima et al. (June 10, 2001) Human Gene Therapy (United States) 12/9:1063-77.
  • CTL4 cytotoxic lymphocyte antigen 4
  • a murine model of type II collagen-induced arthritis was successfully treated via intraarticular inj ection of the vector.
  • U.S. Patent 6,225,290 provides methods and constructs whereby intestinal epithelial cells of a mammalian subject are genetically altered to operatively inco ⁇ orate a gene which expresses a protein which has a desired therapeutic effect.
  • Intestinal cell transformation is accomplished by administration of a formulation composed primarily of naked DNA, and the DNA may be administered orally.
  • Oral or other intragastrointestinal routes of adminisfration provide a simple method of administration, while the use of naked nucleic acid avoids the complications associated with use of viral vectors to accomplish gene therapy.
  • the expressed protein is secreted directly into the gastrointestinal fract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein.
  • the transformed intestinal epithelial cells provide short or long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.
  • U.S. Pat. 6,187,305 provides methods of gene or DNA targeting in cells of vertebrate, particularly mammalian, origin. Briefly, DNA is introduced into primary or secondary cells of vertebrate origin through homologous recombination or targeting of the DNA, which is introduced into genomic DNA of the primary or secondary cells at a preselected site.
  • U.S. Pat. 6,140,111 (issued October 31, 2000) describes retroviral gene therapy vectors.
  • the disclosed retroviral vectors include an insertion site for genes of interest and are capable of expressing high levels of the protein derived from the genes of interest in a wide variety of transfected cell types.
  • retroviral vectors lacking a selectable marker, thus rendering them suitable for human gene therapy in the treatment of a variety of disease states without the co-expression of a marker product, such as an antibiotic.
  • These retroviral vectors are especially suited for use in certain packaging cell lines.
  • the ability of retroviral vectors to insert into the genome of mammalian cells has made them particularly promising candidates for use in the genetic therapy of genetic diseases in humans and animals.
  • Genetic therapy typically involves (1) adding new genetic material to patient cells in vivo, or (2) removing patient cells from the body, adding new genetic material to the cells and reintroducing them into the body, i.e., in vitro gene therapy.
  • Discussions of how to perform gene therapy in a variety of cells using retroviral vectors can be found, for example, in U.S. Pat. Nos. 4,868,116, issued Sep. 19, 1989, and 4,980,286, issued Dec. 25, 1990 (epithelial cells), WO89/07136 published Aug. 10, 1989 (hepatocyte cells) , EP 378,576 published Jul. 25, 1990 (fibroblast cells), and WO89/05345 published Jun. 15, 1989 and WO/90/06997, published Jun. 28, 1990 (endothelial cells), the disclosures of which are inco ⁇ orated herein by reference.
  • GLP-1 is a peptide that regulates insulin secretion. It possesses anti-diabetic activity in human subjects suffering diabetes, especially type II diabetes. Like other peptides, GLP- 1 has a short plasma half-life in humans.
  • the present invention provides fusion proteins with GLP-1 fused to mTf with increased half-life and pharmaceutical compositions of such fusion proteins for the treatment of diseases associated with abnormal glucose levels that can be administered orally.
  • the present invention also provides fusion proteins comprising an GLP-1 analog and mTF.
  • the GLP-1 analog comprises an additional His residue at the N-terminus. The His residue could be added to the N-terminus of GLP-1 or inserted after the His residue at the N-terminus of GLP-1.
  • the GLP-1 analog comprises an amino acid substitution at position 2. For example, the Ala in GLP- 1(7-36) or GLP- 1(7-37) peptide (SEQ ID NO: 6) is substituted with another amino acid.
  • the steps for producing a GLP-1 /mTf fusion protein are described.
  • GLP-1 /mTf fusion protein the amino acid sequence of GLP- 1(7-36) and GLP- 1(7-37) may be used.
  • haegtftsdvssylegqaakefiawlv gr (SEQ ID NO: 7) haegtftsdvssylegqaakefiawlvkgrg (SEQ ID NO: 8)
  • the peptide sequence of GLP- 1(7-36) may be back translated into DNA and codon optimized for yeast:
  • the primers were specifically designed to form 5' Xbal and 3' Kpnl sticky ends after annealing and to enable direct ligation into Xbal/Kpnl cut pREX0052, just 5' of the end of the leader sequence and at the N-terminus of mTf.
  • other sticky ends may be engineered for ligations into other vectors.
  • Top strand SEQ ID NO: 15
  • Bottom strand SEQ ID NO: 16
  • Top strand primer P0056 (nucleotides 7-112 of SEQ ID NO: 15)
  • This vector was designated pREX0094.
  • the cassette was cut out of pREX0094 with Notl and sub-cloned into Notl cut yeast vector, pSAC35, to make pREXOlOO.
  • Insulin is a peptide hormone that is secreted by the islets of Langerhans in the pancreas and that regulates the metabolism of carbohydrates and fats, in particular the conversion of glucose to glycogen. It is given to humans suffering from type I and type II diabetes, as well as to diabetic animals. Currently, insulin must be administered by subcutaneous injection and has a short plasma half-life in humans.
  • the present invention provides fusion proteins of insulin fused to mTf that have increased half-life and pharmaceutical compositions of such fusion proteins for the freatment of diseases associated with abnormal glucose levels that can be administered orally.
  • Saccharomyces constructs were initially made in the base vector pREX0052, comprising an E. coli cloning vector with a cassette for the expression of mTf in yeast, as either inserts between the 5' XballKpnl sites for the N-terminal fusion, or 3' SaWHindlll sites for the C-terminal fusion.
  • constructs form an expression cassette with (5' to 3') the yeast PRB1 promoter, leader sequence directing secretion into the growth media, (N-terminal fusion), mTf sequence, (C-terminal fusion), stop codons and the ADHl terminator sequence.
  • the expression cassettes were recovered as Notl fragments and inserted into Notl digested pSAC35, an E.coli/y east shuttle vector.
  • the insulin sequence used corresponds to that of Genbank Accession No. NM_000207, SEQ ID NOS: 22 (DNA) and 23 (protein), as shown below.
  • the cDNA for the above sequence can be generated in a number of ways, e.g., by RT-PCR, from a cDNA pool, or by overlapping synthetic oligonucleotides. To generate a clone from a cDNA pool, two primers were synthesized and used as PCR primers.
  • a 5' mutagenic primer was used to create a second PCR product using the first PCR product as template. This primer inserted the last 5 amino acids of the leader sequence and the Xbal site. The Kpnl site could not be inserted by this method, as an amino acid change would have resulted from the creation of the Kpnl site. Instead, the PCR product was digested with Xbal/PvuII. A linker was then made of two overlapping oligos with a Pvu ⁇ l 5' end and 3' overhang which would ligate to the Kpnl overhang on Kpnl digested pREX0052.
  • 5' primer 5'-gcttactctaggtctctagataagaggtttgtgaaccaacacctgtgcg-3' (SEQ ID NO: 28)
  • Linkers 5'-ctggagaactactgcaacgtac-3' (SEQ ID NO: 29) 3'-gacctcttgatgacgttg-5' (SEQ ID NO: 30)
  • 5' and 3' mutagenic primers were used to create a second PCR product using the first PCR product as template. This product was then digested with SaWHind ⁇ ll and ligated into SaWHind ⁇ ll digested pREX0052. This resulted in the plasmid pREX0052 C-insulin (SEQ ID NOS: 31 and 32).
  • Hindlll 241 atctgctccc tctaccagct ggagaactac tgcaactaat aagcttaatt tagacgaggg agatggtcga cctcttgatg acgttgatta ttcgaattaa
  • the plasmids pREX0052 N-insulin and pREX0052 C-insulin were digested with Notl and the expression cassettes recovered. These were then ligated into Notl digested pSAC35 to give pSAC35 ⁇ -insulin and pSAC35 C-insulin. These plasmids were then electroporated into the host Saccharomyces yeast strains and fransformants selected for leucine prototrohy on minimal media plates. Expression was determined by growth in liquid minimal media and analysis of superanatant by SDS-PAGE, western blot, ELISA and BIAcore.
  • proinsulin attached to transferrin.
  • Proteases in yeast may convert the proinsulin to insulin as it is being made and secreted, although the final expression product may contain only proinsulin. In that case, the proinsulin can be converted to insulin post-expression using an appropriate purified protease.
  • diabetic rats are first prepared. Female Sprague-Dawley rats are fasted for 24 hours and their blood glucose level determined to establish a baseline. The rats are then injected intraperitoneally with a solution of sfreptozotocin (STZ), 60 mg/ml, at a dosage of 60 mg/kg. I.p. injections of STZ are continued for four more days, and rats with a fasting blood glucose level above 300 mg/dl are selected as diabetic rats.
  • STZ sfreptozotocin
  • Solutions of the fusion protein and of insulin alone are prepared in PBS or sodium bicarbonate to provide dosages of 7 to 80 units of insulin/kg when administered to rats.
  • rats are also freated with PBS alone.
  • the solutions or PBS are administered by oral gavage to rats following a 12 hour fast, and blood samples are collected from the tail after 0, 30 and 60 minutes, and then at 2-hour intervals. Blood glucose levels at 0, 0.5, 1, 3, 5, 7, 9 and 11 hours after dosing are measured with a blood glucose monitoring device designed for diabetics, and the rats are fed again at 11 hours post-dose.
  • the activity of the insulin is determined by measuring the decrease in blood glucose level over time, correcting the decrease by any increases or decreases in the PBS-only samples.
  • the insulin activities of the fusion protein versus unfused insulin are compared.
  • fusion protein and unfused insulin as a control are administered to diabetes induced rats as described above and transport measured using standard sandwich ELISAs and serum samples.
  • 125 I-labeled fusion protein or 125 I-labeled unfused insulin is adminstered to diabetes-induced rats at dosage of 80 U insulin/kg by oral gavage, as described above.
  • Blood samples are collected from the tail after 0, 30 and 60 minutes, then at 2-hour intervals, also as described above, and serum samples are analyzed by HPLC, using, for example a Sephacryl column and eluting samples with PBS.
  • Standards containing I-labeled fransferrin, I-labeled insulin and I-labeled fusion protein are also run on the Sephacryl column to determine their peak elution times and fraction numbers.
  • the radioactivity of each fraction is measured with a gamma counter, and the protein content of each fraction is measured by the absorbance at 280 nm.
  • Serum samples from rats treated with the fusion protein may not show the appearance of the fusion protein immediately, as there may be a delay of a few hours.
  • Therapeutic mTf fusion protein with increased iron affinity may be prepared.
  • modified transferrin fusion proteins with increased iron binding ability the procedure in Example 2 above may be carried out with the following modification. These fusion proteins may be used to facilitate uptake and transfer of the fusion protein across the gastrointestinal epithelium.
  • a cloning vector such as pREX0052, described above, which contains the mTf sequence is cut with a restriction enzyme, or a pair of restriction enzymes, to remove a portion of the mTf gene.
  • this fragment is then subjected to site-directed mutagenesis using primers that introduce a mutation at a position corresponding to nucleotide 723 of SEQ ID NO: 1, converting the codon AAG (Lys) to CAG (Gin) or GAG (Glu).
  • primers are used that introduce mutations at positions corresponding to nucleotides 726 and 728 of SEQ ID NO: 1, converting the codon CAC (His) to CAG (Gin) or GAG (Glu).
  • Primers may also be used that infroduce mutations at all three nucleotide positions, resulting in the substitution of two adjacent amino acids. These nucleotide positions correspond to amino acids 225 and 226 of the protein encoded with the leader sequence and to amino acids 206 and 207 of the mature protein.
  • the mutated fragment is then amplified by RT-PCR and religated into the cloning vector. This vector containing the mutation or mutations is used in a subsequent step for introduction of a DNA molecule coding for the therapeutic protein.
  • the mTf fusion protein sequence may be introduced into yeast expression vectors and transformed into Saccharomyces or other yeasts for protein production.
  • amino acids may also be mutated to to obtain therapeutic mTf proteins with increased iron affinity.
  • Example 4 ⁇ -interferon-transferrin fusion proteins ⁇ -IFN is effective in the freatment of various diseases, such as, but not limited to, multiple sclerosis, brain tumor, skin cancer, and hepatitis B and C. Like most cytokines, ⁇ - IFN has a short circulation half-life.
  • the present invention provides fusion proteins comprising ⁇ -IFN fused to mTf with increased half-life and efficacy in patients. This example describes the steps in generating a ⁇ -IFN/mTf fusion protein that may be administered orally.
  • IFN ⁇ -1 is fused to modified transferrin at either the N- or C- termini.
  • the IFN ⁇ -1 clone was obtained from the ATCC (No. 39517). Specifically designed primers were used to confirm the DNA sequence of the IFN ⁇ -1 clone. These primers were external to the IFN ⁇ -1 DNA sequence and designed to read in from the vector such that the full-length sequence of the clone was obtained. The primers used were:
  • primers were designed for fusion of IFN ⁇ -1 to mTf.
  • the N-terminal fusion was a two step process. A straight fusion using primers with Xbal and Kpnl sites would have desfroyed the Kpnl site and clipped the beginning of mTf.
  • a linker primers P0082 (nucleotides 18-48 of SEQ ID NO: 37) and P0083 (nucleotides 17-39 SEQ ID NO: 38), was designed to create an internal Kpnl site at the 3' end of IFN ⁇ -1, by a single silent mutation of bp 486 from T to G (bold), and with a 5' Xbal overhang and 3 ' GTAC which would anneal with a Kpnl site. The overhang destroyed the existing Kpnl site in pREX0052.
  • linkers were annealed and ligated into pREX0054 cut with XballKp ⁇ l, creating an intermediate vector with mTf untouched and a Kpnl site that could be used to fuse the IFN ⁇ -1 gene at the N-terminus of mTf.
  • a Xbal/ Kpnl digest of this tailored gene removed the last 5 amino acids of IFN ⁇ -1; however, these were already engineered into the intermediate vector.
  • the resulting construct, pREX0048 was created by ligating the IFN ⁇ -1 gene cut with Xbal/Kpnl into the XballKpnl cut intermediate vector.
  • pREX0048 construct was created, it was sequenced to confirm correct insertion.
  • the expression cassette, as a Notl fragment, was then sub-cloned into Notl cut yeast vector, pSAC35, to make the pREX0050.
  • P0086 SEQ ID NO: 44
  • P0087 SEQ ID NO: 45
  • IFN ⁇ -1 to have Sail andHz ' ndlll sites at the 5' and 3' ends, respectively.
  • the newly tailored product was ligated into SalllHindlll cut pREX0052 to create pREX0049.
  • Prex0049 construct was created, it was sequenced to confirm correct insertion.
  • the expression cassette, as a Notl fragment, is then sub-cloned into Notlcut yeast vector, such as pSAC35, to make pREX0051.
  • Notlcut yeast vector such as pSAC35
  • ⁇ -IF ⁇ -1 GenBank Ace. No. NM_002176
  • SEQ ID NO: 46 is made more stable and soluble by mutating Cys 17 (in the mature protein) to Ser.
  • the mutation of Cys 17 to Ser can be performed by routine mutagenic reactions such as a mutagenic PCR reaction using specifically designed primers and the nucleic acid encoding ⁇ -IFN- 1 as the template.
  • the ⁇ -IFN- 1 is modified to prevent glycosylation by modifying the N-linked glycosylation site, NES/T (residues 80 to 82 of SEQ ID NO: 46).
  • N could be mutagenized to Q and S/T could be mutagenized to Ala or other amino acid acids.
  • Such mutagenesis could be performed with mutagenic PCR reaction using specifically designed primers and the nucleic acid encoding ⁇ -IFN- 1 as the template.

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Abstract

La présente invention concerne des compositions pharmaceutiques contenant des protéines hybrides modifiées de la transferrine et des protéines ou peptides thérapeutiques possédant une demi-vie sérique ou une stabilité sérique augmentée. Les protéines hybrides préférées comprennent celles qui sont modifiées de telle manière que le fragment transferrine ne présente que peu ou pas de glycosylation, mais présente une liaison au fer et/ou au récepteur de la transferrine. Les protéines hybrides précitées peuvent être administrées oralement.
PCT/US2003/026778 2001-08-30 2003-08-28 Delivrance orale de proteines hybrides modifiees de la transferrine WO2004019872A2 (fr)

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EP2088154A1 (fr) 2004-03-09 2009-08-12 Ironwood Pharmaceuticals, Inc. Procédés et compositions pour le traitement de troubles gastro-intestinaux
WO2011069038A2 (fr) 2009-12-03 2011-06-09 Synergy Pharmaceuticals, Inc. Agonistes de la guanylate cyclase utiles dans le traitement de l'hypercholestérolémie, de l'athérosclérose, d'une coronaropathie, des calculs biliaires, de l'obésité et d'autres maladies cardiovasculaires
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US8129504B2 (en) 2001-08-30 2012-03-06 Biorexis Technology, Inc. Oral delivery of modified transferrin fusion proteins
US7176278B2 (en) 2001-08-30 2007-02-13 Biorexis Technology, Inc. Modified transferrin fusion proteins
EP2088154A1 (fr) 2004-03-09 2009-08-12 Ironwood Pharmaceuticals, Inc. Procédés et compositions pour le traitement de troubles gastro-intestinaux
EP1814599A2 (fr) * 2004-08-03 2007-08-08 Biorexis Pharmaceutical Corporation Polytherapie basee sur l'utilisation de proteines hybrides de transferrine contenant du glp-1
EP1814599A4 (fr) * 2004-08-03 2008-12-17 Biorexis Pharmaceutical Corp Polytherapie basee sur l'utilisation de proteines hybrides de transferrine contenant du glp-1
CN101511868B (zh) * 2006-07-24 2013-03-06 比奥雷克西斯制药公司 毒蜥外泌肽融合蛋白
WO2008012629A3 (fr) * 2006-07-24 2008-05-08 Biorexis Pharmaceutical Corp Protéines de fusion d'extendine
US8158579B2 (en) 2006-07-24 2012-04-17 Biorexis Pharmaceutical Corporation Fusion protein of an exendin to modified transferrin
US7867972B2 (en) 2006-07-24 2011-01-11 Pharmacia & Upjohn Company, Llc Fusion protein of exendin-4 to a transferrin (Tf) polypeptide
WO2008033395A2 (fr) * 2006-09-14 2008-03-20 Biorexis Pharmaceutical Corporation Protéines de fusion mélanocortine et transferrine
WO2008033395A3 (fr) * 2006-09-14 2008-05-15 Biorexis Pharmaceutical Corp Protéines de fusion mélanocortine et transferrine
US8822639B2 (en) 2006-12-12 2014-09-02 Biorexis Pharmaceutical Corporation Transferrin fusion protein libraries
EP2998314A1 (fr) 2007-06-04 2016-03-23 Synergy Pharmaceuticals Inc. Agonistes de guanylase cyclase utiles pour le traitement de troubles gastro-intestinaux, d'inflammation, de cancer et d'autres troubles
US20110288023A1 (en) * 2007-06-08 2011-11-24 The Regents Of The University Of California Office Of Technology Transfer Cancer drug delivery using modified transferrin
US20090181048A1 (en) * 2007-06-08 2009-07-16 The Regents Of The University Of California Cancer drug delivery using modified transferrin
EP2810951A2 (fr) 2008-06-04 2014-12-10 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utile dans le traitement de troubles gastro-intestinaux, d'une inflammation, d'un cancer et d'autres troubles
EP3241839A1 (fr) 2008-07-16 2017-11-08 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utiles pour le traitement de troubles gastro-intestinaux, inflammatoires, cancéreux et autres
WO2011069038A2 (fr) 2009-12-03 2011-06-09 Synergy Pharmaceuticals, Inc. Agonistes de la guanylate cyclase utiles dans le traitement de l'hypercholestérolémie, de l'athérosclérose, d'une coronaropathie, des calculs biliaires, de l'obésité et d'autres maladies cardiovasculaires
EP2923706A1 (fr) 2009-12-03 2015-09-30 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utiles pour le traitement de l'hypercholestérolémie
US20120004398A1 (en) * 2010-07-02 2012-01-05 University Of Southern California Method for Uses of Protein Precursors as Prodrugs
US20130130967A1 (en) * 2010-07-02 2013-05-23 University Of Southern California Method for Uses of Protein Precursors as Prodrugs
US10513563B2 (en) * 2010-07-02 2019-12-24 University Of Southern California Method for uses of proinsulin transferrin fusion proteins as prodrugs
US9611323B2 (en) 2010-11-30 2017-04-04 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses therefor
US10941215B2 (en) 2010-11-30 2021-03-09 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses thereof
EP4309673A2 (fr) 2012-03-15 2024-01-24 Bausch Health Ireland Limited Formulations d'agonistes de guanylate cyclase c et leurs procédés d'utilisation
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WO2014029924A1 (fr) * 2012-08-23 2014-02-27 Allain Henri Nouvelles applications de la lactoferrine et compositions pharmaceutiques et nutraceutiques
FR2994657A1 (fr) * 2012-08-23 2014-02-28 Henri Allain Nouvelles applications et utilisations de la lactoferrine
WO2014131024A2 (fr) 2013-02-25 2014-08-28 Synergy Pharmaceuticals Inc. Agonistes de la guanylate cyclase et applications associées
EP3718557A2 (fr) 2013-02-25 2020-10-07 Bausch Health Ireland Limited Agoniste du récepteur de la guanylate cyclase sp-333 à utiliser lors du nettoyage du côlon
WO2014151206A1 (fr) 2013-03-15 2014-09-25 Synergy Pharmaceuticals Inc. Agonistes de la guanylate cyclase et leurs utilisations
WO2014151200A2 (fr) 2013-03-15 2014-09-25 Synergy Pharmaceuticals Inc. Compositions utiles pour le traitement de troubles gastro-intestinaux
WO2015054649A2 (fr) 2013-10-10 2015-04-16 Synergy Pharmaceuticals, Inc. Agonistes de guanylate cyclase utiles pour le traitement de troubles induits par les opioïdes
WO2017123634A1 (fr) 2016-01-11 2017-07-20 Synergy Pharmaceuticals, Inc. Formulations et méthodes pour traiter la rectocolite hémorragique
US11753455B2 (en) 2018-06-21 2023-09-12 Novo Nordisk A/S Compounds for treatment of obesity
CN112516168A (zh) * 2020-12-11 2021-03-19 中国人民解放军军事科学院军事医学研究院 用于干预应激性认知障碍的间充质干细胞

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