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WO2008021412A2 - PROTÉINES DE FUSION DE L'INTERFÉRON β ET DE LA TRANSFERRINE - Google Patents

PROTÉINES DE FUSION DE L'INTERFÉRON β ET DE LA TRANSFERRINE Download PDF

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Publication number
WO2008021412A2
WO2008021412A2 PCT/US2007/018099 US2007018099W WO2008021412A2 WO 2008021412 A2 WO2008021412 A2 WO 2008021412A2 US 2007018099 W US2007018099 W US 2007018099W WO 2008021412 A2 WO2008021412 A2 WO 2008021412A2
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Prior art keywords
ifnβ
fusion protein
transferrin
seq
amino acid
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PCT/US2007/018099
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WO2008021412A3 (fr
Inventor
Homayoun Sadeghi
Andrew John Turner
Christopher Philip Prior
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Biorexis Pharmaceutical Corp
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Biorexis Pharmaceutical Corp
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Anticipated expiration legal-status Critical
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Classifications

    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to IFN ⁇ therapeutic proteins or peptides with extended serum stability or serum half-life fused to or inserted in a transferrin molecule modified to reduce or inhibit glycosylation, iron binding and/or transferrin receptor binding.
  • Therapeutic proteins or peptides in their native state or when recombihantly produced are typically labile molecules exhibiting short periods of serum stability or short serum half-lives. In addition, these molecules are often extremely labile when formulated, particularly when formulated in aqueous solutions for diagnostic and therapeutic purposes.
  • PEG Polyethylene glycol
  • Therapeutic proteins or peptides have also been stabilized by fusion to a heterologous protein capable of extending the serum half-life of the therapeutic protein.
  • therapeutic proteins fused to albumin and antibody fragments may exhibit extended serum half-live when compared to the therapeutic protein in the unfused state. See U.S. Patents 5,876,969 and 6,972,322.
  • glycosylated human transferrin Tf
  • Tf glycosylated human transferrin
  • 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 barrier.
  • Transferrin fusion proteins have also been produced by inserting an HIV-I protease sequence into surface exposed loops of glycosylated transferrin to investigate the ability to produce another form of Tf fusion for targeted delivery to the inside of a cell via the Tf receptor (AIi et al. (1999) J. Biol. Chem. 274(34):24066-24073).
  • Serum transferrin is a monomelic glycoprotein with a molecular weight of 80,000 daltons that binds iron in the circulation and transports it to various tissues via the transferrin 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 serum molecules, comprising up to about 5-10% of total serum proteins.
  • Carbohydrate deficient transferrin occurs in elevated levels in the blood of alcoholics and exhibits a longer half life (approximately 14-17 days) than that of glycosylated transferrin (approximately 7-10 days).
  • Carbohydrate deficient transferrin occurs in elevated levels in the blood of alcoholics and exhibits a longer half life (approximately 14-17 days) than that of glycosylated transferrin (approximately 7-10 days).
  • Tf The structure of 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) /. Biol. Chem. 258(6):3543- 3546).
  • Transferrin and antibodies that bind the transferrin 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 vehicle to deliver DNA to cells (Frank et al., 1994; Wagner et al, 1992).
  • Transferrin fusion proteins have not, however, been modified or engineered to extend the serum half-life of a therapeutic protein or peptide or to increase bioavailability by reducing or inhibiting glycosylation of the Tf moiety or to reduce or prevent iron and/or Tf receptor binding.
  • the present invention includes fusion proteins comprising at least one interferon- ⁇ protein (IFN ⁇ ), polypeptide or peptide entity and transferrin (Tf).
  • IFN ⁇ interferon- ⁇ protein
  • Tf transferrin
  • the IFN ⁇ moiety is modified.
  • the present invention includes fusion proteins comprising an IFN ⁇ moiety modified by deletion of an initial methionine residue and/or an amino acid substitution of a free cysteine residue at position 17.
  • the IFN ⁇ moiety is modified to exhibit reduced or no glycosylation.
  • the IFN ⁇ moiety can be modified to exhibit no N-linked glycosylation by mutating an amino acid within or adjacent to the N-glycosylation site NET.
  • the Tf portion is engineered to extend the serum half-life or bioavailability of the interferon- ⁇ molecule.
  • the invention also includes pharmaceutical formulations and compositions comprising the fusion proteins, methods of extending the serum stability, serum half-life and bioavailability of interferon- ⁇ by fusion to transferrin, nucleic acid molecules encoding the fusion proteins and the like.
  • Another aspect of the present invention relates to methods of treating a patient with an interferon- ⁇ and transferrin fusion protein.
  • the fusion proteins comprise a human transferrin moiety that has been modified to reduce or prevent glycosylation and/or iron and receptor binding.
  • the Tf moiety contains A-
  • Figure 1 shows an alignment of the N and C Domains of Human (Hu) transferrin (Tf) with similarities and identities highlighted.
  • Figures 2A-2B show an alignment of transferrin sequences from different species.
  • the light shading represents similarity.
  • the dark shading represents identity.
  • Figure 3 shows the location of a number of Tf surface exposed insertion sites for therapeutic proteins, polypeptides or peptides.
  • Figure 4 is a vector map of pREX0549.
  • Figure 5 is a vector map of pREXOl 97.
  • Figure 6 is a vector map of pREX0435.
  • Figure 7 is a vector map of pcDNA3.1 IFN ⁇ mTF.
  • Figure 8 is a vector map of pREX1020.
  • Figure 9 shows the plasma levels of fusion protein determined by sandwich ELISA assay using antibodies to both transferrin and interferon.
  • Figure 10 shows the neopterin response in cynomolgus monkeys following a single injection of IFN ⁇ /Tf fusion proteins.
  • Figure 11 shows the 2'-5'-OAS response in a cynomolgus monkey following a single injection of BRXl 007 IFN ⁇ /Tf fusion protein.
  • the present invention is based in part on the finding by the inventors that interferon- ⁇ (IFN ⁇ ) can be stabilized to extend their serum half-life and/or activity in vivo by genetically fusing the IFN ⁇ to transferrin, modified transferrin, or a portion of transferrin or modified transferrin sufficient to extend the half-life of the therapeutic protein in serum.
  • the modified transferrin fusion proteins include a transferrin protein or domain covalently linked to a therapeutic protein or peptide, wherein the transferrin portion is modified to contain one or more amino acid substitutions, insertions or deletions compared to a wild- type transferrin 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).
  • TfR Tf receptor
  • the IFN ⁇ therapeutic proteins contemplated by the present invention include, but are not limited to polypeptides, antibodies, peptides, or fragments or variants thereof.
  • the present invention therefore includes IFN ⁇ and transferrin fusion proteins, therapeutic compositions comprising the fusion proteins, and methods of treating, preventing, or ameliorating diseases or disorders by administering the fusion proteins.
  • a transferrin fusion protein of the invention includes at least a fragment or variant of an IFN ⁇ and at least a fragment or variant of modified transferrin, which are associated with one another, preferably by genetic fusion (i.e., the transferrin 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 transferrin) or chemical conjugation to one another.
  • the IFN ⁇ therapeutic protein and transferrin protein once part of the fusion protein, may be referred to as a "portion", "region” or “moiety” of the transferrin fusion protein (e.g., a "IFN ⁇ protein portion' or a "transferrin protein portion”).
  • the invention provides a transferrin fusion protein comprising, or alternatively consisting of, an IFN ⁇ and a modified serum transferrin protein. In other embodiments, the invention provides a fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of IFN ⁇ and a modified transferrin protein. In other embodiments, the invention provides a transferrin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of IFN ⁇ and modified transferrin protein. In further embodiments, the invention provides a fusion protein comprising IFN ⁇ and a biologically active and/or therapeutically active fragment of modified transferrin.
  • the therapeutic protein portion of the transferrin fusion protein is the active form of IFN ⁇ .
  • 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 counterpart.
  • a fusion protein with or without a flexible and/or substantially non-helical linker, substantially exhibits prolonged biological activity if it exhibits one or more biological activities by at least about 2 fold, about 5 fold, about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 200 fold, or at least about 300 fold or more, longer in duration, i.e., period of time, than the same one or more biological activities of unfused IFN ⁇ or native IFN ⁇ , either in vivo or in vitro.
  • a fusion protein with a substantially non-helical linker substantially exhibits prolonged activity if it exhibits one or more biological activities by at least about 2 fold, about 5 fold, about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 200 fold, or at least about 300 fold or more, longer in duration, i.e., period of time, than the same one or more biological activities of a IFN ⁇ and Tf fusion protein lacking a linker sequence or a IFN ⁇ and Tf fusion protein with a flexible linker, either in vivo or in vitro.
  • an "amino acid corresponding to" or an "equivalent amino acid" in a transferrin sequence is identified by alignment to maximize the identity or similarity between a first transferrin sequence and at least a second transferrin sequence.
  • the number used to identify an equivalent amino acid in a second transferrin sequence is based on the number used to identify the corresponding amino acid in the first transferrin sequence. In certain cases, these phrases may be used to describe the amino acid residues in human transferrin compared to certain residues in rabbit serum transferrin.
  • 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 occurring Tf protein or mutant thereof.
  • genes refers to any segment of DNA associated with a biological function.
  • genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression.
  • Genes can also include nonexpressed 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 incorporation 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, semisynthetic, 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 polypeptide fusion.
  • 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 —terminal end of Tf, attachment to the C-terminal end of Tf, and/or insertion between any two amino acids within Tf.
  • modified transferrin refers to a transferrin molecule that exhibits at least one modification of its amino acid sequence, compared to wildtype transferrin.
  • modified transferrin fusion protein refers to a protein formed by the fusion of at least one molecule of modified transferrin (or a fragment or variant thereof) to at least one molecule of a therapeutic protein (or fragment or variant thereof), preferably an antibody variable region.
  • nucleic acid or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. 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 occurring 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 at. (1992); Rossolini et al. (1994) MoI. Cell. Probes 8:91-98).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • DNA segment is referred 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.
  • potency refers to the ability of IFN ⁇ to induce a biological response in vitro or in vivo.
  • potency can be used to refer to the ability of IFN ⁇ to inhibit proliferation of infected cells and stimulate the immune system.
  • the in vivo potency of an IFN ⁇ fusion protein of the invention is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 30 fold, about 40 fold, or about 50 fold or more compared to unfused IFN ⁇ .
  • the in vivo potency of an IFN ⁇ fusion protein containing a substantially non-helical linker is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 30 fold, about 40 fold, or about 50 fold or more compared to an IFN ⁇ fusion protein without a substantially non-helical linker, for instance, a fusion protein containing a flexible linker.
  • promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
  • the term "subject" can be a human, a mammal, or an animal.
  • the subject being treated is a patient in need of treatment.
  • the term “recombinant” refers to a cell, tissue or organism that has undergone transformation with recombinant DNA.
  • substantially non-helical linker or “rigid linker” refers to a linker that physically separates the IFN ⁇ and transferrin moieties of a fusion protein. "Substantially non helical” means that linker peptide exhibits little or no helical or spiral shape or secondary structure. For instance, a substantially non-helical structure can comprise less than about 20% helical or spiral shape or secondary structure.
  • a typical alpha-helical peptide is right-handed (twists in a clockwise direction), comprises the amino acid R groups extending to the outside of the helix, the helix making a complete turn at every 3.6 amino acids and the carbonyl group of each peptide bond extends parallel to the axis of the helix and points directly at the N-H group of the peptide bond 4 amino acids below it in the helix with a hydrogen bond forming between them.
  • the non-helical linkers typically contain at least about 5% , at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% amino acids which disrupt any alpha-helix formation, such as amino acids that induce kinks in the polypeptide chain.
  • Kinks can be introduced into a naturally occurring peptide by modifying amino acid residues.
  • Amino acids which cause kinks in the polypeptide chain include, for instance, proline and glycine amino acid residues.
  • proline and glycine amino acid residues For example, the addition of a proline or a glycine at or about the middle of a straight ⁇ helical barrel will cause the protein to bend, i.e., kink.
  • the introduction of a proline residue will generally cause a greater kink than the introduction of a glycine residue.
  • the introduction of a proline or glycine residue anywhere in a linker peptide can cause a kink.
  • a linker peptide can contain one or more amino acid residues which induce kinks.
  • a substantially non-helical linker can have at least about 5% proline content, at least about 10% proline content, at least about 20% proline content, at least about 30% proline content, at least about 40% proline content, at least about 50% proline content, at least about 60% proline content, at least about 70% proline content, at least about 80% proline content, at least about 90% proline content, at least about 95% proline content or about 100% proline content.
  • Substantially non-helical linkers include, but are not limited to, PEAPTD (SEQ ID NO.: 77), PEAPTDPEAPTD (SEQ ID NO.: 78), PEAPTDPEAPTDPEAPTD (SEQ ID NO.: 79), IgG hinge (SEQ ID NO.: 80-82), PEAPTD + IgG hinge (SEQ ID NOS.: 83-92), PPPPPPPPPPPP (SEQ ID NO.: 93), GEAPTDPEAPTD (SEQ ID NO.: 94), PEAGTDPEAPTD (SEQ ID NO.: 95), PEAPTDGEAPTD (SEQ ID NO.: 96), PEAPTDPEAGTD (SEQ ID NO.: 97), PQAPTNPQAPTN (SEQ ID NO.: 98), and PEAPEAPEAPEA (SEQ ID NO.: 99).
  • PEAPTD SEQ ID NO.: 77
  • PEAPTDPEAPTD SEQ ID NO.: 78
  • substantially non-helical linkers have at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10; at least about 1 1, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or at least about 21 or more amino acids.
  • linker length there is no upper limit on linker length.
  • a targeting entity, protein, polypeptide or peptide refers to such molecules 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 transferrin fusion protein or compound (drug, or cytotoxic agent) to that cell type specifically.
  • therapeutic protein refers to IFN ⁇ proteins, polypeptides, peptide fragments or analogs or variants thereof, having one or more therapeutic and/or biological activities.
  • the terms peptides, proteins, and polypeptides are used interchangeably herein.
  • therapeutic protein may refer to the endogenous or naturally occurring correlate 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 IFN ⁇ 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.
  • therapeutically effective amount refers to that amount of the transferrin fusion protein comprising a therapeutic molecule which, when administered to a subject in need thereof, is sufficient to effect treatment.
  • the amount of transferrin 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 of ordinary skill in the art having regard to his/her own knowledge and to this disclosure.
  • the term "toxin” refers to a poisonous substance of biological origin.
  • transformation refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
  • genetic transformation refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.
  • transformant 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 or variant 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 transferrin 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.
  • the term "vector” refers broadly to any plasmid, phagemid or virus encoding an exogenous nucleic acid.
  • the term is also to 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 purpose.
  • 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. ScL U.S.A. 94:12744-12746).
  • Examples of viral vectors include, but are not limited to, a recombinant vaccinia virus, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, 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).
  • Examples of 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 occurring.
  • the present invention provides fusion proteins comprising IFN ⁇ and transferrin or modified transferrin. Any transferrin may be used to make modified Tf fusion-proteins of the invention.
  • Wild-type human Tf is a 679 amino acid protein, of approximately 75kDa (not accounting for glycosylation), with two main domains, N (about 330 amino acids) and C (about 340 amino acids), which appear to originate from a gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847 and S95936, all of which are herein incorporated by reference in their entirety, as well as SEQ ID NOS: 1, 2 and 3. 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 atoms. These are complexed in the space between the Nl and N2, Cl and C2 subdomains resulting in a conformational change in the molecule. Tf crosses the blood brain barrier (BBB) via the Tf receptor.
  • BBB blood brain barrier
  • the iron binding sites comprise at least of amino acids Asp 63 (Asp 82 of SEQ ID NO: 2 which comprises 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); His 585 (His 604 of SEQ IDNO: 2), 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., the carbonate binding sites comprise at least of amino acids Thr 120 (Thr 139 of SEQ ID NO: 2); Thr 452
  • the fusion protein includes a modified human transferrin, 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 (see Figure 2 for a representative set of Tf sequences). 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) 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 transferrin (lactoferrin; GenBank Ace: NM 002343) and melanotransferrin (GenBank Ace. NM_013900, murine melanotransferrin).
  • lacto transferrin lactoferrin; GenBank Ace: NM 002343
  • melanotransferrin GenBank Ace. NM_013900, murine melanotransferrin
  • Lactoferrin 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 polymorphonuclear 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.
  • Melanotransferrin 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 serum transferrin, human lactoferrin, and chicken transferrin (Brown et al, 1982, Nature, 296: 171-173; Rose et al., Proc. Natl. Acad. Sci., 1986, 83: 1261-1265). However, unlike these receptors, no cellular receptor has been identified for melanotransferrin.
  • the transferrin portion of the fusion protein of the invention includes a transferrin splice variant.
  • a transferrin splice variant can be a splice variant of human transferrin.
  • the human transferrin splice variant can be that of Genbank Accession AAA61140.
  • the transferrin portion of the fusion protein of the invention includes a lactoferrin splice variant.
  • a human serum lactoferrin splice variant can be a novel splice variant of a neutrophil lactoferrin.
  • the neutrophil lactoferrin splice variant can be that of Genbank Accession AAA59479.
  • the neutrophil lactoferrin splice variant can comprise the following amino acid sequence EDCIALKGEADA (SEQ ID NO: 5), which includes the novel region of splice- variance.
  • the fusion protein of the present invention 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.
  • the fusion proteins may also be made using a single Tf domain, such as an individual N or C domain.
  • the use of a single or double N domain is advantageous as the Tf glycosylation sites reside in the C domain and the N domain, on its own, does not bind iron or the Tf receptor.
  • fusions of IFN ⁇ to a single or double C domain may be produced, wherein the C domain is altered to reduce, inhibit or prevent glycosylation, iron binding and/or Tf receptor binding. See U.S. Provisional Application 60/406,977, which is herein incorporated by reference in its entirety.
  • a C terminal domain or lobe modified to function as anN-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 corresponding 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.
  • the transferrin portion of the fusion protein includes at least two N terminal lobes of transferrin. In further embodiments, the transferrin portion of the fusion protein includes at least two N terminal lobes of transferrin derived from human serum transferrin.
  • the transferrin portion of the fusion protein includes, comprises, or consists of at least two N terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, and His249 of SEQ ID NO: 3.
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin N-terminal lobe mutant having a mutation at Lys206 or His207 of SEQ ID NO: 3.
  • the transferrin portion of the fusion protein includes, comprises, or consists of at least two C terminal lobes of transferrin. In further embodiments, the transferrin portion of the fusion protein includes at least two C terminal lobes of transferrin derived from human serum transferrin.
  • the C terminal lobe mutant further includes a mutation of at least one of Asn413 and Asn ⁇ l 1 of SEQ ID NO: 3 which does not allow glycosylation.
  • the transferrin portion of the fusion protein includes at least two C terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant retains the ability to bind metal.
  • the transferrin portion of the fusion protein includes at least two C terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a reduced ability to bind metal.
  • the transferrin portion of the fusion protein includes at least two C terminal lobes of transferrin having a mutation in at least one amino acid residue selected from the group consisting of Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO:3, wherein the mutant does not retain the ability to bind metal and functions substantially like an N domain.
  • the Tf or Tf portion will be of sufficient length to increase the serum stability, in vitro solution stability or bioavailability of IFN ⁇ compared to the serum stability (half-life), in vitro stability or bioavailability of IFN ⁇ 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 IFN ⁇ .
  • the fusion proteins comprising modified transferrin exhibit a serum 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 corresponding 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 serum half-life of the fusion protein and/or IFN ⁇ (to produce asialo-, or in some instances, monosialo-Tf or disialo-Tf).
  • mutations maybe to the adjacent residues within the N-X-S/T glycosylation site to prevent or substantially reduce glycosylation. See U.S. Patent 5,986,067 of Funk et al. It has also been reported that the N domain of Tf expressed in Pichia pastoris becomes O-linked glycosylated with a single hexose at S32 which also may be mutated or modified to prevent such glycosylation.
  • the fusion protein includes a modified transferrin molecule wherein the transferrin exhibits reduced glycosylation, including but not limited to asialo- monosialo- and disialo- forms of Tf.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant that is mutated to prevent glycosylation.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant that is folly glycosylated.
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin mutant that is mutated to prevent glycosylation, wherein at least one of-Asn413 and Asn ⁇ l 1 of SEQ ID NO:3 are mutated to an- amino acid which does not allow glycosylation.
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin 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.
  • the Tf portion of the fusion proteins of the invention may also be engineered to not bind iron and/or not bind the Tf receptor.
  • the iron binding is retained and the iron binding ability of Tf may be used in two ways, one to deliver IFN ⁇ to the inside of a cell and/or across the BBB.
  • These embodiments that bind iron and/or the Tf receptor will often be engineered to reduce or prevent glycosylation to extend the serum 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 transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant does not retain the ability to bind metal.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a weaker binding avidity for metal than wild-type serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a stronger binding avidity for metal than wild-type serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant does not retain the ability to bind to the transferrin receptor.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a weaker binding avidity for the transferrin receptor than wild-type serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a stronger binding avidity for the transferrin receptor than wild-type serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant does not retain the ability to bind to carbonate.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a weaker binding avidity for carbonate than wild-type serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant transferrin mutant having a mutation wherein the mutant has a stronger binding avidity for carbonate than wild-type serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin 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.
  • a recombinant human serum transferrin mutant having a mutation in at least one amino acid residue selected from the group consisting of Asp63, Gly65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr5l4, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a reduced ability to bind metal.
  • a recombinant human serum transferrin 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.
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin mutant having a mutation at Lys206 or His207 of SEQ ID NO: 3, wherein the mutant has a stronger binding avidity for metal than wild-type human serum transferrin (see U.S. Patent 5,986,067, which is herein incorporated by reference in its entirety).
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin mutant having a mutation at Lys206 or His207 of SEQ ID NO: 3, wherein the mutant has a weaker binding avidity for metal than wild-type human serum transferrin.
  • the transferrin portion of the fusion protein includes a recombinant human serum transferrin mutant having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the mutant does not bind metal.
  • Any available technique may be used to produce 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 transferrin portion of the fusion protein, e.g., a modified Tf 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 transferrin 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 transferrin domains or linking a transferrin protein and therapeutic protein or peptide or soluble toxin receptor 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 portions of the 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 and/or no or reduced glycosylation.
  • Iron binding and/or receptor binding may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues corresponding 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. Iron binding may also be affected by mutation to amino acids Lys206, Hys207 or Arg632.
  • Carbonate binding may be reduced or disrupted by mutation, including deletion, substitution or insertion into, amino acid residues corresponding to one or more of Tf N domain residues Thrl20, Argl24, Ala 126, GIy 127 and/or C domain residues Thr 452, Arg 456, AIa 458 and/or GIy 459.
  • 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 corresponding to one or more of TfN domain residues described above for iron binding.
  • glycosylation may be reduced or prevented by mutation, including deletion, substitution or insertion into, amino acid residues corresponding to one or more of Tf C domain residues around the N-X-S/T sites corresponding 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 GIu residues.
  • glycosylation, iron and/or carbonate ions may be stripped from or cleaved off of the fusion protein.
  • available de-glycosylases 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.
  • Additional mutations may be made with Tf to alter the three dimensional structure of Tf, such as modifications to the hinge region to prevent Tf folding 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 to or around the flanking regions of these sites to alter Tf structure and function.
  • the transferrin portion of the fusion protein can function as a carrier protein to extend the half life or bioavailability of IFN ⁇ as well as in some instances, delivering IFN ⁇ inside a cell and/or across the blood brain barrier.
  • the fusion protein includes a modified transferrin molecule wherein the transferrin does not retain the ability to cross the blood brain barrier.
  • the fusion protein includes a modified transferrin molecule wherein the transferrin molecule retains the ability to bind to the transferrin receptor and transport the IFN ⁇ inside cells.
  • the transferrin fusion protein includes a modified transferrin molecule wherein the transferrin molecule does not retain the ability to bind to the transferrin receptor and transport IFN ⁇ inside cells.
  • the fusion protein includes a modified transferrin molecule wherein the transferrin molecule retains the ability to bind to the transferrin • . receptor and transport the fused IFN ⁇ inside cells, but does not retain the ability to cross the blood brain barrier.
  • the fusion protein includes a modified transferrin molecule wherein the transferrin molecule retains the ability to cross the blood brain barrier, but does not retain the ability to bind to the transferrin receptor and transport the fused IFN ⁇ inside cells.
  • the fusion proteins of the invention may contain one or more copies of IFN ⁇ attached to the N-terminus and/or the C-terminus of the Tf protein.
  • IFN ⁇ is attached to both the N- and C-terminus of the Tf protein and the fusion protein may contain one or more equivalents of IFN ⁇ on either or both ends of Tf.
  • IFN ⁇ is inserted into known domains of the Tf protein, for instance, into one or more of the loops of Tf (see AIi et al. (1999) J. Biol. Chem. 274(34):24066-24073).
  • IFN ⁇ is inserted between the N and C domains of Tf.
  • IFN ⁇ is inserted anywhere in the transferrin molecule.
  • the fusion protein of the invention may have one modified transferrin-derived region and one IFN ⁇ region. Multiple regions of each protein, however, may be used to make a fusion protein of the invention. Similarly, one or more therapeutic protein in addition to IFN ⁇ may be used to make a fusion protein of the invention of the invention, thereby producing a multi-functional fusion protein.
  • the fusion protein of the invention contains an IFN ⁇ protein or portion thereof fused to a transferrin molecule or portion thereof. In another embodiment, the fusion protein of the invention contains IFN ⁇ fused to the N terminus of a transferrin molecule. In an alternate embodiment, the fusion protein of the invention contains IFN ⁇ fused to the C terminus of a transferrin molecule. In a further embodiment, the fusion protein of the invention contains a transferrin molecule fused to the N terminus of IFN ⁇ . In an alternate embodiment, the fusion protein of the invention contains a transferrin molecule fused to the C terminus of IFN ⁇ . - • • .
  • the modified transferrin molecule contains the C terminus of a transferrin molecule fused to what would be the N terminus of IFN ⁇ . In an alternate embodiment, the modified transferrin molecule contains the N terminus of a transferrin molecule fused to the C terminus of IFN ⁇ .
  • the transferrin fusion protein of the inventions contains IFN ⁇ fused to both the N-terminus and the C-terminus of transferrin or modified transferrin.
  • the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins, and one of the therapeutic proteins is IFN ⁇ .
  • the therapeutic proteins, of which one is IFN ⁇ may be used to treat or prevent the same disease, disorder, or condition.
  • the therapeutic proteins, of which one is IFN ⁇ may be used to treat or prevent diseases or disorders which are known in the art to commonly occur in patients simultaneously.
  • the second therapeutic protein may be used to target the IFN ⁇ activity to a particular organ, tissue or cell type.
  • the fusion protein of the invention may also be produced by inserting IFN ⁇ or a fragment or variant thereof into an internal region of transferrin or modified transferrin.
  • Internal regions of transferrin or modified transferrin include, but are not limited to, the iron binding sites, the hinge regions, the bicarbonate binding sites, or the receptor binding domain.
  • insertions may be made within the loops comprising Tf amino acids 32-33, 74-75, 256-257, 279-280 and 288-289 (AIi et ai, supra) (See Figure 3).
  • 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.
  • 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 preferred embodiment- Such fusions may include a linker region, such as but not limited to a poly-glycine stretch, to separate the therapeutic protein 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 six 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.
  • IFN ⁇ may be complexed with iron and loaded on a modified transferrin fusion protein 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 IFN ⁇ to a particular cell type, e.g., a cancer cell.
  • IFN ⁇ cytokines
  • the present invention provides IFN ⁇ /transferrin fusion proteins with increased IFN ⁇ 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 autoimmune diseases such as multiple sclerosis, polymyositis and rheumatoid arthritis, 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.
  • ⁇ -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 ⁇ - Ia (SEQ ID NO.: 100) and IFN ⁇ - Ib (SEQ ID NO.: 101).
  • 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.
  • the IFN ⁇ moiety can also be modified to exhibit increased or reduced glycosylation.
  • the N-linked glycosylation site of IFN ⁇ l contains one N- glycosylation site at the sequence NET (amino acid residues 80-82 of SEQ ID NO.: 100).
  • the IFN ⁇ moiety contains a mutation within or adjacent to the N-glycosylation site NET.
  • the IFN ⁇ moiety contains a mutation at T82 within the NET site (SEQ ID NO.: 100).
  • amino acid residues from IFN ⁇ can be grafted over the glycoslyation site of IFN ⁇ to prevent glycosylation.
  • amino acid residues 77-87 IFN ⁇ (SEQ ID NO.: 142 can be grafted over amino acid residues 79-89 of IFN ⁇ (SEQ ID NO.: 100) by SOE PCR to prevent or reduce glycosylation.
  • the IFN ⁇ can be modified by the attachment, of one or more oligosaccharide groups.
  • IFN ⁇ may be modified to exhibit altered glycosylation by methods known in the art. For instance, altered glycosylation may result by introducing glycosylation sites by mutagenesis.
  • U.S. Patent Application 2006/0083715 describes the introduction of a glycosylation site and pegylation of IFN ⁇ .
  • the IFN ⁇ moiety has been modified to increase production of the fusion protein in yeast.
  • amino acid residues 34-47 of IFN ⁇ (SEQ ID NO.: 100) can be be replaced with residues 34-46 of IFN ⁇ (SEQ ID NO.: 142) to increase secretion in yeast.
  • modification of the IFN ⁇ moiety increases secretion by about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold or about 15 fold or more.
  • the IFN ⁇ moiety has been modified to further increase antiviral activity, antiproliferative activity and/or immunomodulatory activity.
  • amino acid residues 34-47 of IFN ⁇ can be be replaced with residues 34-46 of IFN ⁇ to increase antiviral activity, anti-proliferative activity and immunomodulatory activity.
  • the IFN ⁇ moiety has been modified to increase antiviral activity by about 5 fold, about 10 fold, about 15 fold, about 20 fold, about 30 fold, about 40 fold, about 50 fold, about 60 fold or about 70 fold or more compared to IFN ⁇ which has not been modified.
  • the IFN ⁇ .
  • the moiety has been modified to increase anti-proliferative activity by about 10 fold, about 20 ' fold, about 30 fold, about 40 fold, about 50 fold, about 60 fold, about 70 fold, about 80 fold, about 90 fold, about 100 fold, about 110 fold, about 120 fold, about 130 fold, about 140 fold, about 150 fold, about 160 fold or about 170 fold or more compared to IFN ⁇ which has not been modified.
  • the IFN ⁇ moiety has been modified to increase immunomodulatory activity by about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 15 fold, about 20 fold or about 30 fold or more compared to IFN ⁇ which has not been modified.
  • any IFN ⁇ molecule may be used as the fusion partner to Tf according to the methods and compositions of the present invention.
  • the IFN ⁇ exerts one or more therapeutic effects in vivo or in vitro.
  • a therapeutic, i.e., 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 one or more disease symptoms or a modulation, alleviation or cure of the underlying defect to produce an effect beneficial to the treated subject.
  • the fusion protein of the invention includes at least a fragment or variant of IFN ⁇ and at least a fragment or variant of modified serum transferrin, which are associated with one another, preferably by genetic fusion or chemical conjugation.
  • the fusion protein can contain IFN ⁇ peptide fragments or peptide variants of proteins wherein the variant or fragment retains at least one IFN ⁇ biological or therapeutic activity.
  • the fusion proteins can contain IFN ⁇ peptides 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 transferrin or modified transferrin.
  • the fusion protein of the present invention may contain multiple IFN ⁇ peptides.
  • the fusion protein of the invention contains IFNp and a second therapeutic peptide. Increasing the number of peptides may enhance the function of the peptides fused to transferrin and the function of the entire fusion protein.
  • the fusion protein of the invention contains an IFN ⁇ portion that is the full length protein.
  • the IFN ⁇ portion is an IFN ⁇ polypeptide having one or more residues deleted from the amino terminus of the amino acid sequence.
  • the invention includes an IFN ⁇ moiety lacking an initial methionine amino acid residue.
  • the IFN ⁇ portion of the fusion protein includes an IFN ⁇ polypeptide having one or more residues deleted from the carboxy terminus of the amino acid sequence.
  • the present invention also includes fusion proteins containing an IFN ⁇ portion with one or more amino acids deleted from both the amino and carboxy termini.
  • the fusion protein of the invention contains an IFN ⁇ portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference IFN ⁇ protein set forth herein, or fragments thereof.
  • the fusion molecules contain an IFN ⁇ 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 fusion molecules contain an IFN ⁇ 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 IFN ⁇ . Fragments, of these polypeptides are also provided.
  • polypeptide-having an amino acid sequence at least, for example, 95% "identical" to a query IFN ⁇ amino acid sequence of the present invention it is intended that the amino acid sequence of the subject polypeptide is identical to the query IFN ⁇ 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 amino acid sequence of the subject polypeptide is identical to the query IFN ⁇ 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.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.
  • IFN ⁇ reference sequence may occur at the amino- or carboxy-terminal positions of the reference IFN ⁇ amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference IFN ⁇ sequence, or in one or more contiguous groups within the reference IFN ⁇ sequence.
  • any particular polypeptide is at least about 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to, for instance, the IFN ⁇ amino acid sequence of a fusion protein of the invention or a fragment thereof (such as the IFN ⁇ portion), can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Bru ⁇ ag-et al. (Comp. App. Biosci 245- (1990)).
  • the IFN ⁇ 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, VaI, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and GIu; 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 Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and GIy.
  • 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 transferrin, and/ modified transferrin 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 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-tra ⁇ slational 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 formation, demethylation, formation of covalent cross-links, formation of cysteine, glyeosylation, 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 arginylaltion, and ubiquitination.
  • IFN ⁇ is regarded as an active principle not only in the treatment and prophylaxis of viral diseases including, but not limited to, herpes, influenza and hepatitis, but also in the treatment of tumoral conditions such as encephaloma and leukemia.
  • IFN ⁇ is used to treat multiple sclerosis, rheumatoid arthritis, polymyositis, 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. (See Nakajima et al. (2003) Acta. Med. Okayama. 57(5): 217-25; van Holten et al. (2004) Arthritis Res. Ther. 6(3): R239-49; and Dresseland Beuche (2002) J. Neurol. Neurosurg. Psychiatry. 72: 676).
  • 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. Additionally, 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 STATl and STAT2
  • STATl and STAT2 is known to upregulate and downregulate a wide variety of genes, most of which are involved in the antiviral immune response.
  • IFN ⁇ is induced by the presence of dsRNA, both DNA and RNA viruses are sensitive to the effects of IFN ⁇ (Biron, Seminars in Immunology, 10: 383-390 (1998)).
  • IFN ⁇ is generally produced in response to a viral infection. 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 etal, 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).
  • 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). However, 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 Although at the end of a successfully defeated infection T cells die by apoptosis as the immune system returns to a homeostatic balance, some T cells must avoid apoptosis and enter a Go/Gi 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.
  • the former apoptotic pathway is blocked by IFN ⁇ dependent upregulation of Bcl-x, an apoptotic inhibitor. Fas ligation- induced apoptosis occurs much too quickly to be blocked by upregulation of a gene, so IFN ⁇ must block that apoptotic pathway by different means (Scheel-Toellner et al, European Journal of Immunology 29:2603-2612 (1999)). The existence of a second blocking mechanism is supported by the results of Marrack et al. (Journal of Experimental Medicine 189:521-529(1999)), who found that IFN ⁇ prevented T cell apoptosis without increased production of Bcl-x.
  • 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.
  • MS multiple sclerosis
  • 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 ⁇ - Ia a eukaryotic, glycosylated form obtained from hamsters
  • IFN ⁇ - 1 a is produced by recombinant DNA technology.
  • Interferon beta- 1 a 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 maybe 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 ⁇ w.s-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- Ib).
  • IFN ⁇ may also inhibit the induction of inducible nitric oxide synthase (INOS) expression by IL-I and IFN- ⁇ . Production of nitric oxide by INOS in astrocytes has been implicated as a factor in the parthenogenesis of MS (Hua et al. 1998. Beta inteferon prevents nitric oxide/peroxynitrate from damaging the central nervous system).
  • INOS inducible nitric oxide synthase
  • 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 fusion 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 skill 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.
  • the present invention also provides nucleic acid molecules encoding fusion proteins comprising a transferrin protein or a portion of a transferrin protein covalently linked or joined to IFN ⁇ .
  • 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 transferrin protein or portion thereof and the therapeutic protein, preferably 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 preferred. Many vectors and host cells are known in the art for such purposes. It is well within the skill of the art to select an appropriate set for the desired application.
  • DNA sequences encoding transferrin, portions of transferrin and IFN ⁇ 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 Transferrin Sequences from Different Species. Comp. Biochem. Physiol. 106B/1:203-218 and all references cited therein, which are hereby incorporated by reference in their entirety).
  • 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.
  • 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).
  • Preferred 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. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, etal, Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, et al., J. Molec. Biol. 215:403 (1990)). The degree of similarity or identity referred to above is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the degree of identity between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch (1970) Journal of Molecular Biology 48:443-453). For purposes of determining the degree of identity between two nucleic acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3. Codon Optimization
  • the degeneracy of the genetic code permits variations of the nucleotide sequence of a transferrin 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 incorporated 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. In particular, available methods may be used to alter the codons encoding a given fusion protein with those most readily recognized by yeast when yeast expression systems are used.
  • the degeneracy of the genetic code permits the same amino acid sequence to be encoded and translated in many different ways.
  • leucine, serine and arginine are each encoded by six different codons
  • valine, proline, threonine, alanine and glycine are each encoded by four different codons.
  • the frequency of use of such synonymous codons varies from genome to genome among eukaryotes and prokaryotes.
  • synonymous codon-choice patterns among mammals are very similar, while evolutionarily distant organisms such as yeast (S. cerevisiae), bacteria (such as E. coli) and insects (such as D.
  • the preferred 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 nucleotide sequence of IFN ⁇ . •• ⁇ - • >- • ⁇
  • 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 fusion protein comprising transferrin protein or a portion of a transferrin protein joined to a DNA sequence encoding IFN ⁇ , and a transcriptional terminator.
  • a transcriptional promoter operably linked in a 5' to 3' orientation
  • a transcriptional promoter a secretory signal sequence
  • a DNA sequence encoding fusion protein comprising transferrin protein or a portion of a transferrin protein joined to a DNA sequence encoding IFN ⁇
  • a transcriptional terminator any arrangement of IFN ⁇ 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.
  • Suitable yeast vectors for use in the present invention are described in U.S. Patent 6,291,212 and include YRp7 (Struhl et al, Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEpI 3 (Broach et al, Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, pC4 and derivatives thereof.
  • Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and the Pichia vectors available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, ⁇ RS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, 7RPI, LEU2 and URA3.
  • Plasmids pRS413 ⁇ 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 transformants 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 POTl (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. MoI. 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, N.Y., 1982; Ammerer, Meth. Enzymol. 101 : 192-201 j : l 983).
  • particularly preferred promoters are the TPIl 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 preferred transcriptional terminator is the TPIl terminator (Alber and Kawasaki, ibid.).
  • 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 etal., EMBO J. 4: 2093-2099, 1985) and the tpiA promoter.
  • An example of 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, preferably a transferrin fusion protein comprising a modified Tf.
  • Preferred promoters include viral promoters and cellular promoters.
  • Viral promoters include the major late promoter from adenovirus 2 (Kaufman and Sharp, MoI. Cell. Biol. 2: 1304-13199, 1982), the cytomegalovirus promoter (Nelson, J.A. et al., Mole. Cell. Biol. 7: 4125-4129) and the SV40 promoter (Subramani et al, MoI. Cell. Biol. 1: 854-864, 1981).
  • Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., Science 222: 809-814, 1983) and a mouse V6 (see U.S. Patent 6,291,212) promoter (Grant et al., Nuc. Acids Res. 15: 5496, 1987).
  • the mouse Y H (see U.S. Patent 6,291,212) promoter can also be used with the fusion protein of the invention.
  • 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 transferrin fusion protein. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
  • polyadenylation signal located downstream of the coding sequence of interest.
  • Polyadenylation signals include the early or late polyadenylation signals-from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 ElB region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981).
  • a particularly preferred polyadenylation signal is the VH (see U;S. Patent 6,291,212) gene terminator.
  • 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.
  • Preferred 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 adenovirus VA RNAs. - ⁇ . / ⁇ ...- .- ⁇
  • Cloned DNA sequences comprising 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.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
  • the selectable marker may be an amplif ⁇ able selectable marker.
  • a preferred amplifiable selectable marker is the DHFR gene.
  • a particularly preferred 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 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.
  • Exemplary genera of yeast contemplated to be useful in the practice, of the present invention as hosts for expressing the fusion protein of the inventions are Pichia (formerly classified as Hansenul ⁇ ), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaecharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopyis, and the like.
  • Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.
  • Kluyveromyces spp. are K. ftagilis, K. lactis and K. marxianus.
  • a suitable T ⁇ rulasppra species is T. delbrueckii.
  • Pichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomal ⁇ ) and P. pastoris.
  • yeast Saccharomyces cerevisiae are another preferred 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.
  • the host strain carries 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 constructs 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 construct by, for example, drug selection or deficiency in an essential nutrient which are complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.
  • Yeast cells for example, are preferably grown in a chemically defined medium, comprising 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 6.5.
  • Methods for maintaining a stable pH include buffering and constant pH control, preferably through the addition of sodium hydroxide.
  • Preferred 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 preferred 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 serum- containing or serum-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. Drug selection is then applied to select for growth of cells that are ⁇ expressing the selectable marker hi a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.
  • Baculovirus/insect cell expression systems may also be used to produce the fusion proteins of the invention.
  • the BacPAKTM Baculovirus 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.6 viral DNA.
  • the BacPAK ⁇ DNA is missing an essential portion of the baculovirus genome.
  • the DNA recombines with the vector, the essential element is restored and the target gene is transferred to the baculovirus genome.
  • a few viral plaques are picked and purified, and the recombinant phenotype is verified.
  • the newly isolated recombinant virus can then be amplified and used to infect insect cell cultures to produce large amounts of the desired protein.
  • 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 incorporated 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 fusion proteins of the invention.
  • One such secretory peptide that may be used in combination with other secretory peptides is the third domain of the yeast Barrier protein.
  • 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. Preferred 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 IFN ⁇ of the 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, for instance, for binding to its cognate receptor.
  • the linker peptide may consist of amino acids that are flexible or more rigid.
  • the linker can be less than about 50, 40, 30, 20, or 10 amino acid residues. However, there is no upper limit on the size of a linker peptide of the invention.
  • the linker can be covalently linked to and between the transferrin protein or portion thereof and the therapeutic protein.
  • IFN ⁇ is linked to Tf or mTf via a substantially non-helical linker.
  • rigid linkers include PE, PEA, PEAPTD (SEQ ID NO.: 77), (PEAPTD) 2 (SEQ ID NO.: 78), (PEAPTD) 3 (SEQ ID NO.: 79), or (PEAPTD) n (SEQ ID NO.: 102), wherein n is an integer.
  • the present invention also provides the IgG hinge linker, the CEx linker, i.e., C-terminal extension to Exendin-4, (SSGAPPPS; SEQ ID NO.: 103), the IgG hinge linker in conjunction with the PEAPTD linker and the IgG hinge linker in conjunction with the CEx linker. Detection of Fusion Proteins
  • Assays for detection of biologically active modified transferrin-fusion protein may include Western transfer, protein blot or colony filter as well as activity based assays that detect the fusion protein comprising transferrin and therapeutic protein.
  • a Western transfer filter may be prepared using the method described by Towbin et al. (Proc. Natl. Acad. Sd. USA 76: 4350-4354, 1979). Briefly, samples are electrophoresed in a sodium dodecyl sulfate polyacrylamide gel. The proteins in the gel are electrophoretically transferred 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 preferred. 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 preferred buffer comprises 25 mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.
  • Fusion proteins of the present invention may be labeled with a radioisotope or other imaging agent and used for in vivo diagnostic purposes.
  • Preferred radioisotope imaging agents include iodine-125 and technetium-99, with technetium- 99 being particularly preferred.
  • 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 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).
  • Detection of a fusion protein of the present invention can be facilitated by coupling ⁇ i.e., physically linking) IFN ⁇ to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolumiriescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinyl amine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luc ⁇ ferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 I, 35 S or 3 H.
  • 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
  • the binding of the fusion protein is detected by detecting a label on the fusion protein.
  • the fusion protein is detected by detecting binding of a secondary antibody or reagent that interacts with the fusion protein.
  • the secondary antibody or reagent is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • Fusion proteins of the invention may also be detected by assaying for the activity of the therapeutic protein moiety. Specifically, fusion proteins of the invention may be assayed for functional activity (e.g., biological activity or therapeutic activity) using assays known to one of ordinary skill in the art. Additionally, one of skill in the art may routinely assay fragments of IFN ⁇ of a fusion protein of the invention, for activity using well-known assays. Further, one of skill in the art may routinely assay fragments of transferrin protein for activity using assays known in the art.
  • functional activity e.g., biological activity or therapeutic activity
  • 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.
  • 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 transferrin 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.
  • the present invention further provides methods for producing a 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 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 transform 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.
  • 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 preferred.
  • yeast systems that have been modified to reduce native yeast glycosylation, hyper-glycosylation or proteolytic activity may be used.
  • Secreted, biologically active 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 preferred purification method is affinity chromatography on an iron binding or metal chelating column or an immunoaffinity chromatography using an antigen directed against the transferrin or therapeutic protein of the polypeptide fusion.
  • the antigen is preferably immobilized or attached to a solid support or substrate.
  • a particularly preferred substrate is CNBr-activated Sepharose (Pharmacia LKB Technologies, Inc., Piscataway, N.J.).
  • Particularly useful methods of elution include changes in pH, wherein the immobilized antigen has a high affinity for the transferrin fusion protein 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. Delivery of a Drug or Therapeutic Protein to the inside of a Cell and/or across the Blood Brain Barrier (BBB)
  • BBB Blood Brain Barrier
  • the fusion proteins may be used as a carrier to deliver IFN ⁇ complexed to the ferric ion of transferrin to the inside of a cell or across the blood brain barrier.
  • the transferrin will typically be engineered or modified to inhibit, prevent or remove glycosylation to extend the serum half-life of the . transferrin fusion protein and/or therapeutic protein.
  • the addition of a targeting peptide is specifically contemplated to further target the transferrin 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 fusion protein of the invention.
  • Ferric-sorbitol-citrate 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 transferrin 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 fusion proteins comprising transferrin or modified transferrin and IFN ⁇ may be administered to a patient in need thereof using standard administration protocols.
  • the 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 fusion proteins of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal and buccal routes.
  • an agent maybe 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.
  • 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 or about 100-200 mg of protein/dose. 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 include, 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 pharmaceutical composition of the present invention can be in unit dosage form, ' e.g. as tablets or capsules.
  • the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient;
  • the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids.
  • the unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.
  • the dosage to be used in the treatment must be subjectively determined by the-physician.
  • the fusion proteins 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 coadministered 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, or in vitro.
  • transgenic non-human animals that contain a fusion construct with increased IFN ⁇ serum half-life, increased IFN ⁇ serum stability or increased IFN ⁇ bioavailability of the instant invention is contemplated.
  • lactoferrin may be used as the Tf portion of the fusion protein so that the fusion protein is produced and secreted in milk.
  • 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. Biochem. 49:113 [1992]).
  • Other methods for the production of transgenic animals include the infection of embryos with retroviruses or with retroviral vectors. Infection of both pre- and post-implantation mouse embryos with either wild-type or recombinant retroviruses has been reported (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]; Janenich et al, Cell 24:519 [1981]; Guatemalamann et al, Proc. Natl. Acad. Sci.
  • An alternative means for infecting embryos with retroviruses is the injection of virus or virus-producing cells into the blastocoele of mouse embryos (Jahner, D. et al, Nature 298:623 [1982]).
  • the introduction of transgenes into the germline of mice has been reported using intrauterine retroviral infection of the midgestation mouse embryo (Jahner et al., supra [1982]).
  • Infection of bovine and ovine embryos with retroviruses or retroviral vectors to create transgenic animals has been reported.
  • 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.
  • U.S. 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.
  • fusion constructs for gene therapy wherein a modified transferrin protein or transferrin domain is joined to IFN ⁇ is contemplated in one embodiment of this invention.
  • the fusion constructs with increased IFN ⁇ serum half-life or IFN ⁇ serum stability of the instant invention are ideally suited to gene therapy treatments.
  • U.S. Patent 6,225,290 provides methods and constructs whereby intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate 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 administration 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 tract 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. 6,140,1 11 (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 have 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 JuI. 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 incorporated herein by reference.
  • kits containing fusion proteins comprising transferrin and IFN ⁇ which can be used, for instance, for the therapeutic or non-therapeutic applications.
  • the kit comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which includes a transferrin fusion. protein that is effective- for therapeutic or non-therapeutic applications, such as described above.
  • the active agent in the composition is the therapeutic protein.
  • the label on the container indicates that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • ⁇ -IFN is effective in the treatment 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 ⁇ -IFN/mTf fusion protein.
  • IFN ⁇ -1 was fused to modified transferrin at either the N- or C- terminus.
  • the IFN ⁇ -1 clone was obtained from 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 sequence of the clone were 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 destroy the Kpnl site and clip the beginning of mTf.
  • the linkers were annealed and ligated into pREX0054 cut with XbaMKpnl creating an intermediate vector with mTf untouched and a. Kpnl site that was used to fuse the IFN ⁇ -1 gene at the N-terminus of mTf.
  • a XbaVKpnl 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, pREX5048 was created by ligating the IFN ⁇ -1 gene cut with XbaVKpnl into the XbaVKpnl cut intermediate vector.
  • the IFN ⁇ -1 a sequence was modified by site directed mutagenesis to eliminate the free cysteine, C17S (tgt to agt), and to eliminate the N-linked glycosylation site, T82A (act to get) using the primers as shown.
  • the fusion leader (FL) was replaced by the transferrin secretion signal (nL) for improved expression in yeast and for mammalian expression.
  • N-terminal fusion this was achieved by use Single Overlapping Extension PCR (SOE-PCR) with the primers P0245 and P 1025 and using pREX0048 as the template.
  • SOE-PCR Single Overlapping Extension PCR
  • the product from this PCR was digested with the restriction enzymes Afl ⁇ l and BamHI and ligated into A ⁇ ll+BamKl digested pREX0549 ( Figure 4) to give ⁇ REX0549 IFNb mTf.
  • the plasmid pREX0049 was partially digested with the restriction enzyme BstEll and with ifmdIII to recover the 559bp fragment of the 3' end of mTf and the complete IFN ⁇ sequence and ligated into J?s/EII+HzrcdIII digested pREX0197 ( Figure 5) to give pREX0435 ( Figure 6).
  • Ex ample 4 Expression of IFN ⁇ constructs in mammalian cells
  • the vector pcDNA3.1 was used and ligation was via directional blunt end TOPO® cloning (pcDNATM3.1 Directional TOPO® Expression Kit. Catalog nos. K4900-01, K4900-40. Invitrogen).
  • DNA encoding a linker (PEAPTD) 2 between the mTf and IFN ⁇ sequences was introduced into the various constructs by SOE-PCR with appropriate primers, as exemplified for a C-terminal fusion with primers P0887 and PO888.
  • S17C was constructed with the addition of the N-terminal methionine added at position 1 using pREX1007 as a template with the following primers:
  • IFNX430 was shown to result in increased antiviral activity of 25-62x, antiproliferative activity of 31-157x, and immunomodulatory activity of 5-26x when compared to IFN ⁇ (US 4,769,233).
  • the IFN ⁇ sequence in the mTf fusion constructs was modified to remove the glycosylation site NET by mutation of the codon encoding T to A. Removal of glycosylation in IFN ⁇ exposes a hydrophobic region on the surface of IFN ⁇ , the equivalent region in IFN ⁇ is mostly hydrophilic. Grafting over residues 79-89 of IFN ⁇ with residues 77-87 of IFN ⁇ removes the glycosylation site whilst making the region more hydrophilic. The result may be better production due to less aggregation.
  • the IFN ⁇ 34-46 residues were grafted into the IFN ⁇ sequence over residues 34-47 by SOE-PCR with the primers P0518 and P0519.
  • the IFN ⁇ 77-87 residues were grafted into the IFN ⁇ sequence over residues 79-89 by SOE PCR with the primers P0521 and P0522.
  • AAGGAGGACGCCGCATTGAC (SEQ ID NO.: 133) P0519 CTGGTTGCCAAACTCCTCCTGGGGAAATCCAAAGTCATGCCTGTCCTT
  • GAGGCAATATTCAAGC (SEQ ID NO.: 134) PO521 GATGAGACCCTCCTAGACAAATTCTACACTGAAGTCTATCATCAGATA
  • AACCATCTGAAG (SEQ ID NO.: 135) PO522 TTCAGTGTAGAATTTGTCTAGGAGGGTCTCATCCCAGCCAGTGCTAGA
  • Example 8 In vivo and in vitro evaluation of IFN ⁇ Tf fusion proteins
  • IFN ⁇ Tf fusion proteins were produced in CHO cells, purified by affinity chromatography using an anti-IFN ⁇ antibody column and then tested for antiviral activity, pharmacokinetics and pharmacodynamics. .. .
  • VSV vesicular stomatitis virus
  • BRXlOOl 250 ⁇ g/kg
  • BRX1007 (175 ⁇ g/kg)
  • BRX1030 ⁇ 222 ⁇ g/kg
  • Plasma levels of fusion protein were determined by a sandwich ELISA assay using antibodies to both transferrin and interferon; thus the assay was specific for fusion protein.
  • the elimination half-life of the fusion proteins was approximately 20hr ( Figure 9), whereas the published half-life for Betaseron® is 1.9hr.
  • Neopterin is synthesized by macrophages in response to stimulation by interferon and, as such, is a good biomarker of interferon activity in vivo.
  • .Cynomolgus monkeys were injected subcutaneously with either BRX0760 (130 ⁇ g/kg), BRXlOOl (250 ⁇ g/kg), BRX1007 (175 ⁇ g/kg), BRX1030 (222 ⁇ g/kg) or 1 MIU/kg IFN ⁇ -la and blood was drawn at the indicated time points. Plasma neopterin levels were determined using a commercially available ELISA kit (Alpco). The IFN ⁇ Tf fusion proteins produced a robust neopterin response (Figure 10).

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  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des protéines de fusion de la transferrine et de l'IFNβ dont la durée de vie ou la stabilité du sérum est accrue. Des protéines de fusion préférée comprennent celles qui sont modifiées de sorte que la fraction de la transferrine ne présente pas de glycosylation ou présente une glycosylation réduite, se liant au fer et/ou au récepteur de la transferrine.
PCT/US2007/018099 2006-08-14 2007-08-14 PROTÉINES DE FUSION DE L'INTERFÉRON β ET DE LA TRANSFERRINE Ceased WO2008021412A2 (fr)

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US83732306P 2006-08-14 2006-08-14
US60/837,323 2006-08-14

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WO2008021412A3 WO2008021412A3 (fr) 2008-04-24

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030823A (zh) * 2010-06-21 2011-04-27 中国人民解放军军事医学科学院放射与辐射医学研究所 一种肝脏靶向性的基因工程干扰素及其制备方法
US20110229518A1 (en) * 2008-11-28 2011-09-22 Statens Serum Institut Optimized influenza vaccines
US9611323B2 (en) 2010-11-30 2017-04-04 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses therefor

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8412564D0 (en) * 1984-05-17 1984-06-20 Searle & Co Structure and properties
US6825037B1 (en) * 1991-02-08 2004-11-30 University Of Vermont Recombinant transferrins, transferrin half-molecules and mutants thereof
US7176278B2 (en) * 2001-08-30 2007-02-13 Biorexis Technology, Inc. Modified transferrin fusion proteins
WO2005003296A2 (fr) * 2003-01-22 2005-01-13 Human Genome Sciences, Inc. Proteines hybrides d'albumine
JP2008531059A (ja) * 2005-03-04 2008-08-14 バイオレクシス ファーマシューティカル コーポレーション 改変トランスフェリン融合タンパク質

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110229518A1 (en) * 2008-11-28 2011-09-22 Statens Serum Institut Optimized influenza vaccines
US9764024B2 (en) * 2008-11-28 2017-09-19 Statens Serum Institut Optimized influenza vaccines
CN102030823A (zh) * 2010-06-21 2011-04-27 中国人民解放军军事医学科学院放射与辐射医学研究所 一种肝脏靶向性的基因工程干扰素及其制备方法
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

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